LIPID DEPENDENCE AND BASIC KINETICS OF THE PURIFIED 1,2-DIACYLGLYCEROL 3-GLUCOSYLTRANSFERASE FROM MEMBRANES OF ACHOLEPLASMA-LAIDLAWII

UDP-glucose: 1,2-diacylglycerol 3-glucosyltransferase (EC 2.4.1.157), catalyzes the transfer of glucose from UDP-glucose to diacylglycerol ( DAG) to yield monoglucosyldiacylglycerol (MGlcDAG) and UDP, MGlcDAG is the first glucolipid along the glucolipid pathway, and a major (nonbi layer-prone) lipid in the single membrane of Acholeplasma laidlawii, M GlcDAG is further glucosylated to give the major diglucosyldiacylglyce rol (DGlcDAG), The bilayer fractions of these lipids are crucial for t he metabolic maintenance of phase equilibria close to a potential bila yer-nonbilayer transition and a nearly constant spontaneous curvature, The glucolipid syntheses are also balanced against the phosphatidylgl ycerol pathway, competing for the common minor precursor phosphatidic acid, to retain a constant lipid surface charge density, The 1,2-diacy lglycerol 3-glucosyltransferase was purified to homogeneity from deter gent solubilized A. laidlawii cells by three column chromatography met hods (enrichment approximate to 9 000 x), and identified as a minor 40 kDa protein by using SDS-polyacrylamide gel electrophoresis. In CHAPS detergent, mixed micelles, a cooperative dependence on anionic lipids for activity was confirmed, Dependence of the enzyme on UDP-glucose f ollowed Michaelis-Menten kinetics while the other hydrophobic substrat e dioleoylglycerol stimulated the enzyme by an activating potentially cooperative mechanism, Physiological concentrations of the activator l ipid dioleoyl-phosphatidylglycerol influenced the turnover number of t he enzyme but not the interaction with UDP glucose, as inferred from v ariable and constant values of the apparent V-max and K-m, respectivel y, Dipalmitoylglycerol was a better substrate than the oleoyl species, supporting earlier in vivo and crude enzyme data, The responses of th e purified 1,2-diacylglycerol 3-glucosyltransferase indicated that (i) the regulatory features of the MGlcDAG synthesis is held by the catal ytic enzyme itself, and (ii) this strongly corroborates the ''homeosta sis'' model for lipid bilayer properties in A, laidlawii proposed earl ier.