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PubMed1983

Interconversions between different sulfhydryl-related kinetic states in glucokinase.

Rat liver glucokinase (EC 2.7.1.2) undergoes two distinct sulfhydryl-related reversible kinetic transitions. During normal assays in the presence of both substrates but without added reducing agents, the activity decays ("kappa" decay) over time to a new steady-state rate. The half-time for this decay is essentially constant at glucose levels from 2 to 200 mM and averages 6.2 +/- 2 min. Glucokinase in this kappa steady state displays an increased Km for glucose but has the same Vmax as normal, sulfhydryl-activated glucokinase. The kappa form does not itself exhibit kinetic cooperativity with glucose. In contrast, glucokinase incubated with neither glucose nor sulfhydryl reagents decays (mu decay) to a form whose Vmax is near zero. The t 1/2 for this transition is about 0.5 min at 0 or very low (0.5 mM) glucose concentrations. For both decays, incubations of enzyme with intermediate levels of reducing agents give steady-state mixtures of activated and either kappa and/or mu forms, depending on conditions during the decay. Enzyme at intermediate stages of the kappa decay displays an unchanged Vmax, intermediate (increased relative to activated enzyme) glucose S0.5 values, and diminished glucose cooperativity. In contrast, enzyme at intermediate steady-state mixtures of activated and mu forms has a normal glucose S0.5 and cooperativity but a diminished Vmax from the activated states. The enzyme at any stage of each decay may be fully reactivated by the addition of sulfhydryl reducing agents such as dithiothreitol, dithioerythritol, glutathione, or mercaptoethanol. A model is proposed to account for this complex behavior in glucokinase kinetics which proposes different enzymatic states (kappa and mu) locked in by sulfhydryl oxidation of different conformations dictated by glucose concentration. These sulfhydryl-related transitions may be important in regulation of glucokinase activity, since glucokinase is very sensitive (at least 20-fold differential activity) to concentrations of glutathione within the physiological range, perhaps allowing the normally variable glutathione levels or cytosolic redox potential to modify the rate of uptake and storage of blood glucose through control of glucokinase activity.

Animals↗
PubMed1984

myo-Inositol-1-phosphate synthase from pine pollen: sulfhydryl involvement at the active site.

myo-Inositol-1-phosphate synthase [EC 5.5.1.4; 1L-myo-inositol-1-phosphate lyase, (isomerizing)] from Pinus ponderosa pollen has been partially purified and characterized. It has a pH optimum between 7.25 and 7.75. The km for D-glucose 6-phosphate (NAD+ constant at 1 mM) is 0.33 mM. Inhibition by p-chloromercuribenzoate and N-ethylmaleimide, and partial protection against this inhibition by D-glucose 6-phosphate in the presence of NAD+, suggests that there is sulfhydryl group involvement at the substrate binding site.

Binding Sites↗
PubMed1984

Reactions of the sulfhydryl groups of alanyl-tRNA Synthetase.

The six sulfhydryl groups in each subunit of the alanyl-tRNA synthetase of Escherichia coli react with sulfhydryl reagents with at least four different rates. One reacts very rapidly with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and a second reacts somewhat less rapidly with this reagent. These two groups are required for transfer activity, which is lost in proportion to the extent of derivatization. Two other groups react more slowly, with a consequent loss of exchange activity. The remaining two sulfhydryl groups do not react with DTNB until the protein is denatured. The inactivations are reversed by dithiothreitol. Two sulfhydryl groups react with N-ethylmaleimide (NEM) and with a spin-label derivative of NEM. These reactions resemble the modification of two sulfhydryl groups with DTNB, in that they also inactivate the transfer reaction but not the ATP:PPi exchange. The two spin labels are incorporated at similar rates but are in very different environments, one highly exposed and one highly immobilized. These groups do not interact with Mn2+, which is bound to the enzyme in the absence of ATP.

Alanine-tRNA Ligase↗
PubMed1985

Substrate-induced changes in sulfhydryl reactivity of bacterial D-amino acid transaminase.

D-Amino acid transaminase from Bacillus sphaericus strain ATCC 14577 is a dimer with eight cysteinyl residues per molecule (T.S. Soper, W.M. Jones, and J.M. Manning (1979) J. Biol. Chem. 254, 10,901-10,905). The reaction of the cysteinyl residues with a variety of sulfhydryl reagents has been explored to gain insight into the physical environments around these cysteinyl residues in the absence or the presence of substrates. The native enzyme, in the pyridoxal-P conformation, appears to be a symmetrical dimer, whose SH groups react in pairs with anionic reagents such as 5,5'-dithiobis(2-nitrobenzoic acid) or the halo acids. Two SH groups react with either reagent without altering enzymatic activity. Two additional SH groups react with DTNB with loss of catalytic activity. Positively charged reagents such as beta-bromoethylamine are much more effective in inactivating the pyridoxal-P conformation of the enzyme with almost five of the eight SH groups reacting and this results in a significant loss in catalytic activity. The neutral reagent dithiodipyridine is able to detect some asymmetry in the pyridoxal-P conformation. Upon addition of a D-amino acid substrate, the enzyme is transformed into the pyridoxamine-P conformation. This conformation is much more reactive with anionic reagents and much less reactive with cationic reagents, suggesting that there is a significant change in the net charge around one of the SH groups in the pyridoxamine-P conformation. Also, titration with DTNB indicates that the enzyme is a much more asymmetric dimmer in the pyridoxamine-P conformation than in the pyridoxal-P conformation. Thus, upon binding of a D-amino acid substrate, D-amino acid transaminase is transformed into the pyridoxamine-P conformation. This results in a significant change in the environment of four of the sulfhydryl groups of the enzyme. We conclude that the enzyme is transformed from a symmetrical dimer into an asymmetrical dimer and that the net charge of one of the pairs of cysteinyl groups is changed from a net negative charge into a net positive charge. These results suggest that there is a significant conformational change that occurs during the transition from the pyridoxal-P into the pyridoxamine-P form of this transaminase.

Bacillus↗
PubMed1985

Purification and properties of a quinone-dependent p-nitrophenylphosphatase from Clostridium sticklandii.

A highly specialized phosphatase that depends on both a quinone (e.g., 2-methyl-1,4-napthoquinone) and a sulfhydryl compound for activity was purified to homogeneity from extracts of Clostridium sticklandii. Selective adsorption to Cibacron Blue-Sepharose 4B followed by elution with p-nitrophenylphosphate was an effective enrichment procedure. An affinity matrix containing vitamin K5 (4-amino-2-methyl-1-naphthol) covalently attached to Sepharose 4B selectively retained the enzyme and was also used in its purification. The only known substate for the enzyme, p-nitrophenylphosphate, is hydrolyzed to equivalent amounts of orthophosphate and p-nitrophenol. Although a protein phosphotyrosine residue seemed a likely candidate as the natural substrate, the enzyme failed to hydrolyze 32P-labeled phosphotyrosine residues in casein, in vinculin, or in denatured glutamine synthetase. Also, free O-phosphotyrosine and numerous phosphate esters that serve as substrates for common phosphomonoesterases were not hydrolyzed. The molecular weight of the native enzyme, estimated by Sephacryl-S-200 gel chromatography, is 27,600. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed a single component with a molecular weight of 28,600. From the amino acid composition, a minimum molecular weight of 28,000 was calculated.

4-Nitrophenylphosphatase↗
PubMed1991

Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia.

2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dichlorocatechol. NADPH was preferred over NADH. The enzyme had Km value of 14 microM for 2,4-dichlorophenol, and 100 microM for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg2+ and Zn2+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity.

Apoenzymes↗
PubMed1992

Purification and properties of an aminopeptidase from rat-liver cytosol.

An aminopeptidase was purified from the rat-liver cytosolic fraction to apparent electrophoretic homogeneity. The enzyme is a monomeric protein of 95 kDa, having an isoelectric point of 4.9. Amino acid analyses indicate that the enzyme is rich in acidic amino acids and is poor in cysteine. The enzyme hydrolyzed a broad spectrum of amino acid beta-naphthylamides at a neutral pH. The enzyme also hydrolyzed di-, tri-, and oligopeptides, including physiologically active peptides such as enkephalins and Met-Lys-bradykinin. The enzyme was inhibited by metal-chelating agents, sulfhydryl-reactive reagents, N-P-tosyl-L-phenylalaninechloromethyl ketone, N-P-tosyl-L-lysinechloromethyl ketone, and puromycin but not by protease inhibitors of microbial origin. The enzyme was activated by the addition of Co2+ and sulfhydryl compounds. The aminopeptidase enhanced proteolysis when the enzyme was added to the protease assay system with purified rat-liver cytosolic neutral protease, suggesting the cooperative action of aminopeptidase in the overall process of protein degradation.

Amino Acid Sequence↗
PubMed1977

Oligomycin sensitivity of mitochondrial sulfhydryl groups.

Dithionitrobenzoate has been used to titrate sulfhydryl groups of rat liver mitochondria in glutamate buffer, pH 7.4. Reaction with oligomycin and different SH reagents preceded the SH titration. Under these conditions it was found that 2-mercaptopropionylcne and N-ethylmaleimide reacted in an oligomycin-sensitive manner, so that the control values (in the absence of SH reagent) were obtained. Similar concentrations of mersalyl and of N-(N-acetyl-4-sulfamoylphenyl) maleimide, in the presence of oligomycin, enhanced reactivity toward Nbs2. The concentration range of oligomycin-sensitive SH groups was thus defined between approx. 5 and 9 nmol reagent/mg mitochondrial protein. In this way, a differentiation between SH grops, which are implicated in phosphate transport antd those, which react in an oligomycin-sensitive manner, and which are probably connected with the coupling mechanism was achieved.

Animals↗
PubMed1990

Significance and redox state of SH groups in pyruvate carrier isolated from bovine heart mitochondria.

The role and properties of -SH groups of purified pyruvate (monocarboxylate) carrier were investigated. After isolation, this protein has all -SH groups in the oxidized state. Upon reduction, the carrier can be labelled with eosin-5-maleimide. The shift in apparent Mr after the labelling points to the presence of at least two cysteine residues. Pyruvate uptake in the reconstituted system is inhibited by both permeable (eosin-5-maleimide at 1 mM concentration) and impermeable (mersalyl, p-chloromercuribenzoate) -SH group reagents. Phenylarsine oxide inhibits pyruvate transport only slightly (20%), but the inhibition is enhanced after preincubation with the substrate.

Animals↗
PubMed1976

Possible relationship between membrane proteins and phospholipid asymmetry in the human erythrocyte membrane.

After incubation of human erythrocytes at 37 degrees C in the absence of glucose (A) for 24 h, (B) for 4 h with 8 mM hexanol or (C) for 3 h with SH reagents, phosphatidylethanolamine becomes partly susceptible to hydrolysis by phospholipase A2 from Naja naja. The presence of glucose during the pretreatments suppresses this effect, except in the case of SH reagents that inhibit glycolysis. After incubation with tetrathionate, up to 45% of the phosphatidylethanolamine is degraded by the enzyme, an amount considerably in excess of the 20% attacked in fresh erythrocytes. Pancreatic phospholipase A2, an enzyme unable to hydrolyse the phospholipids of intact erythrocytes, partially degrades phosphatidylcholine and phosphatidylethanolamine of erythrocytes pretreated with hexanol or SH reagents. Reagents capable of oxidizing SH groups to disulfides (tetrathionate, o-iodosobenzoate and hydroquinone) even render susceptible to pancreatic phospholipase A2 phosphatidylserine, a phospholipid supposed to be entirely located in the inner lipid layer of the membrane. Alkylating or acylating SH reagents have no such effect. It is postulated that disulfide bond formation between membrane protein SH groups leads to an alteration in protein-phospholipid interactions and consequently induces a reorientation of phospholipids between the inner and the outer membrane lipid layer.

Biological Transport↗
PubMed1980

Studies on (Na+ + K+)-activated ATPase. XLVI. Effect of cation-induced conformational changes on sulfhydryl group modification.

(1) (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.1.6.3) contains 34 sulfhydryl groups on the catalytic subunit, and two on the glycoprotein subunit. Under native conditions, only sulfhydryl groups on the catalytic subunit are accessible to modifying reagents. (2) The degree of inhibition of (Na+ + K+)-ATPase activity by N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoic acid) depends on the cations present in the reaction medium. Mg2+ strongly enchances the inhibitory effects of both sulfhydryl reagents. The effects of Mg2+ on the inhibition by 5,5'-dithiobis(2-nitrobenzoic acid) are counteracted by the addition of Na+ or K+. Na+ has no more effect than choline on the inhibition by 5,5'-dithiobis(2-nitrobenzoic acid), but it enhances the inhibitory effect of N-ethylmaleimide at low Na+ concentrations (less than 10 mM). Low concentrations of K+ (less than 10 mM) slightly protect the enzyme against modification. (3) Titration of residual sulfhydryl groups reveals that these ions do not only influence modification of essential sulfhydryl groups, but also that of sulfhydryl groups which are not essential for the enzyme activity. (4) These results indicate that Na+, K+ and Mg2+ have marked effects on the conformation of the catalytic subunit of (Na+ + K+)-ATPase. Various enzyme conformations can be induced, depending on the concentration and the kind of cation added. The largest effects are observed after addition of Mg2+.

Animals↗
PubMed1980

Effects of thiol-reactive agents on amino acid transport by sheep erythrocytes.

(1) Sheep erythrocytes possess a facilitated diffusion transport system selective for small neutral amino acids of intermediate size (C-system). The effects of seven thiol-reactive agents on this system were investigated. (2) L-Alanine influx by this route was inhibited by HgCl2, p-chloromercuriphenylsulphonate (PCMBS), azodiacarboxylic acid bisdimethylamide (diamide), N-ethylmaleimide and t-butylhydroperoxide. Iodoacetamide and 5,5'-dithiobis(2-nitrobenzoate) had no effect. (3) Detailed analysis of these inhibitor effects suggested the presence of three distinct classes of cellular thiol groups essential for normal transport function. (4) Class 1 thiols react with PCMBS and are located on the outer surface of the cell membrane in the region of the transport site. Class 2 thiols react with N-ethylmaleimide and diamide but are not affected by t-butylhydroperoxide. Class 3 thiols are oxidized during t-butylhydroperoxide treatment and are presumably also attacked by diamide and N-ethylmaleimide. (5) Class 3 thiols are either intracellular GSH or reactive thiols which readily form mixed disulphides with GSSG. Any direct involvement of GSH in amino acid transport is not mediated by the gamma-glutamyl cycle.

Amino Acids↗
PubMed1980

125I-Labeled alpha-bungarotoxin and [3H]quinuclidinylbenzilate binding to rat brain membranes. Effects of physical, chemical and enzymatic treatments.

The effects of various physical, chemical and enzymatic treatments of rat brain membranes were investigated with respect to 125I-labeled alpha-bungarotoxin ([125I]alpha BuTx) and [3H]quinuclidinylbenzilate ([3H]QNB) binding. Binding appeared relatively stable to autolysis, mechanical shearing, freeze-thawing, and divalent cation addition (Sr2+) or removal (EGTA, EDTA). Binding for [125I]-alpha BuTx was slightly reduced by trypsin digestion of the membranes while both [125I]alpha BuTx and [3H]QNB binding were reduced by phospholipase A2 digestion (Crotalus adamantus phospholipase A2 and beta-bungarotoxin). Treatment of the membranes with the disulfide reducing agent, dithiothreitol, resulted in additional [125I]alpha BuTx binding but showed little effect on [3H]QNB binding. Binding of the cholinergic agonists, nicotine and carbamylcholine, was studied by observing their concentration-dependent ability to inhibit [125I]alpha BuTx and [3H]QNB binding, respectively. Membrane sulfhydryl group reduction and endogenous cation removal by EGTA or EDTA resulted in a lowered affinity for nicotine bindng. Alkylation of membranes with N-ethylmaleimide resulted in an increase in carbamylcholine affinity. Other treatments had little or no effect on nicotine or carbamylcholine binding.

Animals↗