Purification and Partial Amino Acid Sequences of a Phospholipase C-Associated GTP-Binding Protein from Calf Thymocytes

Abstract

We recently identified a phosphoinositide-specific phospholipase C (PI-PLC)-stimulating GTP-binding protein (G protein) in calf thymocyte cytosol (Wang, P., Toyoshima, S., & Osawa, T. (1987) J. Biochem. 102, 1275-1287; and (1988) 103, 137-142). In this study we completely purified a G protein whose properties are quite similar to the G protein mentioned above from the calf thymocyte membrane and determined partial amino acid sequences of it. The purification was achieved by first treating the membrane with GTP _γS, followed by sequential column chromatographies on DEAE-Sepharose CL-6B, Sephacryl S-200, Mono Q, and Mono S. The G protein was purified in a GTP _γS-binding form and assayed as to the radioactivity of the [³⁵S]GTP _γS-bound PI-PLC-associated G protein standard obtained from calf thymocyte cytosol. The purified G protein could stimulate the activity of a partially purified PI-PLC for phosphatidylinositol 4, 5-bisphosphate hydroly-sis. From approximately 5μg of membrane protein we obtained about 5 fig of a purified sample. The purified G protein showed a molecular weight of 21 kDa on SDS-PAGE and one of 25 kDa on gel filtration. The partial amino acid sequences were determined by treating the purified sample with lysylendopeptidase, purifying the resultant peptide fragments on a HPLC-reverse phase column and then sequencing the peptide fragments with a sequencer. Comparison of the obtained sequences with those of known lower molecular weight GTP-binding proteins suggested that, although structurally similar to rho gene products, this is a novel G protein.