Resolution of rat mitochondrial matrix proteins by two-dimensional polyacrylamide gel electrophoresis.

The mitochondrial matrix subfractions from rat liver, kidney cortex, brain, heart, and skeletal muscle were isolated and their protein components were resolved by two-dimensional polyacrylamide gel electrophoresis, revealing between 120 and 150 components for each matrix subfraction. Excellent resolution was obtained utilizing a pH 5 to 8 gradient in the first dimension and in 8 to 13% exponential acrylamide gradient in the second dimension, increasing the number of mitochondrial matrix proteins observed 3-fold over one-dimensional systems. Protein components tentatively identified by co-migration with pure enzymes and by known tissue distributions are carbamoyl-phosphate synthetase (EC 2.7.2.5), ornithine transcarbamylase (EC 2.1.3.3), glutamate dehydrogenase (EC 1.4.1.3), pyruvate carboxylase (EC 6.4.1.1), citrate synthase (EC 4.1.3.7), fumarase (EC 4.2.1.2), aconitase (EC 4.2.1.3), alpha-ketoglutarate dehydrogenase (EC 1.2.4.2), dihydrolipoyl transsuccinylase (EC 2.3.1.12), lipoamide dehydrogenase (EC 1.6.4.3), glutamate-aspartate aminotransferase (EC 2.6.1.1), and the two subunits of pyruvate dehydrogenase (EC 1.2.4.1). Protein components unambiguously identified by peptide mapping are citrate synthase, aconitase, and pyruvate carboxylase. The inner membrane subfraction from rat liver mitochondria was also resolved two dimensionally; the alpha and beta subunits of ATPase (F1) (EC 3.6.1.3) were identified by peptide mapping.


Purification and characterization of branched chain alpha-ketoacid dehydrogenase from bovine liver mitochondria.

Branched chain alpha-ketoacid dehydrogenase (EC 1.2.4.3(4)) was solubilized and purified from bovine liver mitochondria for the first time. Decarboxylation of alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, and alpha-ketoisocaproate was catalyzed by this multienzyme complex and this activity was co-purified for each substrate. Three enzymatic functions were contained in the complex including decarboxylation of the above ketoacids, transacylation of their simple acid derivatives, and reduction of NAD+ as an overall reaction. Product stoichiometry of these three reactions was 1 CO2:1 acyl-CoA:1 NADH. Activity depended upon the addition of thiamin pyrophosphate, CoASH, and NAD+ which were dissociable cofactors. Physically, two active forms of the enzyme complex were found: a 275,000-dalton unit and a 2 x 10(6)-dalton component. Both showed a characteristic flavin spectra and catalyzed all functions of the complex, implying that 10 small units aggregated into the larger unit. The soluble complex as visualized by electron microscopy had a diameter ranging from 12 to 24 nm corresponding to a molecular weight of 2 x 10(6). The size of the native membrane-bound component remains to be determined.


Preferential affinity of high molecular weight high mobility group non-histone chromatin proteins for single-stranded DNA.

We have subjected proteins dissociated from chicken erythrocyte or calf thymus chromatin by 0.35 M NaCl to sequential chromatography on columns containing immobilized double-stranded DNA and single-stranded DNA. At 0.2 M NaCl, 1 mM Tris . Cl (pH 7.5), the high molecular weight, high mobility group proteins (HMG-1, HMG-2, and HMG-E), were not retained by double-stranded DNA columns, but were retained by single-stranded DNA columns. Thus, in that solvent, those proteins exhibit selective affinity for single-stranded DNA. This suggests that the functions of the high molecular weight, high mobility group proteins might involve destabilizing the DNA double helix by virtue of their preferential affinity for single-stranded DNA.


Studies on the oxidation of isobutyrylcarnitine by beef and rat liver mitochondria.

Mitochondria from beef liver oxidize isobutyrylcarnitine at approximately 50% the rate of succinate in the presence of rotenone. However, the oxidation rate of isobutyryl coenzyme A in the presence of l(-)-carnitine is very low and can be negligible in both rat and beef liver mitochondria. The limited stimulation of isobutyryl-CoA oxidation by l(-)-carnitine appears to be due to inhibition of isobutyrylcarnitine translocation rather than lack of formation of isobutyrylcarnitine. This conclusion is supported by the fact that: 1) isobutyrylcarnitine oxidation is inhibited by l(-)-carnitine; 2) some oxidation of isobutyryl-CoA is obtained when a low concentration (50 microM) of l(-)-carnitine is used; and 3) under conditions of high isobutyryl-coenzyme A and l(-)-carnitine concentrations (1 mM), isobutyryl-carnitine is produced in near theoretical amounts by these rat liver mitochondria. Other studies demonstrated that less than 25% of the carnitine isobutyryl transferase activity of beef liver mitochondria and rat liver mitochondria is located on the cytosol side of the acylcoenzyme A barrier of these mitochondria.


Enhancement of hormonal stimulation in intact cells. Potentiation of GTP-dependent activation of adenylate cyclase.

The ability of prostaglandin E1 (PGE1) and cholera toxin to increase cyclic AMP levels is potentiated 6-fold when normal rat kidney (NRK) cells are treated with picolinic acid or histidinol, or grown in isoleucine-deficient medium. The response to (-)-isoproterenol is increased 2-fold in NRK cells treated with picolinic acid but not in cells subjected to isoleucine deprivation. The increase in agonist responsiveness is time-dependent, reaches its maximum at 40 h, and is quickly reversed following removal of picolinic acid or addition of medium with normal amounts of isoleucine. The cholera toxin response is also increased about 7-fold in simian virus 40-transformed NRK cells and Moloney sarcoma virus-transformed NRK cells treated with picolinic acid. GTP-stimulated, but not fluoride-stimulated, adenylate cyclase activities are increased in membranes from NRK cells treated with picolinic acid or starved for isoleucine, indicating that the increased response is due, at least in part, to a specific potentiation of GTP-dependent functions of the adenylate cyclase system. The results demonstrate that GTP-dependent events in hormonal stimulation of adenylate cyclase can be altered in intact cells to modulate hormonal enhancement of cyclic AMP production.


Isomerization of the muscarinic receptor . antagonist complex.

The mechanism of binding of two antagonists, 3-quinuclidinyl benzilate and N-methyl-4-piperidinyl benzilate, to the muscarinic receptor was studied. The pseudo-first order rate constant of association showed a hyperbolic dependence on the concentration of the antagonist(s) indicating that the interaction involves two equilibria. The first binding equilibrium is reached rapidly and is characterized by dissociation constants 2.7 +/- 0.4 nM and 6.7 +/- 2.5 nM in phosphate buffer (0.05 M, pH = 7.4) for 3-quinuclidinyl benzilate and N-methyl-4-piperidinyl benzilate, respectively. The first binding equilibrium is followed by a slower isomerization step of the receptor . antagonist complex. The equilibrium constants for the isomerization step of the complex for both ligands were about 0.15. The overall constant of binding obtained as the product of the above constants shows good agreement with the results of equilibrium binding studies.


Evidence for the existence of discrete activator and substrate sites for CO2 on ribulose-1,5-bisphosphate carboxylase.

When incubated with CO2 and Mg2+, ribulose-1,5-bis-phosphate carboxylase forms a ternary complex of enzyme . CO2 . Mg. This complex was prepared using high specific activity [14C]O2 and injected into a solution containing a large (50- to 112-fold) molar excess of [12C]O2 and sufficient ribulose 1,5-bisphosphate to permit the catalytic site to turn over several times. The enzyme was then rapidly separated from the other components by gel filtration and its radiospecific activity was determined to be 30 to 60 times that of the medium. If the CO2 activator and the CO2 substrate sites were one and the same, then, following turnover, the enzyme should have been in isotopic equilibrium with the medium. The finding that this was not the case, by a factor of about 40, indicates that the CO2 activator site is physically distinct from the CO2 substrate site.


The effect of prothrombin fragment 2 on the inhibition of thrombin by antithrombin III.

The effect of prothrombin fragment 2 on the inhibition of thrombin by antithrombin III has been studied. Fragment 2 was found to slow the rate of inhibition of thrombin by antithrombin III about 3-fold. The effect of prothrombin fragment 2 on antithrombin III inhibition was examined by comparing its action in the presence of either thrombin or meizothrombin (des fragment 1). The second order rate constants for antithrombin III inhibition of thrombin with saturating fragment 2 and antithrombin III inhibition of meizothrombin (des fragment 1) were the same. Prothrombin fragment 2 had no effect on either antithrombin III inhibition of meizothrombin (des fragment 1) or Factor Xa. The effect of the fragment on the reaction mechanism of thrombin inhibition was evaluated to see if the fragment altered binding of antithrombin III to thrombin or inhibited the formation of the covalent complex. The fragment was found to have no inhibitory effect on the rate of covalent complex formation, indicating that the protective effect of the fragment is by inhibiting binding of antithrombin III to thrombin. These data suggest that prothrombin fragment 2 may be an important factor in controlling the localization of clot formation by regulating the interaction between thrombin and antithrombin III.


Purification of proline-rich proteins from parotid glands of isoproterenol-treated rats.

Prolonged isoproterenol treatment of rats is known to cause hypertrophy and hyperplasia of the parotid glands. Our results show that a dramatic increase in the synthesis or accumulation in the parotid glands of a series of proteins rich in proline also occurs with isoproterenol treatment. After 10 days of treatment (5 mg of isoproterenol/day) these proline-rich proteins (PRPs) comprise more than 50% of the total soluble proteins in parotid gland homogenates. The PRPs are rapidly labeled in vivo by a single intraperitoneal injection of [3H]proline with maximum incorporation occurring at about 3. More than 90% of the [3h]proline found in parotid gland homogenates is incorporated into PRPs with less than 1% of the radioactivity in alpha-amylase. Tritium incorporated into PRPs was isolated as [3H]proline after acid hydrolysis. One acidic and six basic 3H-labeled PRPs were isolated from the 100,000 x g supernatant fraction of parotid gland homogenates by Sephadex G-100 and ion exchange chromatography. The six basic proteins accounted for about 90% of the total PRPs isolated.


Genetic regulation of coumarin hydroxylase activity in mice. Biochemical characterization of the enzyme from two inbred strains and their F1 hybrid.

Hydroxylation of coumarin to 7-hydroxycoumarin by liver microsomes from control or phenobarbital-pretreated mice is 5- to 10-fold higher in the DBA/2J strain compared to the AKR/J strain, while activities of nine other cytochrome P-450 mediated oxidations show only minor differences. Mixing experiments with whole liver homogenates and subcellular fractionations do not reveal the presence of enzyme activators or inhibitors or competing enzyme reactions in either strain. Comparisons of pH optima (pH 7.6), heat stability at 52 degrees C (6 to 8 min for 50% inactivation), and Km values (0.45 to 0.50 microM coumarin) for coumarin hydroxylase show no significant differences in the two strains of mice or their F1 hybrid. Similarly, only minor differences in inhibition of coumarin hydroxylase by carbon monoxide, SKF-525A, menadione, and several other inhibitors of microsomal mixed function oxidase reactions are observed in the two strains. In contrast to these data, aniline and metyrapone, two compounds which bind to the heme iron of cytochrome P-450 to form ferrihemochromes, show differential and opposite patterns of inhibition of enzyme activity in the DBA/2J and AKR/J mouse strains. This latter observation suggests that a structurally different cytochrome P-450 may hydroxylate coumarin in these two inbred mouse strains.


Concanavalin A inhibition of alpha-bungarotoxin binding to a nonfusing muscle cell line.

Incubation of a nonfusing muscle cell line, BC3H1, with concanavalin A (Con A) results in a maximum decrease of 35% in the cell's ability to bind alpha-bungarotoxin (alpha-BuTx). The Con A-induced inhibition of 125I-alpha-BuTx binding is reversible and the degree of inhibition parallels the degree of saturation of Con A binding sites on the cell surface. The maximum level of Con A-induced inhibition of 125I-alpha-BuTx binding is not affected by increasing the time of incubation in Con A, using higher concentrations of Con A or by increasing the time of incubation in the presence of 125I-alpha-BuTx. In addition, all BC3H1 cells in culture are sensitive to the Con A-induced inhibition of 125I-alpha-BuTx binding. A comparison of the pseudo-first order rate constants for 125I-alpha-BuTx binding to untreated (8.6 x 10(4) M-1 S-1) and Con A-treated (5.4 x 10(4) M-1 S-1) BC3H1 cells, however, shows that those acetylcholine receptors in Con A-treated cells which bind 125I-alpha-BuTx do so with a lowered apparent affinity. Partial inhibition of toxin-binding capacity is not a consequence of two classes of acetylcholine receptors on the cell surface. Furthermore, individual receptors experience partial inhibition of their binding capacity by Con A, resulting in receptors with at least one binding site blocked and at least one site available for alpha-BuTx binding.


Phenol sulfotransferases.

Two phenol sulfotransferases have been purified from rat liver by conventional techniques coupled with affinity chromatography on Affi-Gel blue and ATP-agarose. Both enzymes are homogeneous by the criterion of sodium dodecyl sulfate gel electrophoresis. Each enzyme has a molecular weight of approximately 65,000 and consists of two subunits of apparently equal size. The enzymes are also similar in specificity and in their kinetic parameters but differ in amino acid composition and in their elution from DEAE-cellulose. With adenosine 3'-phosphate 5'-phosphosulfate as donor, a large variety of phenolic compounds serve as sulfate acceptor; sterols, simple alcohols, bile acids, and hydroxamates do not serve as substrates. The transferases may be considered as detoxification enzymes which catalyze the conjugation of xenobiotics containing a phenol group or of phenolic compounds generated by endogenous oxidation. The enzymes act on 3-hydroxyindole to yield indican, suggesting that their in vivo function may include the production of this normal tryptophan metabolite.


The conformation of the chromatin core particle is ionic strength-dependent.

We have examined the susceptibilities of the histones within the HeLa chromatin core particle to covalent modification by a diol-epoxide derivative of the carcinogenic polycyclic aromatic hydrocarbon benzo(a)-pyrene. Core-particle histones exhibit substantial variation in their relative susceptibilities to modification, depending upon the ionic strength of the environment. In contrast, the relative susceptibilities of either purified histones or histones in urea-denatured core particles are insensitive to changes in ionic strength. The variations in the pattern of modification of core particle histones occur primarily at ionic strengths at which the histones remain associated with core-particle DNA (0 to 0.6 M NaCl). Non-histone proteins influence the ionic strength-dependent variations in histone modification. The results imply that the ionic strength of the environment affects the conformation of the core particle and that the nucleoprotein has a flexible structure.


Structures of the homologous series of monoalkene mycolic acids from Mycobacterium smegmatis.

The positions of localization of the single carbon-carbon double bond in homologs of C series mycolic acids from Mycobacterium smegmatis have been established by 1) combined ammonia chemical ionization mass spectrometry/gas-liquid chromatography of aldehyde ozonolysis products, and 2) high resolution electron impact mass spectral analysis of vicinal glycol derivatives as their trimethylsilyl ethers. These studies have revealed that each homolog is a mixture of two isomers differing in double bond location. In each of the three homologs examined, approximately equal amounts of two isomers were present as follows: (formula: see text).


Enzymic activity of cholera toxin. I. New method of assay and the mechanism of ADP-ribosyl transfer.

We tested various methods of assaying the ADP-ribosyltransferase activity of cholera toxin using artificial acceptors of the ADP-ribosyl group. Any of several proteins or poly(L-arginine) could be used with [adenine-14C]NAD+ as ADP-ribosyl donor, but this method was not ideal because of the heterogeneity of potential acceptor groups and the necessity of using costly labeled NAD+. We, therefore, developed an alternative assay using a synthetic low molecular weight acceptor, 125I-N-guanyltyramine (125I-GT). 125I-GT was specifically ADP-ribosylated by thiol-treated cholera toxin or its A1 peptide in the presence of beta-NAD. ADP-ribosyl-125I-GT was quantified after separation from unreacted 125I-GT by batch absorption of the latter to cation exchange resins. Analysis of the kinetics of ADP-ribosylation of 125I-GT indicated that the reaction proceeds by a sequential rather than a ping-pong mechanism. The Km values for NAD+ and 125I-GT were 3.6 mM and 44 microM, respectively. L-Arginine was a competitive inhibitor of 125I-GT (KI = 75 mM), but was at least 1000-fold less active than 125I-GT as an ADP-ribose acceptor.


Interaction of the chiral pyruvate analog, 2-keto-3-bromobutyrate, with pyruvate lyases. 2-Keto-3-deoxygluconate-6-phosphate aldolase of Pseudomonas putida.

The enzyme 2-keto-3-deoxygluconate-6-P aldolase of Pseudomonas putida is inactivated by one of the chiral forms of 2-keto-(3RS)-3-bromobutyric acid (bromoketobutyrate). The inactivation shows saturation kinetics and competition with pyruvate. The minimal inactivation half-time is 4 min and that concentration of bromoketobutyrate half-saturating the enzyme is 2 mM. (3RS)-[3-3H]bromoketobutyrate is catalytically detritiated during enzyme inactivation. A kinetic analysis of rates gave data consistent with both catalysis and inactivation occurring at a single protein site, the catalytic site. The enzyme only detritiates one of the two optical isomers of bromoketobutyrate, and that form which is detritiated also alkylates the catalytic site. The inactive isomer of reagent degrades, with inversion, to L-lactate so that the chiral form specific for the enzyme is 2-keto-(3S)-3-bromobutyrate. Thus, as is the case with bromopyruvate, the enzyme catalyzes protonation of the re face at C-3 of the enzyme-reagent eneamine. As a result, bromoketobutyrate could serve as a chiral probe for stereochemical constraints of selected pyruvate-specific lyase active sites.


Inhibition of ribose-5-phosphate isomerase by 4-phosphoerythronate.

Hoping to exploit the special affinity of enzymes for unstable intermediates in substrate transformation, we have determined the effectiveness of possible analogs of ene-diolate intermediates as inhibitors of spinach ribose-5-phosphate isomerase. 4-Phosphoerythronic acid was found to be a very strong competitive inhibitor, with a Ki value almost 3 orders of magnitude lower than the Km value of ribose 5-phosphate, and very much lower than the Ki value of any other inhibitor that was examined.


A Raman spectroscopic study of the interaction of divalent metal ions with adenine moiety of adenosine 5'-triphosphate.

Raman spectra of ATP at various pH values are affected by addition of equimolar solution of divalent metal ions such as Ca2+, Mg2+, Co2+, Cu2+, and Hg2+. The changes in frequency and intensity have been used to construct models describing the nature of metal-adenine and metal-triphosphate interactions under different conditions. The metal ions are found to co-ordinate the triphosphate group in the entire pH range studies (pH to 12). Calcium (II) and magnesium (II) interact strongly with the phosphate moiety at neutral pH, although a weak interaction with the ring occur at low pH values. Around neutrality, several Raman spectral changes are observed to implicate the interaction of cobalt (II) ion with the five-membered ring of the adenine. The changes in Raman frequency are too small to suggest a direct Co(II)-N7 binding. At least six different Cu(II)-ATP species are identified between pH 3 and 12. At pH approximately 7.0 Raman data are explained better by Cu(II) interacting with N7 simultaneously with the amino group of the adenine ring. However, a Cu(II) binding to N3 at pH 10 to 11 is indicated by the enhancement of the 760 and 1360 cm-1 vibrations. At neutral pH, mercury (II) ion shows a direct coordination at N1 while at low pH with N1 blocked by protonation, mercury (II) does not interact with the adenine moiety.


Purification and subunit structure of propionyl coenzyme A carboxylase of Mycobacterium smegmatis.

Propionyl-CoA carboxylase (EC 6.4.1.3) has been purified from Mycobacterium smegmatis. It has a molecular weight of about 500,000. On sodium dodecyl sulfate gels it dissociates into two subunits with molecular weights of 64,000 and 57,000. There are 3.8 mol of biotin/500,000 g of protein. The biotin is associated entirely with the heavier subunit. The enzyme also used acetyl-CoA as a substrate. No other acetyl-CoA carboxylase could be detected in this organism.


Iodoestrogens, syntheses, and interaction with uterine receptors.

The rationale for undertaking the present study was to evaluate the utility of iodoestradiol analogs made highly radioactive with iodine isotopes in (a) the non-invasive differentiation of estrogen-dependent from estrogen-independent breast tumors, (b) spread of metastases containing estrogen receptors, and (c) potential application in therapeutic irradiation of target tissues. In the present paper, the model syntheses of a number of nonradioactive 127I-estrogen analogs are described. The analogs were tested for their ability to displace (compete with) [3H]estradiol from receptor sites. The most active compounds, 16beta-iodoestra-1,3,5(10)-triene-3,17 beta-diol (17) and 6-iodoestra-1,3,5(10),6-tetraene-3,17 beta-diol (10b), showed a relative binding affinity of 0.57 and 0.49, respectively.


Purification to homogeneity and enzymatic characterization of an alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase from porcine submaxillary glands.

By means of affinity chromatography on CDP-hexanolamine-agarose, a CMP-N-acetylneuraminate: alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase (EC 2.4.99.1) has been purified 117,000-fold to homogeneity from Triton X-100 extracts of porcine submaxillary glands. The enzyme consists of several electrophoretic forms that can be partially resolved by chromatography on Sephadex G-200, the largest of which has a molecular weight of approximately 160,000 as estimated by sodium dodecyl sulfate-gel electrophoresis. Periodate oxidation studies show that the linkage formed by this enzyme with ovine submaxillary asialo-mucin as the acceptor substrate is NeuAc alpha 2 leads to 6GalNAc alpha 1 leads to O-Thr/Ser. On the basis of initial rate studies and the patterns of inhibition observed with alternate acceptor substrates, the transferase is proposed to have either a random equilibrium kinetic mechanism or an ordered steady state mechanism with the acceptor substrate binding first. Among a wide variety of oligosaccharides, glycoproteins, and simple glycosides (including p-nitrophenyl-alpha-N-acetylgalactosaminide), the only acceptor substrates for this enzyme are those glycoproteins containing the structure, R leads to 3GalNAc alpha 1 leads to O-Thr/Ser, where R may be H or a beta-galactoside.


Quantitation of milk proteins and their mRNAs in rat mammary gland at various stages of gestation and lactation.

The content of alpha-lactalbumin and three species of caseins, 42K, 29K, and 25K, have been measured along with the levels and activities of their mRNAs in the rat mammary gland. Changes in these values were followed during gestation and lactation. An increment of 3- to 4-fold over the virgin level was observed for both alpha-lactalbumin and 42K casein during the 1st day of gestation. From this point on, the level of 42K remained unchanged during the 1st week of gestation and increased thereafter. After the increment of the 1st day, the alpha-lactalbumin content decreased rapidly during the 2nd day of gestation, continued to decrease more slowly until the 12th day, and then started to increase thereafter. During the 2nd and 3rd week of gestation. the amounts of alpha-lactalbumin within the gland increased continuously but not uniformly and caseins accumulated rapidly with a tendency to plateau around the 13th to 16th day of gestation. The relative proportions remained, respectively, 42K greater than 29K greater than 25K greater than alpha-lactalbumin until parturition. At the onset of lactation, both alpha-lactalbumin and casein content increased sharply, the relative proportion for caseins changed to 42K greater than 25K greater than 29K greater than alpha-lactalbumin and remained so throughout the lactation period. alpha-Lactalbumin and casein mRNA activity, as judged by the wheat germ translational system, remained unchanged during the 1st week of gestation, then showed a steady but not uniform increase from the 7th day of gestation until parturition. These activities increased sequentially during lactation, alpha-lactalbumin reaching a plateau by the 1st week, caseins between the 1st and 2nd week, and other mRNAs by the end of the 2nd week of lactation. By the 21st day of lactation, the activity of all mRNA had declined. The levels of alpha-lactalbumin mRNA and 16 S doublet casein mRNA sequences measured with the cDNA probes increased by about 8-fold for alpha-lactalbumin mRNA and 6-fold for casein mRNA during the 1st week of gestation. These levels declined slightly early in the 2nd week and then continued to increase until parturition with a shoulder in the levels around the 13th to 16th day. During lactation, these levels increased until the 8th to 12th day and from then on declined. The content of alpha-lactalbumin and caseins, as well as the measurement of sequences and activities of their mRNAs, showed that in the rat mammary gland these differentiated functions are already expressed at the onset of gestation. Both concentration and activity of mRNA are out of phase with protein levels during the 1st week of gestation but they remain in phase thereafter.


A compact form of rat liver mitochondrial DNA stabilized by bound proteins.

A highly folded, rapidly sedimenting form of rat liver mitochondrial DNA has been released from the organelles wiht BRIJ 58 and sodium deoxycholate in the presence of 0.5 M NaCl and isolated by sedimentation velocity in sucrose gradients. Under these conditions a majority of the mitochondrial DNA labeled in vitro sedimented beyond 39 S, the sedimentation coefficient of a highly purified mitochondrial DNA supercoil, and appeared as a stable, heterogeneous population of species ranging in s values between 42 S and about 70 S. Under formamide-spreading conditions most of the rapidly sedimenting forms appeared in the electron microscope as single genome length rosettes constrained at the center in a dense core. Except for an occasional D-loop, no extraordinary structural features were evident along the smooth loops projecting radially from the central core. In sucrose gradients containing various amounts of ethidium bromide, the sedimentation velocity of the folded DNA changed in a biphasic fashion in response to increasing amounts of dye. At a dye concentration of 0.5 microgram per ml the DNA species present reached s value minima, but two major peaks sedimenting at 32 S and 42 S were present at this point. Thus, although these species were similar in superhelix density, there appeared to be additional constraints superimposed upon their tertiary structure that folded these forms to differing degrees of compactness. Direct chemical analyses showed that proteins were bound to the folded DNA at a protein to DNA ratio of about 0.3. Separation of the bound proteins on SDS-polyacrylamide gels revealed an array of proteins ranging in molecular weight between 11,000 and 150,000. Several of the lower molecular weight proteins co-migrated with proteins from the inner mitochondrial membrane, but the major DNA-bound band (Mr = 58,000) was undetectable among the proteins from any other submitchondrial fraction. Digestion of the compact DNA structure with proteinase K under various conditions indicated that the DNA was maintained in the compact conformation by the tightly bound proteins and that the portions of these proteins directly involved in stabilizing the folded DNA were proteinase insensitive unless digestion was carried out in the presence of a disulfide reductant at elevated temperatures.


Temperature and compositional dependence of the structure of hydrated dimyristoyl lecithin.

Differential scanning calorimetry and x-ray diffraction techniques have been used to investigate the structure and phase behavior of hydrated dimyristoyl lecithin (DML) in the hydration range 7.5 to 60 weight % water and the temperature range -10 to +60 degrees C. Four different calorimetric transitions have been observed: T1, a low enthalpy transition (deltaH approximately equal to 1 kcal/mol of DML) at 0 degrees C between lamellar phases (L leads to Lbeta); T2, the low enthalpy "pretransition" at water contents greater than 20 weight % corresponding to the transition Lbeta leads to Pbeta; T3, the hydrocarbon chain order-disorder transition (deltaH = 6 to 7 kcal/mol of DML) representing the transition of the more ordered low temperature phases (Lbeta, Pbeta, or crystal C, depending on the water content) to the lamellar Lalpha phase; T4, a transition occurring at 25--27 degrees C at low water contents representing the transition from the lamellar Lbeta phase to a hydrated crystalline phase C. The structures of the Lbeta, Pbeta, C, and Lalpha phases have been examined as a function of temperature and water content. The Lbeta structure has a lamellar bilayer organization with the hydrocarbon chains fully extended and tilted with respect to the normal to the bilayer plane, but packed in a distorted quasihexagonal lattice. The Pbeta structure consists of lipid bilayer lamellae distorted by a periodic "ripple" in the plane of the lamellae; the hydrocarbon chains are tilted but appear to be packed in a regular hexagonal lattice. The diffraction pattern from the crystalline phase C indexes according to an orthorhombic cell with a = 53.8 A, b = 9.33 A, c = 8.82 A. In the lamellae bilayer Lalpha strucure, the hydrocarbon chains adopt a liquid-like conformation. Analysis of the hydration characteristics and bilayer parameters (lipid thickness, surface area/molecule) of synthetic lecithins permits an evaluation of the generalized hydration and structural behavior of this class of lipids.


delta-Aminolevulinic acid synthetase from rat liver mitochondria. Purification and properties.

delta-Aminolevulinic acid synthetase has been purified from liver mitochondria of young, uninduced rats. After nonionic detergent solubilization of mitochondrial inner membrane-matrix fractions, the enzyme was purified to a specific activity of approximately 2,000 nmol of delta-aminolevulinic acid formed/h/mg of protein at 30 degrees C, by means of ammonium sulfate precipitation, diethylaminoethyl cellulose chromatography, Sephacryl chromatography, and preparative gel electrophoresis. The purified enzyme preparation thus obtained was apparently homogeneous as judged by its migration as a single band with a molecular weight of 58,000 +/- 6,000 upon electrophoresis in sodium dodecyl sulfate polyacrylamide gels. The native enzyme probably exists as a dimer with a molecular weight of approximately 120,000. A pH optimum of 7.5 and an isoelectric point of 4.5 were also determined. Both monovalent cations and hemin strongly inhibited the activity of the purified enzyme.


Biosynthesis of N-glycosidically linked glycoproteins during gastrulation of sea urchin embryos.

Embryos of the sea urchin, Stronglyocentrotus purpuratus, synthesize several classes of sulfated and non-sulfated glycoproteins during gastrulation. The antibiotic tunicamycin, which is a specific inhibitor of the N-glycosylation of proteins, inhibits the synthesis of lipid-linked oligosaccharides in these embryos at concentrations which have little effect on the biosynthesis of other classes of glycolipids or on protein synthesis. As a consequence of this inhibition, glycoproteins with oligosaccharide side chains of the general type (Man)5-7-(GlcNAc)2 are not synthesized. In addition, the biosynthesis of a novel class of sulfated glycoproteins is inhibited. In contrast, no effect upon the synthesis of sulfated glycosaminoglycans is seen. The morphogenetic consequence of tunicamycin treatment is that development of embryos from the mesenchyme blastula to the gastrula stage is arrested. The results provide evidence that during development glycoproteins containing both unsulfated and sulfated N-glycosidically linked oligosaccharide chains are synthesized via the lipid-linked pathway. The biosynthesis of these molecules appears to be a prerequisite to the differentiation and morphogenesis that occurs during gastrulation.


Enzymological characterization of DNA polymerase alpha. Basic catalytic properties processivity, and gap utilization of the homogeneous enzyme from human KB cells.

This report describes the results of our initial enzymological characterization of a homogeneous preparation of DNA polymerase alpha that we have purified from cultured human KB cells. Although the enzyme is most reactive with duplex DNA substrates that contain short gaps (optimally activated) in incubations that require Mg2+, the polymerase possesses the intrinsic capacity to copy the initiated ribohomopolymer template, (A)-n, (dT)-200, at low rates in the presence of Mn2+. Because of the preponderance of DNA polymerase alpha in actively multiplying vertebrate cells, it is probable that this low level of activity comprises the majority of the ribopolymer copying activity that can be detected in crude tissue extracts. The presence of contaminating or associated deoxyribonuclease activities can be excluded from the purified enzyme to levels of 10(-4) to 10(-7) of the polymerase activity. The mechanism of polymerization on activated DNA under optimum conditions is moderately processive, with 11 +/- 5 nucleotides incorporated per polymerization cycle. The polymerase is unable to work at nicks or at short gaps of approximately 20 to 30 nucleotides in length, and it measures a surprisingly invariant effective template length on optimally activated DNA and on DNA molecules that have been gapped to varying extents with Escherichia coli exonuclease III. In the "Appendix" we present an amplification of the theoretical formulation of Bambara et al. (Bambara, R. A., Uyemura, D., and Choi, T. (1978) J. Biol. Chem. 253, 413--423) that permits the use of DNA polymerases with significant associated 3' leads to 5'-exonuclease activities for the accurate measurement of average template lengths (gap sizes) and titration of usable 3'-hydroxyl primer termini in gapped, duplex DNA substrates.


The detection of smooth muscle desmin-like protein in BHK21/C13 fibroblasts.

Three distinct proteins, actin (42,000 daltons), the principal form of fibroblast 10 nm filament protein (55,000 daltons), and a protein with a molecular weight of 52,000 and a pI of 5.8 were detected in nonionic detergent-insoluble cytoskeletal and 10 nm filament preparations of control BHK21/C13 and line 9 hamster fibroblasts. Cytoskeletal preparations of other hamster fibroblast cell types, such as NIL8 and primary embryo fibroblasts, contained the 55,000-dalton component but lacked the 52,000-dalton protein. A Rous sarcoma virus transformant of the BHK21/C13 line and an adenovirus transformant of line 9, resembled the NIL8 and other fibroblast types in that they contained only the 55,000- and 42,000-dalton polypeptides. The identity of the 52,000-dalton protein in BHK21/C13 cells was studied. This protein co-electrophoreses on both one- and two-dimensional polyacrylamide gels with the predominant muscle form of 10 nm filament protein. Further, one-dimensional peptide maps of the hamster smooth muscle 10 nm filament protein and the hamster fibroblast 52,000-dalton protein are identical to one another and distinct from the peptide maps of both the 42,000- and the 55,000-dalton components of the fibroblast cytoskeletal preparations. We conclude that BHK21/C13 cells contain both the fibroblast and the muscle form of 10 nm filament protein.


Reactive sulfhydryl groups of the band 3 polypeptide from human erythroycte membranes. Location in the primary structure.

Human erythrocyte membranes contain a major transmembrane protein, known as Band 3, that is involved in anion transport. This protein contains a total of five reactive sulfhydryl groups, which can be assigned to either of two classes on the basis of their susceptibility to release from the membrane by trypsin. Two of the groups are located in the region COOH-terminal to the extracellular chymotrypsin-sensitive site of the protein and remain with a membrane-bound 55,000-dalton fragment generated by trypsin treatment. The three sulfhydryl groups NH2-terminal to the extracellular chymotrypsin site are released from the cytoplasmic surface of the membrane by trypsin. All three groups are present in a 20,000-dalton tryptic fragment of Band 3. Two of these groups are located very close to the sites of trypsin cleavage that generate the 20,000-dalton fragment. The third reactve group is probably located about 15,000-daltons from the most NH2-terminal sulfhydryl group. Two other well defined fragments of the protein do not contain reactive sulfhydryl groups. They are a 23,000-dalton fragment derived from the NH2-terminal end that is also released by trypsin from the cytoplasmic surface of the membrane and a 19,000-dalton membrane-bound region of the protein that is produced by treatment with chymotrypsin in ghosts. The 20,000-dalton tryptic fragment may, therefore, constitute a sulfhydryl-containing domain of the Band 3 protein.


Faithful transcription of eukaryotic genes by RNA polymerase III in systems reconstituted with purified DNA templates.

The virus-associated (VA) RNAI gene in human adenovirus 2 DNA has been shown by Wu (Wu, G. J. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 2175--2179) to be transcribed by RNA polymerase III in a human KB cell-free extract. In the present report we have examined the fidelity of transcription of adenovirus 2 DNA and Xenopus oocyte 5 S DNA templates by RNA polymerase III in extracts derived from cultured human, murine, and amphibian kidney cells, Size and sequences analysis of the discrete transcripts synthesized in these homologous and heterologous systems indicate that they result from accurate transcription of the corresponding genes. The specific transcripts identified include both the adenovirus VA RNAI and VA RNAII, Xenopus 5 S RNA, and VA RNAI and 5 S RNA species with elongated 3' termini. The extracts derived from the various cell types differ in the ability to discriminate between the two VA RNA genes or between the heterogeneous 5 S RNA genes in the cloned DNA fragment. Wherease the human cell extracts transcribe the VA RNAI and VA RNAII genes of adenovirus at a relative frequency close to that observed in isolated nuclei, the amphibian cell extract appears to transcribe only the VA RNAI gene. The amphibian cell extract transcribes primarily that 5 S RNA gene (within 5 S DNA) which encodes the dominant oocyte 5 S RNA, whereas the human cell extract transcribes at least two distinct 5 S RNA genes. Additionally, it is shown that the VA RNAI and VA RNAII genes have separate promotor sites. The kinetics of the transcription reactions have been examined and conditions optimal for specific transcription have been established by examining the effects of salt, metal ion, and template concentrations on both total and specific RNA synthesis. It is also shown that components in the cell-free extract (from human cells) are active in directing the accurate transcription of adenovirus DNA by purified RNA polymerase III.


Stoichiometry of sodium-calcium exchange in cardiac sarcolemmal vesicles. Coupling to the sodium pump.

Vesicles isolated from cardiac muscle exhibited Na,Ca exchange activity which can be measured by 45Ca influx or efflux of by 22Na efflux. The stoichiometry of Na,Ca exchange was 3 Na:1 Ca. These vesicles also exhibited ATP-dependent 22Na transport which was inhibited by ouabain indicating that this activity is due to the sodium pump, an activity which is thought to reside only in the sarcolemma. The addition of calcium caused rapid efflux of 22Na from vesicles loaded by ATP-dependent 22Na uptake indicating that the Na,Ca exchange is located in the same vesicles as the sodium pump and is thus also a sarcolemmal activity.


Hepatic microsomal epoxide hydrase. Chemical evidence for a single polypeptide chain.

Highly purified hepatic microsomal epoxide hydrase, which had been purified in the presence of proteolytic enzyme inhibitors, was subjected to carboxypeptidase Y digestion, automated Edman degradation, and carbohydrate analysis. Carboxypeptidase Y digestion resulted in the near stoichiometric release of leucine, the COOH-terminal amino acid. Automated Edman degradation permitted the identification of the first 20 amino acid residues of epoxide hydrase. Methionine was identified as the NH2-terminal residue. The NH2-terminal region of epoxide hydrase is similar in hydrophobicity to the NH2-terminal precursor segments of several secretory proteins and the NH2-terminal regions of several microsomal cytochromes P-450. Carbohydrate analyses of the enzyme revealed the presence of 0.5 to 1.0 mol of mannose/50,000 g of protein. These results provide evidence for the presence of a single polypeptide chain in our purified enzyme preparations and suggest that there may be only one enzymic form of epoxide hydrase in microsomes from phenobarbital-treated rats.


Effects of proteases and protease inhibitors on the 4.5 S and 8 S androgen receptor.

The size of androgen receptors from rat ventral and dorsal prostate, dorsal prostate (Dunning) tumor, testis, epididymis, and seminal vesicle was determined using Sephadex G-200 chromatogrpahy and sucrose gradient centrifugation. The protease inhibitor diisopropyl fluorophosphate (DFP) was used to minimize receptor breakdown. An 8-9 S, 85 to 106 A receptor (Mr = 280,000 to 365,000; f/fo = 1.9 to 2.4) observed in unfractionated cytosol prepared in low ionic strength buffer with or without DFP is in equilibrium with a 4.5-5 S, 58 A form (Mr = 117,000; f/fo = 1.8) observed at salt concentrations greater than 0.1 M KCl. Receptor partially purified using (NH4)2SO4 or phosphocellulose chromatography in the absence of DFP was present as smaller fragments of 3.6 S, 37 A and 3.0 S, 23 A. Similar fragments could be generated from the 4.5 S or 8 S receptor by mild trypsin treatment. In addition, ventral prostate contains a DFP-insensitive enzyme which specifically converts the 4.5 S, 58 A receptor to the 3.6 S 37 A fragment. The DFP-insensitive enzyme is partially inhibited by rabbit bile and appears similar to the enzyme seminin, a secretory protein of human prostate. Androgen receptor isolated in the presence of DFP from nuclei labeled in vivo is predominantly 4.5 S, 58 A, with smaller forms (37 and 23 A) appearing in the absence of DFP. The 4.5 S, 58 A nuclear receptors were also in equilibrium with a large 8 S form. Receptor breakdown by DFP-insensitive and sensitive proteases appears to be an in vitro phenomenon. Furthermore, the size of the androgen receptor is not significantly changed during receptor migration from cytoplasm to nucleus.


Acetylcholine receptor degradation in adult rat diaphragms in organ culture and the effect of anti-acetylcholine receptor antibodies.

Acetylcholine receptor located at the neuromuscular synapse of normal innervated adult muscle fibers is extremely stable metabolically. We have studied the kinetics of receptor degradation in both normal innervated and denervated rat diaphragms in organ culture. These studies show that degradation of receptor-bound 125I-alpha-bungarotoxin is a valid measure of junctional receptor degradation. Degradation of junctional receptor is similar or identical to degradation of extrajunctional receptor in many ways: 1) both require energy, 2) both are inhibited by specific lysosomal protease inhibitors, 3) both are inhibited by treatment with colchicine, and 4) both are stimulated by treatment with anti-acetylcholine receptor antibodies. The one important distinction between degradation of junctional and extrajunctional receptor is a 10-fold difference in rate constant for the process.


Degradation of acetylcholine receptor in diaphragms of rats with experimental autoimmune myasthenia gravis.

The degradation of acetylcholine receptor observed in denervated and innervated normal rat diaphragms in organ culture is stimulated by exogenous antireceptor serum. In this paper we demonstrate that diaphragms from rats with experimental autoimmune myasthenia gravis contain reduced amounts of acetylcholine receptor. Acetylcholine receptor from myasthenic, but not from normal, rats has antibody bound to it and is degraded at an accelerated rate. We conclude that in the chronic phase of experimental autoimmune myasthenia gravis increased acetylcholine receptor degradation can be accounted for by a mechanism involving antigenic modulation, and that such a process can contribute to the clinical symptoms of impaired neuromuscular transmission.


The binding of calcium to the activation products of bovine factor IX.

Binding isotherms of Ca2+ to the bovine Factor IX activation intermediates and products, i.e. Factor IXalpha, Factor IXa alpha, and Factor IXa beta have been examined. At pH 7.4, Factor IX alpha possesses at least two strong Ca2+ sites, with an average KD of 0.1 mM, and an additional 11 weaker sites, with an average KD of 3.7 mM. Bovine Factor IXa alpha also contains at least two Ca2+ binding sites, with an average KD of 0.1 mM, and an additional 11 weaker sites, with an average KD of 1.3 mM. Factor IXa beta, the ultimate activation product of Factor IX, in the intrinsic system, likewise contains at least two strong Ca2+ sites, of average KD 0.1 mM, as well as seven additional weaker sites, possessing an average KD of 1.0 mM. The Ca2+-binding properties of the above proteins are similar to those of their precursor molecule, Factor IX, which we have earlier shown to possess at least two strong Ca2+ sites, with an average KD of 0.1 mM, and 11 weaker sites, of average KD 1.3 mM (Amphlett, G.W., Byrne, R., and Castellino, F.J. (1978) J. Biol. Chem. 253, 6774-6779). Circular dichroism analysis of all of the above proteins was consistent with the molecules possessing a low alpha-helical content, and a high quantity of beta structure and random coil conformations.


Basal activities and hormone responsiveness of osteoclast-like and osteoblast-like bone cells are regulated by glucocorticoids.

Isolated bone cells demonstrate cell-type specific responses to glucocorticoids. Osteoclast-like (OC) cells exhibit a large decrease in basal hyaluronate synthesis at physiological doses of glucocorticoids and resistance to further inhibition by pharmacological doses up to 10(-4) M. This effect is not accompanied by decreases in protein synthesis. In contrast, osteoblast-like (OB) cell metabolism is not inhibited by physiological doses of glucocorticoids. However, in OB cells both citrate decarboxylation and collagen synthesis are decreased at pharmacological doses of glucocorticoids and these effects are accompanied by a decrease in general protein synthesis. In addition to these effects on basal and general cell activities, physiological doses of glucocorticoids modulate the hormonal sensitivity of OC and OB bone cells such that lower concentrations of bovine parathyroid hormone (PTH) are necessary to elicit measurable biochemical changes. As a result, the presence of glucocorticoids permits significant responses to PTH to be detected at doses as low as 2 x 10(-13) M in OC and OB bone cells.


Structure of the carbohydrate moieties of bovine rhodopsin.

The sugar chains of bovine rhodopsin were released from the polypeptide moiety by hydrazinolysis and reduced with NaB[3H]4 after N-acetylation. The radioactive oligosaccharides thus obtained were fractionated into three components by paper chromatography. The structures of these components were elucidated as GlcNAc beta 1 leads to 2Man alpha 1 leads to 3 (Man alpha 1 leads to 6)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, GlcNAc beta 1 leads to 2Man alpha 1 leads to 3(Man alpha 1 leads to 3 and 6 Man alpha 1 leads to 6)Man beta leads to 4GlcNAc beta 1 leads to 4GlcNAc, and GlcNAc beta 1 leads to 2Man alpha 1 leads to 3(Man alpha 1 leads to 3 (Man alpha 1 leads to 6)Man alpha 1 leads to 6)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, by sequential exoglycosidase digestion, methylation analysis, and endo-beta-N-acetylglucosaminidase D digestion. The unusual features of the sugar chains of rhodopsin molecule seem to support the proposed processing pathway for the biosynthesis of asparagine-linked sugar chains of glycoproteins.


The carbohydrate of bovine prothrombin. Occurrence of Gal beta 1 leads to 3GlcNAc grouping in asparagine-linked sugar chains.

Bovine prothrombin contains three asparagine-linked sugar chains in 1 molecule. The sugar chains were quantitatively released from the polypeptide backbone by hydrazinolysis. All of the oligosaccharides thus obtained contain N-acetylneuraminic acid. Sialidase treatment of these acidic oligosaccharides released three isomeric oligosaccharides, N-1, N-2 and N-3. N-3 was a typical complex type asparagine-linked sugar chain widely found in other glycoprotein, while N-1 and N-2 were unique, because they contain Gal beta 1 leads to 3GlcNAc grouping in the outer chain moiety. By comparing the data of methylation analysis of the acidic oligosaccharides before and after sialidase treatment, the structures of the sugar chains of bovine prothrombin were confirmed as a mixture of NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc leads to Asn, NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc leads to Asn, NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc leads to Asn and their partially desialized forms.


Stereochemistry and mechanism of reactions catalyzed by indolyl-3-alkane alpha-hydroxylase.

The reaction of tryptamine with indolyl-3-alkane alpha-hydroxylase is shown to remove stereospecifically the pro-S hydrogen at C-2 of the side chain and to give hydroxytryptamine of "R" configuration. The reaction therefore proceeds stereospecifically with net inversion of configuration at C-2 of the tryptamine side chain. In the reaction of L-tryptophan methyl ester, the enzyme also catalyzes stereospecific removal of the pro-S hydrogen at C-3, but the product 3-hydroxytryptophan methyl ester is racemic at C-3. The unreacted tryptophan methyl ester is shown to incorporate solvent hydrogen into the pro-S position at C-3 in an at least partially stereospecific manner, suggesting that the reaction of L-tryptophan methyl ester is reversible. The hydrogens at C-1 of the tryptamine side chain and the alpha-hydrogen of L-tryptophan methyl ester are shown to be retained in the reactions. The results support the notion that the enzyme catalyzes stereospecific 1,4-dehydrogenation of 3-substituted indoles to the coresponding alkylidene indolenines as the primary reaction, followed by stereospecific or nonstereospecific hydration of these intermediates as a secondary process. Substrate specificity studies with a number of tryptophan analogs are in excellent agreement with such a mechanism.


Lipolysis and lipid movement in a membrane model. Action of lipoprotein lipase.

The action of purified bovine milk lipoprotein lipase on tri[3H]oleoylglycerol and the effect of albumin on movement of lipolytic products at an argon-water interface were studied in a specially designed tricomparted trough. The amount of trioleoylglycerol applied was 14 times that needed to cover the surface of the aqueous subphase (0.1 M Tris . HCl, pH 7.4) with a monolayer. It is concluded that trioleoylglycerol was present in lenses on the surface of the aqueous subphase, that hydrolysis by lipoprotein lipase occurred in or near the lipid/argon-water interface, and that lipolytic products immediately located and spread throughout the interface, displacing substances with lower spreading pressures from the interface. Addition of albumin to the aqueous subphase accelerated markedly the desorption of oleic acid and monooleoylglycerol from the interface and thereby enhanced lipolysis. When albumin was not contiguous with the site of hydrolysis, oleic acid and monooleoylglycerol readily moved in the interface to the area of contact with albumin where they were desorbed from the interface. These findings support the hypothesis of transport of lipolytic products by lateral movement in cell membranes.


The calcium binding properties of phosphorylated and unphosphorylated cardiac and skeletal myosins.

Porcine left ventricular cardiac myosin and rabbit white skeletal myosin were phosphorylated by rabbit skeletal myosin light chain kinase and their Ca2+ binding properties were examined by equilibrium dialysis techniques. No significant effect of phosphorylation on the Ca2+ binding properties of these myosins was observed. Both types of striated muscle myosins bound approximately 2 mol of Ca2+/mol of myosin with similar affinities of 3 x 10(7) M-1. In the presence of 3 x 10(-4) M Mg2+ the myosins bound Ca2+ with a reduced affinity of 3 to 4 x 10(5) M-1. Assuming competition between Mg2+ and Ca2+ for the binding sites on myosin, the changes in Ca2+ binding can be accounted for by a Mg2+ affinity of 2.5 to 3.0 x 10(5) M-1.


Metmyoglobin reductase. Identification and purification of a reduced nicotinamide adenine dinucleotide-dependent enzyme from bovine heart which reduces metmyoglobin.

Beef heart muscle has been found to contain an enzyme which will rapidly and directly reduce metmyoglobin in vitro. Reduction rates are far greater than any previously reported for nonspecific or nonenzymatic systems. The enzyme is NADH-dependent and requires the presence of ferrocyanide ion for in vitro assay. The artificial electron carriers, dichlorophenolindophenol and methylene blue, are not required. Nonenzymatic reduction of metmyoglobin, which has previously been reported, was not encountered under the assay conditions described herein. Demonstration of enzymatic activity is dependent on a suitable myoglobin substrate, NADH, and ferrocyanide. An equimolar amount of cytochrome b5 was more effective than ferrocyanide in the enzymatic reduction of metmyoglobin. The methods for preparation of beef heart myoglobin and for purification of the enzyme are presented. The enzyme has been purified over 2000-fold. The enzyme has a pH optimum about 6.5 and a Km of 5.0 x 10(-5) M, and is unaffected by the absence of O2. Sodium dodecyl sulfate-gel electrophoresis revealed a molecular weight around 30,000. Purified enzyme does not react with lipoamide. The reaction is markedly influenced by the composition of the buffering milieu. Enzyme activity is inhibited by p-chloromercuriphenyl sulfonic acid, quinacrine dihydrochloride, and N-ethyl-maleimide. Activity was slightly stimulated by FMN. The characteristics of the enzymatic activity and the assay system are similar to those reported by Hegesh et al. (J. Lab. Clin. Med. 72, 339-344, 1968) for erythrocyte methemoglobin reductase.


Comparison of formation, activation, and nuclear translocation of receptor . estradiol (R . E2) complex at 0 degrees C and 37 degrees C in intact uterine cells.

The present study was undertaken to establish whether molecular events leading to binding, transformation-activation, and nuclear translocation of cytoplasmic uterine estrogen receptor described for cell-free systems also occur in intact uterine cells. Cell suspensions were incubated at 0 degrees C or 37 degrees C with estradiol (E2) and specific binding to intracellular receptors was measured. The data demonstrate that saturation of specific estrogen binding sites occurs within 60 min at 37 degrees C and within 22 h at 0 degrees C, with a total of approximately 24,000 to 30,000 receptor sites per cell. At equilibrium, the total number and subcellular distribution of receptor . estradiol (R . E2) complexes formed in cells incubated at 0 degrees C or 37 degrees C were identical. Scatchard analysis of the equilibrium binding data yielded the same association constants for cytoplasmic and nuclear R . E2 formed in intact cells incubated at either temperature. Sucrose density gradient analysis of nuclear and cytoplasmic R . E2 formed in intact cells at 0 degrees C or 37 degrees C showed that at both temperatures, the nuclear R . E2 had a 5 S sedimentation coefficient; at both temperatures, a 5 S cytosol R . E2 was detected; only in the 0 degrees C incubation, an additional 4 S cytosol R . E2 was found. These results suggest that the molecular interactions regulating the dynamics of estrogen binding in the intact cell are similar at both physiological and low temperatures.


Calmodulin. Production of an antibody in rabbit and development of a radioimmunoassay.

Calmodulin, a heat-stable Ca2+-binding protein (Mr = 16,700) found in all eukaryotes, is a multifunctional modulator, mediating many of the effects of Ca2+ in cellular functions. The protein was derivatized with 1-fluoro-2,4-dinitrobenzene (DNB) to give 3 mol of DNB/mol of calmodulin (DNB3-calmodulin). The dinitrophenylated protein was almost as active as native calmodulin in stimulating bovine brain Ca2+-dependent phosphodiesterase. Incorporation of the dinitrophenyl groups renders calmodulin highly antigenic in the rabbit; native calmodulin is a weak antigen. Rabbits immunized with DNB3-calmodulin produced specific antibody against both DNB3-calmodulin and calmodulin. Using the immunized serum, a radioimmunoassay was developed for calmodulin, the sensitivity for DNB3-calmodulin and calmodulin being approximately 0.2 and 2 pmol, respectively. Although the sensitivity of the radioimmunoassay for calmodulin is comparable to the enzyme assay of calmodulin with Ca2+-dependent phosphodiesterase, the radioimmunoassay affords the detection of calmodulin on the basis of antigenic determinants, and thus measures calmodulin in terms of polypeptide structure instead of its ability to stimulate an enzyme. Further, the accuracy of the radioimmunoassay is not affected by the presence of a heat-labile inhibitor protein, which affects the enzyme assay to give an apparent underestimation.


Biological activities of the two major components of tunicamycin.

Tunicamycin, an antibiotic that inhibits the transfer of N-acetyglucosamine-1-phosphate from UDP-N-acetylglucosamine to dolichol monophosphate and thereby blocks the formation of protein-carbohydrate linkages of the N-glycosidic type, is not a single compound but a mixture of homologous antibiotics. Two major and eight minor homologs have been identified, all of which possess the ability to inhibit protein glycosylation. The biological activities of the two major components of tunicamycin were investigated and found to differ in their ability to inhibit protein glycosylation and in their effectiveness to inhibit protein synthesis. When completely blocking mannose incorporation into protein, one homolog inhibited protein synthesis by 50% while the other had only a negligible effect. The results demonstrate that differences in biological activity can be discriminated among tunicamycin homologs.


Structure of the carbohydrate chain of free secretory component from human milk.

Secretory component from human milk was found to contain 23.4% carbohydrate, which includes galactose, mannose, fucose, glucosamine, and sialic acid. Secretory component could be degraded by pronase or base-borohydride to yield the same, single type of carbohydrate chain. In the glycopeptide produced by pronase digestion, aspartic acid was the only amino acid present in molar quantities after amino acid analysis, which suggests that the carbohydrate moiety is linked to the polypeptide chain at asparagine residues. The positions of links between the various sugar units were studied by methylation analyses of: secretory component, periodate-oxidized and reduced secretory component, the fragment produced by base-borohydride treatment, and the pronase glycopeptide after treatment with specific glycosidases. Sugars released from the glycopeptide by various glycosidases were also quantitated. From the results of these studies a branched chain structure was assigned to the carbohydrate chain of secretory component.


Evidence for an essential histidyl residue at the active site of pyridoxamine (pyridoxine)-5'-phosphate oxidase from rabbit liver.

Pyridoxamine (pyridoxine)-5'-phosphate oxidase (EC 1.4.3.5) from rabbit liver is inactivated by diethylpyrocarbonate in an all-or-none fashion with first order kinetics with respect to modifier concentration. The rate of inactivation increases with pH and reflects a group with a pKa of 7.5. Inactivated enzyme is in the holo form with intact FMN. Four histidyls and a cysteinyl residue are modified by excess reagent. The restoration of enzymatic activity by hydroxylamine, the spectrophotometric and colorimetric amino acid analyses, and our previous studies on cysteine modification (Tsuge, H., and McCormick, D.B. (1979) in Flavins and Flavoproteins (Yamano, T., and Yagi, K., eds) Japan Scientific Societies Press, Tokyo, in press) all suggest that inactivation occurs solely by modification of histidine. Analyses by kinetic and statistical methods indicate that three histidines are modified slowly and are not critical for activity, while one histidine is modified nine times more rapidly and accounts for the observed inactivation. Inactivated enzyme shows no significant perturbations in structure, as evidenced by absorption, CD, fluorescence, and gel filtration, but is unable to bind the product, pyridoxal 5'-phosphate. Furthermore, the substrate-competitive inhibitor, pyridoxal 5'-phosphate oxime, protects from inactivation. Hence, diethylpyrocarbonate inactivates this enzyme by modifying a crucial histidyl residue at the substrate/product-binding site.


Side chain cleavage of some cholesterol esters.

For some time it has been known that the side chain of cholesterol sulfate is cleaved by the cleavage enzyme system present in bovine adrenal mitochondria without prior hydrolysis of the sulfate moiety. In this work, other inorganic esters as well as some organic esters of cholesterol were tested as substrates for this enzyme system. The results revealed that cholesterol nitrate, cholesterol phosphate, and a series of acyl esters of cholesterol can also be cleaved by the enzyme system to their respective pregnenolone derivatives without first being hydrolyzed to cholesterol. The rate of oxidation of the carboxylic acid esters decreased as the size of the acyl groups increased. Cholesterol stearate and cholesterol phosphate were demonstrated to be inhibitors of the side chain cleavage of cholesterol. While digitonin, as might be expected, inhibits the cleavage of cholesterol, it accelerates the oxidation of both cholesterol sulfate and cholesterol nitrate. The results reported in this paper add support to the previously proposed hypothesis that more than one cholesterol side chain cleavage enzyme system exists in adrenal mitochondria.


Biosynthesis of trans-4-hydroxy-L-proline by Streptomyces griseoviridus.

Radioisotopic experiments have revealed that free trans-4-hydroxy-L-proline is an intermediate synthesized from L-proline during formation of the peptide-bound cis-4-hydroxy-D-proline residue in the antibiotic, etamycin. This conclusion was based on the fact that 1) both radiolabeled L-proline and trans-4-hydroxy-L-proline are precursors of the bound D-imino acid as noted previously by Hook and Vining ((1973) J. Chem. Soc. Chem. Commun. 185-186; (1973) Can. J. Biochem. 51, 1630-1637), 2) the unlabeled trans isomer specifically inhibited the incorporation of radiolabel from proline into the antibiotic, 3) the 14C-hydroxyimino-acid was isolated from the intracellular pool and medium following incubations with L-[14C]proline during antibiotic biosynthesis and when etamycin synthesis was blocked by D-leucine. By means of chromatographic and enzymatic analyses, it was established that the free imino acid possesses the trans-L configuration.


Synthesis and translation of mRNA containing 5'-terminal 7-ethylguanosine cap.

Reovirus mRNA synthesis in vitro by the virion-associated RNA polymerase was only slightly (10 to 15%) diminished in the presence of 2 mM S-adenosylethionine. However, methyl group transfer from S-adenosylmethionine (0.05 mM) to the 5'-terminal cap structure, m7GpppGm in this mRNA was markedly inhibited (80%) under these conditions. Replacement of S-adenosylmethionine by S-adenosylethionine (5 mM) yielded mRNAs containing mainly (70%) 5'-terminal e7GpppGe and e7GpppG, but some of the products were unalkylated (5'-GpppG, ppG). The ethylated mRNAs, but not the unalkylated molecules, bound to wheat germ ribosomes and were translated essentially as well as the corresponding methylated mRNAs in wheat germ extracts and in nuclease-treated rabbit reticulocyte lysates. Protein synthesis directed by ethylated mRNAs in wheat germ extract was 80% decreased by 0.1 mM m7GMP. Under conditions of limited initiation, methylated mRNA bound to wheat germ ribosomes preferentially as compared to ethylated mRNA. The results document for the first time the synthesis of ethylated mRNA and support the hypothesis that N7-alkylation of the 5'-guanosine in caps, rather than methylation itself, is important for the enhancing effect of cap on the initiation of eukaryotic protein synthesis.


Domain structure of fibronectin and its relation to function. Disulfides and sulfhydryl groups.

Hamster cell fibronectin is a glycoprotein consisting of two 230,000-dalton subunits in a disulfide-bonded dimer. The molecule is composed of domains which can be separated by partial proteolytic cleavage. The carbohydrates, disulfide bonds, and a single free sulfhydryl group per chain are distributed nonuniformly among these regions. All the interchain disulfides are within 10,000 daltons of the end of the molecule and are removed by mild proteolysis which also generates 200,000- and 25,000-dalton fragments which do not contain interchain disulfides. The 200,000-dalton fragment contains all or most of the carbohydrate side chains, and the free sulfhydryl group, but is relatively poor in cystine. The 25,000-dalton fragment is carbohydrate-free and cystine-rich but has no free sulfhydryl groups. There is heterogeneity in carbohydrate content among the monomeric chains of intact fibronectin and the 200,000-dalton fragments. The gelatin binding site of fibronectin is in the 200,000 fragment. Intact disulfide bonds are required for binding of fibronectin to cells and to gelatin and blockage of the free sulfhydryl groups prevents binding of fibronectin to cells, suggesting that intermolecular disulfide bonding may be important.


Inhibition of fatty acid oxidation by 2-bromooctanoate. Evidence for the enzymatic formation of 2-bromo-3-ketooctanoyl coenzyme A and the inhibition of 3-ketothiolase.

Incubation of rat liver mitochondria with 10 microM DL-2-bromooctanoate causes complete and irreversible inactivation of 3-ketothiolase I (acyl-CoA:acetyl-CoA C-acyltransferase). Evidence is presented that mitochondria convert bromooctanoate to 2-bromo-3-ketooctanoyl-CoA, an alpha-haloketone which is probably the active form of the inhibitor. The inactivation is accompanied by incorporation of radioactivity from [1-14C]bromooctanoate into the enzyme. Bromooctanoate does not affect the activities of the other enzymes of beta-oxidation, except for 3-ketothiolase II (acetyl-CoA:acetyl-CoA C-acetyltransferase), which becomes partially inhibited. Evidence is also presented that various enzymes of beta-oxidation can use 2-bromooctanoyl-CoA and its beta-oxidation products as substrates.


The chemical characterization of favin, a lectin isolated from Vicia faba.

We have determined the subunit structure of the glucose- and mannose-binding lectin favin, from Vicia faba. The molecule is composed of two nonidentical polypeptide chains held together by noncovalent interactions. We have determined the complete amino acid sequence of the smaller alpha chain (Mr = 5,571) and shown that it is homologous to the alpha chain of the lectins from lentil and pea and to residues 72 to 120 of concanavalin A (Con A). The larger beta chain (Mr = 20,000) contains carbohydrate and is homologous to the beta chain of lentil, pea, soybean, peanut, and red kidney bean lectins and is homologous to a portion of the Con A molecule beginning at residue 122. Favin also contains a minor component, beta' (Mr = 18,700), that closely resembles the beta chain but lacks carbohydrate and may, on the basis of apparent molecular weight, lack some part of the COOH-terminal region of the polypeptide chain. Although favin is similar to Con A, it, like the lentil and pea lectins, appears to lack residues corresponding to positions 1 to 71 of Con A. Because these residues contribute significantly to the carbohydrate binding site of Con A, the lack of this region in the otherwise homologous lectin favin suggests that the carbohydrate binding site of favin differs from that of Con A or that the region represented by residues 1 to 71 of Con A is located in a different portion (i.e. in the beta chain) of the favin molecule.


Membrane and bubble oxygenators compared by preservation of myocardial function.

Membrane oxygenators reputedly preserve erythrocytes, platelets, kidneys, brain and lungs better than bubble oxygenators; preservation of ventricular function by the two types of oxygenator is compared in isolated hearts, extremely sensitive to imperfections of perfusion, by isovolumic tests which are simple and accurate, especially for evaluating compliance. Canine hearts were perfused for three hours with either disposable bubble (Temptrol) or membrane (Lande-Edwards) oxygenators. Values at a standard point on regression slopes os isovolumic contractile force, velocity and compliance (volume) against end-diastolic pressure were used to express final functions as percentages of initial ones. Terminal proportional ventricular weight was an index of oedema. The final mean percentages with standard error measurements of initial values for the 12 hearts perfused on buble oxygenators and the 10 on membrane oxygenators were, respectively: 97 +/- 11.5% and 87 +/- 10.7% for contractile force, 117 +/- 23.1% and 88 +/- 10.44% for contractile velocity, and 97.2 +/- 8.48% and 117.3 +/- 12.5% for ventricular compliance, which was the function nearest to showing a significant difference with P less than 0.1. There was no significant difference in weights. This membrane oxygenator, as cheap and simple as conventional ones, probably has similar advantages for the myocardium as for other tissues.


Angioplasty of the profunda femoris in revascularisation of the lower extremity.

Excellent results with respect to preservation of the limb and improvement of claudication were obtained with profundaplasty in 29 patients with Fontaine's stage III or IV chronic obliteration. The method is regarded as one of choice for saving the limb, but not as a replacement for other methods of direct revascularisation of the femoropopliteal axis in cases where complete functional recovery is also required. A comparison is made between the results of profundaplasty and reconstruction of the axis in a personal series.


Spinal cord injury following aortorenal bypass.

Spinal cord ischemia following abdominal aortic aneurysmectomy is believed to be caused by hypotension, aortic clamping, division of lumbar arteries, and emboli. In this report a patient underwent aortorenal bypass complicated by temporary urinary and rectal sphincter paralysis appearing four days postoperatively. Angiographic studies suggest that the cord damage was caused by microemboli traversing iliolumbar collateral arteries.


Consumption coagulopathy associated with arterial aneurysms.

Consumption coagulopathy resulting from arterial aneurysm is an uncommon entity. Two patients, one with a femoral artery aneurysm and one with an abdominal aortic aneurysm containing fresh blood clot associated with a coagulopathy are presented. Reasonable preoperative control of the bleeding disorder with appropriate blood products followed by aneurysmectomy is recommended. Except to treat severe bleeding disorders associated with arterial aneurysms, heparin therapy is rarely necessary, as spontaneous reversal of the coagulopathy usually occurs. A high index of suspicion for the presence of an underlying arterial aneurysm as the cause for the consumption coagulopathy is necessary when no other etiology is obvious.


The anatomy of the lumbar sympathetic trunks in man (with special reference to the question of regeneration after sympathectomy).

Although possible to decentralize the sympathetic outflow to the leg, below the knee, by resecting only the third lumbar sympathetic ganglion, the likelihood of restoration of normal vasoconstrictor activity warrants a more extensive resection to include at least the second and third lumbar ganglia, and preferably the second, third, and fourth lumbar ganglia. When cross-over fibers are suspected or demonstrated following unilateral sympathectomy, the denervation should include the fourth and fifth lumbar ganglia. The first lumbar ganglion should be spared at least unilaterally if interference with reproductive function is to be avoided.


Effect of lumbar sympathectomy on the lower extremity.

1. Lumbar sympathectomy creates a state of vasodilatation equivalent to physiological vasodilatation of daily living; however, after sympathectomy, vasodilatation is permanent and irreversible. 2. This vasodilatation is characterized at rest by a preferred distribution of blood to the skin and to subcutaneous cellular tissue of the lower extremity, foot and toes. 3. This vasodilatation, although permanent, does not interfere with increased output to muscles at work. They benefit by a particular mechanism of metabolic vasodilatation (Fig. 6). 4. Lumbar sympathectomy has a beneficial effect on the collateral compensatory circulation of an obliterated large artery. This effect is induced by suppression of the peripheral resistance, and thus, by increasing the pressure gradient. 5. The vasodilatation obtained by intravenous injection of pentothal is qualitatively the same as that which follows a lumbar sympathectomy. The test with pentothal, because of this action, is an excellent method for prognosis and control of lumbar sympathectomy effects.


Effect of lumbar sympathectomy on the muscles.

With the physiological data concerning microcirculation and sympathetic control, the author foresees the effects that one must obtain with lumbar sympathectomy. The improvements of cutaneous circulation are usually well known while those of muscular circulation are more debated. To prove it, the author has studied the muscular flow after sympathectomy using Xenon radioactive method and the non invasive electromagnetic flowmetry method. Despite the background of a short experience in this matter, the results seems to point out a beneficial effect of lumbar sympathectomy in muscular circulation at rest and exercise, in patients having obstructive arteriopathies of the lower extremities.


Experimental study of sensitization to catecholamines after lumbar sympathectomy.

In a first time, the authors have shown a real supersensitivity to the action of catecholamines after lumbar sympathectomy in dog. Then, they have established a protocol which can be applied to the exploration of those phenomena in arteritic patients. Same doses of epinephrine and norepinephrine (which correspond to a slight hypercatecholamine blood level under stress) are injected intravenously, comparatively before and after lumbar sympathectomy. The modifications of E.C.G., arterial blood pressure and rheographic record, caused by those injections seem to prove the existence of a sensitization which is still lasting 4 months after the operation, but which does not seem to be noxious. Therefore, this sensitization generates compensating reflex reactions to arterial hypertension, leading to a favorable increase of the blood flow in the denervated territories.


Evaluation of effectiveness of lumbar sympathectomy by non invasive diagnostic techniques.

Out of a total of more than 1,200 patients in whom lumbar sympathectomy was carried out, two groups of 96 and 50 consecutive and non selected patients were evaluated before and after the operation by non invasive physiologic tests (digital plethysmography, post-occlusive reactive hyperemia, segmental blood pressure at rest and after exercise, Strandness test). Results were compared with angiography and clinical data. The statistical study of results corroborates the following points. 1. Lumbar sympathectomy is essentially effective in atherosclerotic arterial disease, but much less so in diabetic patients and in cases of widespread disease. 2. Patients with obstructive disease of the femoral artery had more improvement (t = 4.25) than patients with obstruction of peripheral arteries (t = 2.4). 3. Walking time was significantly improved after sympathectomy (p = 0.02). 4. Only one parameter, rheographic quotient Rq was statistically improved (p = 0.01). 5. The post occlusive reactive hyperemia test is a good method to predict the results of lumbar sympathectomy (correlation r = 0.22). We feel that this operation is indicated in cases of: isolated obstruction of the femoral artery with claudication superior to 200 meters, reconstruction with an associated obstruction of the superficial femoral artery.


Plasma testosterone and the diagnosis of panhypopituitarism in women.

Plasma testosterone was shown to be a very sensitive index of panhypopituitarism in women. In 18 women, testosterone levels were found to be 0.06 +/- 0.04 ng/ml (mean +/- SD; normal, 0.40 +/- 0.10 ng/ml). In all patients, the values were below 2 SD of testosterone levels found in normal women. Dihydrotestosterone concentrations were similarly modified. Such an important decrease in plasma testosterone was probably due to the additional effect produced by the functional suppression of both sources of testosterone precursors: the adrenals and the ovaries.


Stimulatory effect of acute baclofen administration on human growth hormone secretion.

The effect of acute administration of the gamma-amino-butyric acid (GABA) derivative, baclofen, on human GH secretion was tested. In eight of the nine volunteers, both 5 and 10 mg baclofen (Lioresal) significantly basal GH secretion. Previoulsy, it has been reported that subacute baclofen treatment inhibits the GH response to insulin hypoglycemia and arginine. Thus, the present study shows that baclofen is able to modify GH secretion via different mechanisms, depending on the test situation and duration of treatment. As a putative GABA agonist, the effect of baclofen may be mediated via GABA-ergic pathways. Because of variable results, the evaluation of a possible physiological role of GABA in GH secretion requires further study.


Direct radioimmunoassay of human renin.

Antibodies were raised in rabbit against pure human renin. The antisera obtained are highly specific for human renin versus hog, dog and rat renin. They do not cross-react with acid proteases such as pepsin and human cathepsin D. A direct radioimunoassay is described for human renin in plasma and kidney extracts. 30 to 50 pg of enzyme (2.5 to 4 x 10(-5) Goldblatt units) are detected.


Prostaglandins: modulators of renal function and pressor resistance in chronic liver disease.

Prostaglandins may modulate renal function and play a role in the hyperreninism and angiotensin pressor resistance of chronic liver disease. To study this possibility, we evaluated 12 patients with alcoholic cirrhosis and ascites. Urine immunoassayable prostaglandin E in 5 female patients was 3.3 +/- 0.5 micrograms/day [normal, 0.3 +/- 0.1 (SE)], renin was 14.6 +/- 3.7 ng/ml.h, and aldosterone was 76 +/- 19 ng/dl. After either indomethacin (200 mg) or ibuprofen (2000 mg) for 1 day, urine immunoassayable prostaglandin E fell to 0.8 +/- 0.4 micrograms/day, renin to 8.0 +/- 2.4 ng/mol.h, and aldosterone to 54 +/- 14 ng/dl (all P less than 0.01). Pressor sensitivity increased dramatically, and creatinine clearance transiently fell from 73 +/- 10 to 32 +/- 7 cc/min (P less than 0.01). Because a primary effect on renin might explain the renal impairment, an additional study used propranolol to lower renin activity. Renal function was unaltered by propranolol. We conclude that prostaglandins play a supportive role in maintaining renal function and are involved in the hyperreninism and pressor resistance of patients with liver disease.


Vitamin D bioavailability: serum 25-hydroxyvitamin D levels in man after oral, subcutaneous, intramuscular, and intravenous vitamin D administration.

Bioavailability of vitamin D (D) was assessed by competitive protein-binding assay of serum 25-hydroxyvitamin D (25OHD) levels in normal adult volunteers after a single oral, sc, or im dose of commercially available D pharmaceuticals or after a single iv injection of ethanol-propylene glycol solution containing D. Similar increases in serum 25OHD levels were noted after either iv D2 or D3 (100 micrograms/kg), suggesting that the 25-hydroxylation of D2 and D3 is comparable in man. Absolute increases in serum 25OHD levels were similar in subjects receiving D in iv doses of 100 and 250 micrograms/kg; however, subjects receiving the larger dose had higher basal 25OHD levels. This finding suggests an inverse relationship in man between basal serum 25OHD concentrations and relative serum 25OHD increments after administration of pharmacological doses of D. Oil depot sc and im injection of D (200 micrograms/kg) resulted in delayed serum 25OHD increases compared to oral and iv dosing (100 micrograms/kg). Studies after im oil depot injection of [3H]D3 into rats showed that D administered in this manner had delayed bioavailability but remained unaltered in situ. Differences in D pharmaceutical bioavailability should be considered in treatment or prophylaxis with this sterol.


A procedure for combining menstrual cycle data.

A procedure is presented which overcomes most of the conceptual and statistical problems associated with the combining of data from heterogeneous menstrual cycles for graphical or statistical analyses. This procedure is based upon an initial normalization of the raw data to eliminate extraneous between-cycle variability, followed by the assignment of the data to a set of discrete cycle phases using a weighted-average technique. The efficacy of this procedure is compared to that of seven other published categorization methods by examining the proportion of variance accounted for and the P values from analyses of variance computed for 17 beta-estradiol and olfactory sensitivity measures. A major advantage of the proposed procedure is that it allows for the grouping of data from entire cycles (including menses) on the same figure without exhibiting points from heterogeneous sectors of individual cycles and without changing the sample size as distance from the midcycle LH surge increases. Thus, this procedure provides equal sample sizes for all phases of the cycle, allowing repeated-measures parametric statistical analyses to be performed. Data are presented which suggest that the categorization of menstrual cycle data without carefully established cycle phases can lead to quite erroneous conclusions.


Conventional supervoltage irradiation is an effective treatment for acromegaly.

Forty-seven acromegalic patients were treated with conventional supervoltage irradiation (4000-5000 rads) and were followed-up at 2, 5, and 10 yr after treatment. The mean fall in the GH concentrations 5 years after treatment was 77%, and GH was less than 10 ng/ml in 73% of the cases. In the majority, GH continued to fall over the 5- to 10-yr interval after treatment, so that by 10 yr, plasma GH was less than or equal to 10 ng/ml in 81% and less than or equal to 5 ng/ml in 69% of the patients. Also, the subjective (acral and facial changes) and the objective (metabolic) effects of GH excess all showed significant improvement with time, and the improvement roughly paralleled the fall in plasma GH concentrations. After therapy, no patient developed extrasellar extension, and headaches improved in the majority of patients. The prevalence of hypopituitarism, which was low before treatment, increased progressively throughout the follow-up period. Significant loss of vision was observed in only one patient, who received 5600 rads and had severe systemic sarcoidosis. The only other complication was temporal hair loss. Conventional super-voltage irradiation by 5-10 yr produces the same fall in plasma GH as all other forms of treatment for acromegaly, including transphenoidal microsurgery. The rate of fall in plasma GH after conventional irradiation is comparable to that of more aggressive forms of irradiation and is slower than that achieved by surgical methods but probably conventional irradiation has fewer serious side effects.


Effects of androgen on sexual behavior in hypogonadal men.

Despite the widespread use of androgen in the treatment of hypogonadal men, its efficacy in restoring sexual behavior to hypogonadal patients has not been established in appropriately controlled behavioral studies. Accordingly, testosterone enanthate or vehicle was injected once every 4 weeks im in a double blind experiment. The subjects were six adult males, aged 32-65 yr, two with gonadal failure and four with secondary hypogonadism. Two doses of testosterone (100 and 400 mg) were administered for approximately 5 months, with the treatments varied at random within and among subjects. Details of sexual activity and experience were followed by the use of daily logs. Frequencies of erections, including nocturnal erections and coitus, showed significant dose-related responses to androgen treatment which closely followed the fluctuations in the circulating testosterone level. As indicated by the Profile of Mood States test, behavioral responses did not appear to be mediated by changes in mood. We concluded that the stimulatory effects of testosterone on sexual activity are rapid, reliable, and not due to a placebo effect. To maintain plasma testosterone and adequate sexual function within normal levels, even high doses of testosterone enanthate should be given no less frequency than once every 3 weeks.


Further comparisons of the [125I]somatomedin A and the [125I]somatomedin C radioreceptor assays of somatomedin peptide.

The GH dependency of somatomedin (SM) peptide content in plasma was assessed using the placental membrane radioreceptor assay (RRA) to measure whole and acid-chromatographed plasma using both [125I]SM A and [125I]SM C. Plasmas from 10 normal children and adults, 11 GH-deficient children before and after therapy with 0.1 U/kg GH im daily for 5 days, and 10 acromegalic patients were assayed as well as a standard plasma pool from 20 healthy adult males. The results of these assays revealed that the SM content of plasma is GH dependent and that assay of whole and acid-chromatographed plasma with the [125I]SM A RRA and assay of acid-chromatographed plasma with the [125I]SM C RRA give comparable measurements of SM peptide content. There was a positive correlation of the [125I]SM A RRAs of whole and acid-chromatographed plasma from each patient (r = 0.87; n = 41; P much less than 0.001). An even stronger positive correlation was observed when the [125I]SM A and [125I]SM C RRAs of acid-chromatographed plasma were compared (r = 0.94; n = 41; P much less than 0.001). Furthermore, it was demonstrated that precise measurement of SM peptide content in plasmas from subjects with abnormal GH states and from normal subjects is enhanced by acid chromatography of the plasma before assay in either the [125I]SM A or [125I]SM C RRA.


Demonstration by radioligand-binding assay of the structural similarity of a partially synthetic growth hormone recombinant molecule to its natural analog and to native human growth hormone.

The recombinant resulting from the noncovalent interaction of a natural amino-terminal fragment of reduced and carbamoylmethylated human GH (residues 1-134) with a synthetic carboxyl-terminal fragment of reduced and carbamoyl-methylated human GH (residues 141-191) reacts in seven immunoassay systems and two receptor assay systems in a similar manner and with comparable potency to its naturally occurring analog, but with less avidity overall than does the native hormone. This similarity of reactivity is strong evidence for similarity of conformation among these molecules. The demonstration that a synthetic fragment can restore radioligand-binding properties to a proteolytic fragment of human GH implies that partially synthetic variant GH molecules have biological activity.


Comparative effectiveness of dextrothyroxine and levothyroxine in correcting hypothyroidism and lowering blood lipid levels in hypothyroid patients.

Data reported here establish that treatment regimens of 4 mg dextrothyroxine and 0.15 mg levothyroxine in hypothyroid subjects produce similar degrees of lowering of serum TSH, cholesterol, triglycerides, and phospholipid levels and equal stimulation of metabolic rate. The Murphy-Pattee total T4 determination applied to blood samples drawn 24 h after the last dose of dextrothyroxine can be used to assess adequacy of treatment. Correction of hypothyroidism requires high serum levels of dextrothyroxine than of levothyroxine. Serum T3 levels increase in patients treated with dextrothyroxine. In the treatment of hypothyroidism, the cholesterol-lowering and metabolic rate-stimulating effects of dextrothyroxine do not appear to be dissociated. Further studies are needed to determine whether such an effect can be demonstrated in euthyroid hypercholesterolemic subjects with doses established herein as equivalent in terms of the stimulating effect on metabolic rate.


The metabolic clearance of aldosterone decreases similarly during infusion of angiotensin II in patients with essential hypertension and in normal subjects.

To determine if the abnormally large increases in levels of plasma aldosterone which occur during infusion of angiotensin II in some patients with essential hypertension are due to abnormal decreases in the metabolic clearance of aldosterone, we measured clearances of aldosterone before and during infusion of angiotensin II in 12 patients with essential hypertension and in 10 normal subjects. The metabolic clearance of aldosterone in the patients and that in the normal subjects were the same before angiotensin II was infused and the clearances decreased similarly during infusion of angiotensin II. In agreement with our previous observations, the plasma aldosterone responses to angiotensin II were greater than normal in most of the patients with low renin essential hypertension. Thus, the brisk increases in levels of plasma aldosterone during infusion of angiotensin II in patients with essential hypertension reflect abnormally large increases in the secretion of aldosterone. These results add further support to the idea that adrenal sensitivity to angiotensin II is increased in some patients with essential hypertension.


Leydig cell responsiveness to single and repeated human chorionic gonadotropin administration.

A single im injection of 1500 IU hCG significantly increased plasma testosterone levels for at least 96--120 h in normal men (n = 7), patients with isolated gonadotropin deficiency (n = 6), and boys with delayed puberty (n = 7); the maximum values [1315 +/- 309, 370 +/- 177, and 963 +/- 249 ng/100 ml (mean +/- SD), respectively] were achieved after 72 h in each group. Repeated daily injections of 1500 IU hCG for 3 days increased plasma testosterone levels in the same subjects at 72 h after the start to levels (1342 +/- 412, 407 +/- 199, and 1052 +/- 449 ng/100 ml, respectively) similar to those found in the single dose experiment. The levels achieved at 24 and 48 h also did not differ significantly in the two experiments. The data indicate the lack of additional leydig cell stimulation by repeated hCG injections given within 48 h after a single dose.


Pattern of the response of testosterone and its precursors to human chorionic gonadotropin stimulation in relation to age in infants and children.

The pattern of response to 14-day hCG stimulation was studied in 100 apparently normal infants and children and in 2 boys with total adrenal insufficiency. Response was estimated on the levels (nanograms per dl) of testosterone (T), delta4-androstenedione (delta4), and 17-hydroxyprogesterone (17OHP) before and after hCG. Basal levels of T, delta4, and 17OHP increased significantly after 9--10 yr of age and before the onset of puberty. It would thus appear that the changes of adrenarche are also expressed by the secretion of delta4 androgens. After hCG, levels of T (629 +/- 218) did not vary with age in infants or prepubertal children but increased significantly at the onset of puberty (1265 +/- 253). Post-hCG levels of delta4 and 17OHP increased with age, but their increments (respectively, 31.1 +/- 14.6 and 108.6 +/- 42.2) did not vary between 2--12 yr of age. On the other hand, the rise of delta4 after hCG tended to be higher in infancy and early pubescence. These data suggest that 1) testicular unresponsiveness to gonadotropins does not seem to be responsible for the postnatal decline of testicular activity; 2) during the prepubertal period there are no age-related changes in the absolute rises of T, delta4, or 17OHP levels after hCG stimulation; and 3) increasing sensitivity of the testis to gonadotropic stimulation appears to be one of the very first changes occurring at the onset of puberty.


Plasma melatonin, luteinizing hormone, follicle-stimulating hormone, prolactin, and corticoids in two patients with pinealoma.

Plasma melatonin, LH, FSH, PRL, and corticoids were measured in two patients with pineal tumors. Plasma melatonin was not detectable (less than 7 pg/ml) in either patient while gonadotropin and cortisol levels were within the normal range. One patient exhibited low PRL levels and the other patient, a prepubertal boy, had elevated levels. The clinical value of the measurement of melatonin as a potential marker for all pineal tumors must be questioned.


LH, FSH and skin temperaure during the menopausal hot flash.

Six postmenopausal women, who were experiencing frequent hot flashes, had an 8 h continuous recording of skin temperature over the dorsum of the finger as an objective index of hot flashes. Frequent blood samples were obtained during the time of the recording for the measurement of serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels. During the 48 h of recording 34 significant temperature elevations were recorded and 32 were associated with a subjective hot flash. 3l pulses of LH release were also observed with 26 occurring simultaneously with the temperature rises. Correlation analysis of simultaneous skin temperature and circulating LH levels showed a significant positive correlation (p less than 0.01). FSH levels showed no consistent relationship with skin temperature. These data suggest that LH or the factors that trigger its pulsatile release are related to the mechanism responsible for the initiation of hot flashes.


Effect of melatonin on L-tryptophan- and apomorphine-stimulated growth hormone secretion in man.

The effect of subacute melatonin (250 mg every 8 h over a period of 40 h) and placebo treatment on L-tryptophan- and apomorphine-stimulated GH secretion was tested in 13 volunteers. Melatonin premedication significantly reduced the GH response to peroral L-tryptophan loading, suggesting that melatonin affects GH secretion in man by interfering with serotonergic transmission. In the apomorphine test, no significant difference in GH response was observed after melatonin when compared to placebo treatment. This suggests that dopaminergic mechanisms do not have a central role in mediating actions of melatonin on the hypothalamic-pituitary axis. It is further proposed that melatonin influences GH secretion at a suprahypophyseal level.


The significance of elevated levels of plasma 18-hydroxycorticosterone in patients with primary aldosteronism.

Plasma 18-hydroxycorticosterone (180HB) concentration was measured in 23 patients with primary aldosteronism. After overnight recumbency, the levels were markedly elevated and were 6 times higher in patients with aldosterone-producing adenomas than in patients with hyperplasia. A value of 100 ng/dl or greater at 0800 h after overnight recumbency distinguished an adenoma from hyperplasia. There was no overlap of values, as was observed with plasma aldosterone concentration (PAC) at 0800 h and after 4 h of upright posture at 1200 h. There was a significant negative correlation between the ratio of 180HB to PAC and the potassium concentration in patients with primary aldosteronism due to hyperplasia. Potassium repletion with potassium chloride and/or spironolactone in patients with aldosterone-producing adenomas decreased the 180HB:PAC ratio by decreasing 180HB and increasing PAC. 180HB is an effective discriminator of an adenoma and may be a useful marker of the events in late aldosterone synthesis.