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γ-Glutamyltranspeptidase activity was demonstrated in the membrane fraction of rabbit erythrocytes. The activity observed (with glutathione and various amino-acid acceptors) was similar in magnitude to that of the γ-glutamylcyclotransferase and γ-glutamylcysteine synthetase activities found in the soluble fraction of the cell. No transpeptidase activity was observed with either γ-glutamyl p -nitroanilide or oxidized glutathione in contrast to the rabbit-kidney enzyme for which these compounds and glutathione serve as substrates. Erythrocyte suspensions and hemolysates formed 5-oxoproline (pyroglutamate; pyrrolidone carboxylate); the rate of 5-oxoproline formation from glutathione by hemolysates was increased by addition of methionine. The findings indicate that 5-oxoproline is an end-product of glutathione metabolism in erythrocytes, and that 5-oxoproline passes out of the erythrocyte and is metabolized in other tissues. The observed rate of 5-oxoproline formation is consistent with the conclusion that the γ-glutamyltranspeptidase-cyclotransferase pathway, together with the synthesis of glutathione from glycine, cysteine, and glutamate, account for a large fraction of the observed amino-acid turnover of erythrocyte glutathione.

Interaction of β-chloro-l-[14C]alanine with l-aspartate-β-decarboxylase from Alcaligenes faecalis leads to inactivation associated with covalent binding of close to 1 mole of the 3-carbon chain of the analog per mole of active site. Evidence was obtained for two different types of binding. Thus, dialysis of the labeled enzyme against 0.05 m acetate (pH 6) led to loss of about half of the vitamin B6 cofactor without loss of 14C from the enzyme; before and after dialysis, about half of the 14C was released from the carboxyl group of the analog by treatment with ceric sulfate indicating the presence of a bound pyruvate residue. After dialysis of the labeled enzyme against 1 m acetate buffer (pH 5) containing 0.1 m l-glutamate, little additional vitamin B6 was lost (although similar dialysis of the holoenzyme releases all of the cofactor), but about half of the bound 14C was released; the 14C that remained bound to the dialyzed enzyme was not readily released by treatment with ceric sulfate. No evidence for nonidentical subunits was found. The data suggest that the two observed types of 14C-binding result from the aldimine-ketimine equilibrium; hydrolysis of the ketimine followed by loss of pyridoxamine 5'-phosphate at some sites competes with stabilization of the aldimine form by further interaction at other sites. The 14C-labeled enzyme was treated with cyanogen bromide and a labeled peptide was isolated. The label was released as β-hydroxy[14C]pyruvate from the peptide by treatment with mild alkali, 6 n HCl (105°, 18 hours), or on prolonged enzymatic digestion. Ammonolysis of the labeled peptide led to formation of glutamine and release of the label. The data indicate that the labeled derivative is β-hydroxypyruvate bound by ester linkage to a glutamic acid residue at the active center of the enzyme.

A highly purified and apparently homogeneous preparation of rat kidney γ-glutamyl transpeptidase was examined with respect to its γ-glutamyl donor and acceptor specificities. Many γ-glutamyl amino acids were active as γ-glutamyl donors, the most active being l-γ-glutamyl-l-glutamine and l-γglutamyl-l-methionine. Glutathione disulfide was about 5% as active as glutathione, while substantial activity was found with S-methylglutathione and ophthalmic acid. In the course of this work convenient spectrophotometric methods for the determination of γ-glutamyl transpeptidase activity were developed in which S-substituted derivatives of glutathione were used, e.g. the S-propanone and S-acetophenone derivatives; these procedures are based on the high ultraviolet absorbance of the corresponding S-substituted cysteinylglycine products. Several of the S-substituted glutathiones were more active in transpeptidation than glutathione itself. Kinetic studies were carried out which indicate a ping-pong mechanism consistent with intermediate formation of a γglutamyl enzyme. Of the 32 amino acids tested, l-glutamine and l-methionine were the best acceptors of the γ-glutamyl group. A number of dipeptides were active as acceptors, and several of these were more active than l-glutamine, e.g. glycylglycine, glycyl-l-alanine, l-methionyl-l-serine, lglutaminyl-l-glutamine, and l-α-aminobutyrylglycine. Aminoacylglycine derivatives were (in the five instances studied) more active than the corresponding free NH2-terminal amino acids; the corresponding aminoacyl-l-alanine derivatives were less active than the aminoacylglycines. The high reactivity of certain dipeptides may reflect their affinity for the "cysteinylglycine" site of the enzyme, but the findings are also consistent with a function of γ-glutamyl transpeptidase in peptide transport as well as in amino acid transport. γGlutamyl transpeptidase is inhibited substantially by the γglutamyl hydrazones of a number of α-keto acids. Inhibition is competitive with respect to the γ-glutamyl donor; of seven α-keto acid γ-glutamyl hydrazones, the derivative of α-ketoglutarate inhibited the most.

Papaya (Carica papaya L.) pulp tissue disks in an incubation medium composed of 0.4 m sucrose evolve ethylene at an optimum pH of 5.25 at 30 C. Disks of young preclimacteric fruit evolve the gas linearly with fruit age until fruit age reaches 4 months. Disks from 5-month-old postclimacteric fruit produce approximately 5-fold more ethylene than disks from 4-month-old fruit. Ethylene evolution by unaged papaya disks is inhibited potently by benzyl isothiocyanate. The compound inhibits production of ethylene by approximately 60% at a concentration of 0.046 mm. However, in aged papaya disks benzyl isothiocyanate causes no inhibition of ethylene production indicating that the compound inhibits the induction of the ethylene-producing system rather than the evolution of the gas per se. Even at a 2-fold higher concentration benzyl isothiocyanate has no effect on respiration of unaged papaya disks. It is proposed that benzyl isothiocyanate may act as an endogenous regulator of ethylene evolution in papaya fruit.

Glutamine synthetase, purified from rat liver, is homogeneous on acrylamide gel electrophoresis and on ultracentrifugation (s20,w, 15.0 S). The enzyme, which consists of eight subunits (subunit molecular weight, 44,000), resembles ovine brain glutamine synthetase in its physical properties, amino acid composition, and substrate specificity. Complete inhibition of the liver enzyme by methionine sulfoximine is associated with the binding of 4 moles of inhibitor per mole of enzyme. The enzyme is activated by α-ketoglutarate (in the presence of Mg++ or Mn++) and is inhibited by glycine, l-alanine, l-serine, and carbamyl phosphate (with Mn++ only). Evidence is presented that inhibition by carbamyl phosphate is produced by the binding of this compound to the active site of glutamine synthetase. Thus, the enzyme can catalyze the synthesis of ATP from carbamyl phosphate and ADP. When glutamine synthetase is incubated with ADP, carbamyl phosphate, and glutamate, glutamine is formed. Enzyme inactivated by treatment with methionine sulfoximine and ATP did not utilize carbamyl phosphate. Liver glutamine synthetase also catalyzes ATP synthesis from ADP and acetyl phosphate; this reaction is competitively inhibited by glutamate. Glutamine synthetases from ovine brain, rat brain, and Escherichia coli also catalyze ATP synthesis from carbamyl phosphate and ADP.

Kerwar, Suresh S. (Oregon State University, Corvallis), Vernon, H. Cheldelin, and L. W. Parks . Valine-isoleucine metabolism in Acetobacter suboxydans and the inhibition of growth by valine. J. Bacteriol. 88: 179–186. 1964.—Extracts of Acetobacter suboxydans can synthesize valine and isoleucine via acetolactate and acetohydroxybutyrate, respectively. The amounts of these amino acids synthesized from different intermediates were determined. The pathways appear to be identical to those described for yeast, Neurospora , and Escherichia coli . When exogenous valine was added to a synthetic growth medium inoculated with A. suboxydans , no growth of the culture was observed. The inhibitory effect of valine was reversed by the addition of isoleucine. The site and mechanism of valine inhibition were investigated. Threonine deaminase was inhibited by valine and isoleucine but not by leucine. Repression of the deaminase by isoleucine but not by valine was indicated. The data reported in this paper suggest that valine prevented growth of the organism through false feedback inhibition of threonine deaminase, thereby limiting isoleucine biosynthesis.

Incorporation of 14C-leucine, injected intraperitoneally into rats, reached a plateau in 30 min in the liver mitochondrial fractions. Part of the mitochondrial soluble protein, amounting to about 6% of the total protein, was labeled more rapidly by 14C-l-leucine than the rest of the protein. Thyroidectomy produced a decrease in the rate of labeling of all the fractions. A single dose of l-thyroxine (0.52 µg per g) stimulated the incorporation in the above rapidly labeled fractions of soluble protein distinctly within 60 min without appreciably affecting the other protein fractions. Respiratory rate during active phosphorylation of ADP (active or State 3 respiration) and after ADP was completely converted to ATP (controlled or State 4 respiration) were proportionately depressed in thyroidectomy, but only controlled respiration was increased after thyroxine treatment.

Studies on the aminomalonate decarboxylase of rat liver indicate that this activity is a property of cytoplasmic serine hydroxymethylase. Thus, throughout purification of the enzyme, aminomalonate decarboxylase, serine hydroxymethylase and allothreonine aldolase exhibited the same relative activities. Competition between the several substrates was observed and resolution of the enzyme by treatment with d-alanine led to loss of all three activities. Cleavage of allothreonine by the enzyme in tritiated water gave S-glycine-2-t (in accord with earlier data which indicate retention of configuration during conversion of l-serine to glycine), but decarboxylation of aminomalonate by the enzyme in tritiated water gave both S- and R-glycine-2-t. Studies with specifically carboxyl-labeled [14C]aminomalonate confirmed the conclusion that the enzyme decarboxylates this amino acid in a nonspecific manner; this result is in contrast to that previously observed with aspartate β-decarboxylase which acts on a specific carboxyl group of aminomalonate. In contrast to cytoplasmic serine hydroxymethylase, mitochondrial serine hydroxymethylase does not catalyze the aldol cleavage of allothreonine or the decarboxylation of aminomalonate; this indicates that there is a significant difference between the active sites of these enzymes.

The specificity of the Pseudomonas phaseolicola toxin for enzyme inhibition and its relationship to toxin-induced chlorosis in bean leaves (Phaseolus vulgaris L.) was examined. The toxin showed no significant inhibitory activity against glutamine synthetase, glutamine transferase, carbamyl phosphate synthetase, aspartate carbamoyltransferase, or arginase at concentrations 100-fold higher than that needed to inhibit ornithine carbamoyltransferase by 50%.Protection from and reversal of toxin-induced chlorosis in bean leaves was attempted with several amino acids. Aside from protection with l-citrulline which was previously reported, only l-arginine-HCl and to a minor extent l-leucine and l-glutamine showed protection from chlorosis. l-Citrulline and l-arginine-HCl (but not l-glutamine and l-leucine) also reversed toxin-induced chlorosis.Ultrastructurally, cells from toxin-treated chlorotic tissues showed no observable changes as compared to nontreated tissues. This, together with the ability of the two amino acids to reverse chlorosis, indicated that the toxin causes a reversible biochemical lesion in treated tissue.While tissues from bean plants inoculated with P. phaseolicola showed a large accumulation of ornithine, toxin-treated tissues showed no accumulation of ornithine. The latter finding indicated that in addition to the ornithine carbamoyltransferase inhibitor, the pathogen may produce inhibitors of other ornithine metabolizing enzymes in inoculated tissues.

The decarboxylation of DOPA by erythrocyte hemolysates differs from DOPA decarboxylation catalyzed by aromatic aminoacid decarboxylases that contain vitamin B 6 in several significant respects. The ability of erythrocyte hemolysates to decarboxylate DOPA is associated with interaction between DOPA and methemoglobin; the ferriheme protein is reduced and DOPA is decarboxylated, probably after oxidation to a quinone intermediate. An analogous reaction takes place between DOPA and other ferriheme proteins, such as metmyoglobin and cytochrome c . This phenomenon may be of significance in relation to the side effects observed in patients with Parkinson's disease who are treated with very large doses of DOPA.

Papaya (Carica papaya L.) pulp tissue disks in an incubation medium composed of 0.4 m sucrose evolve ethylene at an optimum pH of 5.25 at 30 C. Disks of young preclimacteric fruit evolve the gas linearly with fruit age until fruit age reaches 4 months. Disks from 5-month-old postclimacteric fruit produce approximately 5-fold more ethylene than disks from 4-month-old fruit. Ethylene evolution by unaged papaya disks is inhibited potently by benzyl isothiocyanate. The compound inhibits production of ethylene by approximately 60% at a concentration of 0.046 mm. However, in aged papaya disks benzyl isothiocyanate causes no inhibition of ethylene production indicating that the compound inhibits the induction of the ethylene-producing system rather than the evolution of the gas per se. Even at a 2-fold higher concentration benzyl isothiocyanate has no effect on respiration of unaged papaya disks. It is proposed that benzyl isothiocyanate may act as an endogenous regulator of ethylene evolution in papaya fruit.

The soluble liver mitochondrial protein fraction obtained from thyroidectomized rats injected with thyroxine manifests a rapid (within 3 hr) thyroxine-dependent increase in amino acid incorporation, and has the capacity to stimulate State 4 respiration in mitochondria isolated from thyroidectomized rats. This effect mimics the response observed in mitochondria isolated from thyroxine-injected animals. Cycloheximide blocks the thyroxine-induced increase in State 4 respiration, and also inhibits amino acid incorporation into the thyroxine-responsive protein fraction, coincident with a decline in its stimulatory effect on State 4 respiration. Injection of thyroxine also produced an increase in the steady state respiration following the respiratory jump associated with low-level Ca†† uptake, i.e., the State 4 (Ca) respiration was increased. The addition of the thyroxine responsive protein fraction to liver mitochondria obtained from thyroidectomized rats increased State 4 (Ca) in a manner similar to that obse...

The specific activities of the crude and the pH 5.0 precipitated tRNA methylase preparations from thyroidectomized rats were about 30‰ and 66‰, respectively, of those from normal animals. The activity of the crude preparation returned to normal level within 3 hr after thyroxine treatment, but the activity did not change in the pH 5.0 precipitate fraction. The pH 5.0 supernatant fraction inhibited the activity of the pH 5.0 precipitated enzyme. There was a 2-fold increase in the concentration of the natural inhibitor in thyroid deficiency. (Endocrinology95:1185, 1974)

γ-Glutamyl transpeptidase catalyzes transfer of the γ-glutamyl moiety of glutathione (and other γ-glutamyl compounds) to amino acid and peptide acceptors; this reaction probably involves ( a ) formation of a γ-glutamyl enzyme and ( b ) reaction of the γ-glutamyl-enzyme with an acceptor. Maleate decreases the latter reaction and markedly increases hydrolysis of the γ-glutamyl donor, apparently by affecting the enzyme so as to facilitate reaction of the γglutamyl enzyme with water. Transpeptidase catalyzes γ-glutamyl hydroxamate formation from many γ-glutamyl compounds and hydroxylamine; this reaction is stimulated 4- to 5-fold by maleate. Glutamine, a poor substrate for transpeptidation as compared to glutathione, is slowly hydrolyzed and converted to γ-glutamyl-glutamine by the transpeptidase; in the presence of maleate, hydrolysis of glutamine is markedly (>10-fold) increased, as is also its conversion to γ-glutamyl hydroxamate in the presence of hydroxylamine. The findings suggest that the previously described “maleate-stimulated phosphate-independent glutaminase” is a catalytic function of γ-glutamyl transpeptidase. Transpeptidase-catalyzed glutaminase activity may play a role in renal ammoniagenesis. The ability of maleate to decrease transpeptidation of γ-glutamyl compounds (and to increase their hydrolysis to glutamate), when considered in the light of earlier findings that treatment of animals with maleate produces aminoaciduria, is consistent with function of transpeptidase and the γ-glutamyl cycle in amino-acid transport.

Human erythrocytes have been found to contain appreciable amounts of DOPA decarboxylase (EC 4.1.1.26) activity. The enzyme activity in erythrocytes from patients with Parkinson's disease who were treated with DOPA was significantly lower than that of untreated patients and of normal individuals. Administration of the drug to mice led to a marked decrease of DOPA decarboxylase in liver and kidney, but not of the brain enzyme. The findings thus indicate that administration of DOPA leads to a decrease in peripheral DOPA decarboxylase, an effect that is expected to be of benefit in DOPA therapy of patients with Parkinson's disease. Peripheral DOPA decarboxylase concentration also decreases in mice after short periods of fasting; the findings suggest that the peripheral enzyme activities may be affected by various nutritional and perhaps hormonal influences, which may be partially responsible for the observed fluctuations in the motor abilities of Parkinsonian patients receiving constant doses of the drug. Study of DOPA decarboxylase activity in erythrocytes may be useful in following changes in patients receiving DOPA therapy and may also be of general interest and value in investigations of catecholamine metabolism in man.

Publisher Summary This chapter presents the assay, purification, and properties of L-aspartate β -decarboxylase from Alcaligenes faecalis. This enzyme, which catalyzes the β -decarboxylation of L-aspartate to L- α -alanine, has been found in a number of microorganisms. Enzyme activity may be determined in a number of ways, such as by manometric assay of CO 2 evolution from aspartate, measurement of ammonia, Cl - , H + , or pyruvate formation from β -chloroalanine, assay of one of the many transamination reactions catalyzed by the enzyme, or by the determination of the sulfite from L-cysteinesulfinate. The chapter discusses the assay based on the determination of the sulfite, which is done according to West and Gaeke. A unit of enzyme activity is the amount that catalyzes the formation of 1 micromole of sulfite per minute at 37°. Specific activity is expressed as units per milligram of enzyme. The isolated enzyme is homogeneous in the analytical ultracentrifuge and on acrylamide-gel electrophoresis. The enzyme also catalyzes the β -decarboxylation of both L-stereoisomers of β -hydroxyaspartate to form L-serine.

The enzymes of the γ-glutamyl cycle have been found in rabbit ciliary body and, except for 5-oxoprolinase, also in the ocular lens. Histochemical studies show that γ-glutamyl transpeptidase is localized mainly in the basal portions of the epithelial cells of the ciliary body; the findings are similar to those observed in the chloroid plexuses. The histochemical staining reaction in the ciliary epithelium is more intense than in the chloroid plexus, intestine, and kidney. γ-Glutamyl transpeptidase staining activity in the epithelium of the intestinal and renal proximal convoluted tubules is confined to the microvillus border. Moderate transpeptidase activity was found in the cytoplasm of nonpigmented epithelial cells of the iris at the posterior pupillary margin. The histochemical and enzyme activity studies are consistent with the thesis that the γ-glutamyl cycle functions in transport of amino acids across the blood-aqueous humor barrier.

Various anatomic regions of rabbit brain have been examined for activities of the enzymes of the γ-glutamyl cycle. While these enzyme activities were widely distributed in the brain, they are present in much higher concentrations in the choroid plexus than in other parts of the brain. The activities observed are of about the same order of magnitude as found in the kidney. These observations and other considerations suggest that the γ-glutamyl cycle may play a significant role in the transport of amino acids between blood and cerebrospinal fluid.

A chlorosis-inducing toxin of Pseudomonas phaseolicola was examined for inhibition of ornithine carbamoyltransferease prepared from acetone powder of bean (Phaseolus vulgaris L.) plants. The enzyme has a pH optimum at 8.5, involves a ternary complex reaction mechanism, and shows Michaelis constants of 5.0 mm and 1.7 mm for ornithine and carbamoylphosphate, respectively. Assuming reversible catalysis, Michaelas constants of 11 mm and 3.3 mm are calculated for citrulline and arsenate. Toxin induces allosteric competitive inhibition in relation to carbamoylphosphate and a noncompetitive mode of inhibition in relation to ornithine, except at high toxin concentrations where uncompetitive inhibition is observed. In the backward assay, competitive inhibition is observed for both arsenate and citrulline. Inhibition is increased with preincubation time and shows saturation kinetics with regard to toxin concentration.

The specificity of the Pseudomonas phaseolicola toxin for enzyme inhibition and its relationship to toxin-induced chlorosis in bean leaves (Phaseolus vulgaris L.) was examined. The toxin showed no significant inhibitory activity against glutamine synthetase, glutamine transferase, carbamyl phosphate synthetase, aspartate carbamoyltransferase, or arginase at concentrations 100-fold higher than that needed to inhibit ornithine carbamoyltransferase by 50%.Protection from and reversal of toxin-induced chlorosis in bean leaves was attempted with several amino acids. Aside from protection with l-citrulline which was previously reported, only l-arginine-HCl and to a minor extent l-leucine and l-glutamine showed protection from chlorosis. l-Citrulline and l-arginine-HCl (but not l-glutamine and l-leucine) also reversed toxin-induced chlorosis.Ultrastructurally, cells from toxin-treated chlorotic tissues showed no observable changes as compared to nontreated tissues. This, together with the ability of the two amino acids to reverse chlorosis, indicated that the toxin causes a reversible biochemical lesion in treated tissue.While tissues from bean plants inoculated with P. phaseolicola showed a large accumulation of ornithine, toxin-treated tissues showed no accumulation of ornithine. The latter finding indicated that in addition to the ornithine carbamoyltransferase inhibitor, the pathogen may produce inhibitors of other ornithine metabolizing enzymes in inoculated tissues.