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

Publication ,  Journal Article
Polakis, PG; Weber, RF; Nevins, B; Didsbury, JR; Evans, T; Snyderman, R
Published in: J Biol Chem
October 5, 1989

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.

Duke Scholars

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

October 5, 1989

Volume

264

Issue

28

Start / End Page

16383 / 16389

Location

United States

Related Subject Headings

  • rac GTP-Binding Proteins
  • Thionucleotides
  • Sequence Homology, Nucleic Acid
  • Restriction Mapping
  • Proteins
  • Pregnancy
  • Placenta
  • Oligonucleotide Probes
  • Molecular Sequence Data
  • Hydroxyapatites
 

Citation

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Polakis, P. G., Weber, R. F., Nevins, B., Didsbury, J. R., Evans, T., & Snyderman, R. (1989). Identification of the ral and rac1 gene products, low molecular mass GTP-binding proteins from human platelets. J Biol Chem, 264(28), 16383–16389.
Polakis, P. G., R. F. Weber, B. Nevins, J. R. Didsbury, T. Evans, and R. Snyderman. “Identification of the ral and rac1 gene products, low molecular mass GTP-binding proteins from human platelets.J Biol Chem 264, no. 28 (October 5, 1989): 16383–89.
Polakis PG, Weber RF, Nevins B, Didsbury JR, Evans T, Snyderman R. 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.
Polakis, P. G., et al. “Identification of the ral and rac1 gene products, low molecular mass GTP-binding proteins from human platelets.J Biol Chem, vol. 264, no. 28, Oct. 1989, pp. 16383–89.
Polakis PG, Weber RF, Nevins B, Didsbury JR, Evans T, Snyderman R. 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):16383–16389.

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

October 5, 1989

Volume

264

Issue

28

Start / End Page

16383 / 16389

Location

United States

Related Subject Headings

  • rac GTP-Binding Proteins
  • Thionucleotides
  • Sequence Homology, Nucleic Acid
  • Restriction Mapping
  • Proteins
  • Pregnancy
  • Placenta
  • Oligonucleotide Probes
  • Molecular Sequence Data
  • Hydroxyapatites