Formation Of Cysteine Research Articles

Cystathionine γ-lyase from Streptomyces phaeochromoǵenes

Publisher Summary This chapter discusses the determination of cystathionine γ- Lyase from streptomyces phaeochromogenes. Cystathionine γ-lyase was first purified and crystallized from rat liver, but is also widely distributed among fungi. The microbial enzyme is purified and crystallized from Streptomyces phaeochromogenes (IFO 3105). Cystathionine β-synthase and cystathionine γ-synthase activities are also detected in crude extracts of S. phaeochromogenes, but cystathionine β-lyase is not. The enzyme is assayed by measuring the rate of formation of either α-ketobutyric acid or cysteine from L-cystathionine, or the formation of α-ketobutyric acid from L-homoserine. Gaitonde's acid/ninhydrin assay is highly specific, there being essentially no reaction with homocysteine. The lack of color development with homocysteine means that the assay is specific for γ-lyase activity and that β-lyase activity does not interfere. The assay is suitable, therefore, for crude extracts with cystathionine as substrate. The purified enzyme catalyzes the α,γ-elimination reaction of L-homoserine and L-cystathionine at 2.60 and 1.90 μmol/min/ mg of protein, respectively.

Enzymatic synthesis of the neuroexcitatory amino acid quisqualic acid by cysteine synthase

Purification of cysteine synthase from the leaves of Quisqualis indica var. villosa reveals the presence of two forms of this enzyme, separated by chromatography on DEAE-Sephadex A-50. Isoenzyme A was purified 10 000-fold and had a specific activity of 10.8 U/mg protein. Isoenzyme B was purified 460-fold with a specific activity of 0.49 U/mg protein. Both isoenzymes have the same M,s (58 000) and dissociate into identical subunits ( M r 29 000). The K m value of isoenzyme A is 1.9 mM for O-acetyl- L-serine and 59 μM for sulphide, while that of isoenzyme B is 7.1 mM for O-acetyl- L-serine and 4.0 mM for 3,5-dioxo-1,2,4-oxadiazolidine. Both isoenzymes catalyse the formation of cysteine from O-acetyl- L-serine and hydrogen sulphide, but only isoenzyme B catalyses the formation of L-quisqualic acid. Other significant differences occur in the substrate specificity of the two isoenzymes. Some properties of the purified cysteine synthase isoenzymes are also described.

Rat renal peritubular transport and metabolism of plasma [35S]glutathione.

More than 80% of the plasma glutathione is extracted during a single pass through the kidney. The peritubular component of this extraction was characterized by in situ arterial infusion of [35S]glutathione and [3H]inulin. The peak of 35S-labeled material recovered in the renal venous effluent was delayed approximately 10 S compared with the peak of [3H]inulin. As a result, the initial fractions exhibited a decreased 35S/3H ratio, indicating that 35S-labeled material is transported out of the postglomerular peritubular capillaries. Later fractions exhibited a normalized 35S/3H ratio greater than 1, consistent with the subsequent addition of a 35S-labeled metabolite to the venous circulation. An identical profile was observed when perfusion experiments were repeated using gamma-[35S]glutamyl-S-methylcysteine and [3H]inulin. Renal venous plasma samples obtained from a rat perfused with [35S]glutathione were reduced with sodium borohydride, reacted with monobromobimane, and analyzed by high-pressure liquid chromatography. More than 70% of the recovered 35S-labeled material was identified as cysteine and 20% was recovered as unmetabolized glutathione. Pretreatment of rats with a single injection of AT-125 resulted in 96% inactivation of renal gamma-glutamyltranspeptidase. Under these conditions, the percent of glutathione converted to cysteine (35%) was significantly less than the observed level of renal extraction (61%). Two injections of AT-125 caused a complete inhibition of cysteine formation. However, the residual level of renal extraction (41%) was still significantly greater than the filtration fraction (26%). The peritubular transport of glutathione is stimulated by prior depletion of renal glutathione with buthionine-L-sulfoximine and is competitively inhibited by simultaneous infusion of gamma-glutamylcysteine.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of microemulsions as vehicles for nucleophilic reagents in cosmetic formulations

Synopsis The modifications of chemical reactivity induced in the human hair during its treatment with oxidative (H(2)O(2)) or reductive (HSO(3)Na) agents via a micellar or a microemulsion system have been investigated. For this purpose, phase diagrams of micellar solutions and microcmulsions with H(2)O(2) or NaSO(3)H have been made in order to find out the corresponding areas of solubility. The properties of conductivity, surface tension and light scattering of various monophasic compositions as a function of their water content, have been studied. As a result of the chemical reactivity data of human hair obtained through the reaction of H(2)O(2) or HSO(3)Na via a micellar or a microemulsion system, it appears reasonable to predict a more effective reaction of such agents with cystine residues existing in keratinic substrates, particularly when they are applied via a microemulsion. The decrease of the water content of the compositions considered, increases chemical reactivity of the keratinic proteins favouring the formation of cysteine and of cysteic acid in the reductive or oxidative treatments respectively.

Specific cation effect in the reaction of nitroprusside with cysteine, acetophenone and sulfite (Legal and Boedeker reaction).

The colour formation of cysteine (I), acetophenone (II) and sulfite (III) with the sodium and tetrabutylammonium (TBA) salt of nitroprusside (NP2-) in aqueous solution was studied. The intensity of colour formation depends strongly on the nature and concentration of the cations and increases in the order TBA less than Li less than Na less than K less than Rb less than Cs (in case of cysteine TBA and Li are interchanged). This specific cation effect was known for the Boedeker reaction (III) and is now also demonstrated for the Legal reaction (I and II). The adduct formation between NP2- and I to III is based on an anion-anion interaction. The role of the cation is to reduce the Coulombic repulsion between the reactants by ion-pair formation. The efficiency of ion-pair formation corresponds with the order given before except for TBA, which behaves divergently.

Selenium Deficiency and Transsulfuration in the Chick

Experiments were conducted to investigate the effect of selenium (Se) deficiency on the efficiency of cysteine formation from methionine (Met) in three strains of chickens. Hubbard, Leghorn, or crossbred (New Hampshire × Columbian) chicks were fed a crystalline amino acid diet that was fortified with both vitamin E and ethoxyquin but was essentially devoid of Se. The sulfur-containing amino acid (SAA) requirement was provided as equal quantities of Met and cystine (Cys-Cys) or as an isosulfurous level of Met alone. When fed diets unsupplemented with Se, all chicks exhibited depressed growth and markedly reduced blood glutathione peroxidase (SeGSH-Px) activities. SAA source had no effect on SeGSH-Px activity. Source of SAA also had no effect on growth rate of Leghorn or crossbred chicks, regardless of Se status. Se-deficient Hubbard chicks, on the other hand, gained faster and more efficiently when fed the Met-Cys-Cys combination than when fed an isosulfurous level of Met alone. Plasma concentrations of Cys-Cys and cystathionine were reduced in Se-deficient chicks of all strains. Homocystine was detected only in plasma of Hubbard chicks fed Se-adequate diets containing Met as the source of SAA. These findings support the view that transsulfuration efficiency may be impaired by Se deficiency in some strains of chickens.

Acifluorfen metabolism in soybean: Diphenylether bond cleavage and the formation of homoglutathione, cysteine, and glucose conjugates

Metabolism of the substituted diphenylether herbicide, acifluorfen [sodium 5-(2-chloro-4-trifluoromethylphenoxy)-2-nitrobenzoate], was studied in excised leaf tissues of soybean [ Glycine max (L.) Merr. ‘Evans’]. Studies with [ chlorophenyl- 14C]- and [ nitrophenyl- 14C]acifluorfen showed that the diphenylether bond was rapidly cleaved. From 85 to 95% of the absorbed [ 14C]acifluorfen was metabolized in less than 24 hr. Major polar metabolites were isolated and purified by solvent partitioning, adsorption, thin layer, and high-performance liquid chromatography. The major [ chlorophenyl- 14C]-labeled metabolite was identified as a malonyl-β- d-glucoside (I) of 2-chloro-4-trifluoromethylphenol. Major [ nitrophenyl- 14C]-labeled metabolites were identified as a homoglutathione conjugate [ S-(3-carboxy-4-nitrophenyl) γ-glutamyl-cysteinyl-β-alanine] (II), and a cysteine conjugate [ S-(3-carboxy-4-nitrophenyl)cysteine] (III).

Status of the mitochondrial pool of glutathione in the isolated hepatocyte.

Using the selective membrane-solubilizing properties of digitonin and a rapid centrifugation method to separate cytoplasmic and mitochondrial components, the metabolic state of mitochondrial glutathione was investigated in isolated rat hepatocytes. Two pools of GSH were released from hepatocytes incubated with increasing concentrations of digitonin. The largest pool (about 85% of cellular total) was released simultaneously with lactate dehydrogenase, the other pool with citrate synthase, indicating cytoplasmic and mitochondrial locations, respectively. The t1/2 of the mitochondrial pool was estimated by linear regression analysis to be 30 +/- 3 h, while the cytoplasmic pool turned over with a t1/2 of about 2 +/- 0.1 h. The rate of incorporation of [35S]methionine or cysteine into the cytoplasmic pool of GSH, when corrected for turnover, was 15 times greater than into the mitochondrial pool. Mitochondrial GSH was not depleted after 60 min with 185 microM diethyl maleate with or without 75 microM bis-1,3-(2-chloroethyl)-1-nitrosourea, a specific inhibitor of glutathione reductase, whereas cytoplasmic levels were reduced to 40% and 10% of control values, respectively. In vivo experiments, using L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid to inactive gamma-glutamyl transpeptidase to limit cysteine formation from plasma GSH, demonstrated that in the absence of label reincorporation, liver glutathione exhibits a biphasic turnover. The rates of decay (half-lives) and percentages of total GSH under these conditions correlate well with the half-lives and pool distribution seen in the mitochondrial and cytoplasmic populations of GSH found in the isolated hepatocytes.

Some properties of a .BETA.-cyanoalanine-forming enzyme of Enterobacter sp. 10-1.

The properties of a purified β-cyanoalanine (β-CNAla)-forming enzyme isolated from Enterobacter sp. 10–1 were studied.It has been found previously that this enzyme catalyzed the formation of cysteine from O-acetyl-l-serine and soldium sulfide, as well as the formation of β-CNAla from O-acetyl-l-serine and sodium cyanide.The affinity of the enzyme for sodium sulfide in the formation of cysteine was one order of magnitude greater than that for sodium cyanide in the β-CNAla formation. In addition, the optimum pH, pH- and thermal stability of β-CNAla-forming activity were similar to those of cysteine-forming activity. The results of the studies indicated that the β-CNAla-forming enzyme of Enterobacter sp. 10–1 was an O-acetylserine sulfhydrylase catalyzing the formation of cysteine from O-acetyl-l-serine and sulfide. The solution of the enzyme was yellow and exhibited an absorption peak at 420 nm. The enzyme itself contained approximately 1 mol of pyridoxal phosphate per 34,000 g of protein.

Purification and crystallization of a .BETA.-cyanoalanine-forming enzyme from Enterobacter sp. 10-1.

A β-cyanoalanine (β-(-CNAla)-forming enzyme, which catalyzed the formation of β-CNAla from O-acetyl-L-serine and sodium cyanide, was isolated in crystalline form from Enterobacter sp. 10-1, a cyanide-resistant strain. The purification procedure involved protamine treatment, ammonium sulfate fractionation, column chromatography on DEAE-cellulose, DEAE-Sephadex A50, Sephadex G-100 and hydroxyapatite, and crystallization with ammonium sulfate. The crystalline enzyme was homogeneous by criteria of the ultracentrifugation and polyacrylamide gel electrophoresis. The enzyme was determined to have a molecular weight of about 68, 000, and consisted of two apparently identical subunits, each having a molecular weight of about 34, 000. The enzyme also catalyzed the formation of cysteine from O-acetyl-L-serine and sodium sulfide, and the cysteine-forming activity was 245 times greater than that of the β-CNAla-forming activity.

7-Chloro-4-nitrobenzeno-2-oxa-1,3-diazole actin as a probe for actin polymerization.

Lysine 372 of N-ethylmaleimide actin was specifically (60%) labeled by 7-chloro-4-nitrobenzeno-2-oxa-1,3-diazole chloride (NBD-Cl), which also reacted with lysines on cyanogen bromide fragment 17 (20%) and other undetermined residues (20%). Isolation of N-ethylmaleimide peptides and two-dimensional peptide mapping demonstrated that 90% of bound N-ethylmaleimide was attached to an adjacent residue, cysteine 373, independent of the polymerization state of actin during the labeling reaction. Formation of NBD cysteine severely inhibited lysine modification. After N-ethylmaleimide blockage of cysteine 373, lysine labeling with NBD was greatly accelerated. The kinetics of formation of fluorescent compounds were biphasic, with fluorescence decreasing upon prolonged incubation of actin in NBD-Cl. Lysine 372 of purified NBD actin reproducibly responded to polymerization by a 2.2- to 2.3-fold enhancement of fluorescence. By contrast, interaction of NBD actin with several actin-binding proteins caused only very small or undetectable changes in fluorescence intensity: 10% enhancement on myosin subfragment 1 binding, about 6% quenching by DNase I, and no change at all by tropomyosin-troponin. Despite its sensitivity to polymerization the probe did not affect it. Native and modified actin polymerized randomly indicating that the rate constants for polymerization remained the same. Labeling actin with NBD did not diminish its cofactor activity for myosin ATPase activity. Contrary to previous reports we observed that myosin subfragment 1 (single myosin heads) caused actin polymerization in the absence of salt.

Formation of Cysteine from Adenosine 5′-phosphosulf ate (APS) in Extracts from Spinach Chloroplasts

Summary In an assay system containing essentially extract from spinach chloroplasts, 3.3 mM glutathione, O-acetyl-L-serine, and reduced ferredoxin, 35 S-cysteine was formed from 35 S-adenosine 5′-phosphosulf ate ( 35 S-APS) at a rate of 1 to 6 nmol per mg protein per hour. Addition of non-radioactive adenosine 3′-phosphate 5′-phosphosulf ate (PAPS) to the reaction mixture did not reduce appreciably the rate of 35S-cysteine formation from 35 S-APS, indicating that APS is not phosphorylated to PAPS prior to reduction. When glutathione was replaced by dithiothreitol 35 S-cysteine was formed at comparable rates. The addition of different spinach thioredoxins did not influence 35 S-cysteine formation. Free 35 S-SO 3 2− was detected in the reaction mixture with either glutathione or dithiothreitol as active thiol.

Regulatory properties of O-acetyl-L-serine sulfhydrylase of Cephalosporium acremonium: evidence of an isoenzyme and its importance in cephalosporin C biosynthesis.

O-Acetyl-L-serine sulfhydrylase catalyzes the final step in the biosynthesis of cysteine from H2S and O-acetyl-L-serine in the fungus Cephalosporsium acremonium, a cephalosporin C-producing organism. We separated this enzyme from the closely related but less specific O-acetyl-L-homoserine sulfhydrylase and showed that O-acetyl-L-homoserine sulfhydrylase also catalyzes the formation of cysteine from O-acetyl-L-serine and H2S. The expression of O-acetyl-L-serine sulfhydrylase was regulated by exogenous methionine. In addition, this enzyme was inhibited by S-adenosyl-L-methionine and 5-formylpteroyl monoglutamic acid. The inhibition of both S-adenosyl-L-methionine and 5-formylpteroyl monoglutamic acid was noncompetitive. Results obtained with gel filtraton experiments in various buffer systems indicate an association-dissociation behavior of O-acetyl-L-serine sulfhydrylase.

Nitrogen metabolite repression of nitrate reductase in Neurospora crassa

The effect of different nitrogen compounds on the induction of reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase was examined in Neurospora crassa. Whereas in the wild-type strain several amino acids and ammonia inhibit the formation of nitrate reductase, only glutamine, cysteine, and histidine are shown to inhibit the synthesis of nitrate reductase in a glutamine-requiring auxotroph. None of the amino acids inhibited nitrate reductase activity in vitro. The effects of cysteine and histidine are nonspecific, these amino acids being inhibitory of the growth of the organism. The effect of glutamine on the induction of nitrate reductase is not due to an inhibition of the uptake of the inducer nitrate. By the use of histidine-, pyrimidine-, and arginine-requiring auxotrophs, it was shown that glutamine appears to act per se and does not seem to be converted to another product in order to be effective in repression. The repression of nitrate reductase by ammonia appears, from the results described herein, to be indirect; ammonia has to be converted first to glutamine in order to be effective in repression.

Use of isolated kidney cells for study of drug metabolism

Isolated kidney cells were prepared from rat kidneys using a recirculating perfusion system with collagenase. The preparation was rapid and provided a high yield of intact metabolically active kidney cells predominantly of tubular origin. The respiration rate was 2.7 μmol O 2/hr per 10 6 cells and was not stimulated by ADP. GSH content was 28.9 nmol/10 6 cells and did not decline during 2 hr of incubation. Cytochrome P450 content was 0.064 nmol/10 6 cells. The cells were characterized for their drug metabolizing activity using paracetamol as substrate. The rate of formation of the glucuronide and sulfate derivatives was linear for 2 hr, but slower than previously reported for rat liver cells. In contrast to incubations with liver cells, no glutathione conjugate was detected. However, formation of both cysteine and N-acetylcysteine derivatives was observed. The rate of formation of total sulfhydryl conjugates was about 50 per cent of that reported for liver cells when expressed on a cytochrome P450 basis. These studies establish the reliability and utility of this cell preparation as a model system for the study of drug metabolism by the kidney.

Stereochemistry of the formation of cysteine by O-acetylserine sulfhydrase.

The enzymatic preparation of (2S, 3R)- and (2S, 3S)-[3-3H] serine from the corresponding stereospecifically tritiated 3-P-glycerates is described. Following conversion into T-acetylserine, these substrates were used to establish the steric course of the O-acetylserine sulfhydrase reaction. The two samples of cysteine formed in this reaction were analyzed for their configuration at C-3. The results indicate that the replacement of the acetoxy by a sulfhydryl group in the O-acetylserine sulfhydrase reaction occurs with retention of configuration at carbon atom 3.

Optical inducation during biomimetic formation of cysteine.

Optical inducation during biomimetic formation of cysteine.

Comparative study of abiogenesis of cysteine and other amino acids catalyzed by various metal ions

The present work pertains to the study of the influence of nickel, cobalt, thorium, vanadium, molybdate, ferrous ions on the formation of cysteine which is synthesized abiogenically together with other amino acids in sterilized aqueous mixtures of ammonium thiocyanate, formaldehyde, potassium dihydrogen phosphate, calcium acetate, and biological minerals after irradiating by artificial light. The effect of these catalysts on cysteine formation was of the order: Fe++ greater than Mo++ greater than Th++++ greater than V++ greater than Co++ greater than Ni.

Reduction of adenosine 5'-phosphosulfate to cysteine in extracts from Chlorella and mutants blocked for sulfate reduction.

Techniques for preparation of enzyme extracts from Chlorella pyrenoidosa chick are described which yield a stable system which converts adenosine 5′‐phosphosulfate to sulfide and/or cysteine in the absence of added active thiols. Since these thiols (such as dithiothreitol) have been shown to yield sulfite, among other products, from the adenosine‐5′‐phosphosulfate sulfotransferase reaction, we have studied the adenosine 5′‐phosphusulfate reducing system in the presence and absence of dithiothreitol. In all cases dithiothreitol increases the activity of the reducing system forming cysteine from adenosine 5′‐phosphosulfate. Mutant Sat−2, which cannot grow on sulfate, is shown to lack thiosulfonate reductase activity but to retain sulfite reductase activity. Extracts from this mutant will form sulfide and cysteine only in the presence of dithiothreitol, while wild‐type extracts will form these products in presence or absence of dithiothreitol, the activity with dithiothreitol being higher. Mutants Sat−1,3–6 which are blocked in the adenosine‐5′‐phosphosulfate sulfotransferase step are blocked in the formation of sulfide and cysteine from adenosine 5′‐phosphosulfate. Binding studies with adenosine 5′‐phosphosulfate indicate that the sulfonyl group is transferred to a carrier to form carrier‐S‐SO−3 and the available evidence suggests that this carrier is a prosthetic group of the thiosulfonate reductase. On the basis of this evidence, two pathways in the cell‐free extracts are thought to be operating. One, in the absence of active thiols, is a pathway consisting of bound intermediates in which the sulfonyl group of adenosine 5′‐phosphosulfate is transferred via adenosine‐5′‐phosphosulfate sulfotransferase to carrier on the thiosulfonate reductase where it is reduced to the thiol level by ferredoxin; the thiol is then transferred via O‐acetyl‐l‐serine sulfhydrylase to form cysteine. The other, or free intermediate pathway operates in the presence of dithiothreitol which releases free sulfite from the adenosine‐5′‐phosphosulfate sulfotransferase reaction. This sulfite is then reduced via sulfite reductase to sulfide which forms cysteine via O‐acetyl‐l‐serine sulfhydrylase. Since mutant Sat−2, which cannot grow on sulfate nor reduce it in vivo, lacks thiosulfonate reductase activity but yields sulfite reductase activity in extracts, we conclude that the bound intermediate pathway is the preferred pathway of sulfate reduction in vivo; the free intermediate pathway is probably operative only when free sulfite is available either from occasional side reactions in vivo or when sulfite is fed to cells from outside.

Cysteine Formation from Methionine in Pseudomonas melanogenum and Mechanism of Methionineinhibition of L-DOPA Production by the Bacterium

Cysteine Formation from Methionine in Pseudomonas melanogenum and Mechanism of Methionineinhibition of L-DOPA Production by the Bacterium