Identification of the ral and rac1 gene products, low molecular mass GTP-binding proteins from human platelets

J Biol Chem. 1989 Oct 5;264(28):16383-9.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Blood Platelets / metabolism*
  • Blood Proteins*
  • Cell Membrane / metabolism
  • Chromatography
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Cloning, Molecular
  • DNA / genetics
  • Durapatite
  • Female
  • GTP-Binding Proteins / blood*
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Guanosine Triphosphate / analogs & derivatives
  • Guanosine Triphosphate / metabolism
  • Humans
  • Hydroxyapatites
  • Molecular Sequence Data
  • Oligonucleotide Probes
  • Placenta / metabolism
  • Pregnancy
  • Proteins / genetics
  • Proteins / isolation & purification
  • Restriction Mapping
  • Sequence Homology, Nucleic Acid
  • Thionucleotides / metabolism
  • rac GTP-Binding Proteins

Substances

  • Blood Proteins
  • Hydroxyapatites
  • Oligonucleotide Probes
  • Proteins
  • Thionucleotides
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Guanosine Triphosphate
  • DNA
  • Durapatite
  • GTP-Binding Proteins
  • rac GTP-Binding Proteins

Associated data

  • GENBANK/M29893