Some differences in the conjugation of o-aminophenol and p-nitrophenol by the uridine diphosphate transglucuronylase of mouse-liver homogenates

1. A study of the catalysis of the formation of the glucuronides of o-aminophenol and p-nitrophenol by the uridine diphosphate transglucuronylase of homogenates of female mouse liver has been made, with reference to the effect of reagents reacting with thiol groups. 2. The synthesis of both glucuronides was completely inhibited by organic mercurials and N-ethylmaleimide. The inhibition was only partial with arsenite and the arsenoxides, iodoacetamide and o-iodosobenzoate. 3. The o-aminophenol system was much more sensitive than that for p-nitrophenol to all the thiol reagents, except N-ethylmaleimide, which was equally active in both systems. 4. At very low concentrations of the organic mercurials, the o-aminophenol system was activated. 5. With o-aminophenyl glucuronide formation, complete protection was given by glutathione and cysteine against the organic mercurials, N-ethylmaleimide and iodoacetamide, and partial protection against the arsenicals. Reversal was complete against the mercurials, and very limited against the arsenicals and iodoacetamide. The effects of N-ethylmaleimide and o-iodosobenzoate were irreversible. Results with p-nitrophenol were very similar. 6. Uridine diphosphate transglucuronylase was partially protected against p-chloromercuribenzoate and lewisite oxide by uridine diphosphate glucuronate, but not by o-aminophenol. 7. Glutathione did not prevent the decline in the rate of conjugation of o-aminophenol when homogenates were aged by incubation at 30°. Cysteine was unable to prevent or reverse inactivation by ultrasonic radiation.     

Contact inhibition within hemoglobin S polymer by thiol reagents

N-Ethylmaleimide, a thiol reagent, increases the solubility of deoxyhemoglobin S. We investigated which of the two reacted beta 93 cysteine residues of the Hb tetramer was responsible for the inhibition of Hb S polymerization. Accordingly we compared the solubility of equal mixtures of HbA + HbS, HbA NEM + HbS and HbA + HbS NEM. Upon deoxygenation these mixtures contain about 50% a stable and asymmetrical hybrid alpha 2A beta A beta S, alpha 2A beta A,NEM beta S or alpha 2A beta A beta S,NEM respectively and 25% parental molecules as confirmed by ion-exchange HPLC performed in anaerobic conditions. Within the hybrid molecule, beta A or beta A,NEM chain has to be present in the alpha beta dimer located in trans to the dimer which contains the only beta 6 valine residue participating in intermolecular contacts (dimer in cis), while beta S or beta S,NEM must be in cis position in the hybrid molecule. The solubility of mixtures increases 4% for HbA NEM + HbS and 20% for HbA + HbS NEM mixtures compared to HbA + HbS mixture, indicating that the inhibitory effect of N-ethylmaleimide is more effective in cis than in trans position. The absence of a major role played by N-ethylmaleimide located in trans was supported by the solubility study of a mixture of HbS + Hb Créteil beta 89 Ser----Asn. The beta 89 residue in trans next to the cysteine beta 93 modified the T structure similarly to N-ethylmaleimide, and did not affect intermolecular contacts. Crystallographic studies of molecular contacts within deoxyHbS crystals suggest that the cis inhibitory effect of N-ethylmaleimide can be explained by direct inhibition of 'external' contacts between double strands involving the CD corner of the alpha chains.     

Torpedo acetylcholinesterase is inactivated by thiol reagents

A number of sulphydryl reagents inhibit AChE of Torpedo california with pseudo-first-order kinetics, and inhibition can be retarded by quaternary ligands which bind at either the catalytic or peripheral anionic binding sites. Colorimetric determination with one of the inhibitory sulphydryl agents, 5,5'-dithiobis (2-nitrobenzoic acid), reveals the presence of a single thiol group per catalytic subunit; our data thus suggest that inhibition is achieved by reaction with the single free sulphydryl group of Cys231.     

Effects of lead on K(+)-para-nitrophenyl phosphatase activity and protection by thiol reagents

Lead (Pb) inhibited K(+)-stimulated para-nitrophenyl phosphatase (K(+)-PNPPase) of rat brain P2 fraction in a concentration-dependent manner with IC50 3.5 microM. Altered pH versus activity demonstrated comparable inhibitions by Pb in buffered acidic, neutral and alkaline pH ranges. Inhibition of enzyme activity was higher at lower temperatures (17-27 degrees C) compared to 37 degrees C. Preincubation of enzyme with sulfhydryl (-SH) agents such as cysteine (Cyst) and dithiothreitol (DTT) but not glutathione (GSH) protected against Pb-inhibition. Uncompetitive type of inhibition with respect to the activation of K+ was indicated by a decrease in Vmax from 16.2 to 8.37 mumoles of para-nitrophenol (PNP)/mg protein/hr and Km from 18.99 to 12.39 mM. Kinetic studies on substrate (p-nitrophenyl phosphate) activation in the presence of Pb (3.5 microM) indicated a significant decrease in Vmax from 8.94 to 4.69 mumoles of PNP/mg protein/hr with no change in Km. Cyst (3 microM) and DTT (10 microM) reversed the Pb-inhibited Vmax from 4.69 to 8.38 and 7.24 mumoles of PNP/mg protein/hr respectively. These results suggest that the critical conformational property of K(+)-PNPPase is sensitive to Pb. The data also indicates that the Pb inhibits Na(+)-K+ ATPase system by interacting with dephosphorylation of the enzyme-phosphoryl complex, while Cyst and DTT protected against Pb-inhibition.     

Modification of the mitochondrial sulfonylurea receptor by thiol reagents.

The purpose of this study was to investigate the effects exerted by thiol-modifying reagents on themitochondrial sulfonylurea receptor. The thiol-oxidizing agents (timerosal and 5, 5'-dithio-bis(2-nitrobenzoic acid)) were found to produce a large inhibition (70% to 80%) of specific binding of [(3)H]glibenclamide to the beef heart mitochondrial membrane. Similar effects were observed with membrane permeable (N-ethylmaleimide) and non-permeable (mersalyl) thiol modifying agents. Glibenclamide binding was also decreased by oxidizing agents (hydrogen peroxide) but not by reducing agents (reduced gluthatione, dithiothreitol and the 2,3-dihydroxy-1,4-dithiolbutane). The results suggest that intact thiol groups, facing the mitochondrial matrix, are essential for glibenclamide binding to the mitochondrial sulfonylurea receptor.     

Xanthine oxidase inactivation by reagents that modify thiol groups

1. The presence of xanthine was required for the inhibition of bovine milk xanthine oxidase by o-iodosobenzoate, iodoacetamide, hydrogen peroxide or p-chloromercuribenzoate. 2. Inactivation by p-chloromercuribenzoate was very rapid, was reversed by cysteine and was less in the presence of FAD. Lineweaver-Burk plots showed that the inactivation by p-chloromercuribenzoate was competitive with substrate. 3. Inactivation by o-iodosobenzoate, iodoacetamide or hydrogen peroxide could not be reversed by cysteine or xanthine. However, the presence of xanthine during the incubation with inhibitor protected the enzyme against o-iodosobenzoate but not against iodoacetamide or hydrogen peroxide. 4. p-Chloromercuribenzoate protected the enzyme against inactivation by hydrogen peroxide.     

Effect of some thiol reagents on erythrocyte adenosine deaminase (ADA) activity

The effect of three thiol reagents on erythrocyte adenosine deaminase (ADA) activity has been studied. Oxidized glutathione and iodoacetate do not alter ADA activity, while the treatment with p-chloromercuribenzoate at similar concentrations results in a reduction of enzymatic activity which is statistically significant only for ADA 1, but not ADA 2-1 phenotype haemolysates.     

Influence of reagents reacting with metal, thiol and amino sites of catalytic activity and l-phenylalanine inhibition of rat intestinal alkaline phosphatase

1. Studies on the inactivation of rat intestinal alkaline phosphatase by several metal-binding agents, namely EDTA, 8-hydroxyquinoline, pyridine-2,6-dicarboxylic acid, αα′-bipyridyl, o-phenanthroline and sodium cyanide, indicated the functional role of a metal, probably zinc, in the catalysis. The metal ligands lowered stereospecific uncompetitive inhibition of the enzyme by l-phenylalanine by an extent that paralleled the decline in enzyme activity. 2. The thiol reagents p-hydroxymercuribenzoate, iodoacetamide and iodine inactivated rat intestinal phosphatase. The enzyme could be protected from inactivation by either cysteine or substrate. The l-phenylalanine inhibition remained unchanged only in the presence of moderately inactivating concentrations of the thiol reagents. 3. Inactivation of the enzyme by the amino-group-blocking reagent, O-methylisourea, provided ample evidence for the participation in the catalysis of the -amino group of lysine. At the same time, l-phenylalanine inhibition remained unaltered even when the enzyme was strongly inactivated. This -amino-group-blocked enzyme exhibited no change in migration in starch gel, in contrast with enzyme treated with acetic anhydride, formaldehyde or succinic anhydride. The Michaelis constant of the enzyme was enhanced by such modifications, but the optimum pH remained the same. 4. d-Phenylalanine acted as a competitive or `co-operative' activator for intestinal alkaline phosphatase after it had been modified by acetylation.     

Kinetics of polymerization of hemoglobin S modified by thiol reagents and by oxidation

The effects of four thiol reagents on the kinetics of polymerization of hemoglobin S have been studied in high phosphate buffer (1.8 M), in the presence (3 mM) or absence of sodium dithionite, depending on the reduction of mixed disulfides of Hb in the presence of this reducing agent. The effect of oxidized forms (methemoglobin) of HbS on the kinetics of aggregation of deoxyHbS was also studied because of the presence of 33% metHbS when HbS was modified by 4-aminophenyl disulfide. In the presence of sodium dithionite, the delay times prior to polymerization of deoxyHbS modified by N-ethylmaleimide, iodoacetamide and 4-aminophenyl disulfide were, respectively, 1.5-, 1.35- and 1.15-times longer than that of native deoxyHbS. The results indicate that the radicals bound to the cysteine beta 93 residue inhibit the contacts in the polymer formation to various extents but do not modify the size of the nuclei.