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.
Highlights
The G25K is distinct from any known GTP-bindingprotein either amino acid sequences of three peptide fragments de- purified or inferred from a cDNA sequence [4].A 21-kDa rived from the 28-kDa protein were identical to re- GTP-binding protein termed ARF that serves as a cofactor gwsrtaiiaoltsnucstDiisooNofnltAah(tAeewda~imtfphr’oi~nmt’oh+eGaaelcxupic)dle.aApcsteiefonuqntlualeolnlfecconDengNetdhAecdholuuinbcmsereaadrnrvyrafa,rtlioavmcneDdNsaiutAbss-imbffoiieearednncthoiosloneleraaatretdohxo(im6n)-o.cgIaentnaleayidtzdyeidt(i5oA)n,D,aanPmd-riutbhloteistycuDldaeNtioAonfelhonwacsodbmieneognleiptcuuhlraai-rs deduced amino acid sequence, compared with simian mass GTP-binding proteins have been chromatographically ral, alsocontainedthe Asp+Glu substitutionalong resolved from extracts of bovine brain crude membranes [7]
Partial is encoded by a cDNA variously termed smg p21 [10] or rupamino acid sequence obtained from stheceond uniden- 1 [11].The 25-kDa brainprotein, designated smg 25A, is tified 25-kDa protein indicatetshat it is the productof apparently encoded by the rub-3 gene [12]. smgp21 and c-ras the rucl gene; a member of a newly identified gene familywhich encode for low molecularmassGTPbinding proteins
The peak fractions containing the Identification of the ral Protein and Cloning of the Human cDNA-Crude membranesprepared from humanplatelets were extracted with sodium cholate,and thesolubilized GTPbinding activity was chromatographed on successive columns of DEAE-Sephacel and Ultrogel AcA34
Summary
Purification of Low Molecular Mass GTP-binding Proteins from 58-68) contains the low molecular mass GTP-binding proteins. The peak fractions containing the Identification of the ral Protein and Cloning of the Human cDNA-Crude membranesprepared from humanplatelets were extracted with sodium cholate,and thesolubilized GTPbinding activity was chromatographed on successive columns of DEAE-Sephacel and Ultrogel AcA34 (see “Experimental Procedures”).
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