Purification and properties of phosphatidylgkycerophosphate synthetase from mammalian liver mitochondria

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Abstract
The enzyme which catalyzes the synthesis of phosphatidylglycerophosphate from sn-glycerol-3-phosphate and cytidine diphosphate diacyglycerol was released from rat or pig liver mitochondrial membranes by extraction with Triton X-100 or Nonidet P-40. The detergent-extracted enzyme, like the activity of intact mitochondria, did not require added cations or lipids. The Triton extracts were fractionated by column chromatography on Bio-Gel A-1.5. The fractions obtained from the columns exhibited little activity in the standard assay system unless divalent cations were included. Additional stimulation (about 2-fold) was observed in the presence of added phospholipids. The cation requirement of the purified enzyme was relatively nonspecific with Mg2+, Ba2+ or Ca2+ providing maximal activity in the 10 mM range. Either Mn2+ or Co2+ were stimulatory at somewhat lower concentrations but higher concentrations were inhibitory. Other cations such as Cd2+, Zn2+, Hg2+ or Cu2+ were ineffective as cofactors, and in the presence of Mg2+ inhibited the reaction at concentrations greater than 0.5 mM. The phospholipid stimulation was obtained specifically with phosphatidylethanolamines from natural or synthetic sources. Other diacylglycerophosphatides or lysophosphatides including lysophosphatidylethanolamine were ineffective.