Purification and partial amino acid sequences of a phospholipase C-associated GTP-binding protein from calf thymocytes.
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 gamma S, 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 gamma S-binding form and assayed as to the radioactivity of the [35S]GTP gamma S-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 hydrolysis. From approximately 5.6 g of membrane protein we obtained about 5 micrograms 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.
Wang
P
P
Division of Chemical Toxicology and Immunochemistry, Faculty of Pharmaceutical Sciences, University of Tokyo.
Nishihata
J
J
Takabori
E
E
Yamamoto
K
K
Toyoshima
S
S
Osawa
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002848:Chromatography, DEAE-Cellulose; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000032:analysis / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D013462:Sulfur Radioisotopes; D013950:Thymus Gland / Q000166:cytology / Q000201:enzymology*; D010738:Type C Phospholipases / Q000032:analysis / Q000302:isolation & purification*
D010446:Peptide Fragments; D013462:Sulfur Radioisotopes; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins