Abstract
Diacylglycerol pyrophosphate (DGPP) phosphatase is a novel membrane-associated enzyme that catalyzes the dephosphorylation of the beta phosphate of DGPP to yield phosphatidate and Pi. DGPP phosphatase was purified 33,333-fold from Saccharomyces cerevisiae by a procedure that included Triton X-100 solubilization of microsomal membranes followed by chromatography with DE53, Affi-Gel Blue, hydroxylapatite, and Mono Q. The procedure resulted in the isolation of an apparent homogeneous protein with a subunit molecular mass of 34 kDa. DGPP phosphatase activity was associated with the 34-kDa protein. DGPP phosphatase had a broad pH optimum between 6.0 and 8.5 and was dependent on Triton X-100 for maximum activity. The enzyme was inhibited by divalent cations, NaF, and pyrophosphate and was relatively insensitive to thioreactive agents. The turnover number (molecular activity) for the enzyme was 5.8 x 10(3) min-1 at pH 6.5 and 30 degrees C. DGPP phosphatase exhibited typical saturation kinetics with respect to DGPP (Km = 0.55 mol %). The Km value for DGPP was 3-fold greater than its cellular concentration (0.18 mol %). DGPP phosphatase also catalyzed the dephosphorylation of phosphatidate, but this dephosphorylation was subsequent to the dephosphorylation of the beta phosphate of DGPP. The dependence of activity on phosphatidate (Km = 2.2 mol %) was cooperative (Hill number = 2.0). DGPP was the preferred substrate for the enzyme with a specificity constant (Vmax/Km) 10-fold greater than that for phosphatidate. In addition, DGPP potently inhibited (Ki = 0.35 mol %) the dephosphorylation of phosphatidate by a competitive mechanism whereas phosphatidate did not inhibit the dephosphorylation of DGPP. DGPP was neither a substrate nor an inhibitor of pure phosphatidate phosphatase from S. cerevisiae. DGPP was synthesized from phosphatidate via the phosphatidate kinase reaction.
Highlights
Diacylglycerol pyrophosphate (DGPP)1 is a novel phospholipid metabolite recently identified from the plant Catharanthus roseus by Wissing and Behrbohm [1]
DGPP phosphatase activity was solubilized from the microsomal fraction with 1% Triton X-100 followed by chromatography with DE53, Affi-Gel Blue, hydroxylapatite, and Mono Q
Hydroxylapatite peak I DGPP phosphatase activity eluted from Mono Q I at a salt concentration of 0.12 M (Fig. 2B), whereas hydroxylapatite peak II DGPP phosphatase activity eluted from Mono Q II at the beginning of the salt gradient (Fig. 2C)
Summary
Diacylglycerol pyrophosphate (DGPP) is a novel phospholipid metabolite recently identified from the plant Catharanthus roseus by Wissing and Behrbohm [1]. DGPP contains a pyrophosphate group attached to diacylglycerol (Fig. 1) This compound was previously observed by several workers [2,3,4] as the phospholipid product of a lipid kinase reaction in plants that was not identified correctly [1]. In addition to being present in plant cells, DGPP phosphatase activity is present in membrane fractions of Escherichia coli, Saccharomyces cerevisiae, and rat liver.. In addition to being present in plant cells, DGPP phosphatase activity is present in membrane fractions of Escherichia coli, Saccharomyces cerevisiae, and rat liver.2 Whereas it is unclear what role DGPP plays in phospholipid metabolism and cell growth, PA, the product of the DGPP phosphatase reaction, plays a major role in lipid metabolism. We demonstrated that S. cerevisiae synthesized DGPP via the PA kinase reaction
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