Differences in hepatic and metabolic changes after acute and chronic alcohol consumption.

Hepatic metabolism of ethanol to acetaldehyde by the alcohol dehydrogenase pathway is associated with the generation of reducing equivalents as NADH. Conversely, reducing equivalents are consumed when ethanol oxidation is catalyzed by the NADPH dependent microsomal ethanol oxidizing system. Since the major fraction of ethanol metabolism proceeds via alcohol dehydrogenase and since the oxidation of acetaldehyde also generates NADH, an excess of reducing equivalents is produced. This explains a variety of effects following acute ethanol administration, including hyperlactacidemia, hyperuricemia, enhanced lipogenesis and depressed lipid oxidation. To the extent that ethanol is oxidized by the alternate microsomal ethanol oxidizing system pathway, it slows the metabolism of other microsomal substrates. Following chronic ethanol consumption, adaptive microsomal changes prevail, which include enhanced ethanol and drug metabolism, and increased lipoprotein production. Severe hepatic lesions (alcoholic hepatitis and cirrhosis) develop after prolonged ethanol consumption in baboons. These injurious alterations are not prevented by nutritionally adequate diets and can therefore be ascribed to ethanol rather than to dietary inadequacy.


Significant pathways of hepatic ethanol metabolism.

Rat liver microsomes oxidized ethanol two to three times faster than propanol when incubated with either an NADPH- or an H2O2-generating system. In addition, solubilized, purified microsomal subfractions were found to contain protein with an electrophoretic mobility identical to rat liver catalase on SDS polyacrylamide gels, suggesting that the separation of catalase from cytochrome P-450 and other microsomal components may not be feasible. These data support the postulate that catalase is responsible for NADPH-dependent microsomal ethanol oxidation. Direct read-out techniques for pyridine nucleotides, the catalase-H2O2 complex, and cytochrome P-450 were utilized to evaluate the specificity of inhibitors of alcohol dehydrogenase (4-methylpyrazole; 4 mM) and catalase (aminotriazole; 1.0 g/kg) qualitatively in perfused rat livers. 4-Methylpyrazole and aminotriazole are specific inhibitors for alcohol dehydrogenase and catalase, respectively, under these conditions. Neither inhibitor nor a combination of them altered the mixed function oxygen of p-nitroanisole to p-nitrophenol as observed by oxygen uptake and product formation. When ethanol utilization was measured over the concentration range 20-80 mM in perfused liver, a concentration dependence was observed. At low concentrations of ethanol, ethanol oxidation was almost totally abolished by 4-methylpyrazole; however, the contribution of 4-methylpyrazole-insensitive ethanol uptake increased as a function of ethanol concentration. At 80 mM ethanol, ethanol utilization was nearly 50% methylpyrazole-insensitive. This portion of ethanol oxidation, however, was abolished by aminotriazole. The data indicate that alcohol dehydrogenase and catalase-H2O2 are responsible for hepatic ethanol oxidation. At low ethanol concentrations (less than 20 mM), alcohol dehydrogenase is predominant; however, at higher ethanol concentrations (up to 80 mM), the contribution of catalase-H2O2 to overall ethanol utilization is significant. No evidence that the endoplasmic reticulum is involved in ethanol metabolism in the perfused liver emerged from these studies.


Action of metiamide on the lower esophageal sphincter.

The action of metiamide, an H2 antagonist, was tested for its effect upon lower esophageal sphincter (LES) function in the anesthetized opossum. Intravenous infusions of metiamide gave a dose-related increase in LES pressure. A maximum increase in pressure of 17 mm Hg was attained at 2.0 mg per kg-hr of metiamide. This dose of metiamide abolished acid secretion in response to gastrin I, but augmented the maximal LES response to this hormone. Studies in vitro showed that metiamide augmented the maximal LES muscle responses to gastrin I or histamine but had no effect on other agonist responses. Metiamide alone did not alter LES muscle tension. An inhibitory response to gastrin I or histamine could be demonstrated in the presence of atropine or diphenhydramine, respectively, on muscle which had first been depolarized with KCl. These inhibitory responses were antagonized by metiamide. These studies indicate that metiamide: (a) increases LES pressure; (b) selectively augments LES muscle excitatory responses to gastrin I or histamine; and (c) selectively antagonizes inhibitory muscle responses to histamine or gastrin I.


Fate of ingested hepatitis B antigen in blood-sucking insects.

The fate of ingested hepatitis B antigen (HBsAg) in two mosquito species and two Hemiptera species was compared with the rate of blood meal digestion by these insects. In both mosquito species HBsAg was detected by radioimmunoassay for only a few hours after ingestion, disappearing well before the time of blood meal digestion. Production of a protease by the mosquito midgut may have been responsible for destruction of the antigen. In contrast, in the bedbug HBsAg remained detectable throughout a 5-week testing period. Moreover, titers rose during the last week, when blood meal digestion was complete, suggesting possible replication of the antigen. At no time was antigen detected in eggs or feces of any species tested, but juvenile bedbugs fed HBsAg when in the fourth or fifth instar stage still contained antigen after molting. These studies suggest that bedbugs may potentially be a more dangerous source of hepatitis B transmission than mosquitoes.


Production of alkaloids and differentiation in a submerged culture of Claviceps purpurea (Fr.) Tul.

The submerged culture of the Claviceps purpurea strain studied was polymorphous. In the process of alkaloid synthesis, cytodifferentiation preceded biochemical differentiation. The onset of the alkaloid phase was characterized by: predominance of chlamydospores in the culture, the presence of vegetative cells with reduced or arrested proliferation, maximum acetylCoA carboxylase activity, the maximum amount of total fatty acids, an over-average cell pool tryptophan level and minimum tryptophan synthetase activity. Intracellular ricinoleic acid was an indicator of differentiation of the culture towards alkaloid formation and also of alkaloid synthesis. The cytodifferentiation period in the initial phases of fermentation, when the cell has several alternative possibilities of development, is regarded as the most sensitive sector of the regulatory mechanisms of alkaloid formation.


Effect of boseimycin on some enzyme systems of Bacillus subtilis.

The effect of boseimycin on the in vitro activity and in vivo synthesis of alkaline phosphatase, aconitase and lactate, isocitrate, glutamate and alanine dehydrogenases was studied in Bacillus subtilis. At a subinhibitory concentration, synthesis of glutamate dehydrogenase was stimulated but alkaline phosphatase, lactate dehydrogenase and aconitase synthesis was inhibited. On the contrary, boseimycin inhibited slightly the activity of lactate dehydrogenase in cell-free extracts. Glutamate dehydrogenase and aconitase activities were not affected.


Regulation of biosynthesis of secondary metabolites. XVII. Purification and properties of malate dehydrogenase (decarboxylating) in Streptomyces aureofaciens.

The process of isolation and purification of malate dehydrogenase (decarboxylating) (EC 1.1.1.40) from the mycelium of the actinomycete Streptomyces aureofaciens has been worked out. The enzyme was purified 35 fold. The kinetic characters of the purified enzyme are very similar to the figures for malate dehydrogenase (decarboxylating) from other sources. Km for L-malate = 2.1 X 10(-3)M, Km for NADP = 4.6 X 10(-5)M (at pH 7.4). The reaction requires metal divalent ions, Mn2+ being more effective than Mg2+. The enzyme reaches its maximal activity at pH 8.75.


Protease activity in cells of Bacillus megaterium during derepression.

A proteolytic activity hydrolyzing denatured proteins of Bacillus megaterium labelled with 35S or 14C amino acids was detected in cells of the asporogenic strain of Bacillus megaterium. The substrate is hydrolyzed by the enzyme or enzymes at optimum pH around 7, their activity being almost completely inhibited by EDTA and o-phenanthroline. PMSF, the inhibitor of serine proteases, is slightly inhibitory. Gel filtration on a Sephadex column separated the protease activity to two or three fractions. The protease activity in cells with the repressed synthesis of protease corresponds to 5-20 mug of substrate degraded per hour by 1 mg of protein at 37 degrees C. It increases five to ten-fold during the derepression. When the intracellular protease activity increases the extracellular enzyme begins to be excreted into the medium. The intracellular protease activity rapidly decreases after the addition of chloramphenicol or of a mixture of amino acids to the derepressed culture. Half or even more of the protease activity is released from the cells during their conversion to protoplasts by means of lysozyme. This "periplasmic" activity remains mostly in the supernatant also after mesosomes have been centrifuged down from the periplasm. A portion of the activity bound in protoplasts sediments together with membrane fraction after their lysis.


Studies on methanol - oxidizing yeast. III. Enzyme.

Oxidation of methanol, formaldehyde and formic acid was studied in cells and cell-free extract of the yeast Candida boidinii No. 11Bh. Methanol oxidase, an enzyme oxidizing methanol to formaldehyde, was formed inducibly after the addition of methanol to yeast cells. The oxidation of methanol by cell-free extract was dependent on the presence of oxygen and independent of any addition of nicotine-amide nucleotides. Temperature optimum for the oxidation of methanol to formaldehyde was 35 degrees C, pH optimum was 8.5. The Km for methanol was 0.8mM. The cell-free extract exhibited a broad substrate specificity towards primary alcohols (C1--C6). The activity of methanol oxidase was not inhibited by 1mM KCN, EDTA or monoiodoacetic acid. The strongest inhibitory action was exerted by p-chloromercuribenzoate. Both the cells and the cell-free extract contained catalase which participated in the oxidation of methanol to formaldehyde; the enzyme was constitutively formed by the yeast. The pH optimum for the degradation of H2O2 was in the same range as the optimum for methanol oxidation, viz. at 8.5. Catalase was more resistant to high pH than methanol oxidase. The cell-free extract contained also GSH-dependent NAD-formaldehyde dehydrogenase with Km = 0.29mM and NAD-formate dehydrogenase with Km = 55mM.


Transport of acyclic polyols in Saccharomyces cerevisiae.

Acyclic polyols (erythritol, xylitol, ribitol, D-arabinitol, mannitol, sorbitol and galactitol) are not metabolized by Saccharomyces cerevisiae. They are taken up by a fast non-active process, reaching 40-70% distribution referred to total cell water. The uptake is insensitive to temperature, pH (between 4 and 8), 2,4-dinitrophenol and uranyl ions. Its initial rate rises linearly with concentration from 10(-5)M to 1M. The process resembles simple diffusion through large pores or the trapping of the whole solution on the surface. Protoplasts behave like whole cells in this respect. Only erythritol shows a second type of uptake which is inhibitor-insensitive but temperature-dependent.


[Studies on the reaction mechanism of a ribonuclease II from Aspergillus oryzae (author's transl)].

Ribonuclease T2 was isolated from an Aspergillus oryzae extract. In order to define the substrate specificity, the hydrolysis of a series of 2',3'-cyclic nucleotides was measured semiquantitatively. Modifications in all positions of the bases are tolerated, as long as the base stays in the anti conformation or has a chance to return to it; bulky substituents at N-3 of the pyrimidine base lower the rate. So far the conclusion seems justified that the enzyme does not react with the substrates by specific bonds to the base, but rather by hydrophobic binding. The conformation specificity and the pH dependence of the activity support this hypothesis. The pH optima with substrates which may be positively or negatively charged are shifted to pH values at which the substrates are uncharged. This strongly indicates a hydrophobic type of interaction between base and enzyme. From the pH dependence of the kinetic parameters Km and k+2, an enzyme group with a pK of 7 (probably histidine) can be postulated. This group should interact in the protonated form with the phosphate anion. Another B.HB-system (probably two carboxylate groups) seems to be involved in the catalysis step, performing the base catalysis at the 2'-OH group and the proton catalysis at the phosphate oxygen simultaneously.


Studies on a 2,3-diaminopropionate: ammonia-lyase from a Pseudomonad.

2,3-Diaminopropionate:ammonia-lyase, an induced enzyme in a Pseudomonas isolate, has been purified 40-fold and found to be homogeneous by disc gel electrophoresis and by ultracentrifugation. Some of its properties have been studied. The optimum pH and temperature for activity are 8 and 40 degrees C, respectively. The enzyme shows a high degree of substrate specificity, acting only on 2,3-diaminopropionate; the D-isomer is only one-eighth as effective as the L-form. L-Homoserine and DL-cystathionine are not substrates, and 3-cyanolalanine does not inhibit its activity. It is a pyridoxal phosphate enzyme which requires free enzyme sulphhydryls for activity. The Km values for L-2,3-diaminopropionate and pyridoxal phosphate are 1mM and 25 muM, respectively. The molecular weight of the enzyme is about 80 000 as determined by gel filtration. On treatment with 0.5M urea or guanidine by hydrochloride, the enzyme dissociates into inactive subunits with an approximate molecular weight of 45 000. One mole of the active enzyme binds one mole of pyridoxal phosphate. The bacterial enzyme seems to be quite different in many of its properties from the rat liver enzyme which also exhibits the substrate specificity of cystathionine gamma-lyase.


Subcellular distribution and properties of kynurenine pyruvate transaminase in rat kidney.

Kynurenine transaminase activity in rat kidney was found in both the mitochondrial and supernatant fractions. These fractions contained (a) kynurenine pyruvate transaminase, which showed a preference for pyruvate as amino acceptor, and had a pH optimum between 8.0 and 8.5, and (b) kynurenine 2-oxoglutarate transaminase, with a preference for 2-oxoglutarate and a pH optimum between 6.0 and 6.5. The apparent Km value of the former enzyme for L-kynurenine was much lower than that of the latter enzyme.


[Glucosidases specific for the cyanogenic glucoside triglochinin. Purification and characterization of beta-glucosidases from Alocasia macrorrhiza Schott].

beta-Glucosidases have been isolated from Alocasia macrorrhiza plants. The enzymes are highly specific for the hydrolysis of the cyanogenic glucoside triglochinin endogenous to this plant. Upon chromatography of protein extracts on cation exchange resins and Sephadex G-200, separation into various enzymatically active bands was observed. The main fractions possess molecular weights of approximately 310000 and 105 000, as shown by preparative ultracentrifugation in a linear saccharose gradient. The beta-glucosidases are composed of subunits (molecular weight 55 000 to 60 000), as revealed by sodium dodecylsulfate gel electrophoresis. The result of alkaline disc electrophoresis and isoelectric focusing in polyacrylamide gel suggest that the beta-glucosidase fraction with molecular weight 105 000 is a dissociation product of the 310 000 molecular-weight species. The isoelectric points of the various beta-glocusidase bands, obtained by isoelectric focusing, vary between pH 4.5 and 5.0. The beta-glucosidases show a pronounced specificity for triglochinin. The Km for this substrate (3 times 10(-5) M) is 50 to 100-fold lower than for all other substrates hydrolyzed. Of the other cyanogenic glycosides, only those with an aromatic aglycone, (S)-configuration at the asymmetric carbon atom of the aglycone and glucose as sugar moiety were hydrolyzed to a measurable extent. The pH optimum of the enzyme reaction is 5.5, the temperature optimum around 50 degrees C. Cu2 ions and glucono-1,5-lactone inhibit beta-glucosidase activity approximately 50% at a concentration of 5 times 10(-4) M, while Hg2,Ag and p-chloromercuribenzoate show the same percent inhibition at 5 times 10(-7) M. Lipophilic solvents (ethanol, ethylene glycol monomethylether) activate the beta-glucosidase activity, preferentially by influencing the V values of the enzymes.


Kinetic studies on microsomal glucose dehydrogenase in rat liver.

Glucose dehydrogenase from rat liver microsomes was found to react not only with glucose as a substrate but also with glucose 6-phosphate, 2-deoxyglucose 6-phosphate and galactose 6-phosphate. The relative maximum activity of this enzyme was 29% for glucose 6-phosphate, 99% for 2-deoxyglucose 6-phosphate, and 25% for galactose 6-phosphate, compared with 100% for glucose with NADP. The enzyme could utilize either NAD or NADP as a coenzyme. Using polyacrylamide gradient gel electrophoresis, we were able to detect several enzymatically active bands by incubation of the gels in a tetrazolium assay mixture. Each band had different Km values for the substrates (3.0 x 10(-5)M glucose 6-phosphate with NADP to 2.4M glucose with NAD) and for coenzymes (1.3 x 10(-6)M NAD with galactose 6-phosphate to 5.9 x 10(-5)M NAD with glucose). Though glucose 6-phosphate and galactose 6-phosphate reacted with glucose dehydrogenase, they inhibited the reaction of this enzyme only when either glucose or 2-deoxyglucose 6-phosphate was used as a substrate. The Ki values for glucose 6-phosphate with glucose as substrate were 4.0 x 10(-6)M with NAD, and 8.4 x 10(-6)M with NADP; for galactose 6-phosphate they were 6.7 x10(-6)M with NAD and 6.0 x 10(-6)M with NADP. The Ki values for glucose 6-phosphate with 2-deoxyglucose 6-phosphate as substrate were 6.3 x 10(-6)M with NAD and 8.9 x 10(-6)M with NADP; and for galactose 6-phosphate, 8.0 x 10(-6)M with NAD and 3.5 x 10(-6)M with NADP. Both NADH and NADPH inhibited glucose dehydrogenase when the corresponding oxidized coenzymes were used (Ki values: 8.0 x 10(-5)M by NADH and 9.1 x 10(-5)M by NADPH), while only NADPH inhibited cytoplasmic glucose 6-phosphate dehydrogenase (Ki: 2.4 x 10(-5)M). The results indicate that glucose dehydrogenase cannot directly oxidize glucose in vivo, but it might play a similar role to glucose 6-phosphate dehydrogenase. The differences in the kinetics of glucose dehydrogenase and glucose 6-phosphate dehydrogenase show that glucose 6-phosphate and galactose 6-phosphate could be metabolized in quite different ways in the microsomes and cytoplasm of rat liver.


[B-chain shortening of matrix-bound insulin by pepsin, I:Preparation and properties of bovine des-pentapeptide(B26-30) insulin (suthor's transl)].

Insulin was adsorbed to a strongly acidic ion exchanger and incubated with pepsin. The digestion of the matrix-bound insulin was found to be restricted to the cleavage of the peptide bond between phenylalanine-B25 and tyrosine-B26. Factionation of the reaction products was achieved by gel filtrationon Sephadex G-50 at pH 8 where des-pentapeptide(B26-30)-insulin does not aggregate. Another way to purify this compound was ion-exchange chromatography, which was easy due to the loss of one positive charge on the modified insulin. Crystallization could be achieved in a phenol-containing buffer. Des-pentapeptide(B26-30)-insulin was found to be molecularly uniform by electrophoresis at pH 2.2 and 8.6, thin-layer chromatography, performic acid oxidation, end group analysis and amino acid analysis. The CD-spectrum indicated conformational changes compared to insulin. The biological activity was considerably reduced: fat cell assay 20%, blood sugar depression 30%.


Glucose phosphate isomerase deficiency with hereditary hemolytic anemia in a Spanish family: clinical and familial studies.

A new case of glucose phosphate isomerase deficiency associated with cogenital nonspherocytic hemolytic anemia is described in a 12-year-old girl of Spanish origin. The parents exhibited erythrocyte glucose phosphate isomerase activity between 50 and 60% of normal. The enzyme of the propositus had normal Michaelis-Menten constants both for F-6-P and G-6-P, but abnormal pH optimum and decreased heat stability at 48 degrees C. On starch-gel electrophoresis the father's enzyme was normal but the mother's showed a cathodic migrating band in addition to the normal one. The enzyme from the propositus exhibited only one band with cathodal mobility of 116% of the main band found in normal subjects. It is postulated that the propositus is double heterozygous for two abnormal alleles, and the mother contributes a mutant allele with abnormal electrophoretic mobility and thermolability at 48 degrees C whereas the father contributes an allele without enzymatic activity.


Inability of thymus cells from newborn donors to restore transplantation immunity in athymic mice.

Passive transfer of thymus cells from congenic donors to athymic mice reconstitutes the recipient's capacity to reject allogeneic skin grafts, provided the donor is immunologically mature and the number of thymus cells from the adult donor is high enough. Passive transfer of thymus cells from adult allogeneic donors induces a mild to severe graft-versus-host disease and the grafts are retained until death. These results are interpreted on the basis of recent findings on the endocrine conditions of congenitally athymic mice and on the previous data on the hormone dependence of thymus cells to acquire immunocompetence. It is proposed that a normal host environment is a prerequisite for the thymus-derived cells to perform in cell-mediated immune reactions.


Variation in internal polysaccharide synthesis among Streptococcus mutans strains.

Five strains, representative of Streptococcus mutans genetic group III antigenic group d, synthesized and degraded less intracellular polysaccharide (IPS) then 17 strains representative of other S. mutans groups. The strains that synthesize IPS degraded it rapidly. The production of acid  in titratable amounts from endogenous IPS was usually complete within 1 h. IPS synthesis in S. mutans increased abruptly at culture glucose concentrations between 0.2 and 0.5% and was quantitated as both iodine-and glucose oxidase-positive material in cell hyrolysates. IPS degradation was measured by acid production in a pH-stat maintained at 7. The existence within group III d of a strain recently shown to be cariogenic in experimental animals suggest that IPS may not be a prerequisite for virulence in these cariogenic bacteria.


Characterization of a dextranase produced by an oral strain of Actinomyces israelii.

A dextranase-producing, gram-positive, anaerobic, rod-shaped bacterium isolated from human dental plaque was identified as Actinomyces israeli. Although the extracellular dextranase (EC 3.2.1.11) formed by this microbe appeared to be constitutively produced, the bacterium did not utilize the reaction products as a carbon source during growth. A striking feature of the dextranase was the formation of two distinct groups of oligosaccharide end products. The two groups presumably correspond to the limit dextran and the released reaction product which appeared to be cleaved from the end(s) of larger dextran molecules. Low levels of dextranase activity were measured by [3H]NaBH4 reduction and alcohol fixation of the large, tritiated end products on filter paper disks. Of the carbohydrate substrates tested, only alpha-1,6-linked glucans were cleaved. The enzyme did not exhibit any metal ion requirements, and its pH optimum was 6.3. It is suggested that the A. israelii dextranase may function as a regulatory factor during extracellular in vivo glucan synthesis from sucrose by various plaque microbes.


Optimal conditions for the fusion of lymphoid cell lines.

A series of hydrolytic enzymes were compared with lysolecithin, glycerol monooleate, and inactivated Sendai virus for their ability to bring about the fusion of several human and mouse lymphoid cell lines. The agents were tried alone and in various combinations, and a variety of incubation conditions were tested to determine those optimal for fusion. Sendai virus was found to produce the best results with the mouse lymphoid cells; lysolecithin plus glycerol monooleate was slightly superior with the human lymphoid cells. A mixture of hyaluronidase plus collagenase produced low (2 to 6%), but significant, fusion of the human lymphoid cells; both the human and mouse lymphoid cell lines were found to contain relatively high amounts of prolyl hydroxylase, the enzyme which forms collagen from protocollagen. The maximum fusion obtained with the human cells was 16%; with a mouse plasmacytoma line, the maximum was 7.5%; and with a mouse leukemic line derived from L5178Y, the maximum was 60%.


The relative potencies and beta2-selectivities of intravenous rimiterol, salbutamol and isoprenaline in asthmatic patients.

The bronchodilating efficacy and the degree of beta2-selectivity of rimiterol, salbutamol and isoprenaline were determined in seven asthmatic patients. Rimiterol, 0.5 (high dose) and 0.05 mug/kg/min (low dose), salbutamol, 0.3 and 0.03 mug/kg/min, isoprenaline, 0.05 and 0.005 mug/kg/min, and placebo were administered by a single intravenous injection over 6 minutes in a double-blind trial. Airway resistance, heart rate, blood pressure and skeletal muscle tremor were measured before and at various times for 2 hours after each injection. The high doses of rimiterol (37%), salbutamol (37%) and isoprenaline (32%) produced immediate and effective bronchodilatation. The duration of action of rimiterol and isoprenaline was similar and shorter than that of salbutamol. For these ventilatory responses there were heart rate increases of 32, 20 and 40 beats/min for rimiterol, salbutamol and isoprenaline, respectively. The three drugs produced similar increases in pulse pressure and tremor. Dose-responses were obtained for each drug with all parameters measured and significant differences at various times found. Isoprenaline was approximately 8 and 5 times as potent as rimiterol and salbutamol, respectively, in bronchodilator action, when equimolar doses were compared. Similarly, isoprenaline was approximately 16 and 12 times as potent in increasing the heart rate as rimiterol and salbutamol, respectively. For an equal bronchodilator action, isoprenaline increased the heart rate 2 and 2.5 times more than rimiterol and salbutamol, respectively. Rimiterol is an effective, short-acting bronchodilator, with similar beta2-selectivity to salbutamol, when administered intravenously to asthmatic patients. The relative potencies and degrees of beta2-selectivity of these drugs depend partly on their route of administration.


Inhibition of phenytoin metabolism by other drugs used in epilepsy.

Phenytoin metabolism is saturable within its normal therapeutic range and, therefore, small changes in the activity of the enzyme can lead to marked changes in serum phenytoin concentrations. The anticonvulsant drugs sulthiame and pheneturide both inhibit the metabolism of phenytoin. The mechanism of this interaction appears to be different for these two drugs. Nortriptyline produces a small increase in serum phenytoin concentrations, but this is unlikely to be of clinical importance. Case reports suggest that both chlorpromazine and chloramphenicol inhibit phenytoin metabolism to a significant degree.


A comparative study of the activity of beta-adrenoceptor antagonists in man.

1. In healthy subjects the plasma concentration of unchanged drug, heart rate response to excerise and attenuation of isoprenaline tachycardia were maximal at one hour after ingestion of a range of doses of propranolol, oxprenolol, practolol, tolamolol, atenolol and metoprolol. 2. There was a within-drug relationship between plasma concentration of unchanged drug and the attenuation of exercise and iosprenaline tachycardia. There was no similar between-drug relationship. 3. There was a similar exponential relationship between oral dose and reduction of exercise tachycardia for all six drugs. 4. The linear relationship between oral dose and antagonism of infused isoprenaline was significantly greater for oxprenolol and propranolol than for the other four drugs. 5. "Cardioselective" activity was the only ancillary pharmacological property which differentiated the activity of these drugs in normal subjects.


Alteration of hemodynamics, blood gases and lung morphology in bromocarbamide intoxication (animal experiments).

The reaction to animals in bromocarbamide intoxication with respect to blood pressure, resistance in the systemic and pulmonary circulation, cardiac output and arterial blood gas values is presented and discussed in reference to morphological changes in the lung. The animals die due to peripheral  circulatory failure if paralysis of respiration is prevented by means of artificial ventilation. The observed morphological lung changes are not the cause of death.


Folic acid conjugase from Plasma. I. Partial purification and properties.

Human plasma conjugase was partially purified by ammonium sulphate and DEAE-cellulose column fractionation. The pH optimum was found to be 4.5. The enzyme followed normal Michaelis-Menten Kinetics. The Km value was found to be 1.4 x 10(-7)M for folylheptaglutatmate assuming this compound to be the predominant form of folate in yeast extract. The enzyme required sulfhydryl compounds for full activity. Differential microbiological assay of the conjugase reaction product using L. casei and S. faecalis as the test organisms revealed that during the initial stage of the reaction considerable portion of the reaction product was at polyglutamyl stage. On prolonged incubation polyglutamates decreased due to further hydrolysis of the gamma-glutamyl residues. The plasma conjugase activity of different species of animals showed wide variations. Goat, dog and rabbit plasma had neglibible activity while monkey, guinea pig and chick plasma had moderate activity. Bovine plasma had low activity while rat plasma had high activity. The plasma conjugase activity of normal Indian adults ranged from 400 to 900 units per ml, and there was no significant sex difference. The values obtained are considerably higher than those reported in literature.


Folic acid conjugase from plasma. II. Studies on the source of the enzyme in blood.

Following the administration of carbon tetrachloride to rats, the liver conjugase level decreased with 4 hours and continued to be low till 24 hours. Plasma conjugase did not change till 6 hours after administration, but at 24 hours showed a significant increase. Histological examination of the livers revealed liver damage even by 6 hours following carbon tetrachloride administration. However, centrilobular liver cell necrosis could be seen only in the livers of animals killed after 24 hours following carbon tetrachloride administration. The properties of plasma and liver conjugases were similar. The pH optimum of the conjugase in the plasma obtained at 24 hours after carbon tetrachloride administration was 4.5 which is the same as that of the liver conjugase and the Km values were also almost the same. Both the enzymes were stimulated by thiol compounds. The inverse relationship between liver and plasma conjugase activities following carbon tetrachloride administration suggested that liver is probably the source of plasma conjugase.


Gamma-glutamyl transpeptidase as an aid in the diagnosis of liver metastases.

Serum gamma-glutamyl transpeptidase (GGTP) activity was examined in 79 patients. Of these, 49 had diverse malignant tumors, some of which had metastasized to the liver, and 18 had benign diseases of the liver, gallbladder and bile ducts. Enzyme activity increased significantly in all but two patients in whom liver metastases were found. All but one of the patients with malignant tumors, but without liver metastases, showed normal GGTP activity. GGTP activity was elevated in all cases of jaundice. In the absence of jaundice, elevated GGTP activity is suggestive of the presence of liver metastases.


[Physiology of the free-skin transplant].

The publications on the physiology of the free full thickness skin autograft are reported with special reference to the newer ones as a synopsis of literature. Emphasis is placed upon the interesting findings on plastic surgery and especially upon the operative treatment of alopecia.


Antibiograms of pathogenic bacteria isolated from laboratory animals.

Study of antibiotic sensitivity patterns of 178 bacterial isolants from laboratory animals revealed that these bacteria in general were sensitive to many commonly used antibiotics; however, there were notable exceptions. This report presents current antibiotic sensitivity patterns of most gram-negative and gram-positive bacterial pathogens common to laboratory animals.


Studies on lankacidin-group (T-2636) antibiotics. X. Microbial conversion of lankacidin-group antibiotics.

Lankacidin C, a component of lankacidin-group (T-2636) antibiotics, was esterified to lankacidin C 8-butyrate in the presence of methyl butyrate by culture broth and by cell-free extract of Bacillus megaterium IFO 12108. In addition, methyl isobutyrate, methyl valerate and methyl isovalerate served as acyl donors for the esterification, and lankacidin C 8-isobutyrate, lankacidin C 8-valerate and lankacidin C 8-isovalerate were formed respectively. Lankacidin C 8, 14-dibutyrate was hydrolyzed to lankacidin C 14-butyrate by the same organism.


Nonperipheral chemoreceptor stimulation of ventilation by cyanide.

To assess the ventilatory responses elicited by changes of tissue hypoxia, sodium cyanide (0.12 mg/kg-min for 10 min) was infused into the upper abdominal aorta of anesthetized dogs. These infusions produced decreases in oxygen consumption, increases in arterial lactate concentration, and increases in arterial lactate/pyruvate ratio. Coincident with these metabolic changes of hypoxia, minute ventilation (VE) increased 228 +/- SE 36% and arterial PCO2 decreased 21 +/- SE 2 mmHg; therefore, pH increased both in arterial blood in and cisternal cerebrospinal fluid. Following infusion of cyanide into the abdominal aorta, small quantities of cyanide (48 +/- SE 14 mumol/liter) appeared in carotid arterial blood. To evaluate the possibility that the observed increases in VE were due to stimulation of peripheral arterial chemoreceptors by the recirculating cyanide, the carotid and aortic chemoreceptors were denervated in four dogs. Nonetheless, after intra-aortic infusion of sodium cyanide (1.2 mg/kg), ventilation in these chemodenervated animals again increased considerably (154 +/- SE 36%). In order to explore the possibility that cyanide infusion can stimulate ventilation by an extracranial mechanism, heads of vagotomized dogs (including the carotid bodies) were perfused entirely by donor dogs. The intra-aortic infusion of sodium cyanide (0.9 mg/kg) into these head-perfused animals still caused large increases in VE (163 +/- SE 19%). It is concluded that intra-aortic cyanide infusions stimulate VE by an extracranial mechanism other than the carotid and aortic chemoreceptors.


Adaptation of brain monoamine synthesis to hypoxia in the rat.

Oxygen is a substrate in the synthesis of the neurotransmitters, norepinephrine, dopamine, and serotonin. Changes in environmental oxygen appear to cause corresponding alterations in brain monoamine synthesis in vivo. The effect of chronic hypoxia was studied by exposing rats to 10% oxygen for up to 36 h. Brain monoamine synthesis, estimated in vivo, decreased initially and then returned to control levels, despite continued exposure to 10% oxygen. During this apparent adaptation to hypoxia, there were no changes in the concentration of brain serotonin, norepinephrine, dopamine, or tryptophan, while brain tryosine increased after 24 h of exposure. Tyrosine hydroxylase activity in vitro was not altered by the exposure to 10% oxygen. Evidence of hypoxic adaptation in these rats, a rightward shift of their hemoglobin dissociation curves, was found after 24 h of exposure. The adaptation of brain monoamine synthesis to hypoxia appeared to correlate with adaptive changes in brain tissue oxygen rather than any change in the intraneuronal regulation of amine synthesis.


Ventilatory interaction between hypoxia and [H+] at chemoreceptors of man.

By measuring ventilation during isocapnic progressive hypoxia, peripheral chemoreceptor sensitivity to acute hypoxia (deltaV40) was measured in five normal young men under four sets of conditions: 1) at sea level at the subject's resting PCO2, 2) at sea level with PCO2 5 Torr above resting PCO2, 3) after 24 h at a simulated altitude of 4,267 m (PB = 447 Torr) at the subject's resting PCO2 measured during acute hyperoxia, and 4) after 24 h at high altitude, with PCO2 elevated to the subject's sea-level resting PCO2. With this experimental design, we were able to systematically vary the PCO2 and [H+] at the peripheral and central chemoreceptors of man. When mean pHa was decreased from 7.424 to 7.377 without significant change in PACO2, the mean deltaV40 increased from 18.0 to 55.9 1/min. Conversely, when mean PACO2 was altered between 33.8 and 41.6 Torr with pHa held relatively constant, the mean deltaV40 did not change. This suggests that it is the H+ and not CO2 which interacts with hypoxia in stimulating the ventilation of man. An additional finding was that the intrinsic sensitivity of the peripheral chemoreceptors to acute hypoxia did not change during 24 h of acclimatization to high altitude.


Hypothermia and rewarming induced by surface and He-O2 inhalate temperature control.

Hypothermia and rewarming were induced by a combination of temperature-controlled surface and inhalate methods in rabbits. To facilitate respiratory heat exchange, inhalate-respiratory tract temperature and humidity gradients and thermal conductivity were increased. In addition, the upper respiratory tract was bypassed by an endotracheal tube. To aid in maintaining satisfactory circulatory dynamics, hypercapnia and hypoxia were induced. The combined surface and inhalate method produced a markedly more effective rate of cooling than surface temperature-controlled method alone. Animals survived core temperatures as low as 20.9 degrees C with no complications. The noninvasive simplicity of this method suggests its potential applicability in many clinical situations.


Effect of carotid body resection on ventilatory and acid-base control during exercise.

To investigate the role of the carotid bodies in exercise hyperpnea and acid-base control, normal and carotid body-resected subjects (CBR) were studied during constant-load and incremental exercise. There was no significant difference in the first-breath ventilatory responses to exercise between the groups; some subjects in each reproducibly exhibited abrupt responses. The subsequent change in Ve toward steady state was slower in the CBR group. The steady-state ventilatory responses were the same in both groups at work rates below the anaerobic threshold (AT). However, above the AT, the hyperpnea was less marked in the CBR group. Ve and acid-base measurements revealed that the CBR group failed to hyperventilate in response to the metabolic acidosis of either constant-load or incremental exercise. We conclude that the carotid bodies 1) are not responsible for the initial exercise hyperpnea, 2) do affect the time course of Ve to its steady state, and 3) are responsible for the respiratory compensation for the metabolic acidosis of exercise.


Gas-blood PCO2 gradients during avian gas exchange.

The avian respiratory system is a crosscurrent gas exchange system. One of the aspects of this type of gas exchange system is that end-expired PCO2 is greater than arterial PCO2, the highest possible value being equal to mixed venous PCO2. We made steady-state measurements of arterial, mixed venous, and end-expired PCO2 in anesthetized, spontaneously breathing chickens during inhalation of room air or 4-8% CO2. We found end-expired PCO2 to be higher than both arterial and mixed venous PCO2, the sign of the differences being such as to oppose passive diffusion. The observation that end-expired PCO2 was higher than arterial PCO2 can be explained on the basis of crosscurrent gas exchange. However, the observation that end-expired PCO2 exceeded mixed venous PCO2 must be accounted for by some other mechanism. The positive end-expired to mixed venous PCO2 gradients can be explained if it is postulated that the charged membrane mechanism suggested by Gurtner et al. (Respiration Physiol. 7: 173-187, 1969) is present in the avian lung.


Awake baboon's ventilatory response to venous and inhaled CO2 loading.

Steady-state ventilatory responses to CO2 in trained awake baboons were studied to determine the response to a venous CO2 load. CO2 was loaded either directly into the venous blood through an arteriovenous shunt or by addition to the inhaled air. The two modes of loading were adjusted to produce the same increase in minute volume. Minute volume, tidal volume respiratory frequency, end-tidal PCO2, PaCO2, and pHa were measured. PaCO2 and PETCO2 increased the same amount during the two modes of CO2 loading; thus, the response to changes in arterial PCO2, deltaVE/deltaPaCO2, was the same. I conclude that the ventilatory response to venous CO2 loading occurs only through the change in mean arterial PCO2 and thus it is unlikely that there are any important venous CO2 receptors.


Mucociliary tracheal transport rates in man.

A new method for measuring mucociliary tracheal transport rates (MTTR's) is described. An aqueous aerosol containing albumin microspheres labeled with 99mTc was inhaled in such a manner that it was deposited in local concentrations in the large airways. These boli of microspheres were transported up the trachea and their MTTR's measured using a gamma camera. MTTR's were measured in 42 healthy nonsmoking adults (32 men and 10 women, mean age 28 yr). The mean MTTR's appeared to be log normally distributed with a geometric mean of 3.6 mm/min and a coefficient of variation of 75%. The MTTR's of men and women were similar. Each individual's short-term coefficient of variation was 25%. Twenty-two repeat studies 1 wk to 15 mo apart showed the variation within individuals was less than between individuals. The parasympatholytic drug, atropine (0.6 mg iv) decreased MTTR's for at least 3 h. Inhalation of the sympathomimetic drug, Th1165a increased MTTR's. Chronic and acute smoking did not appreciably change the MTTR'S.


Site of initial glycosylation of mannoproteins from Saccharomyces cerevisiae.

The cellular site of initial glycosylation of proteins from Saccharomyces cerevisiae has been studied. Short pulses of [U-14C]mannose label the ribosomal fraction of the yeast. Most of the label was associated with polysomes; monosomes contained only a small amount of radioactivity. All of the radioactivity present in the polysomal fraction was accounted by mannose and smaller amounts of glucose and glucosamine. Puromycin treatment detached more than 50% of the radioactivity from the polysomes; treatment of polysomes at pH 10.0 also caused the release of radioactivity. These results indicate that initial sugar binding occurs while the nascent polypeptide chains are still growing on the ribosomes. When the cells were preincubated with 2-deoxy-D-glucose, incorporation of [U-14C]mannose into the polysomes and the cell wall was inhibited, whereas its incorporation into membrane fractions was unimpaired. It was concluded that 2-deoxy-D-glucose inhibited the synthesis of glycoproteins by interference with the initial glycosylation steps at the ribosomal level.


Regulation of the ammonia assimilatory enzymes in Salmonella typhimurium.

The regulation of glutamate dehydrogenase (EC 1.4.1.4), glutamine synthetase (EC 6.3.1.2), and glutamate synthase (EC 2.6.1.53) was examined for cultures of Salmonella typhimurium grown with various nitrogen and amino acid sources. In contrast to the regulatory pattern observed in Klebsiella aerogenes, the glutamate dehydrogenase levels of S. typhimurium do not decrease when glutamine synthetase is derepressed during growth with limiting ammonia. Thus, it appears that the S. typhimurium glutamine synthetase does not regulate the synthesis of glutamate dehydrogenase as reported for K. aerogenes. The glutamate dehydrogenase activity does increase, however, during growth of a glutamate auxotroph with glutamate as a limiting amino acid source. The regulation of glutamate synthase levels is complex with the enzyme activity decreasing during growth with glutamate as a nitrogen source, and during growth of auxotrophs with either glutamine or glutamate as limiting amino acids.


Maltose metabolism of Pseudomonas fluorescens.

Pseudomonas fluorescens W uses maltose exclusively by hydrolyzing it to glucose via an inducible alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20). No evidence for phosphorolytic cleavage or oxidation to maltobionic acid was found in this organism. The alpha-glucosidase was totally intracellular and was most active at pH of 7.0. Induction occurred when cells were incubated with maltotriose or maltose. Induction was rapid and easily detectable within the first 5 min after the addition of the inducer. Glucose and its derivatives did not repress induction. Cells growing on DL-alanine or succinate plus maltose exhibited lower levels of alpha-glucosidase than those grown on maltose alone or maltose plus glucose. Induction required both messenger ribonucleic acid and protein synthesis.


Inhibition of amino acid transport by ammonium ion in Saccharomyces cerevisiae.

The rate of transport of L-amino acids by Saccharomyces cerevisiae epsilon 1278b increased with time in response to nitrogen starvation. This increase could be prevented by the addition of ammonium sulfate or cycloheximide. A slow time-dependent loss of transport activity was observed when ammonium sulfate (or ammonium sulfate plus cycloheximide) was added to cells after 3 h of nitrogen starvation. This loss of activity was not observed in the presence of cycloheximide alone. In a mutant yeast strain which lacks the nicotinamide adenine dinucleotide phosphate-dependent (anabolic) glutamate dehydrogenase, no significant decrease in amino acid transport was observed when ammonium sulfate was added to nitrogen-starved cells. A double mutant, which lacks the nicotinamide adenine dinucleotide phosphate-dependent enzyme and in addition has a depressed level of the nicotinamide adenine dinucleotide-dependent (catabolic) glutamate dehydrogenase, shows the same sensitivity to ammonium ion as the wild-type strain. These data suggest that the inhibition of amino acid transport by ammonium ion results from the uptake of this metabolite into the cell and its subsequent incorporation into the alpha-amino groups of glutamate and other amino acids.


Soluble macromolecular complexes involving bacterial teichoic acids.

Cell wall and membrane teichoic acids from several bacteria formed soluble complexes with polysaccharides and bovine plasma in alkyl alcohol solutions. Polysaccharides which contain different monomeric units and anomeric configurations complexed with the teichoic acids, suggesting that the interaction is relatively nonspecific. Teichoic acids complexed glycogen or bovine plasma albumin in 50 to 97% ethanol solutions. The macromolecular association between teichoic acids and polysaccharides or proteins was independent of teichoic acid size over a threefold molecular weight range. Glycerol phosphates or an acid hydrolysate of teichoic acid would not complex to either glycogen or bovine plasma albumin in ethanol. The optimal interaction between glycogen and the Bacillus subtilis lipoteichoic acid occurred between pH 4.5 and 8.2. The ability of teichoic acids to bind polysaccharides and proteins in moderate dielectric constant solvents suggests that these polymers may serve as complexing agents for hydrophilic molecules found in membranes.


Aminopeptidases of Bacillus subtilis.

Three enzymes with L- and one enzyme with D-aminopeptidase (EC 3.4.11; alpha-aminoacyl peptide hydrolase) activity have been separated from each other and partially purified from Bacillus subtilis 168 W.T., distinguished with respect to their molecular weights and catalytic properties, and studied in relation to the physiology of this bacterium. One L-aminopeptidase, designated aminopeptidase I, has a molecular weight of 210,000 +/- 20,000, is produced early in growth, and hydrolyzes L-alanyl-beta-naphthylamide most rapidly. Another, designated aminopeptidase II, molecular weight 67,000 +/- 10,000, is also produced early in growth and hydrolyzes L-lysyl-beta-naphthylamide most rapidly. A third, aminopeptidase III, molecular weight 228,000 +/- 20,000, is produced predominantly in early stationary phase and most efficiently utilizes L-alpha-aspartyl-beta-naphthylamide as substrate. The synthesis of aminopeptidase III in early stationary phase suggests that selective catabolism of peptides occurs at this time, perhaps related to the cessation of growth or the onset of early sporulation-associated events. A D-aminopeptidase which hydrolyzes the carboxyl-blocked dipeptide D-alanyl-D-alanyl-beta-naphthylamide (as well as D-alanyl-beta-naphthylamide and D-alanyl-D-alanyl-D-alanine) has also been identified, separated from aminopeptidase II, and purified 170-fold. D-Aminopeptidase, molecular weight 220,000 +/- 20,000, is localized predominantly in the cell wall and periplasm of the organism. This evidence and the variation of the activity during the growth cycle suggest an important function in cell wall or peptide antibiotic metabolism.


Membrane-mediated killing of Saccharomyces cerevisiae by glycoproteins from Torulopsis glabrata.

Cell-free supernatants from cultures of Torulopsis glabrata contained glycoprotein toxins that killed sensitive and killer strains of Saccharomyces cerevisiae with single-hit kinetics. Growing S. cerevisiae treated with the toxins showed a leakage of cellular potassium, partial dissipation of the adenosine triphosphate pool, and a coordinate shutdown of macromolecular synthesis. These pool efflux-stimulating toxins have been partially purified and at least three toxic glycoproteins have been separated. Pool efflux-stimulating toxin activity was stable from pH 3 through 7, though killing was maximal close to pH 4.


Glucose-6-phosphate-dependent pyruvate kinase in Streptococcus mutans.

Pyruvate kinase of Streptococcus mutans JC 2 had an absolute and specific requirement for glucose-6-phosphate. Inorganic phosphate was a strong inhibitor. The enzyme required K+ or NH4+ and Mg2+ or Mn2+. S. mutans FIL and E 49, Streptococcus bovis ATCC 9809, and Streptococcus salivarius ATCC 13419 had also glucose-6-phosphate-dependent pyruvate kinases, whereas Streptococcus sanguis NCTC 10904 had an enzyme activated by fructose-1,6-diphosphate.


Properties of the nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase from Blastocladiella emersonii.

The nicotinamide adenine dinucleotide phosphate (NADP)-specific isocitrate dehydrogenase from Blastocladiella emersonii was purified. The enzyme was very unstable. Satisfactory stability was obtained in the presence of 0.2% ovalbumin. The enzyme had a molecular weight of about 100,000. It did not exhibit homotropic cooperativity for any of it substrates and was not affected by the allosteric modifiers citrate and adenosine monophosphate, diphosphate, and tri-phosphate. The substrate saturation studies showed both intercept and slope effects in Lineweaver-Burk plots. The Km values for isocitrate and NADP were found to be 20 and 10 muM, respectively. The product inhibition pattern was compatible with a random sequential reaction mechanism. The enzyme catalyzed the oxidative decarboxylation of isocitrate about six times better than the reductive carboxylation of alpha-ketoglutarate. The enzyme was inhibited by glyoxylate plus oxalacetate. Assays conducted in the presence of low Mg2+ concentrations exhibited a lag. This lag could be abolished by the addition of reduced NADP to the assay mixture.


Conversion of Escherichia coli cell-produced metabolic energy into electric form.

The formation of membrane potential in energized E. coli cells has been investigated by means of ionic penetrants. The fluxes of anions and cations in opposite directions have been observed: anions moved out and cations moved into the cells. The energy-linked uptake of cations was stoichiometrically coupled with the outflow of H+ ions from the cells. The value of a membrane potential in the energized cells calculated from a distribution of permanent cations was in the range of -140 mV (inside minus). The uptake of penetrating cations by deenergized cells has been observed following the non-enzymatic generation of a membrane potential. The influx of synthetic and natural (lactose) penetrants collapsed the non-enzymatic membrane potential. The effect of lactose was sensitive to N-ethyl maleimide. These results are in favour of the conception that in the energized E. coli cells an energy-linked H+-pump generates a membrane potential which is a driving force for the transport of synthetic and some natural penetrants.


Change of mitochondrial buffering capacity induced by propranolol.

Propranolol is able to increase the amount of the titratable groups of mitochondrial membranes. This effect occurs with sonicated particles and with liposomes, too. The phenomenon is only seen in the presence of salt solutions, not in sucrose. Propranolol increases the fluorescence of anilino-naphthalene sulphonate (ANS) in mitochondrial suspensions. The increase is counteracted by increasing concentrations of potassium chloride. It is suggested that the increase of the titratable groups results from a decrease of the aggregation of the phospholipids of the membranes. At the same time the environment of the bound ANS molecules is more hydrophobic in sucrose than in potassium chloride. The amount of the buffering groups and the hydrophilicity are in direct and the amount of the buffering groups and the fluorescence of ANS in inverse correlation.


Human liver alpha-L-fucosidase. Purification, characterization, and immunochemical studies.

Human liver alpha-L-fucosidase has been purified 6300-fold to apparent homogeneity with 66% yield by a two-step affinity chromatographic procedure utilizing agarose epsilon-aminocaproyl-fucosamine. Isoelectric focusing revealed that all six isoelectric forms of the enzyme were purified. Polyacrylamide gel electrophoresis of the purified alpha-L-fucosidase demonstrated the presence of six bands of protein which all contained fucosidase activity. The purified enzyme preparation was found to contain only trace amounts of seven glycosidases. Quantitative amino acid analysis was performed on the purified fucosidase. Preliminary carbohydrate analysis indicated that only about 1% of the molecule is carbohydrate. Gel filtration on Sepharose 4B indicated an approximate molecular weight for alpha-L-fucosidase of 175,000 +/- 18,000. High speed sedimentation equilibrium yielded a molecular weight of 230,000 +/- 10,000. Sodium dodecyl sulfate polyacrylamide gels indicated the presence of a single subunit of molecular weight, 50,100 +/- 2,500. The enzyme had a pH optimum of 4.6 with a suggested second optimum of 6.5. Apparent Michaelis constants and maximal velocities were determined on the purified enzyme with respect to the 4-methylumbelliferyl and the p-nitrophenyl substrates and were found to be 0.22 mM and 14.1 mumol/mg of protein/min and 0.43 mM and 19.6 mumol/mg of protein/min, respectively. Several salts had little or no effect on fucosidase activity although Ag+ and Hg2+ completely inactivated the enzyme. Antibodies made against the purified fucosidase were dound to be monospecific against crude human liver supernatant fluids and the pure antigen. No cross-reacting material was detected in the crude liver supernatant fluid from a patient who died with fucosidosis.


Mechanism of the reductive activation of succinate dehydrogenase.

When succinate dehydrogenase contains oxalacetate in firmly bound form, activity cannof the enzyme results in dissociation of oxalacetate and activation of the enzyme. The course of reductive titrations appears the same whether or not the enzyme contains oxalacetate, and complete reduction as monitored by bleaching of chromophoric groups requires the incorporation of 6 to 7 reducing equivalents in either case. The stoichiometry is that expected from the non-heme iron and flavin content of the enzyme. Activation of the enzyme during reductive titrations occurs predominantly with the incorporation of the second pair of electrons, while determination of activation levels at various poised potentials shows that the group involved is reduced with the uptake of 2 H+ and 2 e-. These characteristics are consistent with titration of the flavin moiety rather than non-heme iron groups. Thus it appears that activation is concurrent with the reduction of flavin to the hydroquinone form. From the measured half-reduction potential for activation, that of the flavin in an oxalacetate-free enzyme has been estimated at -90 to -60 mv at pH 7.


Mutants of Escherichia coli defective in membrane phospholipid synthesis. Properties of wild type and Km defective sn-glycerol-3-phosphate acyltransferase activities.

The sn-glycerol-3-phosphate (glycerol-P) acyltransferase, the first enzyme of membrane phospholipid synthesis in Escherichia coli, was investigated in a wild type and a mutant strain defective in this activity. The mutant strain, selected as a glycerol-P auxotroph, was previously shown to contain a glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than that of its parent or revertants. The membranous mutant glycerol-P acyltransferase but did not appear to be thermolabile in vivo. Revertants no longer requiring glycerol-P for growth, showed glycerol-P acyltransferase activity with thermolability properties similar to the wild type. The second phospholipid biosynthetic enzyme, 1-acylglycerol-P acyltransferase, was not thermolabile in membranes containing a thermolabile glycerol-P acyltransferase activity. The pH optimum for the mutant acyltransferase was over 1 pH unit higher than that of the parental activity. Further, the mutant and wild type glycerol-P acyltransferase differed in their response to magnesium chloride and potassium chloride. The palmitoyl-CoA dependence of the wild type and mutant glycerol-P acyltransferase activities were different. The mutant glycerol-P acyltransferase activity was inhibited greater than 90% by Triton X-100 under conditions where the wild type activity was not affected. These experiments provide novel information about the wild type glycerol-P acyltransferase activity of E. coli and provide six additional lines of evidence for the mutant character of the glycerol-P acyltransferase in the mutant strains.


Mutants of Escherichia coli defective in membrane phospholipid synthesis. Phenotypic suppression of sn-glycerol-3-phosphate acyltransferase Km mutants by loss of feedback inhibition of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Revertants of Escherichia coli mutants defective in the first enzyme of membrane phospholipid synthesis, sn-glycerol-3-phosphate (glycerol-P) acyltransferase, were investigated. These glycerol-P acyltransferase mutants, selected as glycerol-P auxotrophs, contained membranous glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than the parental activity. The glycerol-P acyltransferase activity was also more thermolabile in vitro than the parental activity. Most revertants no longer requiring glycerol-P for growth regained glycerol-P acyltransferase activity of normal thermolability and apparent Km for glycerol-P. However, two novel revertants were isolated which retained an abnormal glycerol-P acyltransferase activity. The glycerol-P dehydrogenase activities of these novel revertants were about 20-fold less sensitive to feedback inhibition by glycerol-P. The feedback-resistant glycerol-P dehydrogenase co-transduced with gpsA, the structural gene for the glycerol-P dehydrogenase. Further transduction experiments demonstrated that the feedback resistant glycerol-P dehydrogenase phenotypically suppressed the glycerol-P acyltransferase Km lesion. The existence of the class of glycerol-P auxotrophs which owe their phenotype to the glycerol-P acyltransferase Km lesion therefore depends on the feedback regulation of glycerol-P synthesis in E. coli.


Stoichiometry of 4-methyl sterol oxidase of rat liver microsomes.

The stoichiometry of 4-methyl sterol oxidase has been investigated by concurrent assays of rates of oxygen consumption, oxidation of reduced pyridine nucleotide, and formation of steroid 4alpha-oic acid, which is the oxidized product of attack of 4-methyl sterol precursors of cholesterol. The basal, steroid-independent rates of oxidation of alpha-NADH and alpha-NADH-dependent oxygen consumption by rat liver microsomes are about 10 to 15% of the rates observed with beta-NADH. Thus, alpha-NADH is substituted for beta-NADH; alpha-NADH oxidation is observed spectrophotometrically. The slow rate of oxygen consumption is measured accurately with a galvanic oxygen electrode that is attached to an offset amplifier. For maximal velocity, 4alpha-hydroxymethyl-5alpha-cholest-7-en-3beta-ol is the steroid substrate, and oxidase activity is induced 2-fold with a dietary bile acid sequestrant. Under these conditions, accurate measurements are obtained for substrate-dependent increments, which are equal to or greater than basal, substrate-independent rates. For each equivalent of hydroxymethyl group oxidized to carboxylic acid, 2 eq each of oxygen and alpha-NADH are consumed. Thus, the stoichiometry is consistent with that expected for two sequential attacks of the 4alpha-hydroxymethyl group by an external mixed function oxidase. In addition to establishing the stoichiometry of the 4-methyl sterol oxidase, the results further demonstrate that the steroidal 4alpha-carboxylic acid is formed from the hydroxymethyl intermediate by catalysis of a mixed function oxidase rather than dehydrogenases.


Diminished 5alpha-reductase activity in extracts of fibroblasts cultured from patients with familial incomplete male pseudohermaphroditism, type 2.

The activity of 5alpha-reductase, the enzyme that converts testosterone to dihydrotestosterone, has been assessed in cell-free extracts of fibroblasts grown from foreskin, labia majora, scrotum, and nongenital skin from control subjects, from patients with developmental defects of the urogenital system, drom two subjects with the type 2 form of familial incomplete male pseudohermaphroditism and from individuals with other forms of hereditary male pseudohermaphoditism. Enzyme activity was shown to be maximal in the pH range of 5 to 6. Substrate specificity studies indicated that the enzyme so assayed is the 5alpha-reductase previously characterized in human foreskin. The activity of the enzyme was low in normal fibroblasts grown from nongenital skin and high in most fibroblasts grown from genital skin. 5alpha-Reductase activity in extracts of foreskin fibroblasts from two subjects with the type 2 disorder was undetectable at pH 5.5. Activity in comparable fibroblast extracts from most patients with other forms of hereditary male pseudohermaphroditism was easily measurable.


Structural states and transitions of carp hemoglobin.

The wide ligand affinity range previously observed for carp hemoglobin is bounded at both extremes by regions of constant affinity. Within these regions, pH, organic phosphates, and the extent of ligand binding have no effect on the measured affinity and the cooperativity of ligand binding is greatly reduced or absent. The rates of CO recombination to fully and partially unliganded carp hemoglobin, under various organic phosphate and pH conditions, are shown to reflect this behavior. Constant kinetic rates are seen to directly correspond to the regions of constant affinity. Therefore, these are taken to be single protein conformations, one of high and one of low ligand affinity. In the simplest view, these conformations represent the R and T states of a two-state model, and most of the properties of carp hemoglobin are explained quite well within this framework. Increases in either hydrogen or phosphate ion concentrations favor the stabilization of the low affinity structure of even fully liganded carp hemoglobin. We have studied the structural transition from high to low affinity by monitoring the absorption spectra of carp hemoglobins at constant pH as a function of organic phosphate concentration. We find that different spectra are induced in both carp methemoglobin and cyanomethemoglobin by inositol hexaphosphate addition. Furthermore, the dependence of the magnitude of the spectral changes on pH and organic phosphate concentration is the close agreement with that predicted from studies of the ligand binding properties of the molecule.


The enediolate analogue 5-phosphoarabinonate as a mechanistic probe for phosphoglucose isomerase.

A stable analogue has been prepared of the enediolate anion believed to occur transiently in the reaction of phosphoglucose isomerase. This compound, 5-phosphoarabinonate, is the strongest known competitive inhibitor of the enzyme (Ki = 3 times 10(-7) M below pH 7). A distinctive pH dependence of binding, also found for two other aldonic acid omega-phosphates, 6-phosphogluconate and 4-phosphoerythronate, involves pertubation of a pKa from 7.0 in the free enzyme to 9.0 in the enzyme-inhibitor complex. This perturbation may reflect a catalytically advantageous increase in basicity which occurs around the transition state of the normal enzymatic reaction.


Mechanistic implications of the pH independence of inhibition of phosphoglucose isomerase by neutral sugar phosphates.

In contrast to the strongly pH-dependent inhibition of phosphoglucose isomerase by substrate analogues with a free carboxyl group, inhibition of this enzyme by neutral sugar phosphates is essentially invariant between pH 7 and 9. Competitive inhibition constants for glucitol 6-phosphate (40 muM), arabinose 5-phosphate (50 muM), and erythritol 4-phosphate (100 muM) were found to be of the same order of magnitude as that reported previously for substrate binding constants (50 to 240 muM). The unique exception is erythrose 4-phosphate whose Ki (0.7 muM, independent of pH) reflects a tightness of binding similar to that found at pH values near or below neutrality for the transition state analogue 5-phosphorarabinonate. The pH independence of inhibition by erythrose 4-phosphate and other neutral sugar phosphates may reflect a mode and locus of binding to phosphoglucose isomerase different from that of the aldonate inhibitors.


Some properties of mammalian cardiac cytochrome c1.

Investigations into the nature of the axial heme ligands, the strength of the heme crevice, the reactivity with cyanide, and the ascorbate reducibility of cytochrome c1 were performed to explore structure-function relationships of cytochrome c1. The existence of an absorbance band at 690 nm, which was quenched by raising the pH with a pK of 9.2 corresponding to a low spin-low transition, suggested that a methionine residue probably functioned as one of the axial heme iron ligands in this cytochrome. Spectral titrations of cytochrome c1 in the low pH range showed a markedly elevated pK for the low spin-high spin transition relative to cytochrome c. Denaturation studies with urea, the absence of any reaction with cyanide, and the evidence from other lines would appear to indicate that the heme group of cytochrome c1 was reduced by ascorbate at approximately 5% of the rate of reduction of cytochrome c but this rate dramatically increased with increasing pH concomitant with the disappearance of the 690 nm absorbance band. Circular dichroic spectra substantiated that elevated pH produced conformational changes localized to the heme crevice and probably also the regions containing aromatic residues. The enhanced rate of ascorbate reduction was perhaps a consequence of the increased accessibility of the heme iron to ascorbate. Major unfolding of the protein in 8 M urea, however, completely abolished the ascorbate reducibility of cytochrome c1. The buried nature of the heme group of cytochrome c1 would probably preclude transfer of an electron from cytochrome c1 to cytochrome c through a direct Fe-Fe or a heme-heme interaction. This poses an important question concerning the mechanism of this electron transfer between these two cytochromes not only in mitochondria but also in solution.


A novel oligoribonuclease of Escherichia coli. I. Isolation and properties.

A new ribonuclease has been isolated from Escherichia coli. The enzyme is present in the 100,000 times g supernatant fraction and has been purified over 200-fold. Studies of the enzyme reveal that: 1. The enzyme shows a marked preference for oligoribonucleotides; indeed, the reaction rate is inversely proportional to the chain length of the substrate. The enzyme does not attack polynucleotides even at high concentrations of enzyme and has no detectable DNase activity. 2. The enzyme is stimulated strongly by Mn2+, less strongly by Mg2+, and not at all by Ca2+ and monovalent cations. 3. The enzyme is purified free of RNase I, RNase II, RNase III, polynucleotide phosphorylase, and other known ribonucleases of E. coli. The enzyme displays identical properties when isolated from mutants of E. coli that are deficient in the above ribonucleases. 4. The enzyme has a marked thermostability, a point of further distinction from RNase II.


Use of N-benzoyl-L-tyrosine thiobenzyl ester as a protease substrate. Hydrolysis by alpha-chymotrypsin and subtilisin BPN.

In the course of searching for specific chromogenic substrates which might be useful in screening for protease-deficient mutants of Bacillus subtilis, we have developed a method for the synthesis of N-benzoyl-L-tyrosine thiobenzyl ester (BzTyrSBzl) in good yield. Spontaneous base hydrolysis of this thiol ester is low, but several serine proteases hydrolyze it readily. Spectrophotometric measurement of the hydrolysis of the ester in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) provides a continuous assay for chymotrypsin as sensitive as any assay reported in the literature. Serine proteases which hydrolyze this substrate may be detected in polyacrylamide disc gels by incubation in the presence of nitro blue tetrazolium. Apparent Km values of 0.02 and 7 mM and kcat values of 37 S-1 and 126 S-1 were observed for the hydrolysis of BzTyrSBzl by alpha-chymotrypsin and subtilisin BPN', respectively. Additionally, 5 mM indole was observed to behave as a strict competitive inhibitor of the alpha-chymotrypsin-catalyzed hydrolysis of BzTyrSBzl but was observed to increase the maximal rate of hydrolysis of p-nitrophenyl acetate by alpha-chymotrypsin by 30%, as previously described. These data, the published data of other workers, and results from studies with molecular models of trypsin and subtilisin BPN' are used as the basis for describing more fully a secondary hydrophobic binding pocket on alpha-chymotrypsin. The pocket is immediately adjacent to the active site serine and is tentatively suggested to be composed of 4 aliphatic side chain residues and 2 glycine residues.


Studies with GMP synthetase from Ehrlich ascites cells. Purification, properties, and interactions with nucleotide analogs.

GMP synthetase has been purified 57-fold from Ehrlich ascites cells. The enzyme was found to be stable and to have an approximate molecular weight of 85,000 (determined by gel filtration). Its activity was stimulated by dithiothreitol and inhibited by 2-mercaptoethanol, p-chloromercuribenzoate, and hydroxylamine. Both ammonia and glutamine could serve as amino group donors. While none of the 10 triphosphate purine and pyrimidine nucleotides studied were able to substitute for ATP as the energy donor for the reaction, all of these compounds were able to bind to the ATP site. The Ki values for CTP, beta-D-arabinofuranosyl-ATP, and 1-N6-ethenoATP were slightly lower than the Km of ATP (0.28 mM). Six monophosphate nucleotides were aminated by this enzyme. Listed in order of their substrate efficiency (Vmax/Km), they are: xanthosine 5'-phosphate(XMP) (28,000); 2'-dXMP (1,200); 8-azaXMP (320); 6-thioXMP (200); beta-D-arabinofuranosyl-XMP (72); 1-ribosyloxipurinol 5'-phosphate (0.5). 6-ThioXMP was a strong alternative substrate inhibitor with a Ki of 5 muM. The aminated products of the reaction (four studied) were competitive inhibitors with respect to XMP.


Hepatic microsomal alcohol-oxidizing system. Affinity for methanol, ethanol, propanol, and butanol.

Oxidation of methanol, ethanol, propanol, and butanol by the microsomal fraction of rat liver homogenate is described. This microsomal alcohol-oxidizing system is dependent on NADPH and molecular oxygen and is partially inhibited by CO, features which are common for microsomal drug-metabolizing enzymes. The activity of the microsomal alcohol-oxidizing system could be dissociated from the alcohol peroxidation via catalase-H2O2 by differences in substrate specificity, since higher aliphatic alcohols react only with the microsomal system, but not with catalase-H2O2. Following solubilization of microsomes by ultrasonication and treatment with deoxycholate, the activity of the microsomal alcohol-oxidizing system was separated from contaminating catalase by DEAE-cellulose column chromatography, ruling out an obligatory involvement of catalase-H2O2 in the activity of the NADPH-dependent microsomal alcohol-oxidizing system. In intact hepatic microsomes, the catalase inhibitor sodium azide slightly decreased the oxidation of methanol and ethanol, but not that of propanol and butanol, indicating a facultative role of contaminating catalase in the microsomal oxidation of lower aliphatic alcohols only. It is suggested that the microsomal alcohol-oxidizing system accounts, at least in part, for that fraction of hepatic alcohol metabolism which is independent of the pathway involving alcohol dehydrogenase activity.


Nuclear magnetic resonance titration curves of histidine ring protons. The effect of temperature on ribonuclease A.

The ionization constants of 3 of the histidine residues of ribonuclease A have beenobtained at 5 temperatures from the nuclear magnetic resonance titration curves of the imidazole C2 proton resonances. Thermodynamic parameters derived from the ionization constants indicate that histidine residues 105 and 119 are fairly well exposed to solvent, while histidine residue 12 is in a somewhat more restricted environment. Measurements of the low pH inflection present in the titration curve of histidine-12 yield a large negative entropy value, indicating that the group givine rise to this inflection is also buried.


Nuclear magnetic resonance titration curves of histidine ring protons. Human metmyoglobin and the effects of azide on human, horse, and sperm whale metmyoglobins.

Four titrating histidine ring C2 and C4 proton resonances are observed in 220 MHz proton NMR spectra of human metmyoglobin as a function of pH. Values of ionization constants determined from the NMR titration data using an equation describing a simple proton association-dissociation equilibrium are curves (1) 6.6, (2) 7.0, (3) 5.8, and (4) 7.4. Four histidine residues have also been found to be solvent-accessible in human metmyoglobin by carboxymethylation studies (Harris, C.M., and Hill, R.L. (1969) J. Biol. Chem. 244, 2195-2203). Two of the titration curves (3 and 4) deviate significantly from the chemical shift values normally observed for histidine C2 proton resonances. Curve 3, with a low pKa, is shifted downfield at high values of pH and also exhibits a second minor inflection with a pKa value of 8.8. On the other hand, the high pKa curve, 4, is shifted upfield at all values of pH. The characteristics of the NMR titration curves with the lowest and highest pKa values (3 and4) are very similar to curves observed previously with sperm whale and horse metmyoglobins (Cohen, J.S., Hagenmaier, H., Pollard, H., and Schechter, A.N. (1972) J. Mol. Biol. 71, 513-519). These results indicate that the histidine residues from which these curves are derived have unusual and characteristic environments in this series of homologous proteins. The NMR spectra of all three metmyoglobins are changed extensively as a result of azide ion binding, indicating conformational changes affecting the environments of several imidazole side chains. The presence of azide ion causes a selective downfield chemical shift for the low pKa curve and a selective upfield chemical shift for the high pKa curve in all three proteins. Azide also abolishes the second inflection seen in the low pKa curve at high pH. In addition to these effects, the presence of azide ion permits the observation of two additional titrating proton resonances for all three metmyoglobins. Increasing the azide to protein ratio at several fixed values of pH yields results which show that a slow exchange process is occurring with each of the metmyoglobins. In the azide titration studies the maximum changes in the NMR spectra occurred at approximately equimolar concentrations. The NMR results for these proteins in the absence and presence of azide ion are related to x-ray crystallographic studies of sperm whale metmyoglobin and the known alkylation properties of the histidine residues. Tentative assignments of the titrating resonances observed are suggested.


Mechanism of action of bovine testicular hyaluronidase. Mapping of the active site.

The reactions of purified, homogeneous bovine testicular hyaluronidase have been studied with radioactively labeled oligomers of hyalobiuronic acid, (GlcUA-GlcNAc)n, as substrates and acceptors. Transglycosylation occurs by transfer of a glycosyl residue with retention of configuration from a leaving group to an acceptor. On the basis of detailed examination of cleavage and transglycosylation patterns for the trimer; comparison of trimer, tetramer, and polymer as substrates; comparison of acceptors; equilibrium binding; and other data, it is proposed that the enzyme's active site consists of five subsites for hyalobiuronate residues. In the terminology of Schechter, I., and Berger, A. ((1966) Biochemistry 5, 3371), these are s2-s1-s' 2-s3, where the reducing terminus is to the right, and cleavage occurs between s1 and s' 1. It is proposed that subsite s'2 has a high affinity for a substrate residue, while s1 and s'1 have low substrate affinity, and s2 and s' 3 are intermediate in affinity. This proposal is seen to have mechanistic implications. The reactions of several substrates show similar bell-shaped pH dependences, with optima in the region of pH 5 to 5.5.


Nicotinamide adenine dinucleotide glycohydrolase from rat liver nuclei. Isolation and characterization of a new enzyme.

A new type of nicotinamide adenine dinucleotide glycohydrolase (NADase) has been isolated from rat liver nuclei. When partially purified chromatin is passed through a Sephadex G-200 column in the presence of 1 M NaCl, enzyme activities catalyzing the liberation of nicotinamide from NAD elute in two peaks. One, which appears in the void volume fraction, hydrolyzes the nicotinamide-ribose linkage of NAD to produce nicotinamide and ADP-ribose in stoichiometric amounts. This activity is not inhibited by 5 mM nicotinamide. The other, which elutes much later, catalyzes the formation of poly(ADP-ribose) from NAD and is completely inhibited by 5 mM nicotinamide. The former, NADase, is DNase-insensitive and thermostable, has a pH optimum of 6.5 to 7, a Km for NAD of 28 muM, and a Ki for nicotinamide of 80 mM, and hydrolyzes NADP as well as NAD. The latter, poly(ADP-ribose) synthetase, is sensitive to DNase treatment and heat labile, has a pH optimum of 8 to 8.5, a Km for NAD of 250 muM and a Ki for nicotinamide of 0.5 mM and is strictly specific for NAD. Further, the former NADase is shown to lack transglycosidase activity, which has been documented to be a general property of NADases derived from animal tissues. These results indicate that the NAD-hydrolyzing enzyme newly isolated from nuclei is a novel type of mammalian NADase which catalyzes the hydrolytic cleavage of the nicotinamide-ribose linkage of NAD.


Kinetic and binding studies of Mn (II) and fructose 1,6-bisphosphate with rabbit liver hexosebisphosphatase.

The separate interaction of the substrate fructose 1,6-bisphosphate and a metal ion cofactor Mn2+ with neutral hexosebisphosphatase has been studied under equilibrium conditions at pH 7.5 with gel filtration and electron paramagnetic resonance measurements, respectively. Binding data for both ligands to the enzyme yielded nonlinear Scatchard plots that analyze in terms of four negatively cooperative binding sites per enzyme tetramer. Graphical estimates of the binding constants were refined by a computer searching procedure and nonlinear least squares analysis. These results are qualitatively similar to those obtained from binding studies involving teh alkaline enzyme, a modified form of hexosebisphosphatase whose pH optimum is in the alkaline pH region. Both forms of the enzyme enhance the proton relaxation rate of water protons by a factor of approximately 7 to 8 at 24 MHz, demonstrating similar metal ion environments. Teh activator Co(III)-EDTA did not affect Mn2+ binding to the neutral enzyme. In the presence of (alpha + beta)methyl-D-fructofuranoside 1,6-bisphosphate, however, two sets--each containing four Mn2+ binding sites--were observed per enzyme tetramer with loss of the negatively cooperative interaction. These results are viewed in terms of four noncatalytic and four catalytic Mn2+ binding sites. Parallel kinetic investigations were conducted on the neutral enzyme to determine specific activity as a function of Mn2+ and fructose 1,6-bisphosphate concentration. A pro-equilibrium sequential pathway model involving Mn2+-enzyme and the Mn2+-fructose 1,6-bisphosphate complex both as substrate and as an allosteric inhibitor satisfactorily fit the kinetic observations. All possible enzyme species were computed from the determined binding constants and grouped according to the number of moles of Mn2+-fructose 1,6-bisphosphate complex bound to the Mn2+-enzyme, and individual rate constants were calculated. The testing of other models and their failure to describe the kinetic observations are discussed.


Acidic protease from human seminal plasma. Purification and some properties of active enzyme and of proenzyme.

A procedure to purify to homogeneity the active form as well as the proenzyme form of the acidic protease of human seminal plasma is described. This involved precipitation with ammonium sulfate, chromatography on diethylaminoethylcellulose, Sephadex G-200, and Sephadex G-100. The molecular weights of the active form and of the proenzyme were determined by electrophoresis and gel filtration to be 35,000 and 42,000, respectively. The proenzyme was more stable than the active form in alkaline solution and can be converted into the active enzyme under acidic conditions. The active form of the acidic protease can hydrolyze hemoglobin, N,N'-dimethylcasein, N-acetyl-L-phenylalanyl-L-diiodotyrosine, and N-benzyloxycarbonyl-L-glutamyl-L-phenylalanine, but cannot hydrolyze bovine serum albumin, ovalbumin, N-benzyloxycarbonyl-L-glutamyl-L-tyrosine. The active form was also inhibited by p-bromophenacyl bromide and 1,2-epoxy-3-(p-nitrophenoxy)propane.


Biosynthesis of bacterial glycogen. Kinetic studies of a glucose-1-phosphate adenylyltransferase (EC 2.7.7.27) from a glycogen-deficient mutant of Escherichia coli B.

An Escherichia coli B mutant, SG14, accumulates glycogen at 28% the rate observed for the parent E. coli B strain. The glycogen accumulated in the mutant is similar to the glycogen isolated from the parent strain with respect to alpha- and beta-amylosis, chain length determination, and I2-complex absorption spectra. The SG14 mutant contains normal glycogen synthase and branching enzyme activity but has an ADP-glucose pyrophosphorylase with altered kinetic and allosteric properties. The mutant enzyme has been partially purified and requires a 12-fold higher concentration of fructose-P2 or a 26 fold higher concentration of pyridoxal-P than the parent type enzyme for 50% of maximal allosteric activation. TPNH, an effective activator of the E. coli B enzyme, does not activate the SG14 ADP-glucose pyrophosphorylase. Other studies show that for the SG14 enzyme the concentrations of ATP and Mg2+ in the synthesis direction and the concentrations of ADP-glucose and PPi in the pyrophosphorolysis direction required to give 50% of maximal activity are 3- to 6-fold higher than those observed for the parent E. coli B ADP-glucose pyrophosphorylase. The Km for alpha-glucose-1-P at saturating to half-saturating concentrations of the activator, fructose-P2, are about the same for both enzymes. However, in the presence of no activator, the concentration of glucose-1-P required for half-maximal activity is about 1.8-fold higher for the SG14 enzyme. Thus SG14 ADP-glucose pyrophosphorylase has lower affinity for its substrates than does the parent enzyme. Previously the SG14 enzyme had been shown to be less sensitive to inhibition by 5'-AMP than the E. coli B enzyme. This ensensitivity to inhibition renders the SG14 enzyme less responsive to energy charge than the E. coli B ADP-glucose pyrophosphorylase. On the basis of the above results and taking into account the reported concentrations of fructose-P2, of pyridoxal-P, and of the adenine nucleotide pool and its energy charge in E. coli strains, it is concluded that furctose-P2 is the important physiological allosteric activator of E. coli ADP-glucose pyrophosphorylase. Furthermore, the 1.7-fold increased rate of accumulation of glycogen observed when E. coli B or SG14 shifts from exponential phase to stationary phase of growth in nitrogen-limiting media can be accounted for by the 2.4-fold increase of the levels of the glycogen biosynthetic enzymes, glycogen synthase, and ADP-glucose pyrophosphorylase. Thus both allosteric regulation of the ADP-glucose pyrophosphorylase as well as the genetic regulation of the biosynthesis of the glycogen biosynthetic enzymes are involved in the regulation of glycogen accumulation in E. coli B.


Metal ion binding in triclinic lysozyme.

The binding sites of Mn2+, Co2+, and Gd3+ have been determined in triclinic lysozyme at pH 4.5 to 4.6. Mn2+ and Co2+ bind a site approximately 2.5 A from 1 of the oxygen atoms of the Glu-35 chain. The occupancy of the Mn2+ site is 0.22, corresponding to 1 bound ion for each 4.6 protein molecules. The occupancy of the Co2+ site is much lower, about 0.048. Gd3+ appears to be bound at two sites, the main one 2.5 A from an oxygen atom of the Glu-35 side chain, the other 3.1 A from an oxygen atom of the Asp-52 chain. The occupancy of both Gd3+ sites is low, 0.036 and 0.016, the latter being so low that the presence of the ion at this site is in doubt. The binding site of Mn2+ in the di(N-acetylglucosamine)-lysozyme complex has also been determined. It does not differ significantly from the Mn2+ binding site in the native protein, but the occupancy is lower, 0.16.


Characterization of an active transport system for calcium in inverted membrane vesicles of Escherichia coli.

The energy-dependent uptake of calcium by inverted membrane vesicles of Escherichia coli was investigated. Methods for preparation and storage of the vesicles were devised to allow for the maximal activity and stability of the calcium transport system. The pH and temperature optima for the reaction were observed to occur at pH 8.0 AND 30 DEGREES, RESPECTIVELY. The eft was found that the extent of the reaction depended on the presence of phosphate or oxalate. Phosphate was found to enter the vesicles at a rate slower than that of calcium. A Ca2+:Pi ratio of approximately 1.5 was found, suggesting formation of Ca3(PO4)2. Monovalent cations stimulated calcium uptake, with the order of effectiveness being K+ is greater than Na+ is greater than Li+ is greater than NH4+. Inhibition was found with certain divalent cations, but these also inhibited the electron transport chain. Of the divalent cations examined only Mg2+ and Sr2+ inhibited calcium transport without a corresponding inhibition of respiration. Calcium transport exhibited biphasic Kinetics, with a low affinity system and a high affinity system. The low affinity system showed a Km of 0.34 mM and a Vmax of 85 nmol/min/mg of protein. The kinetic constants of the high affinity system were 4.5 muM and 2 nmol/min/mg of protein. The energy for calcium transport could be derived from the electron transport chain by oxidation of NADH, D-lactate, and succinate, in order of their effectiveness. Respiration-driven calcium transport was inhibited by inhibitors of the electron transport chain and by uncouplers of oxidative phosphorylation. ATP could also be used to supply enerty for calcium transport. The ATP-driven reaction was inhibited by inhibitors of the Mg2+ATPase and by an antiserum prepared against that protein, demonstrating that that enzyme is involved in the utilization of ATP for active transport in inverted vesicles.


Alkyl isocyanates as active site-directed inactivators of guinea pig liver transglutaminase.

Alkyl isocyanates are effective inactivators of guinea pig liver transglutaminase. Based on the specificity of the reaction the protection against inactivation by glutamine substrate, and the essential nature of calcium for the inactivation reaction, it is concluded that these reagents act as amide substrate analogs and, thus function in an active site-specific manner. Support for the contention that inactivation results from alkyl thiocarbamate ester formation through the single active site sulfhydryl group of the enzyme is (a) the loss of one free--SH group and the incorporation of 1 mol of reagent/mol of enzyme in the reaction, (b) similarity in chemical properties of the inactive enzyme derivative formed to those previously reported for another alkyl thiocarbamoylenzyme and an alkyl thiocarbamoylcysteine derivative, and (c) the finding that labeled peptides from digests of [methyl-14C]thiocarbamoyltransglutaminase and those from digests of iodoacetamide-inactivated enzyme occupy similar positions on peptide maps. Transglutaminase was found to be inactivated neither by urethan anlogs of its active ester substrates nor by urea analogs of its amide substrates. It is concluded on the basis of these findings that inactive carbamoylenzyme derivatives are formed only by direct addition of the transglutaminase active--SH group to the isocyanate C--N double bond, and not, like several serine active site enzymes, by nucleophilic displacement with urethan analogs of substrate, or by nucleophilic displacement with urea analogs of substrate.


Nonequivalent binding sites in cystathionase. Nanosecond and steady fluorescence studies.

The analogs P-pyridoxyl-L-alanine and P-pyridoxyl-L-homoserine bind to the apoprotein of the enzyme cystathionase and inhibit the reactivation of enzymatic activity after addition of pyridoxyl-5-P. The binding of the inhibitors was monitored by measuring the fluorescence emitted by the P-pyridoxyl moiety at 395 nm (excitation 325 nm). The fluorometric titration results indicate the presence of nonequivalent binding sites in the apoprotein. A model based on two classes of independent binding sites fits the fluorometric data reasonably well. The presence of nonequivalent fluorescent sites in reduced cystathionase was also detected by nanosecond spectroscopy. In contrast to the model compound P-pyridoxyl-epsilon-lysine (tau equals 2.6 ns), the P-pyridoxyl residues of cystathionase display multiexponential fluorescence decay. Two fluorescence lifetimes (tau2 equals 4.1 ns and tau2 equals 15 ns) fit the deconvoluted decay results obtained by pulse fluorimetry. It is proposed that the P-pyridoxyl chromophores of reduced cystathionase have different environments.


Protease II from Escherichia coli. Purification and characterization.

We have previously demonstrated the existence of two types of endopeptidase in Escherichia coli. A purification procedure is described for one of these, designated protease II. It has been purified about 13,500-fold with a recovery of 24%. The isolated enzyme appears homogeneous by electrophoresis and gel filtration. Its molecular weight is estimated by three different methods to be about 58,000. Its optimal pH is around 8. Protease II activity is unaffected by chelating agents and sulfhydryl reagents. Amidase and proteolytic activities are stimulated by calcium ion, which decreases the enzyme stability. Like pancreatic trypsin, this endopeptidase catalyses the hydrolysis of alpha-amino-substituted lysine and arginine esters. It appears distinct from the previously isolated protease I, which is a chymotrypsin-like enzyme. The apparent Michaelis constant for hydrolysis of N-benzoyl-L-arginine ethyl ester is 4.7 X 10(-4) M. The esterase activity is inhibited by diisopryopylphosphorofluoridate (Ki(app) equals 2.7 X 10(-3) M) and tosyl lysine chloromethyl ketone (Ki(app) equals 1.8 X 10(-5) M), indicating that serine and histidine residues may be present in the active site. However, protease II is insensitive to phenylmethanesulfonyl fluoride and several natural trypsin inhibitors. Its amidase and esterase activities are competitively inhibited by free arginine and aromatic amidines. The proteolytic activity measured on axocasein is very low. In contrast to trypsin, protease II is without effect on native beta-galactosidase. It easily degrades aspartokinase I and III. Nevertheless both enzymes are resistant to proteolysis in the presence of their respective allosteric effectors. These results provide further evidence that such differences in protease susceptibility can be related to the conformational state of the substrate. The possible implication of structural changes in the mechanism of preferential proteolysis in vivo, is discussed.


Reversible autophosphorylation of a cyclic 3':5'-AMP-dependent protein kinase from bovine cardiac muscle.

Purified cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase of bovine cardiac muscles catalyzes the incorporation of 2 mol of 32P from [gamma-32P]ATP to seryl residues in its cAMP-binding protein. The reaction appears to be catalyzed by the protein kinase itself rather than by a protein kinase kinase and is enhanced by cAMP and by the addition of polyarginine. Phosphorylation of the purified enzyme facilitates its dissociation by cAMP (Erlichman, J., Rosenfeld, R., and Rosen, O.M. (1974) J. Biol. Chem. 249, 5000-5003) but does not affect cAMP binding. At equilibrium, 2 mol of cAMP are bound to both the phospho- and dephospho-enzymes. Phosphorylation of protein kinase is reversible. Upon addition of ADP and Mg2+, phosphate is transferred from the protein to ADP, and ATP is formed. The reverse reaction is optimal at pH 5.5 unlike the forward reaction which has a broad, more alkaline pH activity optimum. It is activated by polyarginine and dependent upon the addition of cAMP to a much greater degree than the forward reaction. The data suggest that the catalytic subunit of protein kinase catalyzes the forward and reverse reactions but do not exclude the possibility that the holoenzyme may also be active. Autophosphorylation by protein kinase and dephosphorylation by phosphrprotein phosphatases of by reverals of the autophosphorylation reaction may regulate the sensitivity of certain protein kinases to activation by cAMP in vivo.


Guanylate cyclase from the rat renal medulla. Physical properties and comparison with adenylate cyclase.

Guanylate cyclase from the rat renal medulla is found in both the soluble and particulate fractions of the cell. Sucrose density gradient centrifugation and gel filtration in H2O and D2O indicate that the enzyme from the soluble cell fraction has the following properties: S20w, 6.3 S; Stokes radius, 54 A; partial specific volume, 0.75 ml/g; mass, 154,000 daltons; f/fo, 1.4; axial ratio (prolate ellipsoid), 7. The addition of 0.1% Lubrol PX to this fraction activates the enzyme and changes thartial specific volume, 0.74 ml/g; mass, 148,000 daltons; f/fo, 1.6; axial ratio (prolate ellipsoid), 11. These findings show that detergent activates the enzyme by changing its conformation and not simply by dispersing nonsedimentable membrane fragments. The dimensions of this guanylate cyclase in detergent are very similar to those of detergent-solubilized adenylate cyclase from the same tissue (Neer, E.J. (1974) J. Biol. Chem. 249, 6527-6531). Guanylate cyclase can be solubilized from the particulate cell fraction with 1% Lubrol PX but has properties quite different from those of the guanylate cyclase in the soluble cell fraction. It is a large aggregate with a value of S20,w of about 10 S, Stokes radius of 65 A, and a mass of approximately 300,000 daltons. However, the peaks of guanylate cyclase activity in column effluents and sucrose density gradients are very broad indicating a mixture of different size proteins. The conditions used to solubilize guanylate cyclase from the particulate fraction also solubilize adenylate cyclase, and the two activities can be separated on the same sucrose gradient. Studies of this sort require a rapid, accurate guanylate cyclase assay. We have developed an assay for guanylate cyclase activity which meets these criteria by adapting the competitive protein binding assay for guanosine cyclic 3':5' monophosphate originally described by Murad et al. (Murad, F., Manganiello, V., and Vaughn, M. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 736-739).