Search PubMed⌕ Search

Biomedical subjects

A Meister

Publications and source records attributed to A Meister.

At least 199 records · Page 11Linked to original sources
PubMed1982

Inhibition of homocysteine sulfonamide of glutamate synthase purified from Saccharomyces cerevisiae.

Glutamate synthase, isolated in apparently homogeneous form (Mr approximately 265,000) from Saccharomyces cerevisiae after 7500-fold purification, is markedly inhibited by homocysteine sulfonamide. Inhibitions competitive with respect to L-glutamine; the apparent Ki value calculated for L-homocysteine sulfonamide is 3.6 microM; the apparent Km value for L-glutamine is 280 microM. The very high affinity of the inhibitor for the enzyme, as well as structural considerations, suggest that homocysteine sulfonamide is a transition state inhibitor. The previously reported growth inhibitory properties of homocysteine sulfonamide (Reisner, D. B. (1958) J. Am. Chem. soc. 78, 5102-5104) may be due, at least in part, to inhibition of glutamate synthase. L-Methionine sulfone is also a potent competitive inhibitor, whereas L-albizziin, L-methionine-SR-sulfoximine, and L-methionine-SR-sulfoxide are much less effective inhibitors. S. cerevisiae glutamate synthase, which is composed of two dissimilar subunits, uses NADH exclusively, and exhibits low but definite activity when NH3 is substituted for glutamine.

Chemical Phenomena↗
PubMed1982

Effect of sulfhydryl group modification on the activities of 5-oxo-L-prolinase.

5-Oxo-L-prolinase was isolated from rat kidney by a new procedure; highly active and apparently homogeneous enzyme was obtained in 50% yield after 1700-fold purification. The enzyme, which couples cleavage of ATP to ADP with that of 5-oxo-L-proline to L-glutamate, is uncoupled by Ca2+, Co2+, or excess Mn2+ as well as by replacement of adenosine 5'-triphosphate or of 5-oxo-L-proline by certain analogs as previously reported. The enzyme has Mr = 325,000 and is composed of 2 apparently identical subunits. It contains 27 sulfhydryl groups/monomer, 6 of which can be titrated in the native enzyme and 2 of which are required for catalysis. One of the sulfhydryl groups that can be titrated with 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide can be protected against modification by ATP or inosine 5'-triphosphate. The findings suggest that at least 1 sulfhydryl group is at or close to the nucleoside triphosphate binding site and is involved in cleavage of NTP. 5'-p-Fluorosulfonylbenzoyl adenosine and 5'-p-fluorosulfonylbenzoyl inosine interact with the sulfhydryl group involved in NTPase activity and also with another amino acid residue of the enzyme. The data provide additional strong evidence that (a) the enzyme can bind 5-oxo-L-proline in the absence of NTP, and that it can bind NTP in the absence of 5-oxo-L-proline, and (b) the L-glutamate synthesis and NTP-cleaving activities are catalyzed by the same protein.

Adenosine Diphosphate↗
PubMed1982

Cystamine-Sepharose. A probe for the active site of gamma-glutamylcysteine synthetase.

gamma-Glutamylcysteine synthetase, previously known to be potently inhibited by cystamine, has been found to bind covalently to cystamine-Sepharose. ATP facilitates, whereas glutamate plus magnesium ions inhibit, binding of the enzyme to cystamine-Sepharose. A large fraction of the enzyme applied to columns of cystamine-Sepharose binds by forming a disulfide bond between cysteamine-Sepharose and a sulfhydryl group at or near the active site of the enzyme. The enzyme may be released by treatment with dithiothreitol. Some of the enzyme applied to such columns is inactivated and not bound covalently to the column. That the enzyme does not bind to columns of S-(S-methyl)cysteamine-Sepharose, whereas free S-(S-methyl)cysteamine is a potent inhibitor, indicates that a cysteamine-S disulfide moiety derived from the external cysteamine residue of cystamine-Sepharose is the critical group recognized by the enzyme. The observed partitioning of the enzyme on columns of cystamine-Sepharose between covalently column-bound enzyme and nonbound inactivated enzyme suggests that the reactive enzyme sulfhydryl group forms a disulfide linkage with the sulfur atom at the immobilized end of cystamine to link the enzyme to the column and to liberate free cysteamine, and also that the enzyme interacts with the external cysteamine moiety of the bound cystamine. The latter may occur if the free cysteamine released is spontaneously oxidized to free cystamine followed by its inhibition of the enzyme, or if there is a direct reaction between the enzyme-reactive sulfhydryl group and the sulfur atom of the external cysteamine moiety of cystamine-Sepharose.

Animals↗
PubMed1982

Interaction of 5-oxo-L-prolinase with nucleoside triphosphates. Evidence suggesting substrate-dependent conformational change.

5-Oxoprolinase catalyzes the coupled hydrolysis of ATP and 5-oxoproline to yield glutamate, ADP, and Pi; the reaction may be partially or completely uncoupled by structural modification of either substrate. In the present work, we found slow 5-oxoproline-dependent changes in the rates of hydrolysis of ITP, GTP, and UTP. For example, in the absence of 5-oxoproline, the enzyme catalyzes the hydrolysis of UTP at a rapid and constant rate. Following addition of 5-oxo-L-proline, the rate of hydrolysis decreases slowly; after about 25 min, a much slower and constant rate of hydrolysis is attained. This change in rate is associated with a decrease in Vmax and an increase in the Km for UTP. In similar studies with ATP, both Vmax and Km increase over a much shorter time period (less than 10 s). The findings indicate that 5-oxoprolinase is a hysteretic enzyme, and are consistent with the hypothesis that in the normal catalytic reaction, the binding of both ATP and 5-oxo-proline to the enzyme induces a conformational change that brings the substrates into a juxtaposition that facilitates the reaction.

Amidohydrolases↗
PubMed1982

On the chemistry and biochemistry of 3-mercaptopyruvic acid, the alpha-keto acid analog of cysteine.

3-Mercaptopyruvate, generally believed to be formed by enzymatic transamination of L-cysteine and by the oxidative deamination of L-cysteine, has been found to exist in solution, in equilibrium with the cyclic dimer, 2,5-dihydroxy-1,4-dithiane-2,5-dicarboxylic acid; the equilibrium markedly favors the cyclic dimer in aqueous solution at pH 5-7. However, in neutral solutions, and especially in alkaline solution, 3-mercaptopyruvate undergoes very rapid and irreversible conversion to an acyclic aldol dimer. The rate of aldol dimerization is about 100 times greater than that of pyruvate dimerization under comparable conditions. Thus, 10 mM solutions of 3-mercaptopyruvate at pH 7.2 lose about 75% of their lactate dehydrogenase reactivity in 90 min at 25 degrees C. The present findings explain the previous observation that 3-mercaptopyruvate exhibits anomalous behavior in transamination with glutamine. Oxidative deamination of L-cysteine by L-amino acid oxidase yields the highly reactive imine, 2-imino-3-mercaptopropionic acid, which can be quantitatively trapped with semicarbazide. In the absence of semicarbazide, almost quantitative formation of ammonia from L-cysteine occurs with only a 10-20% yield of alpha-keto acid, none of which is 3-mercaptopyruvate; however, 3-mercaptopyruvate may be trapped as 3-mercaptolactate when the oxidase reaction is carried out in the presence of lactate dehydrogenase and NADH. One of the physiological functions of glutamine transaminase may be to convert 3-mercaptopyruvate to L-cysteine, thus preventing its loss through further transformations. In the course of this work it was found that hemithioketals (formed by reaction of cysteine with alpha-keto acids) are substrates of L-amino acid oxidase. Evidence for the cyclic dimer in the solid and vapor states of 3-mercaptopyruvate has been found through infrared and mass spectroscopic observations.

Amino Acid Oxidoreductases↗
PubMed1982

Intracellular cysteine delivery system that protects against toxicity by promoting glutathione synthesis.

Depletion of glutathione by inhibition of its synthesis by buthionine sulfoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, leads to increased sensitivity to (i) irradiation and (ii) oxidative stress. In the present work, an intracellular cysteine delivery system was used to promote glutathione synthesis, and this was found to protect against toxicity. Thus, administration of L-2-oxothiazolidine-4-carboxylate protected against acetaminophen toxicity in mice; the thiazolidine, which is converted to L-cysteine by the enzyme 5-oxo-L-prolinase (present in many animal tissues and in plants) promotes the synthesis of glutathione, which is the actual protectant. The effect of this thiazolidine in increasing the level of glutathione is prevented by administration of buthionine sulfoximine. This thiazolidine may be useful in the treatment of other toxicities and in the treatment of certain diseases. It may also be valuable as a component of amino acid mixtures used in therapy and as a safener in agriculture.

Acetaminophen↗
PubMed1982

Interconversion of leukotrienes catalyzed by purified gamma-glutamyl transpeptidase: concomitant formation of leukotriene D4 and gamma-glutamyl amino acids.

The reversible conversion of leukotriene C4 to leukotriene D4 and of the latter to leukotriene E4 were studied with highly purified homogeneous preparations of gamma-glutamyl transpeptidase, dipeptidase, and aminopeptidase M. The conversion of leukotriene C4 to leukotriene D4, catalyzed by gamma-glutamyl transpeptidase, is significantly more rapid when carried out in the presence of an amino acid mixture a closely approximating that found in blood plasma and is accompanied by gamma-glutamyl amino acid formation. Because gamma-glutamyl transpeptidase is bound to the external surface of cell membranes and thus is readily accessible to plasma amino acids, it appears that conversion of leukotriene C4 to leukotriene D4 under physiological conditions is coupled with the formation of gamma-glutamyl amino acids. The apparent Km value for leukotriene C4 in this reaction is about 6 X 10(-6) M, a value close to that found for glutathione. Conversion of leukotriene D4 to leukotriene C4 is effectively catalyzed by gamma-glutamyl transpeptidase in the presence of relatively low concentrations of glutathione. The conversion of leukotriene D4 to leukotriene E4 is catalyzed much more rapidly by renal dipeptidase than by renal aminopeptidase M. Incubation of leukotriene E4 with gamma-glutamyl transpeptidase and glutathione leads to formation of a compound with the properties of gamma-glutamyl leukotriene E4; this reaction is analogous to that shown previously in which gamma-glutamyl cystine is formed by transpeptidation between glutathione and cystine.

Aminopeptidases↗
PubMed1981

5-Oxo-L-prolinase (L-pyroglutamate hydrolase). Studies of the chemical mechanism.

Rat kidney 5-oxo-L-prolinase catalyzes the endergonic hydrolysis of 5-oxo-L-proline (L-pyroglutamate, L-2-pyrrolidone-5-carboxylate) to form L-glutamate; the reaction is driven by and dependent on the stoichiometric concomitant hydrolysis of ATP to ADP and inorganic phosphate. The present studies are concerned with the mechanism by which the free energy of ATP hydrolysis is conserved and made available for 5-oxoproline hydrolysis. Studies with 18O-labeled substrates showed that (a) all three oxygen atoms of 5-oxoproline are recovered in the product glutamate, and (b) the two water molecules consumed in the reaction contribute one oxygen atom to inorganic phosphate and one oxygen atom to the gamma-carboxyl group of glutamate. It was shown that the enzyme also catalyzes the intrinsically exergonic hydrolysis of alpha-hydroxyglutarate lactone, a reaction that is ATP-dependent. Intermediates in the 5-oxoprolinase reaction were not detected by exchange experiments with radioactive ADP and phosphate, nor were they trapped by adding hydroxylamine. In the presence of very high glutamate concentrations, a slow reversal of the 5-oxoprolinase reaction was demonstrated by measuring ATP formation. The findings are consistent with a mechanism in which 5-oxo-L-proline is phosphorylated by ATP on the amide carbonyl oxygen and the resulting intermediate is subsequently hydrolyzed to yield gamma-glutamyl phosphate; the latter is hydrolyzed to glutamate and inorganic phosphate.

Adenosine Diphosphate↗
PubMed1981

gamma-Glutamyl transpeptidase: catalytic, structural and functional aspects.

gamma-Glutamyl transpeptidase catalyzes transfer of the gamma-glutamyl moiety of glutathione to amino acids, dipeptides, and to glutathione itself; the enzyme also catalyzes the hydrolysis of glutathione to glutamate and cysteinyl-glycine. This review deals with the tissue distribution and localization of the enzyme in mammals, the catalytic properties of the enzyme (including its inhibition by reversible and irreversible inhibitors), structural studies on the enzyme, and new findings about its physiological function.

Amino Acids↗
PubMed1981

Conversion of UMP, an allosteric inhibitor of carbamyl phosphate synthetase, to an activator by modification of the UMP ribose moiety.

UMP is known to be an allosteric inhibitor of carbamyl phosphate synthetase, whereas IMP activates the enzyme. Surprisingly, dialdehyde UMP (prepared by periodate oxidation of UMP) was found to be a potent activator of the enzyme. Dialdehyde IMP, like IMP, produced activation. The corresponding dialcohol analogs of UMP and IMP (prepared by borohydride reduction of the dialdehyde analogs) had no effect on activity. These nucleotide interactions were further characterized by sedimentation velocity studies and by examination of the effects of inorganic phosphate on enzymatic activity. Although UMP promotes formation of an enzyme dimer, and IMP promotes formation of a tetramer (Powers, S. G., Meister, A., and Haschemeyer, R. H. (1980) J. Biol. Chem. 255, 1554-1558), the dialdehyde analogs of UMP and IMP both promote formation of mixed species. Low levels (less than 10 mM) of inorganic phosphate decrease the extent of activation by IMP, dialdehyde IMP, and dialdehyde UMP, but increase the extent of inhibition by UMP. The marked activation observed with dialdehyde UMP, and other considerations, suggest that the binding sites on the enzyme for IMP and UMP may overlap substantially. The findings also suggest that physiological levels of inorganic phosphate function in the modulation of the allosteric regulation of this enzyme by nucleotides.

Allosteric Regulation↗
PubMed1981

Evidence that transpeptidation is a significant function of gamma-glutamyl transpeptidase.

gamma-Glutamyl transpeptidase (purified from rat kidney) was incubated with glutathione and a mixture of amino acids that closely approximates the amino acid composition of blood plasma, and the relative extents of transpeptidation and hydrolysis were determined by quantitative measurement of the products formed (glutamate, cysteinylglycine, gamma-glutamyl amino acids). At pH 7.4, in the presence of 50 microM glutathione and the amino acid mixture, about 50% of the glutathione that was utilized participated in transpeptidation. Studies in which the formation of individual gamma-glutamyl amino acids was determined in the presence of glutathione and the amino acid mixture showed that L-cystine and L-glutamine are the most active amino acid acceptors, and that other neutral amino acids also participate in transpeptidation to a significant extent. These in vitro experiments are consistent with a number of other findings which indicate that transpeptidation is a significant physiological function of gamma-glutamyl transpeptidase.

Animals↗
PubMed1981

Glutathione export by human lymphoid cells: depletion of glutathione by inhibition of its synthesis decreases export and increases sensitivity to irradiation.

Glutathione (in the form of GSH) is transported out of cultured human lymphoid cells at rates proportional to the intracellular glutathione levels. Inhibition of glutathione synthesis by buthionine sulfoximine, a potent selective inhibitor of gamma-glutamylcysteine synthetase, leads to exponential decrease in intracellular glutathione, a large fraction of which appears extracellularly, indicating that glutathione turnover is associated with its export. Although cells with 0.09 mM glutathione (4% of controls) were 85% viable, further decrease was associated with marked loss of viability. Cells with 4-5% of control glutathione levels were much more sensitive than control cells to the effects of gamma radiation and of 5,5'-dithiobis(2-nitrobenzoate). Depletion of glutathione by use of buthionine sulfoximine has advantages over other reagents (such as diamide, other oxidizing agents, and diethylmaleate, which affect other cellular components and may increase glutathione disulfide levels) and therefore has potential usefulness in sensitizing cells to the effects of radiation and to therapeutic agents that are detoxified by reactions involving glutathione.

Biological Transport↗
PubMed1981

Stimulation of hepatic glutathione formation by administration of L-2-oxothiazolidine-4-carboxylate, a 5-oxo-L-prolinase substrate.

5-Oxo-L-prolinase, the enzyme that catalyzes the conversion of 5-oxo-L-proline to L-glutamate coupled to the cleavage of ATP to ADP and Pi, also acts on L-2-oxothiazolidine-4-carboxylate (an analog of 5-oxoproline in which the 4-methylene moiety is replaced by sulfur) and ATP to yield cysteine and ADP. The enzyme, which exhibits an affinity for the analog similar to that for the natural substrate, is inhibited by the analog in vitro and in vivo. L-2-oxothiazolidine-4-carboxylate thus serves as a potent inhibitor of the gamma-glutamyl cycle at the step of 5-oxoprolinase. Administration of L-2-oxothiazolidine-4-carboxylate to mice that had been depleted of hepatic glutathione led to restoration of normal hepatic glutathione levels. Since L-2-oxothiazolidine-4-carboxylate is an excellent substrate of the enzyme, it may serve as an intracellular delivery system for cysteine and thus has potential as a therapeutic agent for conditions in which there is depletion of hepatic glutathione.

Amidohydrolases↗