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 (nonbilayer-prone) lipid in the single membrane of Acholeplasma laidlawii. MGlcDAG is further glucosylated to give the major diglucosyldiacylglycerol (DGlc-DAG). The bilayer fractions of these lipids are crucial for the metabolic maintenance of phase equilibria close to a potential bilayer-nonbilayer transition and a nearly constant spontaneous curvature. The glucolipid syntheses are also balanced against the phosphatidylglycerol pathway, competing for the common minor precursor phosphatidic acid, to retain a constant lipid surface charge density. The 1,2-diacylglycerol 3-glucosyltransferase was purified to homogeneity from detergent-solubilized A. laidlawii cells by three column chromatography methods (enrichment Ϸ9 000 ؋), 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 followed Michaelis-Menten kinetics while the other hydrophobic substrate dioleoylglycerol stimulated the enzyme by an activating, potentially cooperative mechanism. Physiological concentrations of the activator lipid dioleoyl-phosphatidylglycerol influenced the turnover number of the enzyme but not the interaction with UDP-glucose, as inferred from variable and constant values of the apparent V max and K m , respectively. Dipalmitoylglycerol was a better substrate than the oleoyl species, supporting earlier in vivo and crude enzyme data. The responses of the purified 1,2-diacylglycerol 3-glucosyltransferase indicated that (i) the regulatory features of the MGlcDAG synthesis is held by the catalytic enzyme itself, and (ii) this strongly corroborates the "homeostasis" model for lipid bilayer properties in A. laidlawii proposed earlier.The glucolipid monoglucosyldiacylglycerol (MGlcDAG), 1 one of the major lipids in the cytoplasmic membrane of the cell wall-less bacterium Acholeplasma laidlawii, is synthesized by glucosylation of diacylglycerol (DAG), i.e. DAG ϩ UDP-GLC 3 MGlcDAG ϩ UDP (1, 2). This is catalyzed by the membranebound enzyme purified and basically characterized in this work, 1,2-diacylglycerol 3-glucosyltransferase (EC 2.4.1.157) (MGlcDAG synthase). MGlcDAG is the first glucolipid in the glucolipid pathway, and is further glucosylated by another enzyme to give diglucosyldiacylglycerol (DGlcDAG) (1-3), the precursor for the syntheses of two (usually minor) phosphoglucolipids. Acylated variants of the two glucolipids are also made under some conditions. The hydrophobic substrate DAG for 1,2-diacylglycerol 3-glucosyltransferase is synthesized from phosphatidic acid (PA) by the enzyme phosphatidic acid phosphatase.2 PA is also the precursor for the separate and competing pathway lead...