Studies on P-450. X. On the coordination structure of hemoprotein P-450.

The spectral properties of a P-450 preparation from adrenocortical mitochondria were compared with those of both other hemoproteins and model compounds to examine the possibility suggested by previous authors that the mercaptide anion (thiolate anion, RS-) of a cysteine residue might be one of the axial ligands of P-45O. 1. The following features were found in addition to the well known anomalous spectral characteristics of P-450. The Soret band of the ferrous form was at anomalously short wavelengths compared with those of other ferrous high-spin hemoproteins. The regularity in the shifts of the Soret maximum observed with various forms of usual hemoproteins was disrupted in the case of P-450 by the maximum of the ferrous form. 2. The complex of methemoglobin with 2-mercaptoethanol, which was the model for P-450 used by previous authors for spectral and ESR studies, was studied more precisely. The model showed an absorption spectrum very similar to that of the imidazole complex of ferric P-450, whereas the complex of methemoglobin with imidazole showed both Soret and visible absorption maxima at shorter wavelengths. The rapid rate of reduction of methemoglobin by dithionite decreased on binding to mercaptoethanol and the slow rate of reduction of P-450 increased on binding to imidazole. Thus the half-reduction times for both complexes were of the same order of magnitude. 3. An absorption spectrum which was very similar to the spectrum of ferric high-spin P-45O could be obtained by quick scanning immediately after ferric protoheme had been dissolved in a 33% solution of mercaptoethanol in water containing Tween 20 and NaOH. 4. Protoheme dissolved in an aqueous solution of Nikkol BL9EX (a non-ionic detergent) containing NaOH, dithionite, and 1 mM mercaptoethanol formed a transient intermediate complex having a Soret maximum at 470 nm. Treatment of this complex with carbon monoxide resulted in the formation of a CO adduct having a Soret maximum at 450 nm and a single band at about 555 nm in the visible region. These maxima were similar to those of the carbon monoxide complex of P-450. Many of the results could be explained readily by assuming that a mercaptide anion was one of the axial ligands, and also that heme was held in a hydrophobic crevice in the P-450 molecule. The coordination structure of P-450 seems to be more flexible than those of usual hemoproteins.


Rat intestinal brush border membrane peptidases. I. Solubilization, purification, and physicochemical properties of two different forms of the enzyme.

Two brush border peptidases have been isolated from the particulate fraction of the rat intestinal mucosa and purified to homogeneity as judged by polyacrylamide gel electrophoresis, starch gel electrophoresis, isoelectric focusing, and double immunodiffusion. For convenience, the peptidases have been designated peptidase F (fast) and S (slow) on the basis of their anodic mobilities. The isoelectric point of peptidase F was 4.76 and of peptidase S, 5.10. Both enzymes are glycoproteins. The amino acid compositions of the two peptidases are similar. The same carbohydrates are found in both enzymes, but there are differences in the molar concentrations of individual sugars. Peptidase S has greater concentrations of mannose and galactose and of hexosamines than peptidase F, while sialic acid is slightly greater in peptidase F. Carbohydrate accounted for approximately 19% and 23% of the weight of peptidases F and S, respectively. Estimates of the molecular weights of both enzymes by gel filtration gave values of 280,000. Electrophoresis of the enzymes under denaturing conditions on sodium dodecyl sulfate polyacrylamide gels indicated that each enzyme is a dimer consisting of two subunits of equal molecular weight, 140,000.


Preparation and properties of partially purified pulmonary cytochrome P-450 from rabbits.

Cytochrome P-450 from rabbit pulmonary microsomes was purified approximately 32-fold. The purification method involved solubilization of microsomes using sodium cholate, and recovery of cytochrome P-450 in the precipitate formed between 25 to 42% saturation of the digested microsomes with ammonium sulfate in the absence of glycerol. Further purification was achieved by chromatography on DEAE-cellulose and hydroxylapatite using Emulgen 913 as an eluent. Partially purified preparations containing up to 7.4 nmol of cytochrome P-450 per mg of protein were essentially free of NADPH-cytochrome c reductase activity and cytochromes b5 and P-420. However, epoxide hydrase was found to co-purify with cytochrome P-450. The CO-difference spectrum of dithionite-reduced purified cytochrome showed the expected peak at 450 nm. However, the magnitude of the peak was dependent on added microsomal lipid fraction in the assay medium. Purified pulmonary cytochrome P-450 formed typical types I and II substrate difference spectra with benzphetamine and pyridine, respectively. Sodium dodecyl sulfate-gel electrophoresis of partially purified cytochrome P-450 gave two major bands when stained with Coomassie blue. The faster moving band which contained peroxidase activity had an estimated molecular weight of 49,000 +/- 1,200. The cytochrome P-450 fraction, when combined with solubilized pulmonary microsomal NADPH-cytochrome c reductase and lipid fractions, was active in the O-deethylation of 7-ethoxycoumarin and the N-demethylation of benzphetamine.


Substitution of lanthanide ions for calcium ions in the activation of bovine prothrombin by activated factor X. High affinity metal-binding sites of prothrombin and the derivatives of prothrombin activation.

The substitution of lanthanide ions for Ca(II) in the Ca(II)-binding sites of prothrombin and the derivatives of prothrombin activation and in the metal-dependent conversion of prothrombin or prethrombin 1 to thrombin was studied at pH 6.8. Gd(III), Tb(III), La(III), Dy(III), Pr(III), Sm(III), and Ce(III) may be substituted for Ca(II) in the generation of thrombin from prothrombin or prethrombin 1 by activated factor X. The rates of thrombin generation in the presence of optimal concentrations of Gd(III) were about 25% for prothrombin and prethrombin 1 compared to the rate of thrombin generation with optimal concentrations of Ca(II). Maximal rates of thrombin generation were observed at 20 muM Gd(III) using prothrombin as substrate, compared to 10 muM Gd(III) when prethrombin 1 was employed. Using the steady state rate-dialysis method, the high affinity metal-binding sites of prothrombin and the products formed during prothrombin activation were characterized using 153Gd(III). Prothrombin has two high affinity binding sites for Gd(III) (Kd = 0.75 muM). Prethrombin 1 and prethrombin 2 each bind one Gd(III) tightly (Kd = 1.10 muM and 0.81 muM, respectively). Fragment 1, the phospholipid-binding portion of prothrombin, has two sites which bind Gd(III) tightly (Kd 0.16 muM). Fragment 2 has no high affinity metal-binding sites, but has intermediate affinity metal-binding sites (Kd greater than 1.6 muM). Thrombin has numerous high affinity binding sites (Kd less than 0.1 muM), suggesting that the conversion of prethrombin 2 to thrombin is associated with a significant change in tertiary structure. These results indicate that Gd(III) binds tightly to the metal-binding sites of these proteins and can substitute for Ca(II) in metal-dependent prothrombin activation. In the activation of prothrombin by activated factor X, these data suggest that Ca(II) is required for metal-dependent factor V and phospholipid binding and not as a cofactor in enzyme catalysis.


Human tryptophan transfer ribonucleic acid synthetase. Composition, function of thiol groups, and structure of thiol peptides.

Human tryptophanyl-tRNA synthetase resembles its counterpart in Escherichia coli in quaternary structure (alpha2), but differs in molecular weight, amino acid composition, the number of thiol groups, and the relationship of the thiol groups to enzyme activity. Nevertheless, one of the thiol groups resides in a heptapeptide sequence homologous to a heptapeptide sequence containing a thiol group in the E. coli enzyme. Each subunit of the enzyme has 6 half-cystine residues, and four thiol groups are readily titrated with 5,5'-dithiobis(2-nitrobenzoic acid). Titration of these four thiol groups inactivates the enzyme, and the inactivation is partially reversible by reduction with dithiothreitol. One thiol group reacts rapidly unless L-tryptophan, ATP, and Mg2+ are present together.


Conversion of a C-20-deoxy-C21, steroid, 5-pregnen-3beta-ol, into testosterone by rat testicular microsomes.

5-[7ALPHA-3H] Pregnen-3beta-ol, a C-20-deoxy analog of pregnenolone, was synthesized and tested as a substrate for the enzyme system occurring in testes that cleaves the side chain of C21 steroids between C-17 and C-20. This C-20-deoxy C21 steroid was incubated with a microsomal preparation obtained from rat testis and was converted into testosterone in 5% yield. Another C-20-deoxy analog of pregnenolone, 5,20-pregnadien-3beta-ol, was not converted into testosterone by this enzyme system. The significance of this finding for the natural processes by which pregnenolone is converted by the same subcellular fraction into the male sex hormone is examined in the light of the hypothesis that intermediates involved in steroidogenesis are transient, reactive complexes of the appropriate reactants (steroids, oxygen, etc.) with specific enzymes.


Identification of separate acyl- CoA:glycine and acyl-CoA:L-glutamine N-acyltransferase activities in mitochondrial fractions from liver of rhesus monkey and man.

The conjugation of glycine to benzoates and the conjugation of L-glutamine to certain arylacetates are catalyzed by two different acyl-CoA:amino acid N-acyltransferases which can be purified separately from liver mitochondrial fractions of either rhesus monkey or man. In both species, one transferase is specific for glycine and the other for L-glutamine. The glycine enzyme utilizes either butyryl-CoA or benzoyl-CoA as acyl donors while the glutamine enzyme uses either phenylacetyl-CoA or indoleacetyl-CoA. Acyl-CoA substrates for one transferase do not serve as substrates for the other. Additional studies with the monkey liver enzymes revealed that acyl-CoA substrates for one transferase inhibit the other, that the apparent Km value is low (10(-6) to 10(-5) M range) for the preferred acyl-CoA substrate as compared to the amino acid acceptor (greater than 10(-2) M) and that both transferases have a molecular weight of approximately 24,000. Hippuric acid and either phenylacetylglutamine or indoleacetylglutamine were characterized as the products formed by the separate enzymes.


Mutations affecting the quaternary structure of the lac repressor.

A new class of point mutations in the lacI gene of Escherichia coli is described which results in a lac repressor that is unable to form its normal tetrameric structure. Many of these mutations result in amino acid substitutions in the neighborhood of the tyrosine at residue 269. An amber mutation derived from the codon normally specifying tyrosine 269 has been suppressed with Sul, which inserts serine in response to the amber codon. The resulting altered lac repressor is also unable to form its normal tetrameric structure and can no longer bind to the lac operator. Two missense mutations which map in the part of the gene specifying amino acid residues 210 to 216 also produce repressors unable to form tetramers.


Prostaglandin biosynthesis and catabolism in the developing fetal sheep brain.

The biosynthesis of prostaglandins E2 and F2alpha as well as the catabolism of [9beta- 3H1]prostaglandin F2alpha by homogenates of whole brain from fetal and neonatal lambs was investigated. The biosynthetic measurements utilized the mass spectrometric deuterium isotope dilution-dual ion monitoring technique (Samulsson, B., Hamberg, M., and Sweeley, C.C. (1970) Anal. Biochem. 38, 301-304; Wolfe, L.S., and Pace-Asciak, C. (1972) in Prostaglandins in Fertility Control (Bergström, S., Green, K., and Samuelsson, B., eds) Vol. 2. pp. 201-207, WHO, Karolinska Institutet, Stockholm), whereas the activity of the catabolizing system was measured by the radiolabel dilution-thin layer chromatographic technique reported previously (Pace-Asciak, C. (1975) J. Biol. Chem. 250, 2795-2800). The structures of all products were confirmed by mass spectrometry. Early fetal brain (gestational age 30 to 32 days) contained the highest activity of NAD-dependent prostaglandin 15-hydroxy dehydrogenase which varied inversely with age of the fetus, dropping to almost undetectable levels by neonatal Day 4. Both prostaglandins E2 and F2alpha, were formed by brain from all ages tested and the total prostaglandins formed rose gradually with age. Thus, the period of maximal prostaglandin catabolism which we term the "critical prostaglandin period" appears in the fetal lamb brain around 30 days gestational age or earlier. These findins together with others previously reported by us in other tissues, support the concept that prostaglandin catabolism might play an important role in protecting the developing organ from possible adverse effects of locally formed or circulating prostaglandins. These adverse effects on the developing brain might relate to the known vasoconstrictor properties of the prostaglandins, as well as to their known effects on the induction of cell differentiation.


Neuroblastoma adenylate cyclase. Role of 2-chloroadenosine, prostaglandin E1, and guanine nucleotides in regulation of activity.

Neuroblastoma adenylate cyclase is activated by 2-chloroadenosine, prostaglandin E1, and 5'-guanylylimidodiphosphate [GMP-P(NH)P]. However, the process of activation by the first two compounds is different from that induced by the third. Prostaglandin E1 and 2-chloroadenosine activation is rapid, producing elevated activities which are constant throughout a 20-min assay. In contrast, GMP-P(NH)P activation is slow and although the activity is elevated within 1 min, it continues to increase for up to 12 min before attaining a maximal constant value. Activation is more rapid when either prostaglandin E1 or 2-chloroadenosine is present with GMP-P(NH)P. Activation of the enzyme by GMP-P(NH)P appears to be retarded by endogenous nucleotides as suggested by the following observations: (a) if the enzyme is incubated at 30 degrees with 5 mM MgCl2 for 5 to 7 min, GMP-P(NH)P then produces maximal activation without a detect able lag; (b) if, during this incubation, nucleotides, a nucleotide regenerating system, or EDTA (instead of MgCl2) are present, subsequent GMP-P(NH)P activation is slow; and (c) in the assays which contain a nucleotide regenerating systm and MgATP as substrate, the Km for GMP-P(NH)P is 6 +/- 2 muM. However, in the assays using MgAMP-P(NH)P as substrate but no nucleotide regenerating system, the Km is 0.5 +/- 0.2 muM. GPD and GTP do not replace GMP-P(NH)P as an enzyme activator in any of our assays systems, and in fact, are potent inhibitors of GMP-P(NH)P enzyme activation. Prostaglandin E1 and 2-chloradensine do not alter significantly the Km for GMP-P(NH)P but do decrease the ensyme's sensitivity of GDP. Proposed is a hysteretic model of neuroblastoma adenylate cyclase, which shows the enzyme responding slowly to rapid changes in GMP-P(NH)P concentration due to the slow displacement of the tightly bound endogenous guanine nucleotides by GMP-P(NH)P. Additionally, prostaglandin E1 and 2-chloroadenosine increase the rate of GMP-P(NH)P activation by decreasing the enzyme's affinity for these endogenous guanine nucleotides.


Origins of circular dichroism bands in Bowman-Birk soybean trypsin inhibitor.

The spectral properties of Bowman-Birk soybean trypsin inhibitor (BBI) were investigated by analyzing difference absorption spectra and difference CD spectra and by comparing them with those of tyrosyl model compounds. The O-acetylation of tyrosyl side chains showed that the ultraviolet CD bands of BBI above 225 nm originate from disulfide side chains and tyrosyl phenolic groups; phenylalanyl residues do not give rise to detectable CD in BBI in this wavelength region. The results of the tyrosyl ionization experiment were consistent with this interpretation. A broad negative CD band centered around 280 nm in BBI arises mainly from disulfide bonds (epsilonL - epsilonR = -0.83 M-1 cm-1 per disulfide). Each of 2 tyrosyl residues gives rise to negative CD in this region; together they contribute approximately 10% of the total CD intensity at 277 nm (epsilonL - epsilonR = -0.36 M-1 cm-1 per tyrosyl). Disulfide bonds in BBI also have a broad positive CD band centered around 240 nm (epsilonL- epsilonR = 0.9 M-1 per disulfide(. Tyrosyl side chains give rise to a sharp positive peak at 231 nm, overlapping with the positive disulfide CD. Dimerization of monomeric BBI did not alter the CD profile. One of two tyrosyl phenolic groups is relatively exposed and can be 0-acetylated by 100- to 1500-fold molar excess of N-acetylmidazole. The other is inaccessible to the reagent even in the presence of 8 M urea, but can be acetylated in the presence of 6 M guanidine hydrochloride. Fully acetylated BBI has the near-ultraviolet disulfide CD and the far-ultraviolet polypetide CD very similar to those of the native inhibitor, indicating the O-acetylation of two tryosyl side chains did not induce much conformational change in BBI. The near-ultraviolet CD of BBI was altered in the presence of 8 M urea of 6 M guanidine hydrochloride, with a greater change brought about by the latter. Dithiothreitol (20 mM) completely abolished the tyrosyl and disulfide CD in this region.


Comparison of initial velocity and binding data for allosteric adenosine monophosphate nucleosidase.

Adensine monophosphate nucleosidase (AMP nucleosidase) from Azotobacter vinelandii is composed of six subunits with similar or identical charge and size and has a molecular weight of approximately 320,000. Binding studies with tritiated tubercidin 5' -PO4 (4-amino-7-(beta-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5' -monophosphate), a competitive inhibitor with respect to the substrate, AMP, indicate the presence of three independent, identical binding sites for the substrate analog. The binding of tubercidin 5' -PO4 is not affected by either Mg2+ or MgATP2-; however, in initial velocity experiments MgATP2- caused from greater than100- to 4,000-fold activation of substrate hydrolysis depending on the concentration of AMP. Binding studies with [14C]ATP are consistent with six interdependent binding sites for MgAT2-. Initial velocity and binding curves for MgATP2- are similar in shape, but reveal a disproportionate increase in initial velocity at low saturation levels of MgAT2-. Binding of MgAT2- is inhibited by increasing concentrations of P1 which acts as a competitive inhibitor of MgATP2- activation in both initial velocity and binding experiments. In the absence of MgATP2-, 32Pi binds at six or more interdependent modifier sites. The simulataneous binding of Mg[14C]ATP2- and 32Pi was studied in experiments where MgATP2- and Pi were held in constant ratio. Extrapolation to infinite concentrations of both MgATP2- and Pi indicated that 3 molecules of each were bound to the enzyme. Thus the binding of the allosteric activator and inhibitor are mutually exclusive. These results are consistent with a single modifier site per subunit at which either MgATP2- or Pi may combine, or with separate activator and inhibitor sites which cannot be filled simultaneously. Comparative initial velocity and binding studies with Pi indicate that the initial rate of AMP hydrolysis depends primarily on the extent of modifier site saturation with MgATP2-. Thus when two sites are filled with MgATP2-, the initial rate is approximately the same as when two additional modifier sites are filled with Pi. Binding of Pi, therefore, does not appear to affect the catalytic effectiveness of the active site when MgATP2- is also present, except by the displacement of MgATP2-.


Characterization of Enzyme-like activity of human hemoglobin. Properties of the hemoglobin-P-450 reductase-coupled aniline hydroxylase system.

Human hemoglobin was characterized as an enzyme in a reconstituted aniline hydroxylase system containing hemoglobin, NADPH, rat liver cytochrome P-450 reductase, aniline and atmospheric O2. This system catalyzed p-aminophenol formation (turnover number 0.2 mol/min/mol of hemoglobin) with an efficiency similar to that which has been reported for either microsomal cytochrome P-450 or cytochrome P-450 solubilized from rat liver. The rate of the reaction was linearly dependent on hemoglobin concentration up to approximately 1 nmol of hemoglobin/ml. This linear range of hemoenzyme concentration is also similar to cytochrome P-450-catalyzed reactions. Unlike the cytochrome P-450 system, the hemoglobin system did not require a lipid cofactor for maximal activity, and much less reductase was needed for maximal activity. Aniline displayed typical Michaelis-Menten saturation kinetics as substrate, and its Km (8 mM) was the same in the absence of presence of the reductase. Catalase essentially completely inhibited p-aminophenol formation in the absence or presence of reductase. In contrast, superoxide dismutase inhibited the reductase-mediated reaction only to a small extent (if at all). No detectable hydrogen peroxide accumulated during the course of the reaction in the absence of catalase. These findings suggested a hypothetical mechanism for hemoglobin-catalyzed hydroxylation of aniline involving a hemoglobin-bound form of hydrogen peroxide (aniline-Hb3+-OOH-) as an intermediate preceding the rate-determining formation of products.


Side chain hydroxylations in biosynthesis of cholic acid. 25- and 26-Hydroxylation of 5beta-cholestane-3alpha, 7alpha, 12alpha-triol by reconstituted systems from rat liver microsomes.

25- and 26-Hydroxylation of 5beta-cholestane-3alpha, 7alpha, 12alpha-triol was studied with reconstituted systems from rat liver microsomes consisting of partially purified cytochrome P -450, NADPH-cytochrome P -450 reductase, a phospholipid, and an NADPH -generating system. Cytochrome P -450 was prepared either by sodium cholate treatment and ammonium sulfate fractionation or by subtilisin and sodium deoxycholate treatment followed by DEAE-cellulose chromatography. No side chain hydroxylation was observed when cytochrome P-450 was omitted. With ammonium sulfate-fractionated cytochrome P-450 25- and 26-hydroxylation was stimulated 5- to 8-fold by addition of NADPH-cytochrome P-450 reductase. With subtilisin-treated cytochrome P-450 an almost absolute requirement for NADPH-cytochrome P-450 reductase was observed. Omission of lipid did not reduce the rate of hydroxylation. Centrifugation of the cytochrome P-450 preparation at 100,000 X g for 1 hour just before incubation increased markedly lipid dependency. A significant difference between 25- and 26-hydroxylation was observed with respect to substrate saturation. The stimulatory effect of phenobarbital treatment on 25-hydroxylation and the inhibitory effect of this treatment on 26-hydroxylation were associated with the cytochrome P-450 fraction. The use of increasing amounts of sodium cholate in the solubilization of cytochrome P -450 resulted in a gradual decrease of 25-hydroxylase activity and a gradual increase of 26-hydroxylase activity. 25- and 26-Hydroxylase activities were separated partially by chromatography of subtilisintreated cytochrome P-450 fraction on DEAE-cellulose. The question whether different species of cytochrome P-450 are involved in 25- and 26-hydroxylation is discussed.


Glycoprotein-enriched vesicles from sheep erythrocyte ghosts obtained by spontaneous vesiculation.

Sheep erythrocyte membranes have been shown in this laboratory to undergo spontaneous vesiculation when incubated at 4 degrees, fractionating into two bands in dextran gradients (R. McGuire and R. Barber, submitted for publication). While vesicles were observed to be formed in several solvent systems, incubation in the presence of complexors to remove divalent cations was found to be the most efficient method for both vesicle formation and their detachment from the residual membrane. We report here on the characterization of these vesicles formed by spontaneous vesiculation. In the presence of a hypotnoic buffer containing 1 mM EDTA, vesicle production proceeds linearly up to 50 hours and declines, reaching its maximum at 72 hours with up to 20% of the total membrane protein found in the upper band. This upper band is shown in electron micrographs to be composed chiefly of closed vesicles, while the particles in the lower band appear morphologically similar to the original ghosts. Total phospholipid phosphorus and cholesterol in the vesicles are enriched to the same extent, giving a lipid to protein ratio of 2 times that found for whole ghosts. The vesicles contain the same individual phospholipids as the ghosts. The protein composition of these vesicles is unique, in that they are almost depleted in the known extrinsic membrane proteins, while containing practically all types of the various glycoproteins of the original membrane. The two main intrinsic membrane proteins (with apparent molecular weights of 160,000 and 100,000) are found almost exclusively in the vesicles, virtually depleted in the residual ghost-like particles. The protein with 160,000 molecular weight is shown here to be a glycoprotein, giving an anomalous molecular weight on sodium dodecyl sulfate gels and having a molecular weight of approximately 50,000 after lipid extraction. This same glycoprotein appears to fractionate with acetylcholinesterase. From the accessibilities of the substrates to the membrane acetylcholinesterase and NADH-diaphorase, it is concluded that the vesicles are right-side-out and sealed to small molecules. There are more membrane sialic acid residues accessible to neuraminidase in the vesicles (in terms of number of residues/mg og membrane protein) than in ghosts, further supporting the conclustion that these vesicles have a normal orientation and are enriched in glycoproteins. The specific activity of acetylcholinesterase in the vesicles is increased 5- to 6-fold over that found in the original ghosts and almost 20-fold over that in the residual ghost-like particles. Consequently, spontaneous vesiculation occurs simultaneously with the enrichement of specific membrane proteins in certain regions of the lipid bilayer. It is postulated that these domains in the membrane, containing clusters of specific intrinsic membrane proteins, bud out and subsequently release glycoprotein-enriched lipid vesicles.


Protein degradation in human fibroblasts (WI-38). Effects of aging, viral transformation, and amino acid analogs.

Protein degradation occurs more rapidly in senescent WI-38 cultures than in phase II cultures or in SV-40 transformed WI-38 cells (VA-13). The first differences are found in early phase III, when short lived but not long lived proteins are degraded more rapidly. At the end of phase III long lived proteins are also degraded more rapidly as shown by both intermittent perfusion and approach to equilibrium methods. By both methods the rates of protein degradation for the virally transformed derivative are the same as those for phase II WI-38, implying that transformation has not altered these characteristics of protein degradation. WI-38 cells incorporate canavanine, an analog of arginine, into protein. This analog, as well as p-fluorophenylalanine and azetidine carboxylic acid, accelerates the degradation of proteins labeled with [3H]leucine in their presence but does not alter the degradation rates of proteins prelabeled with [14C]leucine in the absence of the analogs. These results imply that the analogs increase the intracellular degradation rates of proteins because they render them more susceptible to the degradative system. Late phase III WI-38 cells may not selectively catabolize proteins containing canavanine as rapidly as do phase II and VA-13 cells. These results imply that the phase III protein degradative system becomes partially defective, thereby losing its ability to rapidly catabolize altered protein which leads to increased levels of abnormal proteins and decreased cell function.


Dietary regulation of 6-phosphogluconate dehydrogenase synthesis.

The relative rates of synthesis and degradation for rat liver 6-phosphogluconate dehydrogenase have been determined in animals maintained at several dietary states. Relative rates of synthesis were determined by pulse-labeling the enzyme either in live rats and determining the radioactivity in the purified enzyme or in whole cell suspensions of hepatocytes followed by precipitation of the enzyme with a specific antiserum. The relative rate of synthesis of the enzyme in rats fed a high carbohydrate fat-free diet was approximately 3.7 to 5.6 times greater than that in animals fed a pellet diet or in fasted rats, respectively. In contrast, a half-life of 13 to 19 hours was determined for the degradation of the enzyme in all three nutritional states. We have concluded that nutritional alterations in the levels of this enzyme in rat liver are caused by alterations in the rate of enzyme synthesis. Glucagon has no effect on the rate of synthesis of this enzyme.


Chemical characterization and distribution of ABO blood group active glycoprotein in human erythrocyte membrane.

Occurrence and distribution of glycoprotein H substances in blood type O human erythrocyte membrance were studied using an alpha N-acetylgalactosaminyltransferase (A-enzyme) purified from milk of blood type A women. Erythrocytes of blood group O, nonsecretors and secretors alike, contain H substances that serve as a substrate for this transferase, and the H determinants seem to occur in all three erythrocyte membrane glycoproteins (periodic acid-Schiff (PAS)-1, PAS-2, and PAS-3). Structural studies of the A glycoproteins obtained by the transferase action, suggested that all erythrocyte H glycoproteins contain only type 2 antigenic determinant.


On a possible mechanism of energy conservation in sarcoplasmic reticulum membrane.

The activation of ATP  equilibrium Pi exchange, ITP equilibrium Pi exchange, and the degree of phosphorylation of the membrane of sarcoplasmic reticulum vesicles by ATP, ITP, and Pi were compared under different experimental conditions. In media containing 0.1 mM CaCl2, 6 mM Pi, and 4 mM ATP, during the period of Ca2+ accumulation the rate of ATP equilibrium Pi exchange was very low and the level of membrane phosphorylation by ATP was about 10-fold higher than the level of membrane phosphorylation by Pi. When net Ca2+ accumulation ceased and the Ca2+ concentration of the assay media had fallen to less than 5 muM, the degree of membrane phosphorylation by ATP decreased 4-fold and both the level of membrane phosphorylation by Pi and the rate of ATP equilibrium Pi exchange increase 4- to 6-fold. Contrasting with these data, when ATP was replaced by ITP, the rate of ITP equilibrium Pi exchange and the level of membrane phosphorylation by Pi were already high during the period of Ca2+ accumulation and varied slightly when the Ca2+ concentration of the incubation medium decreased to less than 5 muM. During the period of Ca2+ accumulation, the degree of membrane phosphorylation by Pi varied inversely with the NTP or NDP concentration of the medium, ATP and ADP being more effective than ITP and IDP in inhibiting the membrnae phosphorylation by Pi. Leaky vesicles incubated in media containing a high Ca2+ concentration were still able to catalyze both ATP equilibrium Pi and ITP equilibrium Pi exchange. Although the membrane of leaky vesicle was amply phosphorylated by Pi in media containing 0.1 nM CaCl2 and ITP, a significant rate of ITP equilibrium Pi exchange could only be measured in Ca2+ concentrations higher than 0.5 mM. The Ca2+ concentration required for half-maximal activation of the rate of either ITP equilibrium Pi or ATP equilibrium Pi exchange in leaky vesicles was found to be in the range of 1 to 2 mM. In leaky vesicles, the apparent Km of Pi for the ITP equilibrium Pi exchange was at least 1 order of magnitude lower than for the ATP equilibrium Pi exchange.


Metal ion activation of galactosyltransferase.

Galactosyltransferase, which functions as the catalytic component of lactose synthase and in the glycosylation of glycoproteins, has been previously reported to have an absolute dependence on Mn2+ for activity, with a Kd for Mn2+ (10(-3) M) 2 to 3 orders of magnitude greater than the physiological range of Mn2+ concentrations (v 10(-6) M). Reinvestigation of the metal ion dependence of this enzyme has shown that Zn2+, Cd2+, Fe2+, Co2+, and Pr3+ also produce activation, although with lower activities at saturation than that attained with Mn2+. Velocity against metal ion concentration curves for all metals, including Mn2+, are sigmoid, suggesting the presence of two or more activating metal binding sites on the enzyme. The presence of two sites is confirmed by studies using both Mn2+ and Ca2+. While galactosyltransferase is inactive in the presence of Ca2+ alone, at low concentrations of Mn2+ (10(-5) M), enzyme activity is stimulated by Ca2+. A more detailed investigation by steady state kinetics has revealed that there is a tight binding site for Mn2+ (site I: Kd of 2 X 10(-6) M) from which Ca2+ is excluded, and a site at which Ca2+ can replace Mn2+ (site II: Kd for Ca2+ of 1.76 X 10(-3) M), to which metal binding has a specific synergistic effect on UDP-galactose binding, possibly as a result of the formation of an enzyme-Ca2+-UDP-galactose bridge complex. The site I Mn2+, site II Ca2+-activated enzyme has a maximum velocity similar to that of the Mn2+-activated enzyme, and is the enzyme form that must act in lactose synthesis in vivo. A trypsin-degraded form of galactose transferase (galactosyltransferase-T) (Powell, J.T., and Brew, K. (1974) Eur. J. Biochem. 48, 217-228) appears to lack site I and is activated by Ca2+ in the absence of Mn2+.


A comparison of the interactions of galactosyltransferase with a glycoprotein substrate (Ovalbumin) and with alpha-lactalbumin.

Sedimentation velocity ultracentrifugation and other procedures have been used to investigate macromolecular interactions of bovine colostrum galactosyltransferase with a glycoprotein substrate (ovalbumin) and with the lactose synthase regulatory protein, alpha-lactalbumin. The determination of equilibrium binding constants for these interactions and the effects of ligands and combinations of ligands on the equilibria have clarified several aspects of the mechanism of galactosyltransferase and its regulation by alpha-lactalbumin. 1. The attachment of Mn2+ at the tight binding site on galactosyltransferase (site I, Kd 2.3 muM) is an essential prerequisite for interactions with ovalbumin and with alpha-lactalbumin. 2. The attachment of Mn2+ or Ca2+ at the weaker metal binding site (site II, Kd 1 to 2 mM) does not significantly affect the interaction of galactosyltransferase with either protein. This is consistent with the hypothesis derived from kinetic studies that site II is functionally connected with the binding of UDP-derivatives. 3. While the binding of ovalbumin to galactosyltransferase in the presence of Mn2+ alone can be observed by ultracentrifugation, this interaction is too weak to cause binding of galactosyltransferase to ovalbumin-Sepharose. Binding to ovalbumin-Sepharose could only be detected by affinity chromatography in the presence of both Mn2+ (10 mM) and UDP (0.3 MM). Sedimentation studies showed that the association of galactosyltransferase with ovalbumin is pressure-dependent, and that the presence of UDP in the complex increases the equilibrium association constant by a factor of 46. The enzyme Mn2+-UDP-ovalbumin complex has unusual hydrodynamic properties. 4. The presence of saturating concentrations of UDP-galactose potentiates the binding of alpha-lactalbumin at high concentrations of Mn2+, as shown by a 25-fold increase in the association constant. Competitive inhibition by alpha-lactalbumin, with respect to ovalbumin that is observed by steady state kinetics, is attributed to the mutally exclusive binding of the proteins with an enzyme complex containing Mn2+ and UDP-galactose. 5. Monsaccharides (N-acetylglucosamine and glucose) strongly enhance the binding of alpha-lactalbumin to enzyme complexes containing Mn2+, in the presence or absence of UDP-glucose. The binding of alpha-lactalbumin and monosaccharide to form  enzyme complexes containing both is random, and evaluation of the four associated equilibrium constants shows that the binding is strongly synergistic. 6. Although the significance of some of the many equilibria studied cannot be ascertained, it appears than an element of randomness may be present in reactions catalyzed by galactosyltransferase...


Crystallization of human placental estradiol 17beta-dehydrogenase. A new method for crystallizing labile enzymes.

Estradiol 17beta-dehydrogenase from human placenta has been crystallized by a new technique, herein referred to as electrophoretic diffusion. This is the first crystallization of an enzyme from human placenta as well as the first crystallization of any steroid-converting enzyme of human source. A solution of the enzyme (specific activity 7.1 units/mg) in 1.5 ml of Tris-barbituric acid buffer, pH 7.0, containing 20% glycerol as stabilizer, was placed in an electrophoresis tube and the tube was closed at both ends with a dialysis membrane which permits the passage of substances of molecular weight less than 18,000. The tube was placed in a gel electrophoresis apparatus and the reservoirs filled with the Tris-barbituric acid buffer. A potential of 100 V was applied for 12 hours, then raised to 200 V for another 12 hours, and finally to 300 V until opalescence appeared at the bottom of the tube. Activity measurements showed that more than 90% of the enzyme had concentrated in the bottom 0.15-ml portion of the solution. When this section of the solution was removed and kept overnight at 4 degrees, gross and microscopic examination revealed a heavy crop of crystals which possessed a specific activity of 7.2 units/mg. The specific activity remained constant throughout three recrystallizations. The crystalline enzyme displayed a single band by analytical and sodium dodecyl sulfate-polyacrylamide gel analysis. Crystals of enzyme of high specific activity could also be obtained from an enzyme sample initially possessing a specific activity of only 4.5 units/mg. The new technique should be appliable for the crystallization of other labile enzymes and receptor proteins which have so far resisted crystallization by conventional methods.


Effect of estrogen on gene expression in the chick oviduct. Effect of estrogen on the sequence and population complexity of chick oviduct poly(A)-containing RNA.

Total cellular RNA preparations were isolated from chicken oviducts at three different development stages: (a) immature chicks which were chronically stimulated with estrogen; (b) estrogen-stimulated chicks which were then withdrawn from hormone for 12 days; and (c) laying hens. Total cellular RNA containing 3'-poly(A) sequences (poly(A)-RNA) were than isolated from these preparations using oligo(dT)-cellulose chromatography. The number average nucleotide length of the poly(A)-RNA preparations in each case was approximately 2000 nucleotides. The number average nucleotide length of the poly(A) residues at the 3'-terminal end of each RNA preparation was approximately 70 adenylate residues. Complementary DNA (cDNA) copies to each preparation of poly(A)-RNA were synthesized using avian myeloblastosis virus RNA-directed DNA polymerase. The cDNApoly(A) preparations were then utilized in DNA excess hybridization experiments to analyze the complexity of the DNA sequences from which these RNAs were transcribed. Approximately 22% of each of the total cellular poly(A)-RNAs were transcribed from repeated DNA sequences (average repeat frequency of 35 copies/genome) while the remaining majority were transcribed from single copy or unique sequence DNA. It was possible to estimate the number of different poly(A)-RNA sequences per cell by analyzing the kinetics of hybridization of these cDNApoly(A) preparations to total cellular poly(A)-RNA extracts under conditions of RNA excess. The results revealed that 41% of the poly(A)-RNA from laying hen oviduct consisted of, on the average, three different sequences/cell, each of which was present in approximately 25,000 copies/cell. The remainder of the poly(A)-RNA in this tissue consisted of approximately 25,000 different sequences/cell, which were present largely in only two or three copies/cell. A somewhat similar sequence complexity was found for oviduct cells prepared from estrogen-stimulated chicks. We estimated that there were approximately 20,000 different poly(A)-RNA sequences/cell, each represented in only one to two copies/cell. However, there were five sequences which were present, on the average, in a concentration of 5600 copies/cell. The poly(A)-RNAs from hormone-wtihdrawn tissue, on the other hand, had a lower sequence complexity. There were only approximately 10,000 different poly(A)-RNA sequences/cell, each present in about three copies/cell. Furthermore, the few sequences present in a great abundance in hen and hormone-stimulated tissues were apparently absent in oviduct tissue from hormone-wtihdrawn chicks, suggesting that the intracellular concentrations of these high frequency RNA sequences are dependent on estrogen.


Effect of hypophysectomy on the synthesis of rat liver albumin.

Hypophysectomy of adult rats results in approximately a 50% decrease in the rate of albumin synthesis relative to total liver protein synthesis. This decrease is accompanied by a proportional decline in the number of albumin-synthesizing polysomes, as determined by the binding of 125I-Labeled anti-albumin antibody, and indirect immunoprecipitation of [3H]leucine-labeled albumin-synthesizing polysomes. Furthermore, this decrease is associated with an equivalent reduction in the amount of total membrane-bound polysomes, whereas total free polysomes show little quantitative change. The size of the specific albumin-synthesizing polysomes, as well as the size of the total polysomes, however, appear to be the same following hypophysectomy as in the normal untreated animal. These observations are consistent with the finding that the relative amount of albumin mRNA activity also decreases approximately 50%, as assayed in a heterologous cell-free protein-synthesizing system using exogenous liver RNA prepared from either isolated polysomes or total liver homogenates. The decrease in albumin production in the hypophysectomized rat, therefore, is apparently the result of a reduction in the amount of active albumin mRNA. The concomitant decrease in albumin-synthesizing polysomes appears to reflect a similar reduction in the amount of total membrane bound polysomes. Thus, a major physiological defect in hypophysectomy may be a preferential decline in membrane-bount polysomes accompanied by a reduction in mRNA levels, which is represented by the decrease in albumin synthesis.


Purification and subunit structure of deoxyribonucleic acid-dependent ribonucleic acid polymerase III from the posterior silk gland of Bombyx mori.

DNA-dependent RNA polymerase III was purified from the posterior silk gland of the moth Bombyx mori by chromatography on DEAE-cellulose, DEAE-Sephadex, CM-Sephadex, and phosphocellulose and by sedimentation in sucrose density gradients. The specific activity of this chromatographically homogeneous enzyme was comparable to that reported for other purified eukaryotic RNA polymerases. Sucrose gradient sedimentation analysis suggested a molecular weight of approximately 590,000 to 660,000 for B. mori RNA polymerase III. Analysis of subunit composition by polyacrylamide gel electrophoresis under denaturing conditions showed that the chromatographically purified RNA polymerase III contained subunits with molecular weights of 155,000 (IIIa), 136,000 (IIIb), 67,000 (IIIc), 62,000 (IIId), 49,000 (IIIe), 39,000 (IIIf), 36,000 (IIIg), 31,000 (IIIh), 28,000 (IIIi), and 18,000 (IIIj). Molar ratios were close to unity for all subunits except for IIIj, which was present in an approximate molar ratio of 2. As has been observed for mammalian class III enzymes, the B. mri RNA polymerase III can be resolved into two components upon electrophoresis under nondenaturing conditions. Comparative studies of the class III enzymes from B. mori and from higher eukaryotic cells show that many of the general chromatographic and catalytic properties, as well as the overall subunit compositions, are similar for the various enzymes. However, unlike the mammalian class III enzymes, B. mori RNA polymerase III is completely resistant to high concentrations of alpha-amanitin, and it does not contain an 89,000-dalton subunit. The data are discussed in terms of the function and regulation of RNA polymerase III in lower and higher eukaryotes.


Structure of Peptococcus aerogenes ferredoxin. Refinement at 2 A resolution.

The model for ferredoxin from Peptococcus aerogenes, derived from a 2.8-A resolution map, has been refined with a 2-A resolution data set. The conventional index, R, decreased from 0.449 for the initial model to 0.188 for the unconstrained one and 0.206 for the constrained one. The standard deviations of the iron and sulfur atoms are 0.04 to 0.05 A and for the oxygen, nitrogen, and carbon atoms they range from 0.14 to 0.26 A. One hundred forty-six water oxygen atoms were included in the solvent part of the model and were checked by a highly selective criterion, suggesting that most of them represented water molecules.


RNase III cleavage of single-stranded RNA. Effect of ionic strength on the fideltiy of cleavage.

Ribonuclease III from Escherichia coli has been purified to apparent homogeneity by affinity chromatography on immobilized double-stranded RNA. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate gave one band of protein with a molecular weight of approximately 25,000. Chromatography on Sephadex G-100 is consistent with a molecular weight of 50,000, suggesting that the native enzyme is a dimer. RNase III cuts some single-stranded RNAs, such as bacteriophage T7 early RNA, at specific sites in vivo. This RNA is cut as these same sites by the purified enzyme under all conditions tested. However, at low ionic strength relatively small increases in enzyme concentration produce cuts as secondary sites. At high ionic strength, the enzyme's preference for the sites cut in vivo is more pronounced and secondary cuts are made only at very high enzyme concentrations. Secondary cuts are shown to occur at specific sites and are made in a variety of RNAs even from sources other than E. coli. By cutting RNAs at secondary sites it should be possible to generate RNA fragments which would be useful in a number of studies.


Dissociation of beta-hydroxy-beta-methylglutaryl-CoA reductase activity from the overall rate of cholesterol synthesis in the liver following the intravenous administration of lipid.

After the intravenous administration to the intact rat of triglyceride carried in either intestinal lipoproteins or in an artificial fat emulsion, the enzymatic activities of microsomal beta-hydroxy-beta-methylglutaryl-CoA reductase activity in the liver assayed in vitro became markedly elevated. This elevation of enzyme activity was not associated with a corresponding change in the overall rate of cholesterol synthesis in the rat liver slice as measured by the incorporation of either [3H]water or [1-14C]octanoate into nonsaponifiable lipids or into digitonin-precipitable sterols. The degree of dissociation of hydroxymethylglutaryl-CoA reductase activity from the overall rate of cholesterol synthesis correlated closely with the amount of lipid administered to the animal, the level of circulating lipids, and the level of ketone synthesis manifest in the liver cell suggesting that this phenomenon might be the consequence of a detergent effect of elevated cellular levels of fatty acids. In any event, under these experimental circumstances hydroxymethylglutaryl-CoA redutase activity no longer reflects the rate at which the liver cell is synthesizing cholesterol.


The ovalbumin gene. Partial purification of the coding strand.

Purified ovalbumin messenger RNA was employed to selectively enrich the concentration of the gene coding for ovalbumin from total chick DNA by molecular hybridization. The coding strand of the ovalbumin gene was partially purified from sheared chick DNA by affinity column chromatography using ovalbumin mRNA immobilized on phosphocellulose. The concentrations of the ovalbumin DNA sequence in various DNA fractions were quantitated by measuring their rates of hybridization with 125I-labeled ovalbumin mRNA. When apparent Cot1/2 values of these reactions were compared to the apparent Cot1/2 value obtained from the hybridization reaction between 125I-ovalbumin mRNA and complementary DNA synthesized against ovalbumin mRNA using the enzyme reverse transcriptase, purification of the coding ovalbumin DNA strand over total chick DNA was estimated to be approximately 9,600-fold. There was no apparent degradation of the 4,000 nucleotide strands of chick DNA throughout the purification procedure. Since ovalbumin mRNA has a complexity of 1890 nucleotides, the resulting DNA was more than twice the length of ovalbumin mRNA and thus should contain DNA sequences adjacent to the structural portion of the ovalbumin gene.


The in vivo effect of colchicine on the addition of galactose and sialic acid to rat hepatic serum glycoproteins.

Colchicine inhibits the secretion of plasma protein by rat hepatocytes and causes their intracellular accumulation in Golgi-derived secretory vesicles. This study examines whether colchicine affects secretion before or after galactose and sialic acid have been added to the secretory glycoproteins. D-[G-3H] Galactose was injected into rats and was found to be incorporated into serum glycoproteins contained within Golgi-derived secretory vesicles. The administration of colchicine (25 mumol/100 g, body weight), immediately before the injection of D-[G-3H] galactose, caused an increase in radioactivity of the serum glycoproteins in these cell fractions. D-[G-3H] Glucosamine was incorporated into serum glycoproteins contained within the rough and smooth endoplasmic reticulum and the Golgi cell fractions; however, its incorporation into the sialic acid moieties of these proteins only occurred in Golgi-derived cell fractions. Colchicine administration resulted in an increased incorporation of D-[G-3H] glucosamine into the sialic acid residues of serum glycoproteins contained within the Golgi cell fractions. These data indicate that colchicine inhibits secretion of serum proteins by rat liver after the addition of galactose and sialic acid to the secretory proteins has taken place.


Pre-proparathyroid hormone. Evidence for an early biosynthetic precursor of proparathyroid hormone.

Pre-proparathyroid hormone, discovered previously by the translation of messenger RNA from parathyroid glands in heterologous cell-free systems, is a polypeptide of 115 amino acids consisting of 25 amino acids added to the NH2 terminus of proparathyroid hormone (90 amino acids), the immediate biosynthetic precursor of parathyroid hormone (84 amino acids). We now have detected pre-proparathyroid hormone formation within intact parathyroid cells, thus providing direct evidence that it is a biosynthetic precursor of proparathyroid hormone. A radiolabeled protein synthesized by slices of bovine parathyroid glands during 1 to 10 min of incubation with [35S] methionine was identified that co-migrated on both urea-acetate and urea-SDS acrylamide gels with 3H-labeled pre-proparathyroid hormone prepared by mRNA translation in a cell-free system derived from wheat germ. The amount of radiolabeled protein reached a maximum 3 min after exposure of the tissue to[35S] methionine and decreased during a 3-min chase incubation with unlabeled methionine. It bound to antisera to both proparathyroid hormone and parathyroid hormone and contained 35S-labeled tryptic peptides that migrated identically with peptides of pre-proparathyroid hormone prepared from the wheat germ system. Radiolabeled proparathyroid hormone was identified in the tissue after 1 min of incubation with [35S] methionine. These findings indicate that pre-proparathyroid hormone is an early biosynthetic precursor of proparathyroid hormone in parathyroid tissue and that this precursor is converted to proparathyroid hormone within 1 min after completion of its synthesis on the rough endoplasmic reticulum.


Interaction of streptokinase with plasminogen. Isolation and characterization of a streptokinase degradation product.

When streptokinase is incubated with human or rabbit plasminogen, one event which occurs is a specific fragmentation of streptokinase. At least five major identifiable streptokinase fragments appear with time, and they possess molecular weights of approximately 40,000 (SK 1), 36,000 (SK 2), 31,000 (SK 3), 26,000 (SK 4), and 10,000 (SK 5) under denaturing conditions, as observed on calibrated sodium dodecyl sulfate-polyacrylamide gels, compared to native streptokinase of molecular weight 45,000. The amount of each of the fragments generated at given times of incubation of plasminogen and streptokinase depends upon the species of plasminogen employed. Utilizing rabbit plasminogen and streptokinase, the SK 4 fragment was purified. This fragment arises by proteolysis at both the NH2 and COOH regions of native streptokinase. However, when isolated utilizing dilute aqueous buffers, the SK 4 fragment contained a portion of the original NH2 terminus of native streptokinase noncovalently bound to the molecule (SK 4'). SK 4' is capable of activating human plasminogen to plasmin, albeit more slowly than native streptokinase. However, the SK 4'-human plasmin complex possess only very weak plasminogen-activating activity toward sheep plasminogen. Upon removal of the noncovalently bound small NH2-terminal peptide of native streptokinase from SK 4', SK 4 is formed. This particular fragment possesses practically no human plasminogen-activating activity and cannot be used as an activator of sheep plasminogen, even with added human plasminogen.


Isolation and characterization of the unique prosthetic group of a green hemoprotein from human erythrocytes.

A simplified and streamlined purification scheme has been developed for the large scale isolation of a green hemoprotein from human erythrocytes. The isolation procedure involves hypotonic lysis, freezing of the hemolysate at -60 degrees, centrifugation, direct chromatography of the supernatant on DEAE-cellulose, and subsequent cation exchange, anion exchange, and gel filtration chromatography. Approximately 2 mumol of this anionic hemoprotein were isolated per liter of packed erythrocytes. The previous inability to separate the prosthetic group from the protein by conventional solvent extraction procedures was shown to be a consequence of the highly polar character of the heme and not due to covalent linkage between heme and protein. The polar nature and marked lability of the heme necessitated development of techniques for the extraction, purification, and derivatization of the prosthetic group. The heme was separated from the protein by membrane filtration in the presence of pyridine and alkali or by disc gel electrophoresis in the presence of cyanide. The heme was methylated with trimethyloxonium tetrafluoroborate and further derivatized. The heme derivatives were purified on columns of Sephadex LH-20 or alumina. Chromatography of the heme, heme methyl ester, acetylated heme ester, and the corresponding porphyrin derivatives suggests that the heme contains three carboxyl groups and one or more polar, acetylatable functional groups, probably hydroxyl groups. Spectral characterization of these compounds, as well as the derivatives resulting from reaction of the heme with NH2OH, NaHSO3, and Na2S2O4, show that the prosthetic group is a previously undescribed, formyl-containing heme that can be clearly distinguished from heme a, Spirographis heme, and all other naturally occurring prosthetic groups.


Interactions of oxytocin and vasopressin with bovine neurophysins I and II. Effects of hormone binding on the protein quaternary structure: a simple model.

The effects of hormone binding on the reversible monomer in equilibrium dimer equilibrium of bovine neurophysins I or II in solution have been studied by sedimentation equilibrium measurements performed in conjunction with equilibrium dialysis experiments. Under normal solution conditions saturating amounts of oxytocin displace the neurophysin dimerization equilibrium toward the associated form of the protein to give a dimeric complex with two oxytocin molecules bound per dimer. Vasopressin exerts different influences on this oligomerization process. At low fractional saturation this ligand exhibits a behavior similar to oxytocin with a higher affinity for the neurophysin dimer than the monomer. But in contrast, at higher fractional saturation, vasopressin strongly displaces the aggregation equilibrium toward a monomeric complex bearing two vasopressin molecules. However, in the presence of a high concentration of LiCl two oxytocin molecules are bound per neurophysin protomer (10,000 daltons). These observations, together with earlier data for vasopressin binding, suggest that each neurophysin molecule possesses two structurally distinct hormone binding sites. These observations can be rationalized in a simple schematic model of hormone binding to neurophysin in which oxytocin favors a dimeric form with one hormone binding site available per 10,000 daltons while vasopressin favors the monomeric form with two hormone binding sites available per 10,000 daltons.


Immobilized ATP and actin columns as a tool for the characterization and separation of different myosins and active myosin fragments.

A comparative affinity chromatography study using agarose-ATP columns revealed differences between heavy meromyosin subfragment 1 (HMM S-1) preparations obtained from rabbit white skeletal, rabbit red skeletal, bovine cardiac, and chicken gizzard muscle myosins. The characteristic patterns were markedly affected by Ca2+ and Mg2+ ions in a manner typical for each myosin species. Similar differences were also observed on comparing the intact myosins of red and white muscles. It thus became possible to separate, at least partially, mixtures of myosin (or HMM S-1) of different origins. Muscle "acetone-dried powder" was used as chromatographic medium for active myosin fragments in affinity chromatography columns. At low ionic strength the columns bound appreciable amounts of heavy meromyosin (HMM) and of HMM S-1. Binding was reversible and the myosin fragments could be eluted by ATP or magnesium pyrophosphate. The absorption peak of bovine cardiac HMM S-1 was found to be less symmetrical than that of the rabbit white skeletal analog. Chemical modification by trinitrophenylation of white skeletal HMM S-1 was found to affect the affinity of binding and the shape of the absorption peak, thus enabling a partial separation of trinitrophenylated fragment from the unmodified protein using an actin column. The resolving power of ATP columns for the separation of red and white skeletal myosins was increased after trintrophenylation of the proteins.


Rates of different steps involved in the inhibition of protein synthesis by the toxic lectins abrin and ricin.

The kinetics of protein synthesis inhibition in a cell-free system from rabbit reticulocyte lysate was studied after addition of abrin and ricin and the isolated A chains. The toxin A chains inhibited protein synthesis at a rate proportional to the amount added. When intact toxins were added to the reticulocyte lysate, the kinetics of protein synthesis inhibition indicated that the A chains must be liberated before ribosome inactivation can take place. The splitting of the toxin in the lysate was directly demonstrated by the use of labeled toxins. The amount of abrin and ricin bound to HeLa cells under different experimental conditions was correlated to the concomitant inhibition of cellular protein synthesis. In the presence of lactose, which inhibits toxin binding, much higher concentrations of toxins were required to inhibit protein synthesis than in the absence of lactose. A linear relationship was found between the lactose concentration in the medium and the toxin concentration required to give 50% reduction in protein synthesis after 3 hours. The amount of toxin bound to the cell surfaces in the presence of lactose was either determined directly or calculated from the apparent association constant between toxins and surface receptors at the various lactose concentrations. Under different conditions involving a 300-fold variation in the concentration of toxin required to reduce protein synthesis by 50% after 3 hours, the amount of toxin bound to the cell surface was found to be the same. The toxicity thus appears to be determined by the number of toxin molecules bound to the cell surface. Purified ricin B chain was used to compete with the toxins for the receptor sites. Only after addition of high amounts of B chain was the toxicity of abrin and ricin appreciably reduced. The data do not support the view that receptors with especially high affinity are involved in the uptake of the toxins. When the time required for 50% inhibition was plotted versus the inverse value of the square root of the number of toxin molecules bound per cell, a straight line was obtained, intercepting at about 30 min. The data indicate that the observed lag time cannot be due entirely to the fact that the A chains must be liberated before they can act.


Partial specific volume, expansibility, compressibility, and heat capacity of aqueous lysozyme solutions.

Density measurements have been made on aqueous lysozyme solutions at 20, 25, and 30 degrees. The apparent specific volumes, phi v, and expansibilities, phi e, have been determined from the density measurements and fitted to a function of concentration (weight per cent). Sound velocities and heat capacities have also been measured for various concentrations of lysozyme-water solutions at 25 degrees. From the density, expansibility, heat capacity, and sound velocity data at 25 degrees, the isothermal compressibility, phi k, for the lysozyme solutions have been calculated over a range of concentrations. All the physicochemical properties measured were found to be a linear function of the weight per cent of lysozyme. The number of water molecules hydrated to 1 mol of lysozyme was estimated from the volume and compressibility and found to be 162 at 25 degrees.


Opposing effects of mitogenic and nonmitogenic lectins on lymphocyte activation. Evidence that wheat germ agglutinin produces a negative signal.

In an effort to clarify the mechanism by which certain plant lectins induce lymphocyte activation, we have studied amino acid (amino[14C]isobutyric acid) uptake in purified human lymphocytes exposed to mitogenic and nonmitogenic lectins. In confirmation of earlier work, mitogenic lectins (concanavalin A and two phytohemagglutinins) produced a dose-related, 2- to 10-fold increase in aminoisobutyric acid transport. Changes occurred as early as 2 hours and reached a maximum after 18 hours. The stimulation by concanavalin A was inhibited by alpha-methyl-D-mannoside but not by other selected monosaccharides, indicating that the effect is modulated through specific carbohydrate receptors. In contrast to the stimulation with concanavalin A and phytohemagglutinin, the nonmitogen wheat germ agglutinin inhibited aminoisobutyric acid transport, both in the presence and absence of the mitogenic lectins. The inhibition was seen over a broad wheat germ agglutinin dose range, was prevented by N-acetylglucosamine, a known inhibitor of wheat germ agglutinin binding, and did not appear to be associated with cytotoxicity. Comparative binding studies with radiolabeled concanavalin A and wheat germ agglutinin demonstrated that differences in transport occurred in cells containing comparable numbers of bound lectin molecules indicating that the failure of wheat germ agglutinin to stimulate a response was not a result of ineffective binding. The lack of stimulation by wheat germ agglutinin was not due to its inability to interact multivalently with membrane receptors since this lectin is divalent, produces capping and agglutination, and continues to inhibit aminoisobutyric acid transport even after the minimum valence was increased to 4 by cross-linking with glutaraldehyde. In contrast both divalent and tetravalent concanavalin A produced stimulation. Competitive binding studies with soluble wheat germ agglutinin or lectin attached to 300 A latex spheres revealed little or no competition for binding sites with radiolabeled concanavalin A, suggesting the two lectins are interacting with different receptors. This was further suggested by kinetic studies of aminoisobutyric acid transport which indicated that wheat germ agglutinin was probably affecting both the Vmax and Km of transport, whereas concanavalin A affected only the Vmax. While the mechanism by which concanavalin A and wheat germ agglutinin produce opposing effects on amino acid transport is not clear, since the two lectins appear to be interacting with different surface receptors we would suggest that they are perturbing microanatomically and functionally different domains on the lymphocyte plasma membrane.


Purification of human erythrocyte adenosine deaminase by affinity column chromatography.

Adenosine deaminase (adenosine aminohydrolase EC 3.5.4.4) has been purified 468,000-fold from pooled human erythrocytes. The procedure developed was used to isolate the enzyme from up to 23 liters of packed erythrocytes at one time. An easily prepared affinity column bed material employing adenosine as the ligand was used as the final step in the purification. During elution from the affinity column there was approximately a 3:1 partition of adenosine deaminase between gel bed and column buffer. There was no apparent difference in the partitioning of unresolved or partially resolved preparations of the electrophoretically different forms of the enzyme on the affinity column. Gel filtration and electrophoresis on polyacrylamide gels of increasing concentration revealed no differences in the Mr of these electrophoretically different forms. The four bands resolved by electrophoresis of the different forms on polyacrylamide gels under nondenaturing conditions yielded a single band when electrophoresis was carried out in the presence of sodium dodecyl sulfate and 2-mercaptoethanol. Partially resolved preparations of the different electrophoretic forms of adenosine deaminase also gave rise to a single band of the same mobility when electrophoresed on polyacrylamide gels  under these conditions. The band had the mobility of a protein of Mr of 36,000. This Mr is approximately the same as estimated for the nondenatured enzyme.


Deoxycytidine kinase from calf thymus. Substrate and inhibitor specificity.

Kinetic constants were determined for 34 nucleoside substrates of deoxycytidine kinase (EC 2.7.1.74) from calf thymus. Substrate efficiency was assessed by the ratio of Vmax to Km. Inhibition constants were determined for 61 nonsubstrate nucleosides or nucleoside analogues. The enzyme was relatively specific for the pentose moiety of nucleoside substrates. beta-D-2'-Deoxyribonucleosides were more efficient substrates than the corresponding beta-D-arabinonucleosides. Unexpectedly, the L isomer of the beta-arabinonucleoside of cytosine was a more efficient substrate than was the D isomer. beta-Cytidine and beta-5-azacytidine were the only beta-D-ribonucleosides studied that had detectable substrate activity. alpha-Cytidine was an inhibitor but not a substrate. Nucleosides containing a variety of sugar moieties other than those mentioned above did not have detectable substrate activity. The enzyme was relatively nonspecific for the base moiety of nucleoside substrates. 2'-Deoxyribonucleosides of a variety of pyrimidines, purines, and other heterocycles were substrates. Cytosine was the most preferred pyrimidine moiety. 5-Substitution, except with fluorine, decreased substrate efficiency with nucleosides of cytosine or uracil. 2-Fluoradenine was the most preferred purine moiety. The effects of various purine ring substituents were interdependent. Nucleosides containing bulky, hydrophobic substituents on either the base or the pentose moiety had no substrate activity but were relatively potent competitive inhibitors. This suggested the presence of a hydrophobic region on the surface of the enzyme near the active site.


Studies on the subunit composition of the renal glomerular basement membrane.

The bovine glomerular basement membrane in its S-carboxymethylated form has been fractionated into a large number of polypeptide components ranging from 25,800 to 205,000 in apparent molecular weight by performing polyacrylamide gel electrophoresis in sodium dodecyl sulfate on fractions obtained by DEAE- and CM-cellulose chromatography. While only 20 electrophoretic bands were recognized in the unfractionated S-carboxymethylated membrane the DEAE-cellulose column fractions yielded 43 distinct polypeptide components and an additional 15 were obtained from the CM-cellulose chromatography. Analyses of the purified subunits indicated pronounced compositional diversity even in polypeptide components of the same molecular weight. Great differences in the content of the amino acids characteristic of collagen, namely, hydroxyproline, hydroxylysine, and glycine were evident and these three amino acids varied in a parallel manner. In contrast a reciprocal relationship between the number of lysine and hydroxylysine residues was observed so that the sum of these two remained fairly constant. While the collagen-like polypeptide components had a relatively low content of S-carboxymethylcysteine, tyrosine, aspartic acid, and heteropolysaccharides the more polar subunits were enriched in there constituents. These results are consistent with a structural model for the membrane in which the peptide chains vary greatly in the proportion of helical segments and polar regions which they contain. It is likely that the subunit polydispersity is nor primarily a biosynthetic function, but is due to limited in vivo proteolysis.


The hydrophobic membrane penicillinase of Bacillus licheniformis 749/C. Characterization of the hydrophilic enzyme and phospholipopeptide produced by trypsin cleavage.

The membrane penicillinase of Bacillus licheniformis 749/C is a phospholipoprotein carrying extra residues of asparagine or aspartate, serine, glutamine or glutamate and glycine not present in the exoenzyme (Yamamoto, S., and Lampen, J.O. (1976) J. Biol. Chem. 251, 4095-4101). Cleavage of the membrane enzyme with trypsin yielded a phospholipipopeptide and a hydrophilic penicillinase differing from exopenicillinase only by the absence of the NH2-terminal lysine residue. Phosphatidylserine was isolated from a pronase digest of the phospholipopeptide. The partial sequence of the phospholipopeptide is: phosphatidylserine-(Ser3, Glx5, Asx7, Gly5)-Asp-Gin-Ser-Lys-COOH with the lysine being the NH2-terminal residue of the usual exoenzyme. The fatty acids present in the membrane enzyme and in the phospholipopeptide had essentially the same composition (predominantly n-16:0, ante iso-17:0, n-18:0, and n-18:1). These acids were also found in the total membrane lipids, although in very different proportions; thus, the phosphatidic acid residue of the phosphatidylserine is probably formed by the usual synthetic pathway for membrane phospholipids, but some special feature of the process affects the nature of the component fatty acids.


The plasma membrane of human placenta. Isolation of microvillus membrane and characterization of protein and glycoprotein subunits.

A microvillus plasma membrane-enriched fraction of human placenta was obtained by a combination of differential, isopycnic, and rate-zonal centrifugation techniques. Assays for enzyme markers from mitochondria, lysosomes, peroxisomes, endoplasmic reticulum, and plasma membrane indicated a relative enrichment of plasma membrane between 10- and 20-fold over the most prominent contaminating enzyme markers. Electron microscopy verified the microvillus ultrastructure of the isolated placental membrane and the lack of significant contamination by identifiable organelles. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the microvillus membrane fraction revealed a protein and glycoprotein subunit composition. There were 16 major protein subunits and 10 major glycoprotein subunits, and apparent molecular weights are assigned to these subunits. 32P-labeling of the microvillus membrane-associated alkaline phosphatase indicated that this enzyme is one of the major glycoproteins of the human placental microvillus membrane.


Formyltetrahydrofolate synthetase-catalyzed formation of ATP from carbamyl phosphate and ADP. Evidence for a formyl phosphate intermediate in the enzyme's catalytic mechanism.

Formyltetrahydrofolate synthetase from Clostridium cylindrosporum catalyzes phosphoryl transfer from carbamyl phosphate in ADP to form ATP. The phosphoryl transfer reaction has an obligatory requirement for tetrahydrofolate presumably as a cofactor for a proper conformation of the active site. Carbamyl phosphate is an analog of formyl phosphate-a potential intermediate in the normal enzymic reaction. The ability of the enzyme to promote synthesis of ATP from carbamyl phosphate and ADP supports a stepwise chemical reaction mechanism for the enzyme in which formyl phosphate participates as a tightly bound intermediate.


Probes of initial phosphorylation events in ATP synthesis by chloroplasts.

Rapid mixing and quenching techniques have been used with chloroplasts activated by an acid-base transition or by light to assess the nature and characteristics of the substances initially labeled by inorganic [32P]phosphate during ATP synthesis. With light-activated chloroplast fragments, but not with acid-base-activated preparations, an initial rapid labeling of a small amount of ADP is observed. With the acid-base activated preparations a slower continued labeling of ADP occurs that is uncoupler-sensitive, that does not proceed via [gamma-32]ATP of the medium and for which medium ADP furnishes the AMP moiety. The results point to ADP as the initial acceptor of phosphate for ATP synthesis, with a slow side reaction in which bound ATP phosphorylates bound AMP to give a bound ADP. The phosphorylation of bound ADP by medium [32P]phosphate in the absence of added ADP is confirmed, but the reaction is too slow to serve as an intermediate in photophosphorylation. The appearance of label from [32P]phosphate in ATP in the acid-base transition at 25 degrees shows a lag of only about 3 to 7 ms, consistent with the absence of any phosphorylated intermediate. The lag is followed by a linear rate of [gamma-32]ATP formation that is about as fast as that observed in steady photophosphorylation, consistent with a proton gradient serving for transmission of energy from electron transfer reactions to the ATP-synthesizing complex.


Purification and characterization of a new family of polypeptide neurotoxins from the heteronemertine Cerebratulus lacteus (Leidy).

Mucus secreted from the integument of the marine heteronemertine Cerebratulus lacteus (Leidy) contains two major types of polypeptide neurotoxin: the A toxins (11,000 daltons) which are lethal to a variety of animal species including mammals, and the B toxins (6,000 daltons) which appear to be selectively toxic for crustaceans. Both types of toxin were readily obtained from live worms by stimulation and collection of mucus with 1% acetic acid followed by batch adsorption of the basic polypeptide fraction upon CM-cellulose. Separate A and B toxin fractions were obtained by gel chromatography. Four toxins were purified from the B toxin fraction by CM-cellulose gradient elution chromatography. Each consists of a single polypeptide chain with NH2-terminal alanine and three (B-I) or four (B-II, B-III, B-IV) intrachain disulfide bonds. All four B toxins contain large proportions of lysine and exhibit identical gel electrophoretic mobilities at neutral pH. The B toxins lack cysteine and reducing sugar moieties. Toxin B-I differs considerably from the others in its amino acid composition and its shorter chain length (49 instead of 53 or 54 residues). The other three toxins have similar compositions, but lack proline, methionine, and phenylalanine. All four polypeptides show crustacean-selective toxicities which vary over a 30-fold range; B-II possesses the highest toxicity. As a working hypothesis it is suggested that the Cerebratulus B toxins are isotoxins which act via a common receptor present in crustacean axon membranes.


Comparative chemical analyses of the alloantigenic fragments of HL-A antigens.

Papain-solubilized HL-A antigens have been shown to contain two polypeptide fragments: beta2-micro-globulin with a molecular weight of approximately 12,000 and a larger fragment with a molecular weight of about 34,000. The large fragments isolated from two HL-A preparations carrying different specificities appeared homogeneous both by immunoelectrophoresis and sodium dodecyl sulfate-acrylamide electrophoresis. Both HL-A antigen preparations contained the same NH2-terminal (glycine) and the same COOH-terminal residue (serine). The carbohydrate content of the large fragment was 12.9%, making the carbohydrate-free molecular weight approximately 30,000. Small but significant differences have been found in the amino acid compositions and tryptic peptide maps of the two large fragments containing different specificities.


Activities of thyroid hormones and related compounds in an in vitro thymocyte assay.

Prior studies have demonstrated that the thyroid hormones L-triiodothyronine and L-thyroxine stimulate the rapid uptake of 1-amino-cyclopentane-1-carboxylic acid into isolated rat thymocytes. In the present study the effects of several groups of thyroid hormones and structurally related compounds were investigated to determine the structure-function relations required for stimulation of this membrane process. Particular attention was given to (a) analogues with modifications at the oxygen bridge, the phenolic hydroxyl group, and the group at the 3' position of the outer ring, and (b) the steric orientation of the thyroid hormones. The following were found to be important for maximal activity: (a) the L-isomer configuration, (b) the presence of a 4'-hydroxyl group, (c) the presence of one substituent in both the inner and outer rings (3 and 3' positions), (d) the distal orientation of the 3' substituent in the outer ring of L-triiodothyronine, and (e) the lipophilic character of the 3' substituent. Of lesser importance was the presence of halogen atoms, or an oxygen atom in the ether position. Since these structure-function relations seen in thymocytes parallel in many respects those relations seen in whole-animal studies, it is believed that thymocytes will be a useful tool for further studies of thyroid hormones and their analogues.


Regulation of adenylate cyclase from cultured human cell lines by adenosine.

Broken cell particulate preparations of adenylate cyclase isolated from the human glioma cell line 132-1N1 were stimulated 2-to 3-fold by 30 muM adenosine. This concentration of adenosine produced a maximal stimulation of the cyclase while 3 to 5 muM adenosine produced half-maximal stimulation. Theophylline, at 40 muM, inhibited the adenosine stimulation of the adenylate cyclase by about 40% while 200 muM produced near complete inhibition. The inhibition by theophylline could be overcome by increasing adenosine to a concentration 10-fold that of theophylline, implying that the inhibition was competitive. Basal activity was not inhibited by even 1.0 mM theophylline, nor was the epinephrine stimulated activity. In contrast, 1.0 muM propranolol essentially completely inhibited the 8-fold stimulation of 1.0 muM epinephrine but had no effect on either basal or adenosine-stimulated activity. Adenosine and 2-chloroadenosine were equipotent in stimulating adenylate cyclase from the 132-1N1 line, whereas neither adenine nor guanosine had any detectable effect. GTP, 10 muM, produced a small variable stimulation of the adenylate cyclase while the GTP analogue, 5'-guanylylimidodiphosphate (Gpp(NH)p), produced a marked stimulation fo the cyclase. Preincubation of the adenylate cyclase preparation with the analogue greatly increased its potency and maximal effect. In contrast, both basal and adenosine-stimulated activity decreased markedly with preincubation. The effects of adenosine or epinephrine in combination with Gpp(NH)p were at least additive and often synergistic in comparison to the effects of the compounds alone. The effects of adenosine on intact and broken cell preparations of the human fibroblast lines WI-38 and VA13-2RA were also examined. In the intact VA13-2RA, adenosine produced rapid and large increases in intracellular and extracellular cyclic adenosine 3':5'-monophosphate (cAMP). In the parental fibroblast line, the WI-38, adenosine slightly elevated basal levels of cAMP, but only produced marked elevations in the presence of non-methylxanthine phosphodiesterase inhibitors. The effect of adenosine on the broken cell particulate preparations of adenylate cyclase from the fibroblasts was similar to its action on the cyclase from the 132-1N1; 30 muM adenosine produced a maximal stimulation of the adenylate cyclase, and the stimulation was inhibited by theophylline.


Characterization of ribosome-protected fragments from reovirus messenger RNA.

The 5'-terminal methylated cap (m7G(5')ppp(5')Gm) in reovirus messenger RNA comprises part of the ribosomes binding site, since attachment of 40 S wheat germ ribosomal subunits to reovirus small (s), medium (m), and large (l) RNA classes conferred almost complete protection of the cap against RNase digestion. After joining of the 60 S ribosomal subunits, however, the cap continued to be protected against T1 RNase within the 80 S initiation complexes formed with only some messenger species; namely the three l-messages, one of the m-messages, and one or two of the s-messages. When protected fragments were recovered from 40 S and 80 S complexes and tested for ability to rebind to ribosomes those fragments which retained the cap were able to rebind most efficiently. The protected fragments recovered from 40 S initiation complexes with several of the s- and m-RNA species were larger than the messenger fragments recovered from 80 S complexes. The medium size class of reovirus RNA, which consists of three messenger species, gave rise to three discrete 5'-terminal fragments after digestion of 40 S complexes with T1 RNase, and to three somewhat smaller fragments after T1 RNase digestion of 80 S complexes. Fingerprints of the T1 oligonucleotides derived from these fragments are consistent with the interpretation that each messenger species within the m-RNA class gives rise to a protected fragment of a unique size and that, with each message, there is extensive overlap between the regions of the message protected by 40 S and 80 S ribosomes. The ratio of the three protected fragments recovered from 40 S complexes with m-RNA was highly reproducible under a given set of binding conditions, but could be shifted by varying the messenger/ribosome ratio in the binding reaction. Thus, one of the fragments, which was preferentially recovered when the ribosome concentration was limiting, could be tentatively identified as the binding site of the most efficiently translated message within the m-RNA class.


Cystine metabolism in human fibroblasts. Comparison of normal, cystinotic, and gamma-glutamylcysteine synethetase-deficient cells.

Incubation of normal human skin fibroblasts or fibroblasts derived from patients with erythrocyte deficiency of gamma-glutamylcysteine synthetase (gamma-glutamylcysteine synthetase-deficient) in culture medium containing L-[35S]cystine resulted in incorporation of radioactivity into protein, cysteine, and glutathione, gamma-Glutamylcysteine synthetase-deficient fibroblasts synthesized glutathione from [35S]cystine at 30% the rate of normal cells and contained 30% the normal amount of glutathione. Cystinotic fibroblasts incorporated [35S]cystine into the large intracellular cystine pool not found in normal or gamma-glutamylcysteine synthetase-deficient cells and also appeared to synthesize glutathione more slowly than normal cells. However, the radioactivity recovered as cystine was reduced greatly and the rate of [35S]cystine incorporation into glutathione increased if cystinotic cells were first depleted of their intracellular cystine pool before incubation in [35S]cystine. This suggests that the apparent reduced rate of glutathione synthesis observed in untreated cystinotic cells was a secondary effect caused by dilution of the [35S]cystine by the large pool of nonradioactive cystine. Cystinotic cells depleted of cystine by treatment with mercaptoethylamine reaccumulate 30 to 50% of their initial cystine in 24 hours in the absence of extracellular cystine. Both normal and cystinotic cells lose more than 90% of their intracellular glutathione in 24 hours in cystine-free medium. Both cell types can reutilize cysteine from glutathione for protein synthesis.


Association of hemoglobin Saint Etienne (alpha2beta295F8 His replaced by G1n) with hemoglobins A and F. Synthesis and subunit exchange in vitro.

The unstable hemoglobin (Hb) Saint Etienne (alpha2beta295F8 His replaced by G1n) (betaSE) was found in the red blood cells of an 8-year-old boy. The composition of this hemoglobin was 26% Saint Etienne, 52% A, 3% A2 and 19% HbF. Studies of hemoglobin synthesis indicate: a) a balanced synthesis of alpha and non-alpha chains (alpha=betaA + betaSE + gamma), b) an increased pool of free alpha hemoglobin chains, and c) a rapid exchange of alpha chains between this pool and HbSE. The alpha chain pool resulted from the dissociation of HbSE and the greater instability of betaSE chains than alpha chains upon heating. Hemoglobin F is of the fetal type and is heterogeneously distributed among the red cells. Furthermore, two populations of red blood cells could be separated according to their i antigen content. Analysis of the hemoglobins revealed a heterogeneous distribution. Thus, F hemoglobin was preferentially associated with cells having low i antigen level, while Saint Etienne hemoglobin was increased in cells having a high i antigen level. HbF and HbSE were not present in the parents of the propositus. Study of the genetic markers confirmed the filiation. The parents were normal upon clinical and hematological examination; they exhibited a normal pattern and synthesis of hemoglobin. The Hb Saint Etienne case is compared with Hb Istanbul, which in spite of the same amino acid substitution is not associated with increased HbF level.


Synthesis of functional aldolase tetramers in a heterologous cell-free system.

The present report describes the complete synthesis of a functional oligomeric enzyme in a heterologous cell-free system. Polysomal RNA from chicken skeletal muscle was used to direct the production of functional aldolase tetramers in wheat germ extracts. The aldolase product was (a) specifically precipitated with monospecific antibodies raised against pure muscle aldolase, (b) had the same subunit molecular weight (40,000) as that of native aldolase (as determined by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate), (c) presumably contained a functional active site since it co-purified with authentic muscle aldolase upon substrate elution from phosphocellulose, and (d) had associated into tetrameric units (Mr=160,000) as shown by centrifugation in sucrose gradients. The present work suggests that, within the cell, post-translational processing of aldolase polypeptide chains is not involved in the formation of functional aldolase tetramers.


Cytidine 5'-triphosphate synthetase of calf liver. Size, polymerization, and reaction stoichiometry.

Calf liver CTP synthetase was purified 2000-fold from cytosol. The formation of 1 mol of CTP from UTP was accompanied by the cleavage of 1 mol of ATP to ADP and the formation of 1 mol of L-glutamate from L-glutamine. The stoichiometry of the liver enzyme reaction was identical with that found for the Escherichia coli B enzyme (Levitzki, A., and Koshland, D.E., Jr. (1971) Biochemistry 10, 3365-3371). The liver enzyme was also similar to the bacterial enzyme in that it polymerized in the presence of kinetically saturating levels of ATP and UTP. In the absence of nucleotides the enzyme had a sedimentation coefficient of 6.8 and an average molecular weight of 133,000 as determined by sedimentation and molecular sieving. In the presence of ATP and UTP, the enzyme had a sedimentation coefficient of 10.1 and a molecular weight, as determined by molecular sieving, of 263,000. The molecular weights of the two forms of the liver enzyme are about 25% higher than those of the corresponding forms of the bacterial enzyme (Long. C.W., Levitzki, A., and Koshland, D.E., Jr. (1970) J. Biol. Chem. 245, 80-87).


Glyoxylate aminotransferase in peroxisomes from rat liver and kidney.

An aminotransferase was isolated from peroxisomes that had been separated by isopycnic centrifugation of homogenates from rat liver or kidney. The enzyme was located only in the peroxisomes and in the soluble fraction, presumably from broken peroxisomes. Within the peroxisomes, this aminotransferase was in the soluble matrix. This specific aminotransferase was not found in spinach leaves. The enzyme was relatively specific for glyoxylate as the amino group acceptor. L-Leucine and L-phenylalanine were the preferred amino donors; other amino acids were less efficiently utilized. Rates were 181 nmol X min-1 of peroxisomal protein with leucine, and 134 with phenylalanine. The rate with serine was only 28% as fast and there was no reaction with glutamate. The reactions were essentially irreversible. Treatment of peroxisomes with 0.04% Triton X-100 increased enzyme activity 80%. The enzyme in the peroxisomes was stable at 50 degrees. The enzyme was purified 100-fold. Activities with leucine, phenylalanine, and histidine could not be separated by gel filtration and DEAE-cellulose chromatography. Its molecular weight was estimated to be 72,000. Reaction kinetics were ping-pong. The Km (glyoxylate) was 0.5 mM with leucine and 0.67 mM with phenylalanine. Km (leucine) was 2.5 mM and Km (phenylalanine) was 2.8 mM. Substrate inhibition occurred at over 4 mM glyoxylate but did not occur with the amino donors. pH optima were 8.5 for leucine and phenylalanine and 6.2 for histidine. There was no requirement for exogenous pyridoxal phosphate, but activity was inhibited by phenylhydrazine and isonicotinic acid hydrazide. The glyoxylate aminotransferase developed postnatally and increased with age until rats were 40 days old. There was more activity in female than male rats. About 50% of the activity disappeared if rats were starved overnight. Clofibrate treatment of male rats increased this enzyme activity in isolated peroxisomes. Rats on a high casein diet had slightly higher enzyme activity.


Effect of cobalt on synthesis of heme and cytochrome P-450 in the liver. Studies of adult rat hepatocytes in primary monolayer culture and in vivo.

Synthesis of heme and cytochrome P-450 have been studied in adult rat hepatocytes in primary monolayer culture. Incubation of cells with delta-aminolevulinic acid increases both cellular heme and cytochrome P-450 relative to that found in cells incubated under control conditions. Formation of heme is proportional to the concentration of delta-aminolevulinic acid in the culture medium and is not saturable. By contrast, formation of cytochrome P-450 is saturable; excess intracellular heme appears as a new absorption band at 420 nm in the carbon monoxide-reduced difference spectrum. We have studied the effect of cobalt on heme and hemoprotein formation in this cell system. The metal blocks formation of cytochrome P-450 but fails to affect heme synthesis. In contrast to previous findings with isolated mitochondria, no cobalt protoporphyrin formed in hepatocytes cultured in the presence of the metal. In studies of rats in vivo, it was confirmed that cobalt acts to reduce the amount of [14C]heme in the liver after administration of delta-amino[14C]levulinic acid. However, the present findings suggest that this effect of cobalt represents accelerated breakdown of newly labeled hepatic heme rather than inhibition of synthesis. We conclude that cobalt interferes with formation of cytochrome P-450 not by direct inhibition of heme synthesis but most likely by blocking the association of heme and apocytochrome.


Depletion of 18O from C18O2 in erythrocyte suspensions. The permeability of the erythrocyte membrane to CO2.

The depletion of 18O from CO2, caused by the exchange of oxygen between CO2 and water during the hydration-dehydration cycle, is catalyzed by carbonic anhydrase. This depletion process at chemical equilibrium in the presence of erythrocytes is biphasic, exhibiting a very rapid depletion rate immediately following the addition of cells to an isotonic solution containing 18O-enriched CO2, followed by a much slower depletion rate. It is hypothesized that these depletion characteristics are caused by the diffusion of labeled CO2 into erythrocytes where depletion occurs rapidly due to the large intracellular carbonic anhydrase content. Kinetic equations which describe this hypothesis are solved and a rate constant is obtained which represents the depletion of 18O in CO2 caused by the presence of red cells. These are equilibrium experiments with no net uptake or loss of CO2 in the cells. Consequently, depletion processes are not limited in rate by bicarbonate-chloride exchange or proton distribution across the membrane. The purpose of these measurements is to determine whether the rate of 18O depletion in red cell suspensions is determined by carbonic anhydrase activity in the cell or by the diffusion process by which CO2 enters the cell. This goal is achieved by partially inhibiting carbonic anhydrase with acetazolamide. The rate constant representing 18O depletion caused by the presence of red cells is unchanged, even though up to 90% of carbonic anhydrase is inhibited. From this rate constant the permeability constant of the membrane of rat erythrocytes to CO2 at 25 degrees and pH 7.4 is determined to be (7.6 +/- 1.2) X 10(-3) cm s-1 in the presence of 3.2 mM picrate, a passive anion diffusion inhibitor intended to block HCO3 -flux across the membrane. Using no picrate and allowing HCO3-flux to introduce an error in the measurements, the permeability constant is (1.6 +/- 0.4) X 10(-2) cm s-1. The permeability constants measured by this technique include the diffusion barrier to CO2 not only of the red cell membrane but also of a portion of the intracellular medium.


Hydrophile-lipophile balance and critical micelle concentration as key factors influencing surfactant disruption of mitochondrial membranes.

A systematic approach to selection of surfactants for disrupting biological membranes, for solubilizing their components, and for removing the surfactant by dialysis is described. The two relevant surfactant parameters were the critical micelle concentration (CMC) and the hydrophile-lipophile balance (HLB). Rat liver mitochondria were treated with two series of nonionic surfactants and the extent of extraction of total protein, total lipid, and six enzymes was determined. Within the homologous series of Triton surfactants, maximum protein and phospholipid extraction occurred at HLB values between 12.5 and 13.5. In addition, a single surfactant species solubilized more protein than a mixture of surfactants with the same mean HLB value. In order to examine independently the effect of CMC and HLB on protein extraction, a specialty surfactant, S10-7, was prepared and compared with its structurally similar analog, Brij 56. Above a concentration of 0.35%, both Brij 56 and S10-7 extracted about 70% of the mitochondrial protein. Hence, for optimum extraction of mitochondrial protein and lipids the HLB must be about 13, and the surfactant concentration must be above the CMC. The S10-7 dialyzed almost as rapidly as cholate and far more rapidly than Brij 58 and Triton X-100. It therefore possesses the two most desirable surfactant properties for disruption of membranes, a high CMC for rapid dialysis, and an HLB value of 13.2.


Sphingolipid base metabolism. Stereospecific uptake of proton in the enzymatic conversion of sphinganine 1-phosphate to ethanolamine 1-phosphate.

D-erythro-(2S, 3R)-Sphinganine 1-phosphate was incubated with rat liver microsomes in the presence of tritiated water. [3H]Ethanolamine 1-phosphate was isolated and converted, through a combination of enzymatic and chemical reactions, to [3H]glycine. The labeled glycine was incubated with D-amino acid oxidase, an enzyme which, in the catalysis of the conversion of glycine to glyoxylic acid, specifically removes the hydrogen in the S configuration at carbon atom 2 of glycine. Essentially complete retention of the labeled hydrogen occurred upon conversion to glyoxylic acid. The combined results indicate that the conversion of D-erythro-(2S,3R)-sphinganine 1-phosphate to palmitaldehyde and ethanolamine 1-phosphate, catalyzed by sphinganine-1-phosphate lyase of rat liver microsomes, proceeds with the stereospecific incorporation of 1 atom of solvent hydrogen into the R configuration of ethanolamine 1-phosphate.