Purification and characterization of a GTP-binding protein with a molecular weight of 20,000 in bovine brain membranes. Identification as the rho gene product.
We have determined that there are at least six GTP-binding proteins (G proteins) with Mr values between 20,000 and 25,000 in the crude membrane fraction of bovine brain and have purified one of them with a Mr of about 24,000 (24K G) to near homogeneity (Kikuchi, A., Yamashita, T., Kawata, M., Yamamoto, K., Ikeda, K., Tanimoto, T., and Takai, Y. (1988) J. Biol. Chem. 263, 2897-2904). In this study, we have purified another G protein with a Mr of about 20,000 (20K G) to near homogeneity and have characterized it. 20K G bound maximally about 1.0 mol of [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein, with a Kd value of about 50 nM. [35S]GTP gamma S binding to 20K G was inhibited by GTP and GDP, but not by other nucleotides such as ATP, UTP, and CTP; it was also inhibited by pretreatment with N-ethylmaleimide. 20K G hydrolyzed GTP to liberate Pi, with a turnover number of about 0.01 min-1, and was not copurified with the beta gamma subunits of the regulatory G proteins of adenylate cyclase. 20K G was not recognized by the antibody against the ADP-ribosylation factor for the stimulatory regulatory G protein of adenylate cyclase. Peptide map analysis showed that 20K G was not a proteolytic product of 24K G. The partial amino acid sequence of 20K G was almost identical with that deduced from the rho gene. The amino acid composition of 20K G was similar to that of the rho gene product. These results suggest that 20K G is the rho gene product and that this G protein is present in bovine brain membranes.
D020727:ADP-Ribosylation Factors; D000262:Adenylyl Cyclases / Q000032:analysis; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001048:Aplysia / Q000032:analysis; D001923:Brain Chemistry; D002417:Cattle; D002462:Cell Membrane / Q000032:analysis; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006150:Guanine Nucleotides / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007118:Immunoassay; D008565:Membrane Proteins / Q000032:analysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009711:Nucleotides / Q000494:pharmacology; D013873:Thionucleotides / Q000378:metabolism; D020743:rhoB GTP-Binding Protein
D000596:Amino Acids; D006150:Guanine Nucleotides; D008565:Membrane Proteins; D009711:Nucleotides; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D020743:rhoB GTP-Binding Protein; D000262:Adenylyl Cyclases; D005033:Ethylmaleimide