Regulation by Phospholipids and Kinetic Studies of Plant Membrane-Bound UDP-Glucose Sterol β-d-Glucosyl Transferase

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Abstract
Solubilization and partial purification of the microsomal UDP-glucose sterol glucosyl transferase activity from maize coleoptiles by chromatography on DEAE-cellulose resulted in a highly delipidated (>95%) and inactive enzymic preparation. Addition of sterols revealed part of the activity and subsequent addition of phospholipids further increased the activity. Negatively charged phospholipids were shown to be by far the best activators. The purification step also produced the elimination of two interfering microsomal enzymic activities: UDPase and steryl glucoside acyl transferase. The removal of these two enzymic activities was a prerequisite for kinetic studies including product-inhibition studies, since the substrates of these two latter enzymes are the products of UDPG-SGTase activity. The results of the kinetic studies strongly suggest an ordered bi-bi mechanism for the glucosylation of sterols. Finally the effect of different phospholipids on the kinetic parameters of the reaction was studied. Both phosphatidylcholine and phosphatidylglycerol significantly decrease Km-sterol (and not Km-UDPglucose) and increase the reaction Vmax. The decrease of Km-sterol is similar with both phospholipids whereas the increase of Vmax is much greater with phosphatidylglycerol than with phosphatidylcholine.
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