Diphosphatase Related to Lipid Metabolism and Gluconeogenesis in Cucumber Cotyledons: Localization in Plasma Membrane and Etioplasts but not in Glyoxysomes

Abstract

Abstract Diphosphatase (inorganic pyrophosphatase) activity was localized within compartments of cotyledons of germinating cucumber seeds during the stage of maximal conversion of fat into carbohydrates. At this stage, almost 2 mol pyrophosphate are produced during the formation of one mole sucrose from 0.28 mol triglyceride. When organelles of the 2000 x g pellet or 10,000 x g pellet were separated by density gradient centrifugation and gradient flotation, the diphosphatase activity paralleled the profiles of markers of the plastid stroma but was virtually absent from the glyoxysomes. Within the fraction of small vesicles and membranes, diphosphatase was attributed to the plasma membrane. The main portion of diphosphatase, contained in the plastids, was partially purified by chromatography on anion exchange resin and molecular sieving, leading to a 75-fold enrichment compared to the stroma fraction. Trace amounts of diphosphatase observed in the glyoxysomal fraction were analyzed in the same way. Comparison of the isoelectric points and the activity profile at different pH values and the inhibitory effect of the various cations indicated that the trace amounts of diphosphatase activity in the glyoxysome fraction represented contaminations originating from the plastids. The plasma membrane form of diphosphatase is an integral membrane protein which was solubilized with octylglucoside. It was shown to differ from the plastid form in pH optimum and sensitivity towards bivalent cations. All forms of diphosphatase were clearly distinguished from other phosphohydrolytic activities.