文章基本信息

标题：Tryptic peptide analysis of normal and mutant forms of hypoxanthine phosphoribosyltransferase from HeLa cells
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作者：G Milman ; S W Krauss ; A S Olsen 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1977
卷号：74
期号：3
页码：926-930
DOI：10.1073/pnas.74.3.926
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Hypoxanthine phosphoribosyltransferase (HPRT, IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8 ) can be purified 5-to 10,000-fold from extracts of HeLa (human) cells by a three-step procedure consisting of high-speed centrifugation, adsorption to Sepharose-conjugated HPRT antibody, and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Purified enzyme labeled in vivo with radioactive lysine, arginine, or methionine was digested with trypsin and the tryptic peptides were separated by column chromatography on Bio-Rad cation exchanger Aminex A-5. Less than 50 ng (2 pmol) of HPRT is required to produce a tryptic peptide pattern. A methionine-labeled peptide was identified as the COOH-terminus because it was not labeled with either lysine or arginine. We have compared the tryptic peptide patterns of normal HeLaHPRT and a crossreacting HPRT protein lacking enzyme activity from HeLa mutant H23 [Milman et al. (1976) Proc. Natl. Acad. Sci. USA 73, 4589--4593]. The mutant protein has a new lysine-labeled peptide, but the chromatography patterns of arginine- or methionine-labeled peptides appear identical to those of the normal protein. The appearance in the H23 mutant HPRT protein of a new tryptic peptide provides strong evidence for a mutation in the HPRT structural gene. The tryptic peptide patterns were used to determine the total number of residues of labeled amino acid in the protein, and the values are reasonably consistent with those determined by conventional amino acid analysis pf erythrocyte HPRT.