Effects of cholesterol on the function and thermotropic properties of pure UDP-glucuronosyltransferase

M Rotenberg,D Zakim

doi:10.1016/s0021-9258(20)64300-9

Abstract

The effects of cholesterol on the activity and thermal properties of a pure, delipidated isoform of UDP-glucuronosyltransferase were examined after incorporation of enzyme into unilamellar bilayers of distearoylphosphatidylcholine (DSPC) or dioleoylphosphatidylcholine (DOPC). Cholesterol, in bilayers of DSPC, decreased enzyme activity and lowered the temperature (from 37 to 30 degrees C) for a reversible transition from the active form of the enzyme to a less active form. These effects could be separated from each other in that the effect on reversible inactivation of the enzyme occurred at lower concentrations of cholesterol than the effect on activity of the active form of the enzyme. In addition, cholesterol in bilayers of DSPC stabilized UDP-glucuronosyltransferase against irreversible thermal inactivation. The extent of stabilization increased with increasing concentration of cholesterol in the bilayers. The effects of cholesterol on UDP-glucuronosyltransferase depended, however, on the nature of the bilayer containing cholesterol. Cholesterol had small effects, if any, on the properties of UDP-glucuronosyltransferase in bilayers of DOPC.

Highlights

In untreated microsomes is constrained as compared with activity in modified microsomes (Leuders and Kuff, 1967; Zakim and Vessey, 1971)
Brenner (1986a) have reported that addition of cholesterol to microsomes from guinea pig liver increased the activity of UDP-glucuronosyltransferase at V, and that supplementation of diet with cholesterol, which increased the cholesterol content of guinea pig liver microsomes, ledto similar modifications of the enzyme (Castuma and Brenner,1986b).In the present work, we report the effects of cholesterol on the function of UDP-glucuronosyltransferase, using a pure isoform designated GT, (Magdalou et al, 1982),that was reconstituted into unilamellar lipid vesicles (ULVs).‘ These data are importantbecause they demonstratethe effects of cholesterol on the properties of UDP-glucuronosyltransferase in a relatively simple, reconstituted system
Small that the functional state of UDP-glucuronosyltransferase in ULVs were prepared by sonication (Barenholz et al, 1977).GT,was situ is modulated by lipid-enzyme interactions (Dannenberg reconstituted into ULVs as in Scotto and Zakim (1985,1988).Briefly, this method consists of the spontaneous insertion of pure enzyme

Summary

Introduction

In untreated microsomes is constrained as compared with activity in modified microsomes (Leuders and Kuff, 1967; Zakim and Vessey, 1971). Shown in Fig. 1are the effects of cholesterol on activities at Vmax(app(a)ctivity extrapolated to infinite concentrations of UDP-glucuronic acid at 0.05 mM p-nitrophenol) for enzyme reconstituted into ULVs of DSPC.

Results

Conclusion