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-SGT ase) catalyzes the formation of steryl glucosides from UDP-glucose and free sterols. This enzyme was purified from etiolated oat shoots (Ave na sativa L. cv Alfred) in five steps. UDPG-SGTase was solubilized fro m a microsomal fraction with the detergent n-octyl-beta-D-thioglucopyr anoside and then extracted into diethyl ether. Subsequent removal of t he organic solvent, resolubilization with an aqueous buffer, and two c olumn chromatographic steps on Q-Sepharose and Blue Sepharose resulted in a 12,500-fold overall purification. Sodium dodecyl sulfate-polyacr ylamide gel electrophoresis of the final preparation revealed a 56-kd protein band, the intensity of which correlated with enzyme activity i n the respective fractions. Polyclonal antibodies raised against this 56-kD protein did not inhibit enzyme activity but specifically bound t o 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 (in hibitor constant = 47 mu M). In contrast to the specificity with regar d to the glycosyl donor, UDPG-SGTase utilized all tested sterol accept ers, such as beta-sitosterol, cholesterol, stigmasterol, and ergostero l.