Photoaffinity labeling with GTP of viral p21 ras protein expressed in Escherichia coli

Stein, Robert B.; Robinson, Pierre; Scolnick, Edwardm .

doi:10.1128/jvi.50.2.343-351.1984

Cited by 56 publications

(19 citation statements)

References 42 publications

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“…Monoclonal antibody Y 13-259 used in this study was obtained from Oncogene Science Inc. (Manhasset, NY). Pure fusion protein from the expression of v-Ha-rus in Escherichia coli (Stein et al, 1984) was a generous gift from Dr. J.B. Gibbs, Merck Sharp and Dohme Research Laboratories (West Point, PA).…”

Section: Immunological Reagentsmentioning

confidence: 99%

Cytological effects of the microinjection of antibody to ras p21 in early cleavage Xenopus embryos

Miron

Lanoix

Paiement

1990

Molecular Reproduction Devel

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The presence of two ras-related proteins (22 and 23 kDa) was demonstrated in Xenopus embryonic extracts by selective immunoprecipitation using anti-ras monoclonal antibodies 142-24E05 and Y13-259. We further describe the cytological effects of the microinjection of anti-ras monoclonal antibody Y13-259 into early cleavage blastomeres of Xenopus embryos. Injection of the antibody into a blastomere at the two-, four-, or eight-cell stage caused cleavage arrest in the descendants of the injected blastomere. Light microscopy (LM) of cleavage-arrested cells revealed extensive deformation of the cells as well as heterogeneity of distribution of yolk platelets and pigment granules. LM analysis of serial sections of cleavage-arrested cells revealed the presence of multiple nuclei. Although the nuclei expressed similar morphological properties, indicating that they were probably in the same stage of the nuclear cycle, they revealed highly variable chromatin densities. Electron microscope (EM) analysis of the cytoplasm of cleavage-arrested cells revealed the accumulation of vesicles and large membranous elements coincident with cleavage arrest. Furthermore, endoplasmic reticulum (ER) existed in two forms, as closed, circular profiles and as long, linear arrays. Mitochondria were characteristically aligned in single file on both sides of the two types of ER cisternae. EM analysis of nuclei confirmed variations in chromatin organization and suggested the occurrence of unique nuclear envelope fusion among micronuclei in cleavage-arrested cells. Cleavage arrest and changes in cytological features were not observed in the cytoplasm of cells microinjected with normal rat IgG. Thus the immunochemical data and microinjection experiments suggest that ras-like or ras antigenicity exists within rapidly replicating Xenopus blastomeres and may be involved in the organization of a number of its cytoplasmic elements.

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Section: Immunological Reagentsmentioning

confidence: 99%

Cytological effects of the microinjection of antibody to ras p21 in early cleavage Xenopus embryos

Miron

Lanoix

Paiement

1990

Molecular Reproduction Devel

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“…We have compared the electrophoretic mobilities of ras p21 and G,,-proteins after their transfer to nitrocellulose, using as probes both [32P]GTP ( fig.lA) and a monoclonal antibody that recognizes all rus proteins ( fig.lB). Pure v-H-ras fusion protein (0.1 pg) with an apparent molecular mass of 28 kDa [19] bound little [32P]GTP (fig.lA, lane a) but gave a strong immunoperoxidase signal (fig.lB, lane a). In contrast, rabbit and human platelet membrane protein (75 pg) contained G,proteins that bound far more [32P]GTP ( fig.lA, lanes b,c).…”

Section: Resultsmentioning

confidence: 99%

G_n‐proteins are distinct from ras p21 and other known low molecular mass GTP‐binding proteins in the platelet

Bhullar

Haslam

1988

FEBS Letters

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The 27 kDa platelet membrane protein (G,27) that binds [c+P]GTP on nitrocellulose blots of SDS-polyacrylamide gels [(1987) Biochem. J. 245, 6176201 was compared with other low molecular mass GTP-binding proteins. Platelet membranes also contained 21 kDa proteins that bound anti-raF p21 antibody and 22-23 kDa proteins that could be ADPribosylated by botulinurn neurotoxin type D. These groups of proteins were resolved electrophoretically from each other and from G,27. A low molecular mass GTP-binding protein from bovine brain [(1987) B&hem. J. 246, 4314391 was also resolved from G.27. At the levels normally present in cell membranes, only G.-proteins bound significant amounts of P*P]GTP after transfer of protein from SDS-polyacrylamide gels to nitrocellulose.G,-protein; GTP-binding protein; ras p21 protein; Botulinum neurotoxin; Platelet

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“…Biochemical activity of bacterially expressed Kirsten ras protein. Autophosphorylation of the p21 protein was done essentially as described previously (27). Briefly, the 50-plI reaction mixture contained 10 pu1 of cell lysate fractions, 25 p.Ci of [cx-32P]GTP, 1 mM ATP, 50 mM Tris hydrochloride (pH 8.0), 100 mM NaCl, 1% Triton X-100, and 5 mM MgCl2.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

“…Viral (13,14,27) and human normal and transforming (10,19) Ha-ras proteins and the viral Kirsten (30) ras proteins have been expressed in Escherichia coli. The E. colisynthesized proteins retained their biochemical activities (10,14,19,27). Studies on the E. coli-expressed protein have established that transforming ras proteins possess slower GTP-hydrolyzing activity than their normal counterparts (9,15,19,28).…”

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confidence: 99%

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Expression of the Kirsten ras viral and human proteins in Escherichia coli

Nakano¹,

Rao²,

Perucho³

et al. 1987

J Virol

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The expression vectors pINIII-A and pINIII (Ipp p5) were used to construct plasmids which direct the synthesis in Escherichia coli of the Kirsten ras viral (v-Ki-ras) and human cellular (c-Ki-ras) oncogene products as fusion proteins containing 9 and 10 extra amino acids, respectively, at their N termini. Authenticity of the bacterially produced proteins was determined by immunoprecipitation and immunoblot analyses with ras-specific monoclonal antibodies. After induction with isopropyl-I8-D-thiogalactopyranoside, the viral protein represented approximately 20% of the total cellular protein. The majority of the protein was found in the postsonication low-speed centrifugation pellet. The synthesized viral protein was active in GTP binding, as judged by autophosphorylation and photoaffinity labeling assays. * Corresponding author. t Present address: Creative Biomolecules, Hopkinton, MA 01748. expression vector pINIII-A2 was digested with EcoRI, and the ends were made flush by treatment with DNA polymerase I (large fragment), and the vector was then digested with HindIII. The ras-containing fragment was ligated to the digested pINIII-A2, and the ligated DNA was used to 302 on July 6, 2020 by guest http://jvi.asm.org/ Downloaded from EXPRESSION OF KIRSTEN ras PROTEINS 303 transform JA221 cells. The A2 reading frame of the expres-

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Photoaffinity labeling with GTP of viral p21 ras protein expressed in Escherichia coli

Cited by 56 publications

References 42 publications

Cytological effects of the microinjection of antibody to ras p21 in early cleavage Xenopus embryos

Cytological effects of the microinjection of antibody to ras p21 in early cleavage Xenopus embryos

G_n‐proteins are distinct from ras p21 and other known low molecular mass GTP‐binding proteins in the platelet

Expression of the Kirsten ras viral and human proteins in Escherichia coli

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Photoaffinity labeling with GTP of viral p21 ras protein expressed in Escherichia coli

Cited by 56 publications

References 42 publications

Cytological effects of the microinjection of antibody to ras p21 in early cleavage Xenopus embryos

Cytological effects of the microinjection of antibody to ras p21 in early cleavage Xenopus embryos

Gn‐proteins are distinct from ras p21 and other known low molecular mass GTP‐binding proteins in the platelet

Expression of the Kirsten ras viral and human proteins in Escherichia coli

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Product

Resources

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G_n‐proteins are distinct from ras p21 and other known low molecular mass GTP‐binding proteins in the platelet