Inactivation of rat pineal hydroxyindole-O-methyltransferase by disulfide-containing compounds

Rat pineal hydroxyindole-O-methyltransferase activity in crude homogenates is reduced by treatment with disulfides. Cystamine (IC50 = 128 microM) and selenocystamine (IC50 = 13 microM) are the most potent compounds tested. Reduced cystamine (cysteamine) and diaminohexane are inactive. N,N'-Diacetylcystamine, penicillamine disulfide, and glutathione disulfide are less potent or inactive; but several peptides (oxytocin, vasopressin, and arginine vasotocin) are active. Inactivation by cystamine is time- and temperature-dependent and is accelerated at higher pH. Disulfide treatment of intact pinealocytes also inactivates the enzyme. Addition of dithiothreitol during the enzyme assay completely reactivates inactivated enzyme formed by disulfide treatment of homogenates or intact cells. Rat hydroxyindole-O-methyltransferase is also inactivated in the absence of added disulfides and dissolved O2. This spontaneous inactivation is time-, temperature-, and pH-dependent and can be completely prevented, but not reversed, by dithiothreitol. In contrast to the inhibitory effects of cystamine on the rat enzyme, cystamine does not alter bovine hydroxyindole-O-methyltransferase and increases ovine hydroxyindole-O-methyltransferase activity. The bovine and ovine enzymes do not become inactive in the absence of added disulfides. Together these observations indicate that rat pineal hydroxyindole-O-methyltransferase can be inactivated by a protein thiol:disulfide exchange mechanism. This mechanism may contribute to the physiological regulation of this enzyme in the rat pineal gland but does not appear to be a common feature of pineal hydroxyindole-O-methyltransferase regulation in all species.     

Inactivation of human placenta glutathione S-transferase by SH/SS exchange reaction with biological disulfides

The oxidized glutathione inhibited the activity of glutathione S-transferase purified from human placenta just through competitive inhibition. On the other hand, cystine and cystamine inactivated the activity by pseudo first-order in low concentrations, accompanying the stoichiometric incorporation of the radioactivity of [14C]-cystine to the enzyme protein until a half mole per one subunit. This and the protective effect of glutathione analogues suggested that the SH/SS exchange reaction occurred between the disulfide and the SH group near the glutathione binding site of the enzyme to form a mixed disulfide.     

Ligand-induced conformational states of rabbit liver fructose 1,6-bisphosphatase as revealed by digestion with subtilisin

Fructose 1,6-bisphosphatase undergoes specific conformational changes in the presence of the substrate fructose 1,6-bisphosphate and of the allosteric modifier, AMP and also on activation by cystamine. These changes can be monitored by observing the changes in sensitivity to digestion by subtilisin. In the presence of AMP the enzyme is protected against the action of subtilisin. Some protection is also observed with high concentrations of fructose bisphosphate while low concentrations of this substrate, which are ineffective alone, enhance the protective effect of low concentrations of AMP. The results suggest that AMP induces a resistant conformation, and that fructose bisphosphate promotes the binding of AMP. Divalent cations, although essential for activity, do not protect the enzyme against digestion by subtilisin. The native enzyme is activated by disulfide exchange with cystamine, and the activated enzyme is also more resistant to subtilisin. Thus, the enzyme in both inhibited (AMP) and activated conformations (cystamine) is rendered resistant to modification by proteolysis.     

Cystamine inhibits caspase activity. Implications for the treatment of polyglutamine disorders

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an abnormally expended polyglutamine domain. There is no effective treatment for HD; however, inhibition of caspase activity or prevention of mitochondria dysfunction delays disease progression in HD mouse models. Similarly administration of cystamine, which can inhibit transglutaminase, prolonged survival of HD mice, suggesting that inhibition of transglutaminase might provide a new treatment strategy. However, it has been suggested that cystamine may inhibit other thiol-dependent enzymes in addition to transglutaminase. In this study we show that cystamine inhibits recombinant active caspase-3 in a concentration-dependent manner. At low concentrations cystamine is an uncompetitive inhibitor of caspase-3 activity, becoming a non-competitive inhibitor at higher concentrations. The IC(50) for cystamine-mediated inhibition of caspase-3 activity in vitro was 23.6 microm. In situ cystamine inhibited in a concentration-dependent manner the activation of caspase-3 by different pro-apoptotic agents. Additionally, cystamine inhibited caspase-3 activity to the same extent in cell lines stably overexpressing wild type tissue transglutaminase (tTG), a mutant inactive tTG, or an antisense for tTG, demonstrating that cystamine inhibits caspase activity independently of any effects it may have on the transamidating activity of tTG. Finally, treatment with cystamine resulted in a robust increase in the levels of glutathione. These findings demonstrate that cystamine may prolong neuronal survival and delay the onset of HD by inhibiting caspases and increasing the level of antioxidants such as glutathione.     

Cystamine induces toxicity in hepatocytes through the elevation of cytosolic Ca2+ and the stimulation of a nonlysosomal proteolytic system

Infusion of cystamine into the isolated, perfused rat liver resulted in tissue damage preceded by the formation of cystamine-protein mixed disulfides which were mainly detected in the plasma membrane fraction. Hepatotoxicity was prevented when dithiothreitol was infused after cystamine or when the calcium antagonist, verapamil, was co-infused with the disulfide. In isolated hepatocytes, the formation of cystamine-protein mixed disulfides was associated with an inhibition of plasma membrane Ca2+-ATPase activity and a decreased rate of Ca2+ efflux from the cells. This resulted in intracellular Ca2+ accumulation which was followed by a stimulation of both phospholipid hydrolysis and proteolysis, as indicated by enhanced rates of release of radioactivity from hepatocytes prelabeled with [14C]arachidonate and [14C]valine, respectively. Preincubation of hepatocytes with the calmodulin inhibitor, calmidazolium, or with the phospholipase inhibitors, chlorpromazine and dibucaine, inhibited the stimulation of [14C]arachidonate release by cystamine. However, none of these agents prevented the onset of cystamine toxicity in hepatocytes. In contrast, pretreatment of the cells with antipain or leupeptin, two inhibitors of Ca2+-activated proteases, abolished the stimulation of proteolysis by cystamine and also protected the cells from cystamine toxicity. Our results suggest that the perturbation of intracellular Ca2+ homeostasis by cystamine is caused by the inhibition of Ca2+ efflux associated with the formation of cystamine-protein mixed disulfides in the plasma membrane and that subsequent cytotoxicity results from Ca2+-activation of a nonlysosomal proteolytic system.     

Gamma-glutamylcysteine synthetase. Interactions of an essential sulfhydryl group

gamma-Glutamylcysteine synthetase (isolated from rat kidney) has one sulfhydryl group that reacts with 5,5'-dithiobis-(2-nitrobenzoate). This single exposed sulfhydryl group is not required for enzyme activity. The enzyme is potently inactivated by cystamine, which apparently interacts with a sulfhydryl group at the active site to form a mixed disulfide. 5,5'-Dithiobis-(2-nitrobenzoate) does not interact with the sulfhydryl group that reacts with cystamine. After the enzyme was 90% inactivated by reaction with cystamine, 3.4 mol of 5,5'-dithiobis-(2-nitrobenzoate) reacted per mol of enzyme, indicating that binding of cystamine exposes sulfhydryl groups which are apparently buried or unreactive in the native enzyme. L-Glutamate (but not D-glutamate or L-alpha-aminobutyrate) protected against inactivation by cystamine. In contrast, ATP enhanced the rate of inactivation by cystamine, and the apparent Km value for this effect is similar to that for ATP in the catalytic reaction. Studies on the structural features of cystamine that facilitate its interaction with the enzyme showed that selenocystamine, monodansylcystamine, and N-[2[2-aminoethyl)-dithio)ethyl]-4-azido-2-nitrobenzeneamine are also good inhibitors. Whereas S-(S-methyl)cysteamine-Sepharose does not interact with the enzyme (Seelig, G. F., and Meister, A. (1982) J. Biol. Chem. 257, 5092-5096), S-(S-methyl)cysteamine is a potent inhibitor; 1 mol of this compound completely inactivated 1 mol of enzyme. In the course of this work, a useful modification of the method for isolating this enzyme from kidney was developed.     

Characterization of the interaction of yeast enolase with polynucleotides.

Yeast enolase is inhibited under certain conditions by DNA. The enzyme binds to single-stranded DNA-cellulose. Inhibition was used for routine characterization of the interaction. The presence of the substrate 2-phospho-D-glycerate reduces inhibition and binding. Both yeast enolase isozymes behave similarly. Impure yeast enolase was purified by adsorption onto a single-stranded DNA-cellulose column followed by elution with substrate. Interaction with RNA, double-stranded DNA, or degraded DNA results in less inhibition, suggesting that yeast enolase preferentially binds single-stranded DNA. However, yeast enolase is not a DNA-unwinding protein. The enzyme is inhibited by the short synthetic oligodeoxynucleotides G6, G8 and G10 but not T8 or T6, suggesting some base specificity in the interaction. The interaction is stronger at more acid pH values, with an apparent pK of 5.6. The interaction is prevented by 0.3 M KCl, suggesting that electrostatic factors are important. Histidine or lysine reverse the inhibition at lower concentrations, while phosphate is still more effective. Binding of single-stranded DNA to enolase reduces the reaction of protein histidyl residues with diethylpyrocarbonate. The inhibition of yeast enolase by single-stranded DNA is not total, and suggests the active site is not directly involved in the interaction. Binding of substrate may induce a conformational change in the enzyme that interferes with DNA binding and vice versa.     

Potentiation and inhibition of nicotinic effects on striated muscle by the tetramine disulfide benextramine

In low concentrations (0.3-3 muM) the tetramine disulfide benextramine (BHC; N,N'-bis[6-(o-methoxybenzylamino)-n-hexyl]cystamine) potentiated the contracture of the isolated frog rectus abdominis muscle caused by acetylcholine but in the presence of physostigmine or in a higher concentration (10 muM) it inhibited. Benextramine only inhibited the contracture caused by carbachol or butyrylcholine. The all-carbon analog of benextramine only inhibited the effect of acetylcholine. The inhibitory effects of benextramine and its carbon analog were noncompetitive and readily reversible but the potentiating effect of benextramine was not readily reversible.     

Protease inhibitors do not prevent the killing of cultured hepatocytes by cystamine

A study was made of the conditions of the killing of cultured hepatocytes by the reactive disulfide cystamine. Six to 12 mM cystamine killed up to 60% of the hepatocytes within 3 hours. The cytosolic calcium ion concentration rose prior to the loss of viability. Treatment with EGTA in a Ca2+-free medium lowered the initial Ca2+ concentration and prevented the rise in response to cystamine. However, there was no change in the number of dead cells. Furthermore, the sensitivity of cultured hepatocytes to cystamine was unaffected by the concentration of calcium in the culture medium. Addition to the culture medium of 3 protease inhibitors, leupeptin, antipain, or chymostatin, did not reduce the extent of cell killing by cystamine despite an inhibition of protein degradation. These data do not support the hypothesis that the toxicity of cystamine is necessarily mediated by proteases activated by a rise in the cytosolic calcium ion concentration.     

Inhibition of Phosphorylation of Synapsin I and Other Synaptosomal Proteins by β-Bungarotoxin, a Phospholipase A2 Neurotoxin

Some snake venom neurotoxins, such as beta-bungarotoxin (beta-BuTX), which possess relatively low phospholipase A2 (PLA2) activity, act presynaptically to alter acetylcholine (ACh) release both in the periphery and in the CNS. In investigating the mechanism of this action, we found that beta-BuTX (5 and 15 nM) inhibited phosphorylation, in both resting and depolarized synaptosomes, of a wide range of proteins, including synapsin I. Naja naja atra PLA2, which has higher PLA2 activity, also inhibited phosphorylation but was less potent than beta-BuTX. At 1 nM, beta-BuTX and N. n. atra PLA2 inhibited phosphorylation of synapsin I only in depolarized synaptosomes. Synaptosomal ATP levels were not affected by 5 or 15 nM beta-BuTX or by 5 nM N. n. atra PLA2. Limited proteolysis, using Staphylococcus aureus V-8 protease, indicated that beta-BuTX inhibited phosphorylation of synapsin I in both the head and the tail regions. The inhibition of phosphorylation was not antagonized by nordihydroguaiaretic acid or indomethacin, suggesting that arachidonic acid derivatives do not mediate this inhibition. Furthermore, inhibition of phosphorylation by beta-BuTX and N. n. atra PLA2 was not altered in the presence of the phosphatase inhibitor okadaic acid, suggesting that stimulation of phosphatase activity is not responsible for this inhibition. Inhibition of protein phosphorylation by PLA2 neurotoxins and enzymes may be associated with an inhibition of ACh release.     

Regulation of rat liver microsomal glutathione S-transferase activity by thiol/disulfide exchange

The regulation of purified glutathione S-transferase from rat liver microsomes was studied by examining the effects of various sulfhydryl reagents on enzyme activity with 1-chloro-2,4-dinitrobenzene as the substrate. Diamide (4 mM), cystamine (5 mM), and N-ethylmaleimide (1 mM) increased the microsomal glutathione S-transferase activity by 3-, 2-, and 10-fold, respectively, in absence of glutathione; glutathione disulfide had no effect. In presence of glutathione, microsomal glutathione S-transferase activity was increased 10-fold by diamide (0.5 mM), but the activation of the transferase by N-ethylmaleimide or cystamine was only slightly affected by presence of glutathione. The activation of microsomal glutathione S-transferase by diamide or cystamine was reversed by the addition of dithiothreitol. Glutathione disulfide increased microsomal glutathione S-transferase activity only when membrane-bound enzyme was used. These results indicate that microsomal glutathione S-transferase activity may be regulated by reversible thiol/disulfide exchange and that mixed disulfide formation of the microsomal glutathione S-transferase with glutathione disulfide may be catalyzed enzymatically in vivo.     

Interaction of L- and D-3-amino-1-chloro-2-pentanone with gamma-glutamylcysteine synthetase

The optical isomers of 3-amino-1-chloro-2-pentanone, which are the alpha-chloroketone analogs of L- and D-alpha-aminobutyrate, were synthesized and found to be highly potent irreversible inactivators of gamma-glutamylcysteine synthetase. These chloroketones are 20 to 30 times more active than L-2-amino-4-oxo-5-chlorpentanoate. L- and D-Glutamate, in the presence of Mg2+ or Mn2+, protect the enzyme against inactivation. The enzyme is almost completely inhibited by cystamine under conditions in which 0.5 mol of this compound is bound/mol of enzyme. Treatment of the enzyme with cystamne, which produces inhibition that is reversible by dithiothreitol, prevents the interaction of the new chloroketones, L-2-amino-4-oxo-5-chloropentanoate and methionine sulfoximine with the enzyme. The findings suggest that a sulfhydryl group at the active site interacts with the chloroketones and with cystamine and that the chloroketone inhibitors and cystamine bind to the enzyme as glutamine analogs. The data also suggest that a gamma-glutamyl-S-enzyme intermediate may be formed in the reaction catalyzed by this enzyme.     

Rapid and reversible activation of acetyl CoA hydrolase in intact pineal cells by disulfide exchange

Using intact pinealocytes in suspended cell culture it has been determined that acetyl CoA hydrolase activity can be rapidly increased by treatment with cystamine. Similar results are seen with diacetylcystamine, but not with GSSG, penicillamine disulfide, nor with oxidized DTT. The activation of acetyl CoA hydrolase by cystamine is reversible: after cystamine treatment is terminated, enzyme activity decreases slowly in cell culture. It is also possible to reverse the activation by treating homogenates of cystamine-treated cells with dithiothreitol. These observations are consistent with previous findings indicating that pineal acetyl CoA hydrolase activity can be regulated $\text{via}$ protein thiol: disulfide exchange. The observations presented in this report also indicate that conditions within the cell allow this type of reaction to take place, and raise the possibility that disulfide exchange mechanisms may be physiologically involved in the intracellular regulation of the activity of this and perhaps other enzymes.     

S-METHYLTHIO-CYSTEINE AND CYSTAMINE ARE POTENT STIMULATORS OF THIOL PRODUCTION AND GLUTATHIONE SYNTHESIS

The effects of methylthio-cysteine disulfide (MT-Cy) and cystamine (CAM) on the thiol production and glutathione content of a human T cell line (CEM-SS) have been investigated. MT-Cy per se and CAM in the presence of cystine greatly enhanced thiol production and glutathione content of cells while cystine alone exerted no or slight influence in the first hours. The MT-Cy- or CAM-induced extracellular SH-generation was observed both in a complete nutrient medium and even more in SH-free D-PBS. The acid-soluble thiol level and glutathione content of cells elevated markedly (up to 5–6 fold in two hours) when incubating cells in complete medium. Inhibition of glutathione synthesis by DL-buthionine (S,R)-sulfoximine did not alter the MT-Cy- or CAM-induced extracellular thiol production indicating that glutathione synthesis is not involved in this effect. The results suggest that MT-Cy easily enters the cells thus accelerating the thiol cycle in SH-poor medium while CAM promotes cystine uptake into the cells. Phenylalanine and leucine inhibited both MT-Cy- and CAM-dependent thiol production in D-PBS most effectively suggesting the involvement of the L membrane transport system in these effects. © 1998 Elsevier Science Inc.     

Effect of protein and peptide inhibitors on the activity of protein disulfide isomerase

The protein disulfide isomerase catalyzed reduction of insulin by glutathione is inhibited by peptides of various length and amino acid composition. Peptide inhibitors are competitive against insulin and noncompetitive against GSH, consistent with a sequential rather than a double displacement mechanism. Peptides of unrelated primary sequence that do not contain cysteine inhibit the GSH-insulin transhydrogenase activity of PDI, and the affinity of these peptides toward the enzyme is largely dependent on the peptide length rather than composition, hydrophobicity, or charge. Cysteine-containing peptides are 4-8-fold better inhibitors than non-cysteine-containing peptides of the same length, suggesting a cysteine-specific component to the interaction with the enzyme. Oxidized insulin chain B also inhibits the oxidative folding of reduced ribonuclease in a glutathione redox buffer with an inhibition constant that is comparable to that observed for the inhibition of insulin reduction, suggesting a similar if not identical binding site for the catalysis of oxidative protein folding and the reduction of insulin.     

Cooperative behavior in the thiol oxidation of rabbit muscle glycogen phosphorylase in cysteamine/cystamine redox buffers

Glycogen phosphorylase a and b are irreversibly inactivated by oxidation with the disulfide cystamine. The mechanism is complex and involves oxidation of at least two classes of sulfhydryl groups. The oxidation of one or more of the first class of 4 +/- 1 sulfhydryl groups is reversible, but the equilibrium constant for the oxidation is so unfavorable (1 X 10(-4)) that the micromolar concentrations of cysteamine released stoichiometrically with enzyme oxidation are sufficient to prevent complete oxidation even in the presence of 100 mM cystamine. The rapid phase of inactivation of phosphorylase b, which is first order in cystamine (k = 2.9 +/- 0.3 M-1 min-1), is followed by the oxidation of 5 +/- 1 groups in an irreversible process that is second order in cystamine concentration (k = 3.9 +/- M-2 min-1). Similar behavior is observed for phosphorylase a, although the behavior is complicated by association/dissociation equilibrium. The second-order dependence of the rate of irreversible inactivation on cystamine concentration is interpreted in terms of a "cooperative" model in which a rapidly reversible thermodynamically unfavorable equilibrium oxidation of one or more sulfhydryl groups must precede the irreversible oxidation of one or more additional sulfhydryl groups. The thiol/disulfide oxidation equilibrium constant for the initial reversible reaction is estimated to be at least 10(4) less favorable than that for the reversible oxidation of phosphofructokinase.     

Anti-proliferative activity of L-651,582 correlates with calcium-mediated regulation of nucleotide metabolism at phosphoribosyl pyrophosphate synthetase

L-651,582, 5-amino-[4-(4-chlorobenzoyl)-3,5-dichlorobenzyl]-1, 2,3-triazole-4-carboxamide, is an antiproliferative and antiparasitic agent which inhibits nucleotide metabolism in mammalian cells. The drug equivalently inhibited 3H-hypoxanthine, 14C-adenine, and 14C-formate incorporation into nucleotide pools in Madin-Darby bovine kidney (MDBK) cells, suggesting depletion of the supply of phosphoribosyl pyrophosphate, (PRPP), required for each of these independent pathways. Inhibition of nucleotide metabolism correlated with inhibition of proliferation for three cell types with differing sensitivities toward the drug. L-651,582 inhibited incorporation of 3H-hypoxanthine into nucleotide pools with either glucose, uridine, or ribose as carbon source suggesting a block at PRPP synthetase, rather than a block in a pathway supplying ribose-5-phosphate. PRPP synthetase was not inhibited directly by the compound, indicating regulation of the enzyme in intact cells. Drug treatment did not kill cells but reduced the fraction of cells in S and G2/M while increasing the population in G1. Inhibition of uptake of 45Ca was demonstrated at concentrations identical to those required for inhibition of nucleotide metabolism or proliferation. Inhibition of cellular PRPP biosynthesis rates were also observed using EGTA to lower calcium levels. These data suggest a previously unrecognized link between calcium entry, the regulation of nucleotide biosynthesis at PRPP synthetase, and the rate of proliferation of mammalian cells.     

Inhibition of mammalian 5-lipoxygenase by aromatic disulfides

As a primary step in leukotriene biosynthesis, arachidonic acid is converted into 5-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid by 5-lipoxygenase. This enzyme is studied in the supernatant fraction from sonified RBL-1 cells, a preparation that converts [1-14C]arachidonic acid to 5-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid and several 5,12-dihydroxyeicosatetraenoic acids including LTB4. In order to examine the reversibility of inhibitors, the supernatant fraction can be depleted of low molecular weight constituents by vacuum filtration. The 5-lipoxygenase is irreversibly inhibited by 500 microM N-ethyl-maleimide or 300 microM methyl methanethiolsulfonate, reagents that react covalently with protein sulfhydryl groups. In contrast, diphenyl disulfide reversibly inhibits this enzyme at 1-5 microM, irrespective of the GSH concentration in the supernatant. KCN also inhibits 5-lipoxygenase at 4 mM, suggesting the presence of a metal-containing prosthetic group. These observations imply that diphenyl disulfide and similar molecules with electron-releasing substituents on the aromatic rings could inhibit by binding to an electrophilic metallic center, the binding being stabilized by hydrophobic interactions between the enzyme and the aromatic groups on the flexible disulfide. Even though diphenyl disulfide does not inhibit soybean 15-lipoxygenase or endoperoxide synthase in cell-free systems, this compound does suppress prostaglandin as well as leukotriene synthesis in intact murine peritoneal macrophages and CXBG cells. Since lipoxygenases are susceptible to peroxide activation and peroxidase deactivation, changes in the redox state of the cell may alter arachidonic acid metabolism as effectively as actual enzyme inhibition.     

Oxidation of thiols in the Ca2+-ATPase of sarcoplasmic reticulum microsomes

We recently showed that oxidative stress impairs the function of the sarcoplasmic reticulum to transport and retain calcium. Inhibition results primarily from oxidation of one or more thiol groups in the Ca2+-ATPase. We now report that thiol oxidation does not result in disulfide formation. Oxidative inhibition of Ca2+-ATPase activity was not reversed by dithiothreitol. Also, arsenite, which crosslinks dithiols, only mildly inhibited Ca2+-ATPase activity and protected against inhibition by peroxydisulfate. These data suggest the thiols susceptible to oxidation are not spatially close enough to form a disulfide. Furthermore, these thiols appear to be involved in some aspect of phosphoenzyme formation. ATP, in the presence of calcium and magnesium, protected against inhibition of Ca2+-ATPase activity by both oxidants and thiol-binding agents. Both inhibitors also decreased binding of the nucleotide analogue TNP-AMP after phosphorylation by Pi. Dithiothreitol and arsenite were protective. In conclusion, reversible redox regulation of the Ca2+-ATPase of sarcoplasmic reticulum by thiol-disulfide exchange does not occur. However, some other mechanism of redox regulation may operate because the enzyme is sensitive to oxidants, thiol-binding agents and activity can be enhanced by prolonged exposure to dithiothreitol.     

Recombinant rat liver guanidinoacetate methyltransferase: reactivity and function of sulfhydryl groups

Rat liver guanidinoacetate methyltransferase, produced in Escherichia coli by recombinant DNA technique, possesses five cysteine residues per molecule. No disulfide bond is present. Analysis of the chymotryptic peptides derived from the iodo[14C]acetate-modified enzyme shows that Cys-90, Cys-15, Cys-219, and Cys-207 are alkylated by the reagent in order of decreasing reactivity. Incubation of the enzyme with excess 5,5'-dithiobis(2-nitrobenzoate) (DTNB) in the absence and presence of cystamine [2,2'-dithiobis(ethylamine)] causes the appearance of 4 and 5 mol of 2-nitro-5-mercaptobenzoate/mol of enzyme, respectively. Reaction of the methyltransferase with an equimolar amount of DTNB results in an almost quantitative disulfide cross-linking of Cys-15 and Cys-90 with loss of a large portion of the activity. The methyltransferase is completely inactivated by iodoacetate following nonlinear kinetics. Comparison of the extent of inactivation with that of modification of cysteine residues and the experiment with the enzyme whose Cys-15 and Cys-90 are cross-linked suggest that alkylation of Cys-15 and Cys-90 results in a partially active enzyme and that carboxymethylation of Cys-219 completely eliminates enzyme activity. The inactivation of guanidinoacetate methyltransferase by iodoacetate or DTNB is not protected by substrates. Furthermore, disulfide cross-linking of Cys-15 and Cys-90 or carboxymethylation of Cys-219 does not impair the enzyme's capacity to bind S-adenosylmethionine. Thus, these cysteine residues appear to occur outside the active-site region, but their integrity is crucial for the expression of enzyme activity.