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Titolo:
PROPERTIES OF THE CATALYTIC DOMAIN OF SDC25P, A YEAST GDP GTP EXCHANGE FACTOR OF RAS PROTEINS COMPLEXATION WITH WILD-TYPE RAS2P, [S24N]RAS2P AND [R80D, N81D]RAS2P
Autore:
POULLET P; CRECHET JB; BERNARDI A; PARMEGGIANI A;
Indirizzi:
ECOLE POLYTECH,CNRS,BIOCHIM LAB F-91128 PALAISEAU FRANCE ECOLE POLYTECH,CNRS,BIOCHIM LAB F-91128 PALAISEAU FRANCE CNRS,ENZYMOL LAB GIF SUR YVETTE FRANCE
Titolo Testata:
European journal of biochemistry
fascicolo: 1-2, volume: 227, anno: 1995,
pagine: 537 - 544
SICI:
0014-2956(1995)227:1-2<537:POTCDO>2.0.ZU;2-E
Fonte:
ISI
Lingua:
ENG
Soggetto:
ELONGATION-FACTOR-TU; GTPASE ACTIVATING PROTEIN; AMINOACYL-TRANSFER RNA; SACCHAROMYCES-CEREVISIAE; GUANINE-NUCLEOTIDES; FACTOR-TS; H-RAS; MOLECULAR MECHANISM; KINETIC-PROPERTIES; ESCHERICHIA-COLI;
Keywords:
GDP GTP EXCHANGE FACTOR; RAS PROTEINS; GTP-BINDING PROTEINS; ONCOGENESIS;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
49
Recensione:
Indirizzi per estratti:
Citazione:
P. Poullet et al., "PROPERTIES OF THE CATALYTIC DOMAIN OF SDC25P, A YEAST GDP GTP EXCHANGE FACTOR OF RAS PROTEINS COMPLEXATION WITH WILD-TYPE RAS2P, [S24N]RAS2P AND [R80D, N81D]RAS2P", European journal of biochemistry, 227(1-2), 1995, pp. 537-544

Abstract

The catalytic domain of the Saccharomyces cerevisiae SDC25 gene product, including the last 550 C-terminal residues (Sdc25p-C), was produced as an Escherichia coli recombinant protein fused with glutathione S-transferase. The highly purified (greater than 95%) stable fusion protein, obtained by affinity chromatography, was very active in enhancingthe dissociation rate or the GDP/GTP exchange of the GDP complex of Ras2p or human H-ras p21. This activity was further increased (three times) by glutathione S-transferase cleavage with thrombin. The stimulation of the guanine nucleotide release by Sdc25p-C was stronger for Ras2p.GDP than Ras2p.GTP, an effect that was less pronounced in the case of the p21 complexes. The association rate of the Ras2p.GDP (GTP) complex was also enhanced by Sdc25p-C. Monovalent and divalent salts inhibit the nucleotide-releasing activity of Sdc25p-C. Retention phenomena occurring on gel-filtration chromatography hindered the use of highly purified Sdc25p-C to study the formation of stable complexes with Ras2p. For this purpose, Sdc25p-C was produced as a non-glutathione-S-transferase fusion protein via pTTQ19. Upon partial purification, this product yielded a 54-kDa truncated form of Sdc25p-C (truncated Sdc25p-C) showing the same specific activity as the 64-kDa Sdc25p-C protein. On gel filtration, truncated Sdc25p-C and nucleotide-free Ras2p (or p21) formed a stable 1:1 stoichiometric complex that was dissociated by increasing concentrations of GDP. The proper ties of this complex were analyzed by using the mutant [S24N]Ras2p, the homologue of [S17N]p21 known to induce a dominant negative phenotype, [R80D, N81D]Ras2p, a recessive negative mutant insensitive to the truncated form of Sdc25p-C in vitro. The complex with [S24N]Ras2p was greater than 100-fold less sensitive to the dissociating effect of GDP, whereas [R80D, N81D]Ras2p was unable to form a stable complex with truncated Sdc25p-C. These results strongly suggest that the residues R80 and N81 are situated in or closely associated with the Ras2p specific site binding Sdc25p.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 27/11/20 alle ore 04:44:00