Characterization of a Gly19-->Val mutant of ram p25, a low Mr GTP-binding protein: loss of GTP/GDP-binding activity in the mutated ram p25.
A substitution of Gly for Val at position 19, which corresponds to oncogenic Gly13-->Val mutation of ras p21, was introduced in a low Mr GTP-binding protein, ram p25. The protein was expressed in cytosolic fraction of Escherichia coli and purified by using specific antibody raised against ram p25. The mutated protein had no guanine nucleotide-binding activity although [Val13]ras p21 was reported to have. The analysis of guanine nucleotide composition of the purified [Val19]ram p25 revealed that the protein was free of nucleotide whereas the normal ram p25 bound about 1 mol of GDP per mol of protein. These results strongly suggested that some part(s) of variable regions as well as the consensus regions are important for the biochemical properties of ram p25.
Nagata
K
K
Department of Biochemistry, Gifu University School of Medicine, Japan.
Suzuki
T
T
Okano
Y
Y
Hamaguchi
M
M
Nozawa
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011905:Genes, ras; D005998:Glycine; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009369:Neoplasms / Q000235:genetics; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014357:Trypsin; D014633:Valine; D020691:rab GTP-Binding Proteins; D000075745:rab27 GTP-Binding Proteins
D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000075745:rab27 GTP-Binding Proteins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D014357:Trypsin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C066694:RAB27A protein, human; D020691:rab GTP-Binding Proteins; D014633:Valine; D005998:Glycine