KINETIC-PROPERTIES OF MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE FROM SPINACH CHLOROPLAST ENVELOPE MEMBRANES

We have investigated the functioning of monogalactosyldiacylglycerol ( MGDG) synthase activity partially purified from spinach chloroplast en velope membranes, using mixed micelles containing diacylglycerol (the substrate for MGDG synthase), CHAPS cholamidopropyl)dimethylammonio]-1 -propanesulfonic acid), and phosphatidylglycerol. The presence of this anionic phospholipid was essential for optimal MGDG synthase activity because it strongly improves diacylglycerol solubilization by CHAPS. We have demonstrated that the ''surface dilution'' kinetic model propo sed by Deems ct al. (Deems, R. A., Eaten, B. R., and Dennis, E.A. (197 5) J. Biol. Chem. 250, 9013-9020) is valid for MGDG synthase assayed i n mixed micelles within a narrow range of CHAPS concentration. However , the experimental conditions we have set up in this study led to the description of defined equilibrium and kinetic parameters of the inter action of the envelope MGDG synthase with diacylglycerol. Two-substrat e kinetic studies were performed with varied UDP-galactose molar conce ntrations and varied dioleoylglycerol surface concentrations. The fami lies of reciprocal plots obtained were shown to intersect at a single point of the 1/[substrate] axis thus demonstrating that MGDG synthase is a sequential, either random or ordered, bireactant system. Therefor e, MGDG synthase possesses two distinct and independent substrate-bind ing sites, a hydrophilic one for UDP-galactose and a hydrophobic one f or diacylglycerol. The dependence of kinetic parameters on the diacylg lycerol mel fraction allows a comparison of the affinity of the enzyme for a wide range of diacylglycerol molecular species. The K-m values obtained were ranging between 0.0089 mol fraction (52 mu M) for dilino leoylglycerol (18:2/18:2) to 0.0666 mol fraction (416 mu M for distear oylglycerol (18:0/18:0), but the differences observed were not really related to the unsaturation of the molecule since the K-m value for di linoleoylglycerol was much lower than that (0.040 mol fraction) for di linoleoylglycerol (18:3/18:3). The K-m values for dioleoylglycerol (18 :1/18:1) and for the diacylglycerol molecular species synthesized with in chloroplasts, i.e. containing 18:1/ 16:0, were in the average range , i.e. lower than 0.030 mol fraction (around 170 mu M).