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Title: Photoaffinity labeling of rat liver microsomal morphine UDP-glucuronosyltransferase by ( sup 3 H)flunitrazepam

Abstract

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 gelmore » 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).« less

Authors:
;  [1]
  1. Univ. of Iowa, Iowa City (USA)
Publication Date:
OSTI Identifier:
6422678
Resource Type:
Journal Article
Journal Name:
Molecular Pharmacology; (USA)
Additional Journal Information:
Journal Volume: 38:3; Journal ID: ISSN 0026-895X
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; TRANQUILIZERS; RECEPTORS; TRANSFERASES; ENZYME ACTIVITY; METABOLISM; ANDROSTERONE; BIOCHEMICAL REACTION KINETICS; CHEMICAL RADIATION EFFECTS; CHROMATOGRAPHY; ELECTROPHORESIS; GLUCURONIC ACID; INHIBITION; LABELLING; LIVER; MICROSOMES; MOLECULAR WEIGHT; MORPHINE; RATS; TESTOSTERONE; TRACER TECHNIQUES; TRITIUM COMPOUNDS; ULTRAVIOLET RADIATION; ALDEHYDES; ALKALOIDS; ANALGESICS; ANDROGENS; ANDROSTANES; ANIMALS; BODY; CARBOXYLIC ACIDS; CELL CONSTITUENTS; CENTRAL NERVOUS SYSTEM AGENTS; CENTRAL NERVOUS SYSTEM DEPRESSANTS; CHEMISTRY; DIGESTIVE SYSTEM; DRUGS; ELECTROMAGNETIC RADIATION; ENZYMES; GLANDS; HORMONES; HYDROGEN COMPOUNDS; HYDROXY ACIDS; HYDROXY COMPOUNDS; ISOTOPE APPLICATIONS; KETONES; KINETICS; MAMMALS; MEMBRANE PROTEINS; NARCOTICS; OPIUM; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANOIDS; ORGANS; PROTEINS; PSYCHOTROPIC DRUGS; RADIATION CHEMISTRY; RADIATION EFFECTS; RADIATIONS; REACTION KINETICS; RIBOSOMES; RODENTS; SEPARATION PROCESSES; STEROID HORMONES; STEROIDS; VERTEBRATES; 550201* - Biochemistry- Tracer Techniques; 560120 - Radiation Effects on Biochemicals, Cells, & Tissue Culture

Citation Formats

Thomassin, J, and Tephly, T R. Photoaffinity labeling of rat liver microsomal morphine UDP-glucuronosyltransferase by ( sup 3 H)flunitrazepam. United States: N. p., 1990. Web.
Thomassin, J, & Tephly, T R. Photoaffinity labeling of rat liver microsomal morphine UDP-glucuronosyltransferase by ( sup 3 H)flunitrazepam. United States.
Thomassin, J, and Tephly, T R. 1990. "Photoaffinity labeling of rat liver microsomal morphine UDP-glucuronosyltransferase by ( sup 3 H)flunitrazepam". United States.
@article{osti_6422678,
title = {Photoaffinity labeling of rat liver microsomal morphine UDP-glucuronosyltransferase by ( sup 3 H)flunitrazepam},
author = {Thomassin, J and Tephly, T R},
abstractNote = {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).},
doi = {},
url = {https://www.osti.gov/biblio/6422678}, journal = {Molecular Pharmacology; (USA)},
issn = {0026-895X},
number = ,
volume = 38:3,
place = {United States},
year = {Sat Sep 01 00:00:00 EDT 1990},
month = {Sat Sep 01 00:00:00 EDT 1990}
}