Purification and characterization of a novel GTP-binding protein with a molecular weight of 24,000 from bovine brain membranes.
At least six GTP-binding proteins (G proteins) with Mr values between 20,000 and 25,000 were extracted from the crude membrane fraction of bovine brain by sodium cholate and purified by successive column chromatographies of Ultrogel AcA-44, phenyl-Sepharose CL-4B, hydroxyapatite, and Mono Q HR5/5. One G protein with a Mr of about 24,000 (24K G) was purified to near homogeneity and characterized. 24K G bound maximally about 0.7 mol of [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein with a Kd value of about 46 nM. [35S]GTP gamma S binding to this protein was inhibited by GTP and GDP, but not by other nucleotides such as ATP, UTP, and CTP. 24K G hydrolyzed GTP to liberate Pi with a rate of about 40 mmol of Pi/mol of protein/min. 24K G did not associate with the beta gamma subunit of Go. 24K G was distinguishable on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from the ras protein (ras p21) and ADP-ribosylation factor, the G protein with a Mr of about 21,000 serving as a cofactor for the cholera toxin-dependent ADP-ribosylation of Gs. 24K G was not recognized by the antibody against ras p21, ADP-ribosylation factor, the alpha subunit each of Gs, Gi, and Go, or tubulin. These results indicate that 24K G is a novel G protein and suggest that there are multiple forms of G proteins with small Mr values as is the case with the G proteins which have the alpha subunits with Mr values of about 40,000.
D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002850:Chromatography, Gel; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000302:isolation & purification*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D008566:Membranes / Q000032:analysis; D008970:Molecular Weight; D013873:Thionucleotides / Q000378:metabolism
D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D005033:Ethylmaleimide