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Biomedical subjects

A Meister

Publications and source records attributed to A Meister.

At least 55 records · Page 3Linked to original sources
PubMed1995

Chemical modification of active site residues in gamma-glutamyl transpeptidase. Aspartate 422 and cysteine 453.

gamma-Glutamyl transpeptidase, an enzyme of central significance in glutathione metabolism, is inactivated by iodoacetamide, which esterifies an active site carboxyl group identified here as that of Asp-422. Treatment of the inactivated enzyme with hydroxylamine leads to deesterification and to restoration of enzymatic activity. N-Acetylimidazole, which also inactivates the enzyme, acetylates several amino acid residues. Acetylation exposes Cys-453, which is buried in the native enzyme, to reaction with iodoacetamide. Incubation of the acetylated enzyme with glutamine produces a stabilized gamma-glutamyl-enzyme form which is (a) located exclusively on the light subunit, (b) more labile to base than to acid, (c) destabilized by denaturation of the enzyme with guanidinium ions, and (d) reactive with hydroxylamine to form gamma-glutamylhydroxamate. Stabilization of the gamma-glutamyl-enzyme appears to be associated with acetylation of lysine residues (including Lys-99). These and other findings suggest that the alpha-amino group of the gamma-glutamyl substrate is linked electrostatically to Asp-422 so as to facilitate reaction of the gamma-carbonyl of the substrate with an enzyme hydroxyl group to form a gamma-glutamyl-enzyme.

Acetylation↗
PubMed1995

Mitochondrial changes associated with glutathione deficiency.

Glutathione deficiency produced by giving buthionine sulfoximine (an inhibitor of gamma-glutamylcysteine synthetase) to animals, leads to biphasic decline in cellular glutathione levels associated with sequestration of glutathione in mitochondria. Liver mitochondria lack the enzymes needed for glutathione synthesis. Mitochondrial glutathione arises from the cytosol. Rat liver mitochondria have a multicomponent system (with Kms of approx. 60 microM and 5.4 mM) that underlies their remarkable ability to transport and retain glutathione. Mitochondria produce substantial quantities of reactive oxygen species; this is opposed by reactions involving glutathione. Glutathione deficiency leads to widespread mitochondrial damage which is lethal in newborn rats and guinea pigs, animals that do not synthesize ascorbate. Glutathione esters and ascorbate protect against the lethal and other effects of glutathione deficiency. Ascorbate spares glutathione; it increases mitochondrial glutathione in glutathione-deficient animals. Glutathione esters delay onset of scurvy in ascorbate-deficient guinea pigs; thus, glutathione spares ascorbate. Glutathione and ascorbate function together in protecting mitochondria from oxidative damage.

Animals↗
PubMed1995

Different sites of acivicin binding and inactivation of gamma-glutamyl transpeptidases.

Acivicin is a potent inhibitor of gamma-glutamyl transpeptidase (EC 2.3.2.2), an enzyme of importance in glutathione metabolism. Acivicin inhibition and binding are prevented by gamma-glutamyl substrates and analogs (e.g., serine plus borate), consistent with the previous postulate that acivicin and substrates bind to the same enzyme site. Inactivation of rat kidney transpeptidase by acivicin leads to its binding as an ester to Thr-523. The pig enzyme, which has Ala-523 in place of Thr-523, is inhibited by acivicin with esterification at Ser-405. The human enzyme has Thr-524 (corresponding to Thr-523 in rat); its inactivation leads to esterification of Ser-406 (corresponding to Ser-405 in rat and pig). Hydroxylamine treatment of the acivicin-inactivated enzymes restores activity and releases the acivicin-derived threo-beta-hydroxyglutamate moiety. The findings indicate that there are significant structural differences between the active site region of the rat enzyme and the active site regions of the human and pig. Human mutant enzymes in which Thr-524 and Ser-406 were replaced by Ala, separately and together, are enzymatically active, indicating that these amino acid residues are not required for catalysis. However, esterification of these residues (and of another near the active site) effectively blocks the active site or hinders its function. Acivicin can bind at enzyme sites that are close to that at which gamma-glutamylation occurs; it may bind at the latter site and then be transesterified to another enzyme site.

Alanine↗
PubMed1995

Amino acid sequence of rat kidney glutathione synthetase.

Glutathione (GSH) synthetase [gamma-L-glutamyl-L-cysteine:glycine ligase (ADP-forming), EC 6.3.2.3], an enzyme present in almost all cells, catalyzes the ATP-dependent synthesis of GSH from gamma-L-glutamyl-L-cysteine and glycine. Highly purified preparations of the enzyme have been obtained from rat kidney and several lower forms. The rat kidney enzyme (M(r), 118,000), which contains approximately 2% carbohydrate, is composed of two apparently identical subunits. The cDNA encoding rat kidney GSH synthetase was isolated from a rat kidney lambda gt11 cDNA library by immunoscreening with an antibody prepared against the isolated enzyme. The cDNA contains 1905 nucleotides and an open reading frame of 1422 nucleotides coding for 474 amino acids. The cDNA has a 3' untranslated region of 439 nucleotides, which includes a poly(A) tail. The deduced amino acid sequence (M(r), 52,344) contains all five of the peptide sequences that were independently determined by Edman degradation. The cDNA was expressed in Escherichia coli. The amino acid sequence of the rat kidney enzyme has no significant similarity to that of the enzyme from E. coli and shows some similarity to those deduced for the yeast and frog enzymes. Knowledge of this amino acid sequence is expected to facilitate elucidation of the sequence of the corresponding human enzyme and to lead to studies on the biochemical mechanisms involved in human GSH synthetase deficiency as well as to development of improved methods for prenatal diagnosis of these inborn diseases.

Amino Acid Sequence↗
PubMed1995

Expression of an active glycosylated human gamma-glutamyl transpeptidase mutant that lacks a membrane anchor domain.

A mutant of human gamma-glutamyl transpeptidase (EC 2.3.2.2, a membrane-bound enzyme of importance in glutathione metabolism) that differs from the wild type by deletion of the putative signal peptide/anchor domain (amino acid residues 1-27) was expressed in insect cells using a baculovirus system. In contrast to the wild-type enzyme--which, as expected, was mainly cell-associated--the mutant enzyme was secreted into the medium. The mutant and wild-type enzymes were purified and found to exhibit virtually identical catalytic properties. The mutant enzyme was glycosylated and processed into two subunits, as found for the wild-type enzyme. Brefeldin A inhibited secretion of the mutant enzyme and led to its accumulation in cells. The findings indicate that gamma-glutamyl transpeptidase can be targeted to the endoplasmic reticulum in a manner that does not involve function of an amino-terminal "signal/anchor" domain and that this domain is involved primarily in a membrane anchoring function. Another region of the enzyme may function as a signal domain.

Amino Acid Sequence↗
PubMed1995

L-2-oxothiazolidine-4-carboxylate, a cysteine precursor, stimulates growth and normalizes tissue glutathione concentrations in rats fed a sulfur amino acid-deficient diet.

The efficiency of L-2-oxothiazolidine-4-carboxylate, a cysteine precursor, in stimulating glutathione synthesis and growth was evaluated in growing rats. Animals were fed a sulfur amino acid-deficient diet (0.25% L-methionine and no cysteine) supplemented with L-2-oxothiazolidine-4-carboxylate (0.35%) for 3 wk and compared with age-matched animals receiving the sulfur amino acid-deficient diet alone. Rats fed the sulfur amino acid-deficient diet had lower glutathione concentrations in bronchoalveolar lining fluid, lung, lymphocytes, and liver than rats fed a sulfur amino acid-deficient diet supplemented with L-2-oxothiazolidine-4-carboxylate. Rats fed the supplemented diet had normal tissue and bronchoalveolar lining fluid glutathione levels. Central venous plasma glutathione concentrations, mostly reflecting liver excretion, were less affected by L-2-oxothiazolidine-4-carboxylate supplementation. Rats fed L-2-oxothiazolidine-4-carboxylate supplementation had normal weight gain compared with a much lower weight gain in animals fed the sulfur amino acid-deficient diet alone. Thus, L-2-oxothiazolidine-4-carboxylate increased tissue glutathione concentrations and stimulated growth in rats. The lung glutathione status of the rats was reflected by glutathione concentrations in lymphocytes and the bronchoalveolar lining fluid, but not by the central venous plasma glutathione concentrations.

Amino Acids, Sulfur↗
PubMed1994

Interaction of gamma-glutamyl transpeptidase with acivicin.

Inactivation of gamma-glutamyl transpeptidase by acivicin (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid) is rapid, thought to be irreversible, and associated with binding of close to 1 mol of inhibitor/mol of enzyme. Previous studies with [3-14C]acivicin indicated binding (prevented by substrate) to a specific hydroxyl group (threonine 523) of the rat kidney enzyme. In the present work, we found that such inactivation can be reversed by treating the inhibited enzyme with hydroxylamine. Reactivation (more than 85% complete) is associated with release from the inactivated enzyme of compounds that exhibit the properties of threo-beta-hydroxy-L-gamma-glutamyl hydroxamate and 3-hydroxypyrrolidone-2-carboxylate. We found that the enzyme acts very slowly on acivicin, at a rate that is about 10(-9) that of its normal catalytic rate with glutathione, to form threo-beta-hydroxy-L-glutamate and hydroxylamine. The findings indicate that inhibition by acivicin involves its transformation on the enzyme to an inhibitory species which is attached, apparently by ester linkage, to a specific hydroxyl group of the enzyme. The very slow rate of release of this intermediate appears to account for the observed inhibition.

Animals↗
PubMed1994

Utility of DNA amplified by degenerate oligonucleotide-primed PCR (DOP-PCR) from the total genome and defined chromosomal regions of field bean.

Degenerate oligonucleotide primed (DOP)-PCR has emerged as a simple and rapid method for representative amplification of highly complex genomic DNA from humans, mice and Drosophila. The present paper describes the adaptation of this method for use on a plant species, Vicia faba, with a large genome (2C = 30 pg). Specific low-copy-number sequences as well as highly repeated sequences were detectable among DOP-PCR products obtained from small samples of purified genomic DNA (100 pg), DNA from 10 prophase nuclei, 10 flow-sorted chromosomes or 15 microdissected chromosome segments (satellites) following reamplification with sequence-specific primers and/or Southern hybridization. Biotinylated chromosome-specific DOP-PCR products were used for fluorescent in situ hybridization. All chromosomes showed hybridization signals, with the exception of regions containing Fok elements which are not present in the chromosomal DNA targeted by DOP-PCR.

Base Sequence↗
PubMed1994

Differentiation of field bean heterochromatin by in situ hybridization with a repeated FokI sequence.

The chromosomes of a field bean line with a reconstructed karyotype (ACB) were hybridized in situ with biotinylated probes of a repetitive Fok I sequence, of DOP-PCR (degenerate oligonucleotide primed polymerase chain reaction) amplified DNA from a chromosome that does not contain this sequence, and with probes containing dispersed repetitive sequences. The results were compared with Giemsa banding, DNA late replication and Fok I in situ digestion patterns. This allowed further differentiation between the chromatin types of this species. Centromeric and NOR-associated heterochromatin as well as euchromatin were shown to be free of Fok I sequence repeats. Among the interstitial late replicating Giemsa bands, subdivided into 'marker' and 'additional' bands, most of the marker bands located at mid-arm positions were composed mainly or exclusively of tandemly arranged Fok I repeats. Some of the marker bands and nearly all of the additional bands located in the vicinity of centromeres were free of FokI sequence repeats, of Fok I recognition sites, and possibly also of dispersed repetitive sequences. They are probably composed of specific, not yet defined, repetitive sequences.

Base Sequence↗
PubMed1994

Active deglycosylated mammalian gamma-glutamyl transpeptidase.

gamma-Glutamyl transpeptidase, a highly glycosylated heterodimeric enzyme that is usually attached to the external surface of cell membranes, is of major importance in the metabolism of glutathione. The enzyme, which has been isolated from many animal sources, contains a large amount of carbohydrate, which is linked to both protein subunits. Previous work has not shown whether such carbohydrate is needed for enzyme activity nor indicated its functional role. Notably, gamma-glutamyl transpeptidase isolated from Escherichia coli, which exhibits about 80% amino acid sequence homology with the rat enzyme, has only about 0.1% of its specific enzymatic activity and is not glycosylated. Here we treated the highly glycosylated gamma-glutamyl transpeptidases isolated from rat and pig kidneys with a mixture of glycosidases and then separated two completely active gamma-glutamyl transpeptidase fractions from each species. One fraction was completely devoid of carbohydrate and was fully active as compared with the respective isolated enzymes, but differed in solubility and stability. The other fraction, which contained 10-20% of the initially bound carbohydrate, exhibited a marked increase in susceptibility to proteases. The oligosaccharide chains of gamma-glutamyl transpeptidase may protect against protease action (including self-destruction by the inherent protease activity of the light subunit) during synthesis of the active enzyme from its single chain precursor, as well as after enzyme synthesis.

Animals↗
PubMed1994

Nitrate tolerance in vivo is not associated with depletion of arterial or venous thiol levels.

Results from in vitro experiments suggest that development of nitrate tolerance is due to a depletion of vascular thiol compounds (ie, cysteine and glutathione [GSH]) necessary for the bioconversion of organic nitrates. However, it is unknown whether in vivo tolerance development is associated with changes in thiol levels. This study measures plasma and vessel tissue GSH and cysteine levels in nontolerant rats, nitrate-tolerant rats, and rats treated with the two characteristically different thiol donors N-acetyl-L-cysteine and L-2-oxothiazolidine-4-carboxylic acid (OXO). Chronically catheterized conscious rats received an intravenous infusion of either nitroglycerin (NTG, 0.2 mg/h) or matching placebo for 3 days. At day 3, the hypotensive effect of 2.5 mg NTG/kg was decreased by 74 +/- 6% (mean +/- SEM, P < .05) in the NTG-treated group (n = 7), indicating the development of tolerance. No change in the hypotensive effect of NTG was seen in the placebo group (n = 6, P > .05). Hemodynamic tolerance is not associated with changes in aorta cysteine or GSH levels as compared with the placebo group (cysteine, 77 +/- 14 versus 57 +/- 11 [mean + SEM] nmol/g; GSH, 414 +/- 62 versus 399 +/- 89 nmol/g; P > .05). However, the increase in vascular thiol levels seen after OXO treatment in nontolerant rats is completely absent in nitrate-tolerant animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcysteine↗
PubMed1993

Transport of glutathione diethyl ester into human cells.

Glutathione monoesters in which the carboxyl group of the glycine residue is esterified were previously found, in contrast to glutathione itself, to be effectively transported into various types of cells and to be converted intracellularly into glutathione. Glutathione monoesters are thus useful for prevention of oxidative stress, certain toxicities, and for treatment of glutathione deficiency. Glutathione diethyl ester is rapidly split to the glutathione monoethyl ester by mouse plasma glutathione diester alpha-esterase activity. Thus, as expected, glutathione mono- and diesters have similar effects on cellular glutathione levels in mice. However, human plasma lacks glutathione diester alpha-esterase; thus, it became of interest to compare the transport properties of glutathione mono- and diesters in human cells. We found that human cells (erythrocytes, peripheral blood mononuclear cells, fibroblasts, ovarian tumor cells, and purified T cells) transport glutathione diethyl ester much more effectively than the corresponding monoethyl (glycyl) ester. Human cells rapidly convert glutathione diethyl ester to the monoester, whose intracellular levels rise to levels that are significantly higher than levels found after application of the monoester to the cells. High levels of the monoester provide the cells with a means of producing glutathione over a period of time. We conclude that glutathione diethyl ester is highly effective as a delivery agent for glutathione monoester, and thus for glutathione, in human cells and therefore could serve to decrease oxidative stress and toxicity. Hamster (and certain other animals) also lack plasma glutathione diester alpha-esterase and therefore would be suitable animal models. Previously reported toxicity of certain glutathione ester preparations appears to reflect the presence of impurities rather than effects of the esters.

Animals↗