Metabolism of diacylglycerol pyrophosphate by suspension cultured Catharanthus roseus cells : Identification and characterization of diacylglycerol pyrophosphate phosphatase in plants

Abstract

Diacylglycerol pyrophosphate (DGPP) is a novel phospholipid found in plants that is the product of the reaction catalyzed by phosphatidate kinase. In this study we examined the metabolism of DGPP in Catharanthus roseus cells. Metabolic labeling studies showed that DGPP was rapidly metabolized to phosphatidate and P i. The enzyme catalyzing this reaction was named DGPP phosphatase. DGPP phosphatase activity was 20 times more active than phosphatidate kinase activity. Microsomal and soluble isoforms of DGPP phosphatase were found in C. roseus cells. The microsomal enzyme was primarily associated with intracellular membranes. The pH optima of the microsomal and soluble DGPP phosphatases were 4.5 and 3.5, respectively. Both activities were independent of a divalent cation requirement and were insensitive to EDTA and N-ethylmaleimide. The DGPP phosphatase activities were inhibited by NaF, MgCl 2, MnCl 2, and sphingosine. The dependence of the microsomal and soluble DGPP phosphatase activities on DGPP were dose-dependent with maximum activities obtained at 100 μM (3 mol %) and 600 μM (20 mol %), respectively, using a Triton X-100/DGPP mixed micellar substrate. Pyrophosphate, p-nitrophenylphosphate, and adenosine diphosphate (ADP) were not substrates for the DGPP phosphatase enzymes. DGPP phosphatase activity was also found in a wide range of organisms including Escherichia coli, Saccharomyces cerevisiae, rat, pig, and cattle.