Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0162473 (Frey)
 2,599 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Oligomeric protein complexes containing the nuclear oncogene p53 and the simian virus 40 large tumor antigen (D. I. H. Linzer and A. J. Levine, Cell 17:43-51, 1979), the adenovirus E1B 55-kilodalton (kDa) tumor antigen, and the heat shock protein hsc70 (P. Hinds, C. Finlay, A. Frey, and A. J. Levine, Mol. Cell. Biol. 7:2863-2869, 1987) have all been previously described. To begin isolating, purifying, and testing these complexes for functional activities, we have developed a rapid immunoaffinity column purification. p53-protein complexes are eluted from the immunoaffinity column by using a molar excess of a peptide comprising the epitope recognized by the p53 monoclonal antibody. This mild and specific elution condition allows p53-protein interactions to be maintained. The hsc70-p53 complex from rat cells is heterogeneous in size, with some forms of this complex associated with a 110-kDa protein. The maximum apparent molecular mass of such complexes is 660,000 daltons. Incubation with micromolar levels of ATP dissociates this complex in vitro into p53 and hsc70 110-kDa components. Nonhydrolyzable substrates of ATP fail to promote this dissociation of the complex. Murine p53 synthesized in Escherichia coli has been purified 660-fold on the same antibody affinity column and was found to be associated with an E. coli protein of 70 kDa. Immunoblot analysis with specific antisera demonstrated that this E. coli protein was the heat shock protein dnaK, which has extensive sequence homology with the rat hsc70 protein. Incubation of the immunopurified p53-dnaK complex with ATP resulted in the dissociation of the p53-dnaK complex as it did with the p53-hsc70 complex. This remarkable conservation of p53-heat shock protein interactions and the specificity of dissociation reactions suggest a functionally important role for heat shock proteins in their interactions with oncogene proteins.
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PMID:Purification of complexes of nuclear oncogene p53 with rat and Escherichia coli heat shock proteins: in vitro dissociation of hsc70 and dnaK from murine p53 by ATP. 328 77

Recently, Frey (Cytometry 17:310-318, 1994) demonstrated that TO-PRO-3 iodide (TP3) can be excited indirectly by a 488 nm laser line through energy transfer by propidium iodide (PI). In the present study, we investigated whether PI-TP3 energy transfer can help to overcome spectral cross talk problems associated with the combined use of fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), and PI. Mixtures of keratin 8/18 FITC-labeled, keratin 8/18-PE-labeled, and unlabeled MCF-7 breast carcinoma cells were prepared and stained for DNA with PI (100 microM). The effect of adding a range of TP3 concentrations (0.001 to 16 microM) to these mixtures was evaluated. The combined use of PI and TP3 was further evaluated using mixtures of unlabeled and p53 FITC-labeled COV362.cl4 ovarian cancer cells and mixtures of unlabeled and p53 FITC-labeled COV362.cl4 cells and peripheral blood lymphocytes (PBL), additionally stained for keratin 8/18 (PE). Finally, a human ovarian ascites tumor specimen was triple-stained for keratin 8/18 (PE), vimentin (FITC) and DNA or keratin 8/18 (PE), PCNA (FITC) and DNA. Addition of TP3 allowed complete correction for spectral cross talk of PE/PI into the green fluorescence detector (FL1). Only minimal (FL1 - %FL2) compensation was required at a TP3 concentration of 2.0 microM in the presence of PI (100 microM). The PI spectral cross talk into the orange fluorescence detector (FL2) was reduced by about 50% using the same photomultiplier (PMT) settings. Although addition of TP3 reduced the signal-to-background ratio by about 30%, the advantage gained through full compensation for spectral cross talk resulted in an improved discrimination of p53-positive and -negative subpopulations in a mixture of human PBL and COV362.cl4 cells. Furthermore, vimentin-negative and PCNA-negative cells were better resolved in a human DNA-aneuploid ovarian ascites tumor after staining the DNA with PI/TP3, rather than with PI alone. We conclude that the addition of TP3 to PI improves the combined measurement by single-laser flow cytometry of DNA-ploidy and antigen expression in heterogenous clinical samples.
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PMID:Improved single laser measurement of two cellular antigens and DNA-ploidy by the combined use of propidium iodide and TO-PRO-3 iodide. 926 54