Identification of the ral and rac1 gene products, low molecular mass GTP-binding proteins from human platelets.
Identification of the GTP-binding proteins from human platelet particulate fractions was attained by their purification via successive column chromatography steps followed by amino acid sequencing. To enhance the likelihood of identifying the GTP-binding proteins, two assays were employed to monitor GTP-binding activities: (i) guanosine 5'-(3-O-[35S]thio)triphosphate (GTP gamma S)-binding followed by rapid filtration and ii) [alpha-32P]GTP-binding following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting onto nitrocellulose membranes. The latter assay permitted the isolation of a 28-kDa GTP-binding protein that bound [alpha-32P]GTP prominently but was only poorly detected with the GTP gamma S-binding assay. The amino acid sequences of three peptide fragments derived from the 28-kDa protein were identical to regions of the amino acid sequence deduced from a simian ral cDNA with the exception of one conservative substitution (Asp147----Glu). A full length human ral cDNA was isolated from a placental cDNA library, and its deduced amino acid sequence, compared with simian ral, also contained the Asp----Glu substitution along with two other substitutions and an additional three NH2-terminal amino acids. In addition to the 28-kDa protein, two distinct 25-kDa GTP-binding proteins were purified from platelets. One of these proteins has been previously characterized as G25K, an abundant low molecular mass GTP-binding protein. Partial amino acid sequence obtained from the second unidentified 25-kDa protein indicates that it is the product of the rac1 gene; a member of a newly identified gene family which encode for low molecular mass GTP-binding proteins (Didsbury, J., Weber, R.F., Bokoch, G. M., Evans, T., and Snyderman, R. (1989) J. Biol. Chem. 264, 16378-16382). These results identify two new GTP-binding proteins in human platelets, ral, the major protein that binds [alpha-32P]GTP on nitrocellulose transfers, and rac1, a substrate for botulinum C3 ADP-ribosyltransferase.
Polakis
P G
PG
Department of Pharmacological Sciences, Genentech, Incorporated, South San Francisco, California 94080.
Weber
R F
RF
Nevins
B
B
Didsbury
J R
JR
Evans
T
T
Snyderman
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001792:Blood Platelets / Q000378:metabolism*; D001798:Blood Proteins; D002462:Cell Membrane / Q000378:metabolism; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D017886:Durapatite; D005260:Female; D019204:GTP-Binding Proteins / Q000097:blood*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D006882:Hydroxyapatites; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013873:Thionucleotides / Q000378:metabolism; D020744:rac GTP-Binding Proteins
D001798:Blood Proteins; D006882:Hydroxyapatites; D015345:Oligonucleotide Probes; D011506:Proteins; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D004247:DNA; D017886:Durapatite; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins