Purification of phosphatidylglycerophosphate synthase from Chinese hamsterovary cells

Phosphatidylglycerophosphate (PGP) synthase catalyses the committed step in the biosynthesis of phosphatidylglycerol and cardiolipin in mammalian cell s. Recently we isolated a Chinese hamster ovary (CHO) PGS1 cDNA encoding PG P synthase, In the present study we purified this PGP synthase to near-homo geneity from the mitochondrial fraction of CHO-K1 cells; the final enzyme p reparation gave a single 60 kDa protein on SDS/PAGE. Polyclonal antibodies raised against a recombinant CHO PGS1 protein cross-reacted with the purifi ed 60 kDa protein and with CHO membrane proteins of 60 kDa and 62 kDa that increased after transfection with the PGS1 cDNA. The 60 and 62 kDa protein levels in a PGP synthase-defective mutant of CHO-K1 cells were markedly low er than those in CHO-KI cells. These results indicated that the purified 60 kDa protein was PGP synthase encoded by the PGS1 gene. In addition we foun d that the purified PGP synthase had no PGP phosphatase activity, indicatin g that phosphatidylglycerol was produced from CDP-diacylglycerol through tw o steps catalysed by distinct enzymes, PGP synthase and PGP phosphatase.