Cyanide production from glycine by a homogenate from a Pseudomonas species.

A cell-free preparation with cyanide-producing activity was obtained from a bacterium, strain C, of the genus Pseudomonas. To preserve activity, an oxidizing agent, e.g., phenazine methosulphage (PMS), had to be added to the cell suspension before disruption by sonic treatment. By the procedure described, a total homogenate made from a 15% (wet weight) bacterial suspension in tris(hydroxymethyl)aminomethane-hydrochloride buffer (0.05 M, pH 8.2) and with PMS (0.4mM) exhibited about 8% of the activity obtained from a suspension of untreated bacteria. In the presence of flavine-adenine dinucleotide (0.3 mM) and PMS (0.4mM), the activity was augmented to about 16% of that of the intact cells. By gradient centrifugation the homogenate was separated into three fractions. The main enzyme activity was associated with those fractions which by electron microscopy were found to consist of membranous structures.


Metabolism of prostaglandins E, A, and C in serum.

Three prostaglandin-metabolizing enzymes were detected in human serum. One enzyme was a dehydrase that converted prostaglandin E to prostaglandin A, the second was a prostaglandin A isomerase that converted prostaglandin A to prostaglandin C and the third was a prostaglandin C isomerase that converted prostaglandin C to prostaglandin B. All three were inactivated by sulfhydryl blocking agents. In human serum, only prostaglandin C isomerase had high activity, whereas the two other enzymes had very low activity. In rabbit serum both isomerases were very active, but dehydrase activity could not be detected. Prostaglandin C isomerase activity was also found in crystallized human serum albumin and rabbit serum albumin.


Isolation and characterization of a ribonuclease from human leukemic blood cells specific for ribonucleic acid of ribonucleic acid-deoxyribonucleic acid hybrid molecules.

A ribonuclease that specifically hydrolyzes RNA in RNA. DNA hybrids has been purified more than 100-fold from human acute leukemic white blood cells. The molecular weight of this enzyme has been estimated as 80,000 by glycerol gradient centrifugation. It requires Mg-2plus for activity and is inhibited by N-ethylmaleimide. The optimum activity is observed at pH 8 (37 DEGREES). It is a heat-labile protein, t 1/2 at 50 degrees being 2 min. Among the substrates examined, (A)n X (dT)m, (I)n X (DC)m, and PHIX-174 DNA X RNA were hydrolyzed efficiently. (U)n X (dA)m showed a slight substrate activity, while (c) n X (dG) m and (G)n X (dC)m were not significantly hydrolyzed. The enzyme is an endonuclease and does not require RNA ends in the substrate molecule. It is capable of converting more than 95% of the RNA portions in hybrid substrates into acid-soluble products which are mono- and oligonucleotides terminated in 3'-OH and 5'-phosphate.


Characterization of particulate and soluble guanylate cyclases from rat lung.

Rat lung homogenates contained significant amounts of guanylate cyclase activity in both 100,000 times g (60 min) particulate and supernatant fractions. In the presence of detergent, the particulate fraction contained 40% as much activity as did the supernatant fraction. Detergent-dispersed particulate and partially purified soluble guanylate cyclase preparations were characterized with respect to divalent cation requirements, divalent cation interactions, kinetic behavior, and gel filtration profiles. Both soluble and particulate guanylate cyclases required divalent cation for activity. The soluble preparation was 10 times more active in the presence of Mn-2plus than in the presence of Mg-2plus or Ca-2plus and no detectable activity was seen with Ba-2plus or Sr-2plus. Particulate guanylate cyclase activity was detectable only in the presence of Mn-2plus. Both enzyme preparations required Mn-2plus in excess of GTP for optimal activity at subsaturating amounts of GTP. At near-saturating GTP, the soluble enzyme required excess Mn-2plus, but the particulate enzyme did not. For kinetic analyses the enzymes were considered to require two substrates: metal-GTP and Me-2plus. Apparent negative cooperative behavior was seen with the soluble enzyme when excess Mn-2plus (in excess of GTP) was varied from 0.01 to 0.2 mM; above 0.2 mM excess Mn-2plus classical kinetic behavior was seen with an apparent KMn-2plus of 0.2 mM at near-saturating MnGTP. Similar studies using the particulate preparation yielded only classical kinetic behavior, but the apparent KMn-2plus decreased to near zero when MnGTP was near-saturating. Kinetic patterns for the particulate and soluble enzymes also differed when reciprocal initial velocities were plotted as a function of reciprocal MnGTP concentrations; classical kinetic behavior was seen with the soluble enzyme with an apparent KMnGTP of about 12 muM (at near-saturating excess Mn-2plus), whereas apparent positive cooperative behavior was seen with the particulate preparation (Hill coefficient equals 1.6, S0.5 EQUALS 70 MUM. Ca-2plus "activation" of soluble guanylate cyclase was related to the Mn-2plus:GTP ratio. Activation was most apparent when saturating amounts of Mn-2plus and MnGTP. At relatively high concentrations of Ca-2plus (0.1 to 4 mM), the addition of 10 muM Mn-2plus resulted in a 3- to 5-fold increase in soluble guanylate cyclase activity. In contrast, Ca-2plus sharply inhibited particulate guanylate cyclase activity. Gel filtration profiles of particulate and soluble preparations indicated differences in physical properties of the enzymes. As estimated by gel filtration, particulate (detergent-dispersed)evels. Here, removal of renal tissue is contraindicated. In all renal hy


Effects of cations on guanylate cyclase of sea urchin sperm.

Mn2+ and to some degree Fe2+, but not Mg+, Ca2+, ba2+, Sr2+, Co2+, Ni2+, La3+, or Fe3+ were able to serve as effective metal cofactors for sea urchin sperm guanylate cyclase. The apparent Michaelis constant for Mn2+ in the presence of 0.25 mM MnGTP was 0.23 mM. In the presence of a fixed free mn2+ concentration, variation in mngTP resulted in sigmoid velocity-substrate plots and in reciprocal plots that were concave upward. These positive cooperative patterns were observed at both pH 7.0 and 7.8 and in the presence or absence of Triton X-100. When Mn2+ and GTP were equimolar, Ca2+, Ba2+, Sr2+, and Mg2+ increased apparent guanylate cyclase activity. This increase in enzyme activity at least could be accounted for partially by an increase in free Mn2+ concentration caused by the complex formation of GTP with the added metals. However, even at relatively low GTP concentrations and with Mn2+ concentrations in excess of GTP, Ca2+, Sr2+, and Ba2+ significantly increased guanosine 3':5'-monophosphate production. As the total GTP concentration was increased, the degree of stimulation in the presence of Ca2+ decreased, despite maintenance of a fixed total concentration of Ca2+ and a fixed free concentration of Mn2+, suggesting that the concentration of CaGTP and MnGTP were determining factors in the observed response. The concave upward reciprocal plots of velocity against MnGTP concentration were changed to linear plots in the presence of CaGTP or SrGTP. These results suggest that sea urchin sperm guanylate cyclase contains multiple nucleotide binding sites and that stimulation of guanosine 3':5'-monophosphate synthesis by Ca2+, Sr2+, and perhaps other metals may reflect interaction of a metal-GTP complex with enzyme as either an effector or a substrate.


Activation and control of factor VII by activated factor X and thrombin. Isolation and characterization of a single chain form of factor VII.

Factor VII purified as previously described, was found to consist of two polypeptide chains joined by disulfide bridges. We now report the isolation and 200,000-fold purification of a single chain form of Factor VII. This was accomplished by protecting the molecule against proteolysis by including benzamidine during the entire purification. The purification was essentially as previously reported except that barium cirtate was substituted for barium sulfate as an absorbant for Factor VII as it resulted in a 4-fold increase in yield. Single chain Factor VII is rapidly hydrolyzed by Factor Xa in the presence of calcium ions and phospholipids, and by thrombin, to a two-chain form which possesses at least 85 times the Factor VII clotting activity of the single chain species. The two-chain form of the enzyme requires tissue factor in order to activate Factor X. From the observed rates of activation of Factor VII by Xa in the presence of calcium ions and phospholipids, it was calculated that at approximately physiological concentration, Factor VII activity would increase at an initial rate of 20-fold per min; this reaction is sufficiently rapid to constitute a feedback control mechanism. The action of thrombin is approximately 40-fold slower under these conditions. Diisopropylphosphorofluoridate inactivates the single chain and two-chain forms of Factor VII at approximately equal rates. After inhibition, the single chain species could be cleaved but not activated by proteolysis.


Solvent accessibility in folded proteins. Studies of hydrogen exchange in trypsin.

In a native protein, the exchange of a peptide amide proton with solvent occurs by one of two pathways, either directly from the folded protein, or via unfolding, exchange taking place from the unfolded protein. From the thermal unfolding rate constants, the contribution of unfolding to the over-all kinetics as a function of solvent and temperature has been determined. Exchange involving unfolding of the protein is characterized by a high activation energy, in the range of 50 to 60 Cal per mol. The activiation energy (Eapp) of the rates of exchange directly from the folded protein is approximately 20 to 25 Cal per mol. Because for the proton transfer step, Eapp approximately equal to 20  Cal per mol, the activation energy for any contributing protein conformational process(es) is approximately equal to 0 to 5 Cal per mol. Most, if not all, of the peptide amide protons in a folded protein can exchange directly with solvent without the protein unfolding. The number of "slowly" exchanging protons at a given condition of pH and temperature is not related to a discrete structural unit, but rather to the distribution of observed rates within the broader distribution of actual rates. The large attenuation of hydrogen exchange rates in folded proteins, resulting in a distribution of first order rates over 6 orders of magnitude, is primarily due to the effects of restricted solvent accessibility of labile protons in the three-dimensional structure. Any protein conformational process, such as protein fluctuations, invoked to explain the solvent accessibility must be of low activation energy and attenuated by ethanol and other co-solvents (Woodward, C. K., Ellis, L. M., and Rosenberg, A. (1974) J. Biol. Chem. 250, 440-444).


The solvent dependence of hydrogen exchange kinetics of folded proteins.

The effects of ethanol, ethylene glycol, dioxane, and other organic co-solvents upon the hydrogen exchange rates of randomly coiled oxidized RNase, native RNase, and native trypsin have been measured. The exchange rate of oxidized RNase, the model compound for the proton transfer step in hydrogen exchange, is decreased by all of the co-solvents studied at temperatures in the range 3-20 degrees. This has been ascribed to the combined effects of the disruption of peptide bond solvation due to a reduction in the concentration of water, and of changes in [OH-] ion concentration due to changes in the acid dissociation constant of water, Kw. The solvent dependence for both native RNase and native trypsin is similar in all of the solvents studied. At a low temperature (3-20 degrees), the exchange rates go through a minimum as the solvent concentration is increased. At higher temperatures (20-35 degrees) the exchange rates are increased at all concentrations of the co-solvent. The apparent rate minimum at lower temperatures is due to two opposing effects. Co-solvents decrease the rate of exchange that occurs directly from the folded molecule. At higher concentrations and higer temperature. The decrease in rates for exchange directly from folded protein is primarily due to the effects on the proton transfer step, and not to binding or the solvent effects on protein structure. The solvents used in this study have no apparent effect on conformational processes contributing to the hydrogen exchange process in folded proteins.


Proton magnetic resonance studies of alpha-keto acids.

Pulsed Fourier transform proton magnetic resonance was used to study alpha-ketoglutaramic, and several other alpha-keto acids in aqueous solutions as a function of pH. Most alpha-keto acids were found to exist in equilibrium with the hydrate (gem-doil). The equilibrium position favors the nonhydrated alphs-keto acid at neutral pH, but at low pH values (below the pKa of the alpha-carboxylic acid group) the hydrate predominates. We found evidence that alpha-ketoglutaric acid exists in a third equilibrium form which is assigned to the lactol. alpha-Ketoglutaramic acid (the alpha-keto acid analog of glutamine) which is known to exist predominantly in a cyclic form at pH 7.0 was shown to exist as a cyclic structure over a wide pH range. However, the cyclic form is an equilibrium mixture of 2-pyrrolidone-5-hydroxy-5-carboxylic and 1-pyrrolin-2-one-5-carboxylic acids.


Prostaglandin metabolism. II. Identification of two 15-hydroxyprostaglandin dehydrogenase types.

Homogenates of several mammalian tissues were measured by radioimmunoassay for 15-hydroxyprostaglandin dehydrogenase activity. Two types of enzyme activity were detected. One, which used NAD-plus as cofactor much more effectively than NADP-lus, was found in monkey lung, heart, liver, kidney, and spleen and in chicken heart and dog lung. A second type, which uses NADP-plus as a cofactor more effectively than NAD-plus, was found in monkey and human brain and red blood cells and in swine kidney. These two types of 15-hydroxyprostaglandin dehydrogenase were partially purified from monkey brain and chicken heart. In addition to different cofactor requirements, the two partially purified enzymes could be distinguished by chromatographic properties, their relative affinities for prostaglandin I2 and F2alpha, and their sensitivities to inhibition by reduced pyridine nucleotides, thyroid hormones, and prostaglandin B2.


Reversible modification of arginine residues. Application to sequence studies by restriction of tryptic hydrolysis to lysine residues.

1, 2-Cyclohexanedione reacts specifically with the guanidino group of arginine or arginine residues at pH 8 to 9 in sodium borate buffer in the temperature range of 25-40 degrees. The single product, N-7, N-8-(1,2-dihydroxycyclohex-1,2-ylene)-L-arginine (DHCH-arginine) is stable in acidic solutions and in borate buffers (pH 8 to 9). DHCH-Arginine is converted to N-7-adipyl-L-arginine by periodate oxidation. The structures of the two compounds were elucidated by chemical and physicochemical means. Arginine or arginyl residues can be regenerated quantitatively from DHCH-arginine by incubation at 37 degrees in hydroxylamine buffer at pH 7.0 FOR 7 TO 8 hours. Analysis of native egg white lysozyme and native as well as oxidized bovine pancreatic RNase, which were treated with cyclohexanedione, showed that only arginine residues were modified. The utility of the method in sequence studies was shown on oxidized bovine pancreatic ribonuclease A. Arginine modification was complete in 2 hours at 35 degrees in borate buffer at pH 9.0 with a 15-fold molar excess of the reagent. The derived peptides showed that tryptic hydrolysis was entirely limited to peptide bonds involving lysine residues, as shown both by two-dimensional peptide patterns and by isolation of the resulting peptides. The stability of DHCH-arginyl residues permits isolation of labeled peptides.


Effects of ethidium bromide on the respiratory chain and oligomycin-sensitive adenosine triphosphatase in purified mitochondria from the cellular slime mold Dicyostelium discoideum.

Mitochondria were isolated from the cellular slime mold. Dictyoostelium discoideum, and partially purified by sucrose density gradient fractionation. The most purified mitochondrial fraction from the gradient contained essentially no contaminating lysosomes and minimal amounts of contaminating peroxisomes as determined by the marker enzymes N-acetyl-glucosaminidase and catalase. A mitochondrial fraction with the same amount of lysosomal and peroxisomal contamination was also isolated from cells which had been treated with ethidium bromide for 5 days. The most purified mitochondrial fraction from control and ethidium bromide-treated cells had an identical buoyant density of 1.181 to 1.182 g per ml, suggesting that treatment with the drug does not result in any drastic structural changes in the mitochondrial membrane which would affect its density. In the purified mitochondria from ethidium bromide-treated cells, the content of cytochromes a-a3 was decreased over 80% and that of cytochrome oxidase and oligomycin sensitive ATPase were reduced approximately 50%. By contrast, the specific activities of NADH and succinate dehydrogenases were identical in the purified mitochondria from control and ethidium bromide-treated cells. Previously, we had reported that the specific activities of these two enzymes had nearly doubled in whole cells maintained in ethidium bromide for a time equivalent to six or seven generations after growth had stopped (Stuchell, R. N., Weinstein, B. I., and Beattie, D. S. (1973) Fed. Eur. Biochem. Coc Lett. 37, 23-26). These results suggest that continued formation of new mitochondrial membranes, with an identical complement of succinate and NADH dehydrogenases, must occur despite the cessation of cell growth which occurs as a result of the ethidium bromide induced loss of mitochondrial enzymes. Consequently, the amount of mitochondria, or mitochondrial protein per cell, calculated from the activity of NADH and succinate dehydrogenases has increased nearly 50%. Possible models to explain the control of mitochondrial biogenesis are discussed to explain these results.


Deoxycytidine triphosphate deaminase of Salmonella typhimurium. Purification and characterization.

Deoxycytidine triphosphate deaminase (EC 3.5.4., dCTP aminohydrolase) of Salmonella typhimurium LT2 has been pruified 500-fold. The reaction requires the presence of Mg-2plus, Mn-2plus, Ca-2lus, or Co-2plus. Kinetics of the reaction with varying Mg-2plus concentrations, keeping the concentration of dCTP constant, suggests that the true substrate of the reaction is MgdCTP. The dependence of the rate of reaction on dCTP concentration in the presnece of 5-fold excess of Mg-2plus is sigmoid, with a Hill coefficient of 1.7. The enzyme is specifically inhibited by dTTP and dUTP. In the presence of increasing dTTP concentrations the sigmoidicity of the substrate saturation curves increases. With 0.2 and 0.4 mM dTTP the Hill coefficients are 2.6 and 3.0, respectively. Despite several attempts no dissociation of the substrate site and the inhibitor site of the enzyme was achieved.


Subunit interactions in aspartate transcarbamylase. Characterization of a complex between the catalytic and the regulatory subunits.

The complex formed when excess regulatory subunits (r2) of aspartate transcarbamylase is added to a dilute solution of the catalytic subunit (c3) has been further studied. By stabilizing the complex with saturating levels or r2, it was possible to perform ultracentrifugation in sucrose density gradients. The sedimentation coefficient of the complex (7.7 plus or minus 0.2 S) is intermediate between those of the catalytic subunit (5.8 S) and of the native enzyme (11.7 S). Consideration of the likely hydrodynamic properties of the complex suggests that this sedimentation coefficient may be consistent with the c3r6 structure previously proposed. The formation of c3r6 from c3 and r2 is readily reversible. At nonsaturating levels or r2, conversion to the native enzyme (c3r6) takes place. This conversion is inhibited by high concentrations of r2. The c3r6 complex shows Michaelis-Menten kinetics with a low Km for aspartate and considerable substrate inhibition. The pH activity profile at high aspartate concentrations is almost identical with that of the native enzyme. All of these observations suggest that c3r6 represents the relaxed (R) state of aspartate transcarbamylase. The insensitivity of c3r6 toward CTP or ATP can also be explained by considering c3r6 as a stabilized relaxed state. These properties of c3r6 have significant implications regarding the allosteric mechanism of the native enzyme.


Concomitant changes in adenylate cyclase and cytolytic activities of lymphoid cells during graft versus host reaction.

Spleen cells from adult F1 hybrid mice undergoing the graft versus host reaction due to the inoculation of lymphoid cells of parental origin showed increased adenylate cyclase activity and cytolytic activity. A time-course study revealed that both the adenylate cyclase and cytolytic activities started increasing at Day 4 and reached maximum at Day 8 of initiation of the graft versus host reaction. Furthermore, the magnitude of both adenylate cyclase activation and cytolytic activity were dependent upon the number of parental cells injected into the F1 hybrid recipients. Elevated adenylate cyclase activity was also observed in spleen cells from irradiated animals undergoing the graft versus host reaction in which the nonspecific, but not the specific cytolytic activity was markedly depressed.


Purification and characterization of two cellulases from auxin-treated pea epicotyls.

Two forms of beta-1,4-glucan 4-glucanohydrolase (EC 3.2.1.4) were extracted from growing regions of Pisum sativum epicotyls which had been treated with the auxin, (2,4-dichlorophenoxy)acetic acid. One cellulase is buffer-soluble, the other buffer-insoluble but extractable with high salt concentrations. Both enzymes catalyze endohydrolysis of carboxymethylcellulose with the same pH optimum (5.5 to 6.0). They were purified with the use of DEAE-cellulose chromatography, Sephadex gel filtration, and ultrafiltration. They are distinct proteins as characterized by: electrofocusing and disc gel electrophoresis (pI values = 5.2 and 6.9, respectively); mobility in sodium dodecyl sulfate polyacrylamide gels, fractionation on Sephadex, and sedimentation in the ultracentrifuge (mol wt = approximately 20,000 and 70,000, S values 2.63 and 3.73); and immunological properties. The buffer-soluble enzyme tends to dimerize on purification. Amino acid analyses show that the buffer-soluble enzyme is relatively rich in glycine, alanine, and valine and deficient in cystine, tryosine, and phenylalanine compared to the buffer-insoluble enzyme. The two cellulase activities were generated in approximately equal amounts after auxin treatment. Within 5 days their levels had increased at least 100-fold and they constituted about 0.1% of total cellular protein. Present data indicate that one is not derived from the other.


Soluble cytochrome P-450 from housefly microsomes. Partial purification and characterization of two hemoprotein forms.

Housefly microsomes contain two spectrally different forms of cytochrome P-450 which we have termed P-450 and P-450I. Methods have been developed for the fractionation and chromatographic purification of these two hemoprotein forms. Microsomes are solubilized first with Triton X-100 in the presence of glycerol, dithiothreitol, ethylenediaminetetra-acetic acid, and phenobarbital. Cytochrome P-450 is recovered in a floating pellet after the addition of 25% ammonium sulfate followed by centrifugation, whereas cytochrome P-450I remains in the 25% ammonium sulfate supernatant fluid. Cytochrome P-450 is purified further by Sephadez G-200 and DEAE-Sephadex A-50 column chromatography, which also allows the isolation of cytochrome b5 and NADPH-dependent cytochrome P-450 reductase in good yields and with little cross-contamination. Cytochrome P-450 apparently is free of cytochromes b5 and P-420 as well as of reductase and is obtained in a final yield of approximately 16% with a 6.9-fold purification. Its maximum absorbance is at 45 mn in the CO-difference spectrum and its average extinction coefficient is 103 cm-1 nm-1. Cytochrome P-450I is purified by Sephadex G-25 column chromatography but still contains some cytochromes b5 and P-420 as well as reductase. Its maximum absorbance is at 448.5 nm in the CO-difference spectrum and its extinction coefficient is 83 to 86 cm-1 mM-1. Both cytochromes hydroxylate type I substrates such as aminopyrine. Sufficient amounts of reductase are present in the cytochrome P-450I preparation to sustain activity, but the reductase has to be added to cytochrome P-450 in a reconstituted system for activity. Cytochrome P-450 is fairly stable, whereas cytochrome P-450I can be isolated only when protected by a substrate (phenobarbital). Detergent-solubilized housefly cytochromes P-450 and P-450I seem to correspond to either aggregates or oligomeric proteins. Cytochrome P-450 appears to correspond to a tetramer, each subunit having a molecular weight of 45,000, whereas cytochrome P-450I may correspond to an aggregate of at least 10 subunits. The cytochrome P-450 aggregate is dissociated by 6 M urea, but cytochrome P-450I remains as such.


p-Chlorphenylalanine effect on phenylalanine hydroxylase in hepatoma cells in culture.

We have investigated the p-chlorophenylalanine-dependent loss of phenylalanine hydroxylase activity in cultured hepatoma cells. The similarity of the effect of p-chlorophenylalanine on phenylalanine hydroxylase in the hepatoma cells and that reported from studies in vivo indicates that the loss of phenylalanine hydroxylase activity is due to a direct interaction of the amino acid analogue with the liver. We can find no evidence that the loss of phenylalanine hydroxylase activity is due to: a direct inactivation of the hydroxylase by p-chlorophenylalanine or an inhibitor produced by p-chlorophenylalanine treatment; an effect similar to that of p-fluorophenylalanine; or leakage of enzyme from the cells during p-chlorophenylalanine treatment. The data presented indicate: (a) the p-chlorophenylalanine effect is rather specific for phenylalanine hydroxylase; (b) following p-chlorophenylalanine removal, new protein synthesis is necessary for restoration of the hydroxylase activity; (c) the rate of loss of phenylalanine hydroxylase activity after the addition of p-chlorophenylalanine is much faster than the rate of restoration of the hydroxylase activity after removal of p-chlorophenylalanine; (d) even in the presence of p-chlorophenylalanine, hydrocortisone greatly stimulates the hydroxylase activity; (e) the cell density-dependent increase of phenylalanine hydroxylase activity is blocked by p-chlorophenylalanine. A discussion of the possible mechanisms of p-chlorophenylalanine-dependent loss of phenylalanine hydroxylase is presented. To measure very low leanine-dependent loss of phenylalanine hydroxylase is presented. To measure very low levels of phenylalanine hydroxylase activity, a new procedure, based on isotope dilution, was developed for isolating the tyrosine formed during the enzymatic reaction.


Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase. Proteolytic and thermal inactivation of the membrane component.

Pyridine dinucleotide transhydrogenase of the Rhodospirillum rubrum chromatophore membrane was readily resolved by a washing procedure into two inactive components, a soluble transhydrogenase factor protein and an insoluble membrane-bound factor. Transhydrogenation was reconstituted on reassociation of these components. The capacity of the membrane factor to reconstitute enzymatic activity was lost after proteolysis of soluble transhydrogenase factor-depleted membranes with trypsin. NADP+ or NADPH, but neither NAD+ nor NADH, stimulated by several fold the rate of trypsin-dependent inactivation of the membrane factor. Substantial protection of the membrane factor from proteolytic inactivation was observed in the presence of Mg2+ ions, an inhibitor of transhydrogenation, or when the soluble transhydrogenase factor was bound to the membrane. Coincident with the loss of enzymatic reconstitutive capacity of the membrane factor was a loss in the ability of the membranes to bind the soluble transhydrogenase factor in a stable complex. The membrane component was inactivated by preincubating soluble transhydrogenase factor-depleted membranes at temperatures above 45 degrees. NADP+, NADPH, or Mg2+ ions, but neither NAD+ nor NADH, protected against inactivation. These studies indicate that (a) the binding of NADP+ or NADPH to the membrane factor promotes a conformational alteration in the protein such that its themostability and susceptibility to proteolysis are increased, and (b) the inhibitory Mg2+ ion-binding site resides in the membrane component.


Prenyltransferase from Saccharomyces cerevisiae. Purification to homogeneity and molecular properties.

Prenyltransferase (EC 2.5.1.1) has been purified to homogeneity from the supernatant fraction of yeast by ammonium sulfate fractionation, diethylaminoethyl-cellulose and hydroxylapatite chromatography, and column isoelectric focusing techniques. The active enzyme from isoelectric focusing columns emerged as a single symmetrical peak with specific activities 15- to 35-fold higher than previously reported preparations. The enzyme was found to be homogeneous by continuous polyacrylamide gel electrophoresis at pH 8.4 and discontinuous polyacrylamide gel electrophoresis at pH 6.9 as well as sodium dodecyl sulfate polyacrylamide electrophoresis at pH 7.0. By means of gel chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was shown to be a dimer with a molecular weight of 84,000 plus or minus 10%. The isoelectric point of the enzyme was determined to be 5.3. The enzyme synthesizes farnesyl and geranylgeranyl pyrophosphates from dimethylallyl, geranyl, and farnesyl pyrophosphates. Michaelis constants for the enzyme were 4, 8, and 14 mu M for isopentenyl, dimethylallyl, and geranyl pyrophosphates, respectively.


Purification of Keratan Sulfate-endogalactosidase and its action on keratan sulfates of different origin.

A glycosidase which attacks corneal keratan sulfate was purified from extracts of Pseudomonas sp. IFO-13309. When corneal keratan sulfate was degraded by the purified enzyme, Sephadex G-50 chromatography indicated the presence of a number of oligosaccharides differing in size and sulfate content. The characterization of two major fractions of the oligosaccharides indicated that the point of enzyme attack is limited to the endo-beta-D-galactoside bonds in which nonsulfated D-galactose residues participate. The enzyme, unlike ordinary exo-beta-D-galactosidases, did not catalyze the hydrolysis of phenyl beta-D-galactoside. Moreover, beta-D-galactosyl-(1 leads to 3)-2-acetamido-2-deoxy-beta-D-glucosyl-(1 leads to 3)-beta-D-galactosyl-(1 leads to 4)-D-glucose ("lacto-N-tetraose") was completely refractory to the action of this enzyme, suggesting that a structure of the type, X-(1 leads to 3)-beta-D-galactosyl-(1 leads to 4)-Y, is not the only specificity-determining factor, i.e. neighboring sugars, X and Y, or even larger portions of substrate molecule must have an important effect. Compared with corneal keratan sulfate, keratan sulfates from human nucleus pulposus and shark cartilage were attacked at lower rates with a resultant production of oligosaccharides of relatively large size. The result is in agreement with the view that considerable variations exist in the structure of keratan sulfates of different origin, and further suggests that the enzyme may serve as a useful reagent in studying these variations.


Conformation and spin state in methemoglobin.

The properties of human methemoglobin have been investigated under a wide variety of conditions to determine its conformation and to test for evidence of the T state conformation which has been proposed by Perutz to exist in the presence of high spin ligands and inositol hexaphosphate (IHP). Subunit dissociation was measured as a criterion for the T state since marked differences in the tetramer-dimer equilibrium exist for oxyhemoglobin (R state) and deoxyhemoglobin (T state). In the absence of IHP, complexes of methemoglobin with both high spin ligands (water, fluoride) or low spin ligands (azide, cyanide) show extensive dissociation in 2,2-bis(hydroxymethyl)-2,2',2"-nitriloethanol buffers, pH 6, 0.1 M NaCl, with values of the tetramer-dimer dissociation constant (K4,2) near 10-5 M. The addition of IHP lowers K4,2 to a value near 10-5 M for all forms of methemoglobin. Combination of IHP with methemoglobin promotes a conformational change, but the change is apparently independence of spin state. The conformation acquired in the presence of IHP is not identical with the T state (K4,2 similar to 10-12 M) and can also occur with hemoglobin in the ferrous form, as revealed by a substantial reduction in K4,2 for CO-hemoglobin upon addition of IHP. Subunit dissociation has also been measured using the haptoglobin reaction, since haptoglobin binds only to hemoglobin dimers. The haptoglobin experiments give results that are qualitatively in agreement with the conclusions reached by ultracentrifuge measurements. Similar results are also obtained by estimating the degree of dissociation on the basis of the material which aggregates following mixing with dithionite. The effect of IHP on azide-binding kinetics with methemoglobin has also been examined. Changes in reactivity is observed upon addition of IHP, but the principal effect is observed upon addition of IHP, but the principal effect is an enhancement of the rate of reaction of the beta chains. Changes in the reactivity of the beta93 sulfhydryl group of methemoglobin also accompany addition of IHP, but in a manner which is largely independent of the spin state of the iron. Similar changes are again found with CO-hemoglobin upon addition of IHP. The rate of binding of bromthymol blue also shows some changes upon addition of IHP, but the changes are more pronounced for deoxyhemoglobin than for methemoglobin. Since the results obtained did not appear to indicate a significant role for spin state in the changes observed, additional studies were undertaken using EPR spectroscopy.


The effect of functional differences in the alpha and beta chains on the cooperativity of the osidation reduction reaction of hemoglobin.

Partially oxidized solutions of hemoglobin have been reacted with azide to determine the extent of oxidation, of the alpha and beta chains according to the method of McQuarrie and Gibson  (J. Biol. Chem. (1971) 246, 517-522) In 2, 2'2'' nitriloethanol buffer the fraction of oxidized material represented by the beta chains decreases with  decreasing extent of total oxidation, of the alpha chains. Upon addition of insitol hexaphosphate, the degree of perferntial oxidation in terms of a two-state model similar to the description of oxygenation by Edelstein (nature(1971) 230, 224-227) but with the incorporation of chain heterogeneity. The results indicate that the pH-dependent cooperativity of the oxidation-reduction reaction can be described in terms of a bell curbe of n versus log l, the allosteric somewhat lower and shifted slightly to the left, due in part to an affnity of beta chains for electrons approximately twince that of alpha chains. Because the curve is shifted to the left, oxidation-reduction equilibria at l values corresponding to pH 6 to  lie on the right side of the bell curve where cooperativity the preferntial affity of beta chains for electrons rises to about 4 times that of alpha chains. As a consequence, the coreesponding bell curve is lowered with the Hill coeficient falling to unity or below in the range of l encountered. Thus the principal cause of decreased cooperativity is chain heterogeneity and not stabilization in the t state as suggested by Perutz; under these conditions the molecules of methemoglobin in the t state are only a fractional part of the population.


The dephosphorylation of phosphoglucomutase by nucleophilic reagents.

The phosphoryl group on the serine residue at the active site of phosphoglucomutase is presumed to undergo nucleophilic attack by the monophosphate substrates glucose 1- and glucose 6-phosphate to form glucose 1,6-diphosphate. Fluoride, hydroxylamine, and several thiol compounds have now been shown to serve as effective nucleophiles toward the active phosphate and result in the dephosphorylation of phosphoglucomutase. The more extensively studied nucleophiles, cysteine, hydroxylamine, and fluoride, are effective at a concentration as low as 1 mM with a relative reactivity of 40, 2, and 1, respectively. The reaction proceeds as long as the catalytic activity of the enzyme is maintained. Inactivation of the enzyme abolishes dephosphorylation by all nucleophilic reagents thus far studied. The dephosphorylation reaction shows optimal activity of pH 6.5. The rate of dephosphorylation exhibits saturation kinetics. With fluoride the Km is 534 mM. Dephosphorylation by fluoride is stimulated by some but not all bivalent cations. Cu+ and Co2+ are the most effective. Cu2+ not only augments the reaction with fluoride but also facilitates a nucleophilic attack by water, in the absence of the halogen, to yield inorganic phosphate. No augmentation of the rate of dephosphorylation by bivalent cations can be elicited with either cysteine or hydroxylamine. The products of the fluoride reaction are phosphorofluoridate, a small but variable amount of inorganic phosphate, and a fully active dephosphoenzyme. By constrast, cysteine and hydroxylamine yield inorganic phosphate and a partially inactive enzyme. The dephosphorylation rate varies with temperature. Arrhenius plots for the fluoride reaction reveal two distinct slopes. The heat of activation between 5-37 degrees was found to be 10.2 Cal per mol. Between 0-5 degrees, however, it was considerably greater amounting to 24.3 Cal per mol.


Effects of beta-adrenergic catecholamines on potassium transport in turkey erythrocytes.

Isoproterenol stimulates cellular accumulation of cyclic adenosine 3':5'-monophosphate (cyclic AMP) and produces a 2- to 4-fold increase in bidirectional potassium fluxes in turkey erythrocytes. Ouabain, which does not alter catecholamine-stimulated cellular cyclic AMP, inhibits potassium influx by 50 to 70%, does not alter potassium outflux or isoproterenol-stimulated potassium influx, but increases isoproterenol-stimulated potassium outflux. Stimulation of potassium transport by isoproterenol can be reproduced by adding cyclic AMP to the medium and is inhibited by propranolol or dichloroisoproterenol but not by phentolamine. Theophylline at concentrations which inhibit cyclic nucleotide phosphodiesterase in isolated turkey erythrocyte plasma membranes by greater than 90%, does not augment isoproterenol stimulation of cellular cyclic AMP or of potassium transport but does potentiate stimulation of potassium influx produced by adding cyclic AMP to the medium. Isoproterenol-stimulated cellular cyclic AMP increases steadily for at least 2 hours. Potassium transport, however, increases rapidly, becomes maximal after 20 to 30 min of incubation, and thereafter decreases progressively so that after 2 hours of incubation potassium fluxes are only slightly greater than for the control. Ouabain prolongs the duration of catecholamine-stimulated potassium influx and potassium outflux, reflecting its ability to relieve the refractoriness developed by turkey erythroyctes to endogenous cyclic AMP.


Lipids of bovine adrenal plasma membranes.

The lipid composition of a membrane fraction from bovine adrenal cortex was determined. This preparation has the capacity to bind adrenocorticotropic hormone and is enriched in adenylate cyclase and 5'-nucleotidase. The adrenal plasma membranes have a significantly higher lipid content (54.8%) than bovine liver plasma membranes and a surprisingly low proportion of this lipid is cholesterol (4.2%). The phospholipids comprise 76.4% of the total lipids and their composition if very similar to that of bovine liver membranes with the exception of sphingomyelin which comprises only 4.5% of the phospholipids in the adrenal preparation.


The molecular dissociation of ferrihemoglobin derivatives.

Measurement of the dissociation constants of ferrihemoglobin by light scattering indicates that the quaternary structure is altered by the type of heme ligand. Fluoromethemoglobin and aquomethemoglobin, high spin derivatives with weak ligands, have tetramer-dimer dissociation constants of 80 and 50 muM, respectively. For low spin cyanmethemoglobin the dissociation constants were 1 muM (pH 6.0) and 3 muM (pH 9.0) under the general conditions of 0.1 ionic strength and 25 degrees. Of the ferrihemoglobins studied, alkaline methemoglobin (pH 9.0) has the lowest dissociation constant (0.2 muM). Dissociation constants of mixtures of alkaline and fluoromethemoglobin were significantly higher than that of the alkaline form alone. At pH 9.0 the 55 and 78% fluoride-bound derivatives had tetramer-dimer dissociation constants of 0.7 and 2 muM, respectively. The cyanmethemoglobin quaternary conformation was found to be less affected by pH than the fluoromethemoglobin and aquomethemoglobin conformations. Measurement of the dissociation constant (0.2 muM) for aquomethemoglobin-inositol hexaphosphate indicates stabilization of the tetramer by this organic phosphate. The extent of stabilization by inositol hexaphosphate does not appear to be that found for deoxyhemoglobin as suggested by Perutz (Perutz, M. F. (1972) Nature 237, 495-499) even though inducement of higher spin and iron-heme plane displacement may occur.


The enzymic conversion of protoporphyrinogen IX to protoporphyrin IX. Protoporphyrinogen oxidase activity in mitochondrial extracts of Saccharomyces cerevisiae.

The oxidation of protoporphyrinogen IX to protoporphyrin IX in yeast cells is enzyme-dependent. The enzyme, protoporphyrinogen oxidase, associated with purified mitochondria isolated from Saccharomyces cerevisiae was solubilized by sonic treatment in the presence of detergent and partially purified. The molecular weight of the enzyme was 180,000 plus or minus 18,000. The purified preparation could be stored at -20 degrees in the presence of 20% glycerol for several months without loss of activity. Enzyme activity was destroyed by heating above 40 degrees and by proteolytic digestion and irreversible inactivation occurred outside the pH range of 4.0 to 9.5. The pH optimum of the enzymic reaction was 7.45 and the value of the Michaelis constant was approximately 4.8 muM. Protoporphyrinogen oxidase did not catalyse the oxidation of coproporphyrinogen I or III or uroporphyrinogen I or III and the rate of enzymic oxidation of mesoporphyrinogen IX was less than 20% of that observed with protoporphyrinogen IX. The presence of thiol groups in the enzyme system was indicated but no metal ion or other cofactor requirement was demonstrated. Enzyme activity was insensitive to cyanide, 2,4-dinitrophenol, and azide whereas it was inhibited in the presence of Cu-2+ or Co-2+ ions, high ionic strength, heme, or hemin.


Functional specificity of guanosine 3':5'-monophosphate-dependent and adenosine 3':5'-monophosphate-dependent protein kinases from silkworm.

Adenosine 3':5'-monophosphate-dependent protein kinase partially purified from silkworm pupae shows identical functional activities with those of mammalian protein kinases; the insect and mammalian kinases are completely exchangeable in the phosphorylation of muscle glycogen phosphorylase kinase and glycogen synthetase resulting in the activation and inactivation of the respective enzymes. In contrast, guanosine 3':5'-monophosphate-dependent protein kinase obtained from the same organism is totally inactive in this role and phosphorylates different, mainly seryl and some threonyl, residues of acceptor proteins. Substrates of the latter kinase intimately involved in the regulation of biological processes have remained unknown.


The effect of potassium chloride on the Bohr effect of human hemoglobin.

The normal and differential titration curves of liganded and unliganded hemoglobin were measured at various KCl concentrations (0.1 to 2.0 M). In this range of KCl concentrations, the curves for deoxyhemoglobin showed no salt-induced pK changes of titratable groups. In the same salt concentration range oxyhemoglobin showed a marked change in titration behavior which could only be accounted for by a salt-induced increase in pK of some titratable groups. These results show that the suppression of the alkaline Bohr effect by high concentrations of neutral univalent salt is not caused by a weakening of the salt bridges in deoxyhemoglobin but is due to an interaction of chloride ions with oxyhemoglobin. Measurements of the Bohr effect at various KCl concentrations showed that at low chloride ion concentration (5 times 10-3 M) the alkaline Bohr effect is smaller than at a concentration of 0.1 M. This observation indicates that at a chloride ion concentration of 0.1 M, part of the alkaline Bohr effect is due to an interaction of chloride ions with hemoglobin. Furthermore, at low concentrations of chloride ions the acid Bohr effect has almost vanished. This result suggests that part of the acid Bohr effect arises from an interaction of chloride ions with oxyhemoglobin. The dependence of the Bohr effect upon the chloride ion concentration can be explained by assuming specific binding of chloride ions to both oxy- and deoxyhemoglobin, with deoxyhemoglobin having the highest affinity.


The pH dependence of the hydrolysis of benzoyl-L-arginine ethyl ester in cooled mixed solvents.

Tables of protonic activity (paH) of a number of buffers, determined in mixed solvents and at subzero temperatures, are reported for the following media: water-1,2-propanediol, water-glycerol, and water-dimethylsulfoxide (50:50, in volume). These data with those previously reported allowed us to study enzymic reactions under these conditions. The paH dependence of the tryptic hydrolysis of benzoyl-L-arginine ethyl ester has been studied in the presence of organic solvents (methanol, ethylene glycol, 1,2-propanediol, glycerol, and dimethylsulfoxide, all 50% by volume) between 20 and -20 degrees. The results have allowed us to show the validity of our paH scales in mixed solvents. The paH profiles obtained under these conditions are similar to those observed in pure water at 20 degrees. They are shifted nevertheless by both solvent and temperature. Such shifts are interpreted in terms of the effects of solvents and temperature on pKES on the basis of the conclusions drawn from a study of the effect of these variables on small dissociable molecules. The results obtained under these conditions of solvents and temperature are consistent with the presence at the active site of the enzyme of a histidine residue, and thus provide, concerning the solvent effect, a direct verification of the method of Findlay et al. (Findlay, D., Mathias, A. P., and Rabin, B. R. (1962) Biochem. J. 85, 139-144). On the other hand, the large temperature interval provided by the low temperature procedure, allows us to vary significantly the pK of ionizable groups of the enzymes and thus makes possible their identification, on the basis of their enthalpy of ionization.


Immunochemical evidence for glutamine-mediated degradation of glutamine synthetase in cultured Chinese hamster cells.

The specific activity of glutamine synthetase in cultured Chinese hamster cells is inversely related to the concentration of glutamine in the surrounding solution. Enzyme specific activity increases 8- to 10-fold when glutamine is removed from serum-free F12 growth media. The induction of glutamine synthetase activity occurs only after glutamine removal and not after the removal of other amino acids (methionine, leucine, or isoleucine). The analysis of the glutamine-mediated decrease in glutamine synthetase activity has been simplified by the finding that depression proceeds in nutrient-free buffered saline solution (141 mM NaCl, 5.4 mM KCl and 30 mM Tricine (pH 7.4). Under these conditions, 0.1 mM cyanide blocks glutamine-mediated depression. The cyanide inhibition is reversed by the addition of 1.0 mM glucose which suggests that ATP is required for depression. Glutamine-mediated depression is temperature-dependent, occurring between 25 and 45 degrees with an optimum rate at 37 degrees. Studies of the time course of induction and depression as a function of glutamine concentration suggest that glutamine regulates the rate at which the enzyme is either modified or degraded. We have employed an antibody prepared against homogeneous Chinese hamster liver glutamine synthetase to measure the amount of glutamine synthetase protein in extracts of cells containing induced or depressed levels of enzyme activity. A highly sensitive immunoprecipitation procedure enables quantitation of nanogram amounts of glutamine synthetase protein. Glutamine synthetase in cell extracts containing induced levels of enzyme activity possesses the same molecular specific activity (ratio of activity to antigenicity) as homogeneous Chinese hamster liver glutamine synthetase. The molecular specific activity of glutamine synthetase is almost the same in extracts of cells with depressed levels of enzyme obtained by growth for short (2 hours) and long (24 hours) times in the presence of glutamine. These data suggest that glutamine-mediated depression of glutamine synthetase results from degradation of enzyme molecules.


Ligand binding properties of hemoglobin 3 of the trout, Salmo gairdneri. The occurrence of an acid Bohr effect in the absence of heme-heme interaction.

The four components of hemoglobin from the rainbow trout (Salmo gairdneri) have been isolated. The oxygen affinities of the first two components eluted from the DEAE-cellulose column have much smaller pH dependencies than the last two components. These components have very low O2 affinities at low pH. The effect of pH on the equilibrium and kinetics of ligand binding to the third fraction, the pH-dependent component present in greatest amounts, has been studied. Measurements of ligand binding equilibria demonstrate the presence of both an alkaline and an acid Bohr effect. In the region of the alkaline Bohr effect the value of n in the Hill equation is a function of ligand affinity. For CO binding n decreases as the pH is decreased until at pH 6, the minimum ligand affinity is reached. At this pH there is also a complete loss of cooperative ligand binding. Decreasing the pH further results in an increase of ligand affinity, but this acid Bohr effect is not associated with a reappearance of cooperativity. This suggests that Fraction 3 of S. gairdneri is frozen in the low affinity, deoxygenated conformation at low pH and that the quaternary structure does not change even when fully liganded. However, the properties of the low affinity conformation of this hemoglobin are pH-dependent.


Cross-linking of the components of lactose synthetase with dimethylpimelimidate.

The cross-linking of the two components of lactose synthetase, alpha-lactalbumin and a galactosyltransferase, with dimethylpimelimidate was examined. The extent of the cross-linking at pH 8.1 was found to be dependent upon the presence of substrates or inhibitors for the galactosyltransferase. N-acetylglucosamine and mixtures of either N-acetylglucosamine, Mn-2+ and UDP, or UDP-galactose and Mn-2+ promoted the formation of cross-linked species. Glucose or a mixture of UDP and Mn-2+ were much less effective in promoting cross-linking. Two types of intermolecularly cross-linked species of alpha-lactalbumin and the galactosyltransferase were obtained. Each was a 1:1 cross-linked complex of alpha-lactalbumin and either of the two forms of the transferase with molecular weights of about 42,000 and 48,000, respectively. Cross-linked complexes were not observed with more than 1 molecule each of alpha-lactalbumin and the transferase. The cross-linked complexes were obtained in homogeneous form by gel filtration on Sephadex and absorption of uncross-linked enzyme by affinity chromatography on alpha-lactalbumin-Sepharose in the presence of N-acetylglucosamine. They migrated on gel electrophoresis in sodium dodecyl sulfate with mobilities in accord with their predicted molecular weights as 1:1 complexes of alpha-lactalbumin and the transferase. The amino acid composition of the cross-linked complex was in reasonable agreement with the expected composition of a 1:1 mixture of alpha-lactalbumin and galactosyltransferase. The enzymic properties of the cross-linked and uncross-linked enzymes were compared. The cross-linked complex had a much higher intrinsic lactose synthetase activity than did uncross-linked enzyme although only about 1% of the potential activity of uncross-linked enzyme in the presence of optimal concentrations of alpha-lactalbumin. The lactose synthetase activity of the cross-linked complex, however, was unaffected by exogenous alpha-lactalbumin. In addition, the complex readily catalyzed the transfer of galactose from UDP-galactose to xylose in the absence of exogenous alpha-lactalbumin. The N-acetyllactosamine synthetase activity of the complex was low compared to its activity with other monosaccharides. Ovalbumin, which is a good acceptor for the uncross-linked transferase, was not an acceptor for the cross-linked complex. Kinetic studies of the complex suggest that its modified catalytic activity is not the result of the modification by dimethylpimelimidate but reflects the expected effects of  is provided, and that


Circular dichroism and absorbance properties of nitrotyrosyl chromophores in staphylococcal nuclease and in a model diketopiperazine.

An active derivative of staphylococcal nuclease, in which only tyrosine residue 115 has been nitrated with use of tetranitromethane, has been characterized using absorbance, circular dichroism, and fluorescence spectroscopy. The results show that nitrotyrosine-115 nuclease is indistinguishable from native nuclease with regard to the average secondary structure of the folded polypeptide chain, the susceptibility of the enzyme to heat denaturation, and the local tertiary structure around tryptophan residue 140. Inasmuch as optical properties of nitrotyrosine-115 nuclease from 300 to 500 nm can be unambiguously assigned to nitrotyrosine residue 115 in the active site region, this modified enzyme presents a good model system for studying the circular dichroism properties of this aromatic amino acid in a protein. The spectral properties of nitrotyrosine-115 nuclease have been compared to those of the model compounds, cyclo-(-Gly-Tyr(3NO2)-) and Tyr(3NO2). Circular dichroism spectral changes in nitrotyrosine-115 nuclease due to the binding of deoxythymidine 3',5'-diphosphate and Ca-2+ have been compared to the corresponding nitrotyrosyl-115 absorption spectral changes. This comparison shows that the circular dichroism difference spectrum exhibits an over-all change in the intensity of the observed Cotton effects, whereas the absorption difference spectrum exhibits a blue shift. This finding supports the suggestion that perturbations of aromatic amino acid chromophores in proteins due to ligand binding result in red or blue shifts in absorption difference spectra, but in over-all changes of intensity in circular dichroism difference spectra.


Circular dichroism of C3a anaphylatoxin. Effects of pH, heat, guanidinium chloride, and mercaptoethanol on conformation and function.

Circular dichroism spectra for C3a anaphylatoxins (protein fragments generated enzymatically in serum during activation of the third component of complement (C3)) from human and porcine sources were compared in the region of 190 to 250 nm. The spectra were indistinguishable in this region, although an estimated difference of approximately 20% exists between the primary structures of human and porcine C3a. Calculations indicated a total of 40 to 45% alpha helical content based on either the 208 or 222 nm extremum of the CD spectra. Addition of either mercaptoethanol or 6 M guanidinium chloride to human C3a produced a marked decrease in the mean residue ellipticity centered at 222 nm without irreversibly affecting the biological activity. Simultaneous addition of mercaptoethanol and guanidinium chloride virtually eliminated the CD contribution at 222 nm and resulted in more than 90% inactivation of the anaphylatoxin. Removal of the denaturant and reducing agent restored both the biological activity and the CD spectrum of C3a. Heat treatment or reduction and alkylation of human C3a produced biologically inactive and conformationally modified anaphylatoxin. In contrast, C3a inactivated by enzymatic removal of the COOH-terminal arginyl residue was structurally unchanged as judged by CD measurements. Consequently, it is proposed that in addition to the essential COOH-terminal arginyl residue, a highly ordered conformation is required for biological functionality of the C3a molecule.


Solubilization and partial characterization of rat liver squalene epoxidase.

The microsomal enzyme system from rat liver which catalyzes squalene epoxidation requires a supernatant protein and phospholipids (Tai, H., and Bloch, K. (1972) J. Biol. Chem. 247, 3767). It has now been found that these two cytoplasmic components can be replaced by Triton X-100. The same detergent solubilizes the microsomal squalene epoxidase and the resulting supernatant can be separated into two components, A and B, by DEAE-cellulose chromatography. Neither Fraction A nor B alone has significant squalene epoxidase activity but combining the two affords a reconstituted system 5-fold higher in specific epoxidase activity than that of the original microsomes. FAD and Triton X-100 in addition to molecular oxygen and NADPH are required in the reconstituted system. Subjecting Fraction A to a second DEAE-cellulose chromatography does not change its specific activity but lowers NADH-ferricyanide reductase activity and the protoheme content to 1/25 and 1/4, respectively. When Fraction B was chromatographed on Sephadex G-200, the specific epoxidase activity tested in the presence of Fraction A was increased 3-fold. This procedure also raised the specific activity of NADPH-cytochrome c reductase activity in Fraction B 3-fold. The reconstituted epoxidase system is not inhibited by either carbon monoxide, potassium cyanide, or o-phenanthrolien but Tiron at 1 mM was inhibitory (50%). Erythrocuprein has no effect on epoxidation. No evidence has been found for the participation of hemoproteins (P450 or cytochrome b5) in squalene epoxidation. Component B appears to be identical with the flavoprotein NADPH-cytochrome c reductase. Component A may be a flavoprotein with an easily dissociable prosthetic group.


Studies on the mechanism of Mycobacterium smegmatis L-lactate oxidase. 5-Deazaflavin mononucleotide as a coenzyme analogue.

Apo-lactic oxidase from Mycobacterium smegmatis reconstituted with the deazaisoalloxazine analogue of FMN, 5-deazaFMN, undergoes reduction by L-lactate but not catalytic reoxidation by O2. The rate of deazaFMN-holo-enzyme reduction by substrate is 1.1 min-1. With L-[alpha-3-H]-lactate, direct tritium transfer to enzyme-bound deazaFMN occurs during reduction. No evidence for a stable covalent lactate-deazaFMN adduct has been obtained. The deaza-FMNH2-enzyme is reoxidized extremely slowly by O2, consistent with the sluggish nonenzymatic reaction of deaza-FMNH2 with oxygen. On the other hand, addition of pyruvate to the deazaFMNH2-enzyme causes rapid reoxidation, a process not detected in the absence of enzyme.


Acetylation of amino groups and its effect on the conformation and immunological activity of obalbumin.

A procedure is described for the specific acetylation of the lysine residues of ovalbumin. Six acetylated ovalbumins varying in the degree of modification from 21 to 98% were prepared and were found to be homogeneous by polyacrylamide gel electrophoresis, immunodiffusion, and immunoelectrophresis. As expected, the anodic movement of ovalbumin increased and the isoionic point shifted to lower pH values with progressive acetylation of the protein. Measurements on ultraviolet absorption, fluorescence, tryptic digestion, intrinsic viscosity, gel filtration behavior, and immunological reactivity demonstrated that the native folded conformation of ovalbumin was appreciably altered by acetylation. However, even the maximally modified ovalbumin retained considerable residual structure consisting of regions of ordered structure containing antigenic determinants.


The mechanism of end product inhibition of serine biosynthesis. V. Mechanism of serim inhibition of phosphoglycerate dehydrogenases.

The reduction of enzyme-bound DPN constitutes a half-reaction of phosphoglycerate dehydrogenase and has been investigated fluorometrically. Serine was found to inhibit the half-reaction to the same extent and with the same degree of cooperation as the steady state reaction. This finding identifies the ternary complex conversion as the point in the reaction sequence at which serine inhibition occurs. Delta H determinations for the half-reaction showed no difference whether serine was or was not present and led to the conclusion that the inhibitory effect of serine could only manifest itself through the delta S term in the expression for the formation of the activated transition state complex. DL-3-P[2-2H]glyceric acid showed no primary isotope effect in the half-reaction. This result excludes hydrogen transfer as the rate-limiting step in the half-reaction and confirms that an isomerization step, affected by serine, exists in the ternary complex conversion scheme. The deuterated 3-P-glyceric acid shows an isotope effect of 2 in the steady state reaction.


Effect of nicotinamide adenine dinucleotide on the membrane-associated reduced nicotinamide adenine dinucleotide oxidase of Mycobacterium phlei.

NAD+ had a biphasic effect on the NADH oxidase activity in electron transport particles from Mycobacterium phlei. The oxidase was inhibited competitively by NAD+ at concentrations above 0.05 mM. NAD+ in concentrations from 0.02 to 0.05 mM resulted in maximum stimulation of both NADH oxidation and oxygen uptake with concentrations of substrate both above and below the apparent K-M. Oxygen uptake and cyanide sensitivity indicated that the NAD+ stimulatory effect was linked to the terminal respiratory chain. The stimulatory effect was specific for NAD+. NAD+ was also specific in protecting the oxidase during heating at 50 degrees and against inactivation during storage at 0 degrees. NAD+ glycohydrolase did not affect stimulation nor heat protection of the NADH oxidase activity if the particles were previously preincubated with NAD+. Binding studies revealed that the particles bound approximately 3.6 pmol of [14C1NAD+ per mg of electron transport particle protein. Although bound NAD+ represented only a small fraction of the total added NAD+ necessary for maximal stimulation, removal of the apparently unbound NAD+ by Sephadex chromatography revealed that particles retained the stimulated state for at least 48 hours. Further addition of NAD+ to stimulated washed particles resulted in competitive inhibition of oxidase activity. Desensitization of the oxidase to the stimulatory effect of NAD+ was achieved by heating the particles at 50 degrees for 2 min without appreciable loss of enzymatic activity. Kinetic studies indicated that addition of NADH to electron transport particles prior to preincubation with NAD+ inhibited stimulation. In addition, NADH inhibited binding of [14C]NAD+. The utilization of artificial electron acceptors, which act as a shunt of the respiratory chain at or near the flavoprotein component, indicated that NAD+ acts as at the level of the NADH dehydrogenase at a site other than the catalytic one resulting in a conformational change which causes restoration as well as protection of oxidase activity.


Molecular weight and structure of 7 S nerve growth factor protein.

The molecular weight of 7 S nerve growth factor has been studied in the analytical ultracentrifuge between pH values 6.8 and 7.8. At pH 6.8, where no dissociation is observed, the molecular weight was found to be 137,000 plus and minus 7,000. Between pH values 7.4 and 7.8 there is some dissociation. Using the data from this study and results in the literature, a model of 7 S nerve growth factor, (alpha beta gamma)2, in reversible equilibrium with a subunit complex, (alpha beta gamma), is proposed.


Factors affecting the reassociation and reactivation of the half-molecular weight nonidentical subunits of pigeon liver fatty acid synthetase.

The pigeon liver fatty acid synthetase complex (14 S) is dissociated in low ionic strength buffer containing dithiothreitol to form a half-molecular weight subunits (9 S) which are completely inactive for the synthesis of saturated fatty acids. The dithiothreitol-protected (reduced) subunits are rapidly reassociated and reactivated to form the active enzyme complex, not only by an increase in salt concentration but also by micromolar concentrations of NADP+ or NADPH. Increases in KCl or NADPH concentration result in an increase in the extent of reactivation (equilibrium) with no change in the over-all rate of the reaction or the half-life ofreactivation of the enzyme. The extent (equilibrium) of reactivation of the enzyme is the same in 0.2 M potassium phosphate buffer, pH 7.0; 0.2 M KCl in 5 mM Tris-35 mM glycine buffer, PH 8.3; and 50 muM NADP+ or NADPH in the Tris-glycine buffer. The extent and rate of reactivation of the enzyme is dependent not only on ionic strength and NADPH concentration, but also on pH and temperature. Reactivation with 0.2 M KCl is optimal between pH 7.3 and 8.5. At higher and lower pH values the rate and extent of reactivation are lowered. The rate and extent of reactivation are also decreased as the temperature is lowered below 10 degrees. At 0 degrees there is little reactivation of enzyme activity. However, in the presence of 0.2 M KCl containing 15 to 40% glycerol at 0 degrees, reactivation of the enzyme is about 50% complete. The rate of reactivation of enzyme in the presence of KCl or NADPH conforms to first order kinetics. This result suggests that the subunits first combine to form an inactive complex which is subsequently transformed to an enzymatically active complex. Evidence for the presence of inactive complex was obtained in experiments carried out in 0.2 M KCl at pH 6.0, and in 0.2 M KCl at pH 8.3, at both 6 and 3 degrees. Under these conditions the amount of complex observed upon ultracentrifugation was greater than expected from determinations of enzyme activity. The above findings suggest that ionic and hydrophobic interactions, and possibly the water structure surrounding the interacting sites, are of prime importance in reassociation and reactivation of enzyme. In addition, NADP+ and NADPH have very specific effects in bringing about reassociation and in maintaining the structural integrity of the multienzyme complex.


Crystallization and some properties of chondroitinase from Arthrobacter aurescens.

A strain of Arthrobacter aurescens which secretes a large amount of chondroitinase into a culture broth, was isolated from soil. The chondroitinase was purified 380-fold over culture broth in 24% yield and crystallized. Some properties of the purified enzyme were studied and described: thermal stability (below 45 degrees), pH stability (pH 4.9 to 7.4), optimum temperature (50 degrees), and optimum pH (pH 6.0). Chrondroitin sulfate A and C, chondroitin, and hyaluronic acid were split by the enzyme but dermatan sulfate could not be. The initial rates of enzymic degradation of chondroitin sulfate C, chondroitin, and hyaluronic acid were 1.1, 1.95, and 3.2, respectively, compared to that of chondroitin sulfate A. When the enzyme was allowed to act on chondroitin sulfate A and C, the reducing power and the ultraviolet absorption at 232 nm increased proportionally to the decrease in viscosity of the substrate solution. Finally these substrates were degraded to the extent of 100% to disaccharides. By the enzyme action the main products from chondroitin sulfate A and C were deta 4,5-unsaturated disaccharides, which were identified as 2-acetamido-2-deoxy-3-O-(Beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose and 2-acet-amido-2-deoxy-3-O-(Beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose by paper chromatography, ultraviolet absorption spectroscophy, and infrared spectroscopy. Thus it is suggested that the chondroitinase is a chondroitin sulfate A and C lyase, one of the hyaluronate lyases (EC 4.2.99.1).


The polyadenylate polymerases from yeast.

Poly(A) polymerase activity was first detected in yeast extracts, primarily in association with the ribosomal fraction, by Twu and Bretthauer in 1971 (Twu, J. S., and Bretthauer, RK. (1971) Biochemistry 10, 1576-1582). This activity has now been separated into three distinct enzymes by chromatography on DEAE-cellulose. Each of the three enzymes can catalyze the incorporation of adenylate residues from ATP into a polyadenylate (poly(A)) tract at the 3' terminus of a primer RNA. Enzyme I elutes at 0.07 M ammonium sulfate from the DEAE-cellulose column, utilizes the mixed polynucleotide poly(A,G,C,U) or ribosomal RNA most efficiently in vitro, and may be responsible in vivo for the initiation of the poly(A) tracts found on yeast messenger RNA. Enzyme II elutes from the column at 0.20 M ammonium sulfate, requires poly(A) itself or an RNA primer containing a 3'-oligo(A) tract, and may be responsible in the nucleus for the elongation of tracts initiated by enzyme I. Enzyme III elutes from the column at 0.56 M ammonium sulfate and is present in low amounts in nuclear extracts. It may be involved in adding poly(A) tracts to messenger RNA in mitochondria. These enzymes also have the intrinsic capacity for the incorporation of cytidylate residues from CTP, which correlates with the finding of cytidylate residues in the poly(A) tracts present in the yeast RNA which is rapidly labeled in vivo. About 75% of the total poly(A) polymerase activity of yeast is enzyme I, most of which is present in the soluble protein fraction of the whole yeast extract. About 20% of the total poly(A) polymerase is enzyme II, and 1 to 5% is enzyme III. All three of the yeast poly(A) polymerases require an RNA primer with a free 3'-hydroxyl group, show no requirement for a DNA template, require Mn-2+ for optimal activity, have pH optima of 8.5, and are inhibited by GTP, CTP, UTP, and native yeast DNA. Polymerases I and II have similar molecular weights by gel filtration.


Intracellular localization of fructose 1,6-bisphosphate aldolase.

Submission of a rat liver homogenate made in 250 mM sucrose-1 mM EDTA to centrifugation between 9,500 times g for 10 min and 105,000 times g for 60 min results in the sedimentation of 60 to 70% of the total cellular fructose 1,6-bisphosphate aldolase (EC 4.1.2.13). Under these conditions only about one-quarter of the total triose phosphate dehydrogenase and phosphoglycerate kinase appears in the microsomal fraction. Ultrastructural immunologic localization techniques have demonstrated that the aldolase is associated with the endoplasmic reticulum, in situ. The binding of this enzyme to the membrane is sensitive to changes in pH with an optimum at 6.0, and to increasing concentrations of NaCl and fructose 1,6-bisphosphate, being about 100-fold more sensitive to the ester than to the inorganic salt.


Relationship of the light-induced proton uptake in bovine retinal outer segment fragments to triton-induced membrane disruption and to volume changes.

Light-induced proton uptake in bovine retinal outer segment (ROS) fragments was shown to be closely related to pH, salt concentration, membrane integrity, and perhaps secondarily to the volume of osmotic compartments. The principal findings were as follows: 1. As pH increased, both the discs and the plasmalemma swelled, and proton uptake markedly diminished. 2. As the discs were disrupted by increasing concentrations of Triton, proton uptake at slightly alkaline pH was supplanted by proton release. 3. Increasing the concentration of chloride salts caused increased H+ uptake roughly proportional to osmotic shrinkage of the ROS. Buffering by acetate prevented the measurement of proton uptake in the presence of acetate salts, although osmotic behavior of the ROS was similar to that observed in chloride salts. Although increasing the concentration of sucrose also resulted in osmotic shrinkage of the ROS, it was not accompanied by a systematic increase in the magnitude of proton uptake. 4. Light-induced H+ uptake was accompanied by small but reproducible changes in volume, probably of the discs. The magnitude and direction of these rapid volume changes were subject to influence by pH, solute, and other variables.


Evidence for involvement of protein kinase in the activation by adenosine 3':5'-monophosphate of brain tyrosine 3-monooxygenase.

Addition of adenosine 3':5'-monophosphate (cAMP) to high speed supernatant preparations obtained from rat brain caused a 3- to 4-fold increase in tyrosine 3-monooxygenase (tyrosine hydroxylase) activity. The tyrosine 3-monooxygenase remained in an activated state upon removal of the cAMP by passing the enzyme through a Sephadex G-25 column. Substances which inhibit cAMP-dependent protein kinase, namely, EDTA, ADP, and adenosine, and protein kinase modulator, each antagonized the activation of tyrosine 3-monooxygenase produced by cAMP. Furthermore, addition of partially purified brain cAMP-dependent protein kinase caused a several-fold increase in tyrosin 3-monooxygenase activity. The activation of tyrosine 3-monooxygenase by added cAMP and protein kinase required the presence of ATP and Mg-2+. These data suggests that the cAMP activation of tyrosine 3-monooxygenase may be mediated by a cAMP-dependent protein kinase.


Formation of adenosine triphosphate from Pi and adenosine diphosphate by purified Ca-2+-adenosine triphosphatase.

Ca-2+-ATPase purified from sarcoplasmic reticulum of rabbit muscle forms a phsophoeznyme when exposed to inorganic phosphate in the presence of Mg-2+. On addition of ADP and Ca-2+ virtually all of the phosphate bound to the enzyme is transferred to form ATP. It has been shown previously and confirmed by us that (a) the purified ATPase contains one major polypeptide and about 30% phospholipids; (b) on removal of residual detergent by passage through Sephadex the enzyme forms vesicular membranes; and (c) these vesicles are leaky and incapable of accumulating Ca-2+. Our findings therefore indicate that we have observed ATP generation from ADP and P-i without the formation of an ion gradient across a membrane. We propose that the energy derived from ion-protein interaction drives the formation of ATP.


Acute respiratory failure complicating multiple fractures in the absence of fat embolism. A study of pathogenesis and treatment and a preliminary report of catheterization of the right side of the heart.

Eight patients with multiple fractures were all treated for acute respiratory failure. The investigation included catheterization of the right side of the heart, determinations of alveolar-arterial oxygen gradients and studies of serum lipid and coagulation and of water and protein balance. The magnitude of respiratory failure correlated well with the number of fractures, the duration and degree of hypotension, the quantity of fluids and blood administered for resuscitation, the magnitude and duration of positive water balance, and the degree of serum hypoalbuminemia. In three patients, the pulmonary-artery pressures were normal at the onset of respiratory failure. The essential aspects of successful therapy included the early institution of assisted ventilation and the achievement of early negative negative water balance.


Action of rennin on kappa-casein.

The action of rennin on kappa-casein was studied as a function of time using several methods to measure activity. The first indication of rennin cleavage of kappa-casein is precipitability in .1 M acetate buffer at pH 5.2 and 5 C. A longer exposure to rennin is required to alter kappa-casein so that it forms a precipitate with calcium ions and loses its ability to stabilize alpha s-casein. The least sensitive indication of rennin activity is measurement of nitrogen soluble in 2% trichloroacetic acid. Electrophoresis experiments showed that these methods detect various stages in the conversion of kappa-casein para-kappa-casein.


Physiological effects of 1,3-butanediol fed to cattle.

In four trials with growing cattle we observed effects of 1,3-butanediol on rumen fermentation end products, blood components, growth rates, feed efficiency, and body composition. Diets A and C contained 80% grain: 20% alfalfa pellets and 40% grain: 60% alfalfa pellets; in diets B and D, 1,3-butanediol replaced 4% of diets A and C. Feeding 4% 1,3-butanediol caused no significant differences in rumen pH, volatile fatty acid ratios, blood glucose, or blood ketones. Cattle fed 4% 1,3-butanediol had rates of gain and feed efficiency equivalent to and often better than cattle fed the same diet without butanediol. Body composition was not affected by feeding 4% 1,3-butanediol. Results indicate 4% 1,3-butanediol can be fed to cattle without adverse physiological effects and may improve performance. When 6% or more 1,3-butanediol was fed, blood ketones were elevated considerably, animals were hyperactive, gained less weight, and feed efficiency was lower.


Lipid deficient extender for bovine spermatozoa: its development and use in measuring freezing-induced lipid loss.

To facilitate the measurement of lipid losses from spermatozoa due to freezing, three low-lipid seminal extenders containing lactose, bovine serum albumin, or soybean protein were evaluated as potential cryoprotectants. All extenders were formulated to have an osmotic pressure within the range of 270 to 330 mosmol and a pH of 6.8 to 7.0. Soybean protein (Promine-D) maintained the highest post-thaw motility of spermatozoa with similar survival for spermatozoa frozen in ampules and straws. The extender derived from testing several components consisted of Tris(hydroxymethyl) aminomethane (245 mM), and citric acid monohydrate (78mM), as the buffering compounds; and fructose (69 mM), glycerol (7% vol/vol), and Promine-D (1.5% wt/vol). Post-thaw sperm motility of approximately 40% was not different from the Tris-egg yolk control. Fertility of fresh rabbit semen treated with the extender was normal. After freeze-thawing, protected spermatozoa contained more lipid (1.61 versus 1.20 mug/10-6 sperm) and lost less glutamic oxaloacetic transaminase enzyme (102 versus 108 Karmen units) than when Promine-D was not incorporated. However, even with protection by soybean protein, spermatozoa lipid content decreased from 2.43 to 1.61 mug/10-6 sperm after one freeze-thawing. The lipid status of spermatozoa frozen and thawed in conventional bull seminal extenders containing large amounts of lipids is unknown.


Suppression of histamine-induced pruritus by three antihistaminic drugs.

A double-blind crossover study of inhibition of histamine-induced pruritus by three commonly prescribed antihistamines was conducted on 28 normal subjects. Drugs used included diphenhydramine HCl (Benadryl), cyproheptadine (Periactin), hydroxyzine HCl (Atarax), and a lactose placebo in identical capsules. Intradermal histamine dose-response thresholds of pruritus were obtained before and after pretreatment with the three antihistamines and placebo in each subject. Analysis of data revealed a fivefold increase above baseline of the histamine dose required to produce pruritus following both cyproheptadine and placebo. This compared to a tenfold increase following diphenhydramine and a 750-fold increase following hydroxyzine HCl. The most common side effect was drowsiness, which occurred with all three drugs.


The use of psychoactive drugs in elderly patients with psychiatric disorders: survey conducted in twelve Veterans Administration hospitals.

A survey conducted at 12 VA hospitals included the collection of detailed information on the use of psychoactive drugs in 1,276 elderly psychiatric patients. On the day of the survey, 61 per cent of the patients were receiving psychoactive drugs. Prescription practices relating to the choice of drugs, prevalence of drug use, dosage, combination drug preparations, and antiparkinson agents are discussed in terms of such factors as the patient's age and the diagnosis. Also discussed is the literature on psychoactive drugs, particularly as it pertains to elderly populations.


Drug therapy for cardiovascular disease in the aged.

Cardiovascular diseases and their treatment in the aged are discussed under the headings of ischemic heart disease, hypertension, cardiac failure (with special reference to the use of diuretics and digoxin), infective carditis and thyroid disorders. Advanced age modifies the approach to treatment; the choice of drugs and the dosage must be adjusted accordingly. Possible drug interactions should also be considered. A rehabilitation program is of great benefit for many elderly cardiac patients. It should be planned individually and involve psychologic and environmental factors as well as medical therapy. After successful treatment of the acute episode, even the aged patient can undertake rewarding activities in his remaining lifetime.


Complement activation by interaction of polyanions and polycations. I. Heparin-protamine induced consumption of complement.

Interactions of heparin and protamine in fresh human serum, in amounts far below those required for complement depletion by either agent alone, were found to induce virtually complete depletion of total hemolytic complement activity. This depletion was dependent on time, temperature, pH, divalent cations, and serum concentration. The predominant complement component hemolytic activity depleted was C1; under appropriate reaction conditions C4 and C2 were depleted as well. Equivalent amounts of heparin along induced lesser but substantial depletion of C3-9, whereas equivalent amounts of protamine had no effect upon complement component activities. We conclude that interaction of heparin with protamine, like interaction of antibody with antigen, markedly enhances its ability to interact with the first component of complement and activate the classical complement pathway. It is suggested that complement activation by interactions between certain polyanions and polycations, like interactions between antigens and antibodies, may have a role in the initiation of inflammatory reactions.


Rat-mouse radiation chimeras: characterization of an antibody-mediated graft-vs-host reaction.

A modification of the Jerne hemolysis-in-agar technique was used to demonstrate antibody-mediated graft-vs-host (GvH) hemolytic activity in lymphoid tissues of rat-mouse radiation chimeras. Initial foci of hemolytic activity were detected on the 3rd day in spleens of lethally x-irradiated mice infused with rat spleen cells (950 R-RSp chimeras). The peak of GvH foci response occurred near the end of the 1st week when 70% of 950 R-RSp chimera spleens examined contained an average of 18 to 21 foci per spleen. GvH hemolytic activity was also detected in mice reconstituted with rat bone marrow (950 R-RBM chimeras); however, only 40% of these chimeras contained splenic foci (nine foci per spleen) on day 8, the peak day of response. Mesenteric lymph nodes from 950 R-RSp chimeras, but not 950 R-RBM chimeras, also contained GvH hemolytic foci. Specificity experiments demonstrated that the GvH hemolytic reaction could be inhibited with rabbit anti-rat IgM serum and host (C3BF) antigenic cell fragments. Indirect (IgG) hemolytic activity was also observed in spleens of 950 R-RSp chimeras 6 to 10 days after treatment. At least 90% of indirect foci appeared coincident with direct foci. Rat IgM and IgG containing cells beneath hemolytic foci were observed by combining the fluorescent antibody and foci techniques. To determine whether the foci response reflects and in vivo sensitization of donor cells, spleen cell transfer experiments were done. The results indicate that spleen cells from 950 R-RSp chimeras were more effective in acelerating mortality and anemia and induced a higher incidence of Coomb's positive red cells in lethally irradiated isogeneic recipients than spleen cells from 950 R-RBM chimeras; spleen cells from either chimera were more effective in accelerating GvH indices than spleen and marrow cells from normal rats.


Intracellular events involved in the induction of immune competence in lymphoid cells by a thymus humoral factor.

A humoral factor extracted from calf thymus (THF) restores the immunocompetence of spleen cells from neonatally thymectomized (NTx) mice to induce an in vitro graft-vs-host (GVH) response. This acquistion of immunocompetence consists of a series of biochemical events, the first of which involves an obligatory rapid increase in adenyl cyclase acitivity and in intracellular levels of cyclic AMP. Protein synthesis occurs as a further step in the events leading to induction of immunocompetence by THF and could be blocked by cycloheximide with the consequent abolishment of the immunocompetence of the lymphoid cells tested. The induction of competence by THF is accompanied by a simultaneous reduction in DNA synthesis resulting from the increase in intracellular cyclic AMP levels. These steps have been studied in the absence of antigenic stimulation which is not required for the induction of competence by THF. Spleen extracts prepared by a similar procedure as THF were found to be devoid of the properties described above.


Immunopathology of mouse hepatitis virus type 3mii. effect of immunosuppression in resistant mice.

Normal adult A strain mice are resistant to MHV-3 infection. A strain mice immunosuppressed by 600 rads of x-irradiation or by anti-lymphocyte serum treatment became susceptible to the virus and died with specific lesions of the liver and high virus titers. However, mice immunized with MHV-3 before sublethal x-iraddiation resisted a second injection of virus. Resistant adult (A times C3H) F-1 hybrids undergoing graft-vs-host (GVH) reaction became highly susceptible to MHV-3 injected 8 days after parental cell injection. Virus titer 3 days after injection was 2 logs higher in mice undergoing GVH than in controls. However F-1 hybrid mice resisted virus challenge when the first injection of virus was given 2 weeks before GVH induction. In addition, thymectomy also modified the behavior of resistant animals toward virus infection. It appears, therefore, that cell-mediated immune functions play an important role in resistance of mice to MHV-3.


Thyroglobulin-binding lymphoid cells in obese strain (OS) chickens.

The binding of chicken thyroglobulin-coated chicken red blood cells by splenic, thymic, and bursal lymphoid cells was analysed in Obese strain (OS) chickens with spontaneous autoimmune thyroiditis and normal white Leghorn controls aged 1 week to 2.5 yr. Chicken erythrocytes coated with pneumococcal polysaccharide SIIII served as controls. The specificity of thyroglobulin-rosette-forming cells was verified by inhibition experiments. OS chickens showed significantly higher counts of thyroglobulin-rosette-forming cells in the spleen and thymus as compared to normal controls while no such difference was found for SII-rosette-forming cells. The values obtained with bursal lymphoid cells were in the same range in both OS and normal white Leghorn birds. The appearance of thyroglobulin-rosette-forming cells and their peak values clearly preceded the maximum frequency and severity of thyroiditis and the peak of the thyroglobulin antibody curve. It is concluded that the presence of thyroglobulin rosette-forming cells is a prerequisite for the future development of spontaneous autoimmune thyroiditis in the OS. Inhibition studies with specific turkey anti-chicken bursa and thymus cell sera revealed the B cell nature of active thyroglobulin-rosette-forming cells and suggested that passive rosette-forming cells were of T cell origin.


Platelets fixed with paraformaldehyde: a new reagent for assay of von Willebrand factor and platelet aggregating factor.

Present methods for assay of platelet aggregating agents use freshly prepared platelets. Much time is spent in daily preparation of platelets and standardization presents problems. The preparation of fixed washed platelets (FWP) and their use in two bioassays are described in this report. Washed human platelets were fixed for 48 hours with 4 per cent paraformaldehyde, washed twice in phosphate buffer, pH 6.4, and stored at 4 degrees C. Aggregation of FWP was studied with a macroscopic test and a light absorbance measurement. FWP did not aggregate with adenosine diphosphate, collagen, adrenalin, and thrombin. FWP aggregated with bovine or porcine plasma, poly-L-lysine, and ristocetin with normal human plasma but not with von Willebrand's disease plasma. These observations confirm the direct aggregating effect of these agents. Macroscopic aggregation times were dependent on the amount of aggregating agent (bovine plasma, normal human plasma). A quantitative assay for bovine platelet aggregating factor (PAF) and von Willebrand factor (vWF) with FWP was developed. The ability of FWP to aggregate remained unchanged after 1 month of storage at 4 degrees C. Ristocetin alone caused a decrease in light transmission of FWP suspensions, depending upon the concentration of ristocetin, but did not cause aggregation. FWP constitute a stable reagent suitable for quantitative measurement of PAF and vWF.


Determination of hemoglobin-oxygen affinity on micro samples.

The spectrophotometric determination of hemoglobin-oxygen dissociation on dilute red cell suspensions has been modified by the use of a bicarbonate buffer containing bovine serum albumin. The red cell suspension is equillibrated with known ratios of air and nitrogen and 5 per cent carbon dioxide at atmospheric pressure using two gas pumps. The method enables the hemoglobin-oxygen dissociation curve to be measured on 10 to 20 mul of whole blood, and has given values for the P50 (the oxygen tension at 50 per cent hemoglobin-oxygen saturation) of 25.9 plus or minus  1.5 S. D. which are comparable to the value of 26.0 plus or minus 1.0 S. D. obtained by the mixing technique and by other methods. The addition of bovine albumin (35 mg. per cent or greater) increased the P50 by 3 mm. Hg above that obtained with buffer alone. The P50 value has been compared in bis-Tris and phosphate buffers with and without albumin, and in bicarbonate buffers with the addition of approximately equimolar concentrations of either human or bovine serum albumin or polyvinyl-pyrrolidone (PVP), and buffer alone. The effect of albumin on the P50 value was most marked in bicarbonate buffer, and was statistically significantly greater in the presence of human or bovine albumin than in buffer alone or buffer plus PVP. The addition of albumin could not be explained through an alteration of cell shape or volume, but may be an influence on intracellular pH.


Thiol-disulfide interchange in the binding of bence jones proteins to alpha-antitrypsin, prealbumin, and albumin.

Native light Ig chains of kappa- but not of lambda-type form -S-S linked complexes with prealbumin, alpha1-AT and albumin in vivo. kappa-chains isolated from urines have cysteinyls which are more promptly reacting with dithionitrobenzoate (DTNB) than lambda-chains. Both are monomerized on this reaction. On addition to plasma mixed disulfides between both types of light chains and DTNB form larger amounts of complexes than the native chains. The lower reactivity of native lambda-chains to the plasma proteins can be explained by their higher dimer stability. From the light chain reactions obtained with isolated alpha1-AT and albumin it is concluded that alpha1-AT has a disulfide which efficiently interchanges with monomeric, light chain thiolate ions released from thionitrobenzoate derivates of light chains and that on interchange with the derivatized light chains albumin releases more free light chains into the solution than are bound to albumin. Addition of derivatized light chains to a mixture of alpha1-AT and albumin increases the yield of alpha1-AT complexes and decreases the amount of albumin complexes formed. The relative amount of the different complexes formed in the latter experiments corresponds to the findings in vivo in patients with Bence Jones proteinemia. Prealbumin and alpha1-AT in plasma have a roughly 10-fold stronger tendency to link the light chains than albumin. The complexes are formed through thiol-disulfide interchange though neither the disulfide of native alpha1-AT nor the thiols of prealbumin is available for reaction with DTNB. The three plasma proteins may together constitute a system for linkage and transport of peptides with reactive thiols or disulfides released into the extracellular fluids. The trypsin and elastase binding and inhibiting capacity of alpha1-AT remains after cleavage of the internal -S-S-bridge of alpha1-AT through interchange with a light chain thiol for which reason an intact internal -S-S-bridge of alpha1-AT is not necessary for inhibition and linkdage of the enzymes.


Gas transport and blood acid-base balance in diving sea snakes.

The values of hemoglobin concentration, Hb-O2 affinity and buffering capacity of the blood of six sea snake species considerably overlap values from terrestrial squamates. Decreased blood pH had little effect on the P50 but increased the n-value of Hb-O2 equilibrium curves. The O2 saturation of blood in the dorsal aorta varied between about 30 and 70% during voluntary diving in Acalyptophis peronii and Lapemis hardwickii. Voluntary dives ended when the lung PP02 was about 50 mm Hg and the arterial PO2 about 30 mm Hg indicating that roughly half of the O2 reserves had been used. In conjunction with relatively stable blood lactate concentration and pH, this indicates that voluntary dives occurred largely aerobically. In contrast, forced dives resulted in depletion of O2 reserves and large changes in blood acid-base balance. Long recovery periods following forced dives are inconsistent with field observations and thus suggest that extensive anaerobic metabolism does not normally occur in sea snakes. Bradycardia was not evident during forced dives. Large differences in PO2 between the lung and dorsal aorta indicated considerable right to left shunting either in the heart or in the lung. Venous blood represented over 50% of the systemic flow when there was considerable O2 in the lung. Therefore blood PO2 may remain relatively low despite elevated lung PO2 resulting from diving. In view of substantial capability for extra-pulmonary gas exchange, high shunting reduces the possibility of losing O2 through the skin and also may help prevent decompression sickness following deep dives.


The effect of coenzyme leakage and replacement on the growth and metabolism of two fusobacteria.

Suspensions in water of two species of Fusobacterium leaked several coenzymes when incubated at normal growth temperatures. Chromatography of filtrates from these suspensions revealed the presence of NAD, NADP, FMN, tetrahydrofolic acid and, in one of the two, pyridoxal phosphate. Analyses of some enzymic activities in whole organisms demonstrated deficiencies in coenzymes:glutamate dehydrogenase was virtually inactive in the absence of added NAD; tryptophanase activities were diminished by washing but the extent differed between strains; histidase activity was not decreased by washing or suspension in water or saline. Both lag phase and doubling time increased markedly in severely washed organisms inoculated into fresh medium. Addition of appropriate coenzymes shortened the lag phase for both strains and shortened the doubling time in one.


Extremely thermophilic acidophilic bacteria convergent with Sulfolobus acidocaldarius.

A series of extremely thermophilic acidophilic bacteria has been characterized as closely resembling the species Sulfolobus acidocaldarius except for a totally different guanosine-cytosine content in the DNA; some conceptual consequences of this situation are discussed. Both organisms also share special features, including a very characteristic type of ether lipid, with other extreme acidophilic thermophiles.


Ammonia assimilation by rhizobium cultures and bacteroids.

The enzymes involved in the assimilation of ammonia by free-living cultures of Rhizobium spp. are glutamine synthetase (EC. 6.o.I.2), glutamate synthase (L-glutamine:2-oxoglutarate amino transferase) and glutamate dehydrogenase (ED I.4.I.4). Under conditions of ammonia or nitrate limitation in a chemostat the assimilation of ammonia by cultures of R. leguminosarum, R. trifolii and R. japonicum proceeded via glutamine synthetase and glutamate synthase. Under glucose limitation and with an excess of inorganic nitrogen, ammonia was assimilated via glutamate dehydrogenase, neither glutamine synthetase nor glutamate synthase activities being detected in extracts. The coenzyme specificity of glutamate synthase varied according to species, being linked to NADP for the fast-growing R. leguminosarum, R. melitoti, R. phaseoli and R. trifolii but to NAD for the slow-growing R. japonicum and R. lupini. Glutamine synthetase, glutamate synthase and glutamate dehydrogenase activities were assayed in sonicated bacteroid preparations and in the nodule supernatants of Glycine max, Vicia faba, Pisum sativum, Lupinus luteus, Medicago sativa, Phaseolus coccineus and P. vulgaris nodules. All bacteroid preparations, except those from M. sativa and P. coccineus, contained glutamate synthase but substantial activities were found only in Glycine max and Lupinus luteus. The glutamine synthetase activities of bacteroids were low, although high activities were found in all the nodule supernatants. Glutamate dehydrogenase activity was present in all bacteroid samples examined. There was no evidence for the operation of the glutamine synthetase/glutamate synthase system in ammonia assimilation in root nodules, suggesting that ammonia produced by nitrogen fixation in the bacteroid is assimilated by enzymes of the plant system.