Characterization of nucleoside-diphosphate kinase-associated guanine nucleotide-binding proteins from HeLa S3 cells.
Nucleoside-diphosphate (NDP) kinase-associated [alpha-32P]GTP-incorporating proteins from HeLa S3 cells have been biochemically characterized. Two distinct NDP-kinases (F-I and F-II) had been partially purified from HeLa S3 cells by Sephacryl S-300 gel filtration and DEAE-cellulose column chromatography. The [alpha-32P]GTP-incorporating proteins (approx. Mr 20,000) could be separated from NDP-kinases (approx. Mr 80,000) by 5-25% glycerol density-gradient centrifugation analysis after treatment with 7 M urea in the presence of 1 mM EDTA. [alpha-32P]GTP incorporation into these two proteins (G1 and G2) from NDP-kinases required 5 mM Mg2+ and was highly inhibited by either GDP or GTP analogues, such as guanylyl imidodiphosphate and guanylyl methylenediphosphate. [3H]GDP, but no other nucleoside 5'-diphosphates, was also bound to these two proteins in the presence of Mg2+ (5 mM). Moreover, incubation of [alpha-32P]GTP with either G1 or G2 in the presence of Mg2+ (5 mM) resulted in the formation of [32P]GDP and Pi. The data presented here indicated that the guanine nucleotide-binding activity, the GTPase activity, and the molecular weight (approx. Mr 20,000) of NDP-kinase-associated proteins from HeLa S3 cells are similar to those reported for ras oncogene products (p21 proteins).
D002118:Calcium / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002712:Chlorides / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004492:Edetic Acid / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells / Q000032:analysis*; D006358:Hot Temperature; D006801:Humans; D008094:Lithium / Q000378:metabolism; D018021:Lithium Chloride; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D008970:Molecular Weight; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*
D002712:Chlorides; D046911:Macromolecular Substances; D006160:Guanosine Triphosphate; D008094:Lithium; D004492:Edetic Acid; D010770:Phosphotransferases; D009701:Nucleoside-Diphosphate Kinase; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D018021:Lithium Chloride; D008274:Magnesium; D002118:Calcium