IDENTIFICATION OF 2 ISOFORMS OF PHOSPHOLIPASE-C-ALPHA FROM DIVIDING MURINE FIBROBLASTS BY PROTEIN MICROSEQUENCING

Growth responsive proteins were studied in cultured murine C3H10T1/2 f ibroblasts. Two-dimensional gel electrophoresis (2D PAGE) of radiolabe led cells revealed several proteins apparent during log growth which w ere absent under non-dividing conditions, including two cytosolic 62 k D proteins. Of these two growth responsive proteins, both were present in growing cells but only the more acidic protein was present during the resting state. These two 62 kD proteins were resolved by charge (p I = 6.2 and 6.3) using preparative 2D PAGE, electroblotted, sequenced and identified as two phospholipase C-alpha isoforms. Both proteins sh ared an identical amino terminal sequence for 14 residues which matche d the known predicted sequence for PLC-alpha from cDNA clones. The two isoforms are phosphorylated and dephosphorylated forms of the enzyme. The finding of serine at the amino terminus, 24 residues from the ami no terminus predicted from cDNA, is consistent with the presence of an amino terminal signal sequence and proteolytic processing.