Characterization of recombinant rat brain phospholipase

We have recently cloned a cDNA encoding a phosphatidylcholine-specific PLD from rat brain and named it rPLD (submitted to J. Biol. Chem.) It shows 90 percent amino acid identiy with the human PLD isoform hPLD1B. We have expressed rPLD as a histidine-taGged fusion protein in insect cells using the expression vector bluebasrlfis. The recombinant protein was purified to homogeniy by Ni ² agarose affinity chromatography. The activity and expression of rPLD in Sf9 cells wre detected in both cytos9ol and membrane fractions. Pl(4.5)P ² and PI(3.4.5)P ³activated rPLD equipotentiy, but other acide phospholipids were ineffective, The activity of rPLD was dependent on both M ²⁺ and Ca ²⁺and showed a broad dependence on PH wish optinum activety at pH 6.5-7.5 The enzyme was activated by the small G protens but kinase C and 3 but not activated PLD in a manner that was stimulated by phorbol ester but did not require ATP. The purified rPLD did not show wynergistic interactins betweem ARF and R ho and between these proteins and PKC or Purified PKC and phosptorylated parified PL in vitro and the phosphorylated PLD exhibited a mobility shift on SDS PAGE. Phosphorylation of PLD by PKC Proxo another regutatoryu mechanism for the enzyme.