UDP-glucose sterol beta-D-glucosyltransferase, a plasma membrane-bound enzyme of plants: enzymatic properties and lipid dependence.

UDP-glucose sterol beta-D-glucosyltransferase (UDPG-SGTase) catalyzes the glucosylation of plant sterols. This enzyme has been shown to be membrane-bound, most of its activity being associated with plasma membrane in etiolated maize coleoptiles. After solubilization with detergents, total delipidation and purification, kinetic studies performed with a purified enzyme preparation in the presence of detergent and soybean phosphatidylcholine (PC) strongly suggest an ordered bi-bi mechanism for the glucosylation of sterols. A reduced sulfhydryl group and an arginyl residue were shown to be essential for activity. Lipid dependence studies have been performed on the delipidated enzyme in two systems: a micellar one composed of a mixture of enzyme, detergent and phospholipids and another one where the enzymatic activity was reconstituted in unilamellar lipid vesicles. In both systems it was shown that the UDPG-SGTase activity was stimulated to a large extent by negatively charged phospholipids. Enzymatic assays were performed with membrane fractions originating from plants whose sterol content was profoundly modified following treatment with a sterol biosynthesis inhibitor. Results showed that the sterol glucosylating activity was strongly inhibited in these fractions in accordance with sterol substrate specificity studies. All these results show that the UDPG-SGTase is exquisitely sensitive to its lipid environment. Physiological implications of these data are discussed in the light of the putative role of sterols in the plant cell.

Phospholipid-Dependence of Plant UDP-Glucose Sterol beta-d-Glucosyl Transferase : IV. Reconstitution into Small Unilamellar Vesicles.

The phospholipid dependence of the UDP-glucose sterol glucosyl transferase (UDPG-SGTase) from maize coleoptiles was previously demonstrated using the partially purified and highly delipidated enzyme, in the presence of the detergent Triton X-100 (P Ullmann, P Bouvier-Navé, P Benveniste [1987] Plant Physiol 85: 51-55). We now report the reconstitution of the enzyme activity into unilamellar lipid vesicles. This was achieved by adding phospholipids, sterols and beta-octylglucoside to the solubilized enzyme and passing the mixture through Sephadex G-50. The treatment led to almost complete removal of the detergents. The incorporation of UDPG-SGTase in the lipid vesicles was demonstrated by (a) coelution of the enzyme activity with the labeled lipid vesicles (average diameter: 260A) on a Sephacryl S-1000 column and (b) flotation experiments on metrizamide density gradients. Release of dithiobis-(2-nitro-benzoic acid) (DTNB) from DTNB-preloaded vesicles was very slow, indicating good membrane integrity of the vesicles. Treatment of the intact vesicles with the nonpermeant reagent p-chloro-mercuribenzene sulfonate led to more than 95% inactivation of the total enzyme activity, i.e. the activity measured in the presence of Triton X-100 at permeabilizing concentration. This suggests an outward orientation for the active site of the enzyme. Finally, the enzyme was incorporated into vesicles of various phospholipid compositions and the kinetic parameters of the reactions were determined. Our results clearly show that the reconstituted UDPG-SGTase activity is stimulated to a large extent by negatively charged phospholipids.