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Microsomal UDP‐Glucuronyltransferase in Rat Liver: Oxidative Activation
Author(s) -
Letelier María Eugenia,
Pimentel Alejandra,
Pino Patricia,
Lepe Ana María,
Faúndez Mario,
Aracena Paula,
Speisky Hernán
Publication year - 2005
Publication title -
basic and clinical pharmacology and toxicology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.805
H-Index - 90
eISSN - 1742-7843
pISSN - 1742-7835
DOI - 10.1111/j.1742-7843.2005.pto_96612.x
Subject(s) - microsome , dithiothreitol , chemistry , incubation , glutathione , enzyme , biochemistry , enzyme assay , cysteine , microsoma
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.