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. 1988 Jun;8(6):2647–2650. doi: 10.1128/mcb.8.6.2647

Molecular tools for inactivating a yeast enzyme in vivo.

J R Carlson 1, I L Weissman 1
PMCID: PMC363467  PMID: 3043187

Abstract

As part of an effort to develop a new means of inducibly inactivating cellular proteins in vivo, three monoclonal antibodies which neutralize yeast alcohol dehydrogenase (ADH) activity were isolated and characterized with respect to criteria important for the inactivation strategy. The significance of these criteria is considered, and a general means of generating appropriate antibodies is suggested. All three antibodies described here were specific for ADH I; they did not recognize the closely related isozyme ADH II in a plate-binding assay and did not immunoprecipitate molecules other than ADH from a Saccharomyces cerevisiae extract. Neutralization occurred in a yeast extract and, for two antibodies, was blocked by high concentrations of the coenzyme NAD+. This finding suggests that the antibodies may block enzyme activity by stabilizing an inactive form of ADH lacking bound NAD+. These results provide a foundation for the use of these antibodies to inactivate ADH in vivo.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beier D. R., Sledziewski A., Young E. T. Deletion analysis identifies a region, upstream of the ADH2 gene of Saccharomyces cerevisiae, which is required for ADR1-mediated derepression. Mol Cell Biol. 1985 Jul;5(7):1743–1749. doi: 10.1128/mcb.5.7.1743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beier D. R., Young E. T. Characterization of a regulatory region upstream of the ADR2 locus of S. cerevisiae. Nature. 1982 Dec 23;300(5894):724–728. doi: 10.1038/300724a0. [DOI] [PubMed] [Google Scholar]
  3. Bennetzen J. L., Hall B. D. The primary structure of the Saccharomyces cerevisiae gene for alcohol dehydrogenase. J Biol Chem. 1982 Mar 25;257(6):3018–3025. [PubMed] [Google Scholar]
  4. Carlson J. R. A new means of inducibly inactivating a cellular protein. Mol Cell Biol. 1988 Jun;8(6):2638–2646. doi: 10.1128/mcb.8.6.2638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. HVIDT A., KAEGI J. H., OTTESEN M. EFFECT OF OXIDIZED NICOTINAMIDE-ADENINE DINUCLEOTIDE ON HYDROGEN-DEUTERIUM EXCHANGE OF YEAST ALCOHOL DEHYDROGENASE AS MEASURED BY INFRARED SPECTROPHOTOMETRY. Biochim Biophys Acta. 1963 Sep 24;75:290–292. doi: 10.1016/0006-3002(63)90614-0. [DOI] [PubMed] [Google Scholar]
  6. Koshland M. E. Structure and function of the J chain. Adv Immunol. 1975;20:41–69. doi: 10.1016/s0065-2776(08)60206-0. [DOI] [PubMed] [Google Scholar]
  7. Kronvall G., Grey H. M., Williams R. C., Jr Protein A reactivity with mouse immunoglobulins. Structural relationship between some mouse and human immunoglobulins. J Immunol. 1970 Nov;105(5):1116–1123. [PubMed] [Google Scholar]
  8. Russell D. W., Smith M., Williamson V. M., Young E. T. Nucleotide sequence of the yeast alcohol dehydrogenase II gene. J Biol Chem. 1983 Feb 25;258(4):2674–2682. [PubMed] [Google Scholar]
  9. Shulman M., Wilde C. D., Köhler G. A better cell line for making hybridomas secreting specific antibodies. Nature. 1978 Nov 16;276(5685):269–270. doi: 10.1038/276269a0. [DOI] [PubMed] [Google Scholar]
  10. Vallee B. L., Hoch F. L. ZINC, A COMPONENT OF YEAST ALCOHOL DEHYDROGENASE. Proc Natl Acad Sci U S A. 1955 Jun 15;41(6):327–338. doi: 10.1073/pnas.41.6.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Wills C., Jörnvall H. The two major isozymes of yeast alcohol dehydrogenase. Eur J Biochem. 1979 Sep;99(2):323–331. doi: 10.1111/j.1432-1033.1979.tb13260.x. [DOI] [PubMed] [Google Scholar]
  12. Young E. T., Pilgrim D. Isolation and DNA sequence of ADH3, a nuclear gene encoding the mitochondrial isozyme of alcohol dehydrogenase in Saccharomyces cerevisiae. Mol Cell Biol. 1985 Nov;5(11):3024–3034. doi: 10.1128/mcb.5.11.3024. [DOI] [PMC free article] [PubMed] [Google Scholar]

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