PURIFICATION OF A MEMBRANE-BOUND UDP-GLUCOSE-STEROL BETA-D-GLUCOSYLTRANSFERASE BASED ON ITS SOLUBILITY IN DIETHYL-ETHER

Membrane-bound UDP-glucose:sterol beta-D-glucosyltransferase (UDPG-SGTase) catalyzes the formation of steryl glucosides from UDP-glucose and free sterols. This enzyme was purified from etiolated oat shoots (Avena sativa L. cv Alfred) in five steps. UDPG-SGTase was solubilized from a microsomal fraction with the detergent n-octyl-beta-D-thioglucopyranoside and then extracted into diethyl ether. Subsequent removal of the organic solvent, resolubilization with an aqueous buffer, and two column chromatographic steps on Q-Sepharose and Blue Sepharose resulted in a 12,500-fold overall purification. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final preparation revealed a 56-kd protein band, the intensity of which correlated with enzyme activity in the respective fractions. Polyclonal antibodies raised against this 56-kD protein did not inhibit enzyme activity but specifically bound to the native UDPG-SGTase. These results suggest that the 56-kD protein represents the UDPG-SGTase. The purified enzyme was specific for UDP-glucose (K-m = 34 mu M), for which UDP was a competitive inhibitor (inhibitor constant = 47 mu M). In contrast to the specificity with regard to the glycosyl donor, UDPG-SGTase utilized all tested sterol accepters, such as beta-sitosterol, cholesterol, stigmasterol, and ergosterol.