Microsomal UDP-glucuronyltransferase in rat liver: oxidative activation.
Activation of microsomal UDP-glucuronyltransferase (UDPGT) activity by treatment of hepatic microsomes with either detergents or Fe(3+)/ascorbate pro-oxidant system has been reported; however, definite mechanisms underlying these effects have not been clarified. In this work, we characterize Fe(3+)/ascorbate-induced activation of UDPGT activity prior to solubilization with Triton X-100 and after the oxidation process provoked the solubilization of the enzyme. We observed a time-dependent increase in UDPGT activity up to 20 min. incubation of the microsomes with Fe(3+)/ascorbate (3-times); after 20 min. incubation, however, we observed a time-dependent decrease in this activity to basal levels after 4 hr incubation. Treatment of microsomes with 0.1% Triton X-100 (5 min.) lead to a similar increase in UDPGT activity; higher detergent concentrations produced a dose-dependent decrease in this activity to basal levels with 1% Triton X-100. Interestingly, UDPGT activity was susceptible to activation only when associated to microsomal membranes and the loss of activation correlated with the solubilization of this activity. UDPGT activation by either Fe(3+)/ascorbate or Triton X-100 was correlated with an increase in p-nitrophenol apparent K(m) and V(max) values. This activation was prevented or reversed by the reducing agents glutathione, cysteine or dithiothreitol when it was induced by the Fe(3+)/ascorbate. Furthermore, the latter provoked a significant decrease in microsomal thiol content, effect not observed after treatment with Triton X-100. Our results suggest that the main mechanism responsible for Fe(3+)/ascorbate-induced UDPGT activation is likely to be the promotion of protein sulfhydryl oxidation; this mechanism appears to be different from detergent-induced UDPGT activation.
Letelier
María Eugenia
ME
Laboratory of Pharmacology, Department of Pharmacological and Toxicological Chemistry, Faculty of Chemical and Pharmaceutical Sciences, University of Chile, Santiago, Chile. mel@ciq.uchile.cl
Pimentel
Alejandra
A
Pino
Patricia
P
Lepe
Ana María
AM
Faúndez
Mario
M
Aracena
Paula
P
Speisky
Hernán
H
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology*; D003545:Cysteine / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation; D014453:Glucuronosyltransferase / Q000378:metabolism*; D005978:Glutathione / Q000494:pharmacology; D066298:In Vitro Techniques; D007501:Iron / Q000494:pharmacology*; D015227:Lipid Peroxidation / Q000187:drug effects; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology*; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley; D013438:Sulfhydryl Compounds / Q000378:metabolism
D013438:Sulfhydryl Compounds; D017830:Octoxynol; D007501:Iron; D014453:Glucuronosyltransferase; D005978:Glutathione; D003545:Cysteine; D001205:Ascorbic Acid; D004229:Dithiothreitol