Photoaffinity labeling of rat liver microsomal morphine UDP- glucuronosyltransferase by [3H]flunitrazepam

J Thomassin and TR Tephly

Department of Pharmacology, University of Iowa, Iowa City 52242.

Benzodiazepines have been shown to competitively inhibit morphine glucuronidation in rat and human hepatic microsomes. Flunitrazepam exerted a potent competitive inhibition of rat hepatic morphine UDP- glucuronosyltransferase (UDPGT) activity (Ki = 130 microM). It has no effect on the activity of p-nitrophenol, 17 beta-hydroxysteroid, 3 alpha-hydroxysteroid, or 4-hydroxybiphenyl UDPGTs. Because flunitrazepam is an effective photoaffinity label for benzodiazepine receptors, studied were performed in solubilized rat hepatic microsomes and with partially purified preparations of morphine UDPGT to determine the enhancement of flunitrazepam inhibition and binding to morphine UDPGT promoted by exposure to UV light. Under UV light, flunitrazepam inhibition was markedly enhanced. UV light exposure also led to a marked increase in binding of [3H]flunitrazepam to microsomal protein, which was protected substantially by preincubation with morphine. Testosterone, androsterone, and UDP-glucuronic acid did not protect against UV-enhanced flunitrazepam binding, and morphine did not reverse flunitrazepam binding once binding had occurred. As morphine UDPGT was purified, a good correlation was found between the increases in specific activity of morphine UDPGT and flunitrazepam binding to protein. Chromatofocusing chromatography showed that flunitrazepam bound only to fractions containing active morphine UDPGT, and no binding to 4-hydroxybiphenyl UDPGT was observed. Fluorography of a sodium dodecyl sulfate-polyacrylamide electrophoresis gel of solubilized hepatic microsomes that had been treated with [3H] flunitrazepam under UV light revealed a band with a monomeric molecular weight between 54,000 and 58,000. This monomeric molecular weight compares favorably with the reported monomeric molecular weight of homogeneous morphine UDPGT (56,000). These studies suggest that flunitrazepam binds rather selectively to the morphine binding site of morphine UDPGT and may prove to be a useful probe for this enzyme.

Volume 38, Issue 3, pp. 294-298, 09/01/1990
Copyright © 1990 by American Society for Pharmacology and Experimental Therapeutics

This article has been cited by other articles:

				L. Antonilli, C. Suriano, G. Paolone, A. Badiani, and P. Nencini Repeated Exposures to Heroin and/or Cadmium Alter the Rate of Formation of Morphine Glucuronides in the Rat J. Pharmacol. Exp. Ther., November 1, 2003; 307(2): 651 - 660. [Abstract] [Full Text] [PDF]

				G. R. Rios and T. R. Tephly Inhibition and Active Sites of UDP-Glucuronosyltransferases 2B7 and 1A1. Drug Metab. Dispos., December 1, 2002; 30(12): 1364 - 1367. [Abstract] [Full Text] [PDF]

				C. King, B. Finley, and R. Franklin The Glucuronidation of Morphine by Dog Liver Microsomes: Identification of Morphine-6-O-Glucuronide Drug Metab. Dispos., June 1, 2000; 28(6): 661 - 663. [Abstract] [Full Text]