Detection and partial characterization of the chromatin-associated proteases of yeast Saccharomyces cerevisiae.
The chromatin fraction was prepared from yeast Saccharomyces cerevisiae free from cytoplasmic contamination except for a trace of mitochondria. When the yeast chromatin was incubated with histones as a substrate it showed three peaks of proteolytic activity as approximately pH 4, pH 7 and pH 11. These activities were separated from each other by differential extractions from chromatin and successive gel filtration through Sephadex G-100. Proteases were partially characterized by affinity labeling with [3H]diisopropylfluorophosphate (iPr2P-F) and by various protease inhibitors. The neutral and the alkaline proteases were serine proteases with a molecular mass of 35 kDa and 25 kDa respectively. The acidic protease showed a molecular size larger than 100 kDa on the gel filtration, and was probably an aspartyl protease because it was most strongly inhibited by pepstatin. A iPr2P-F-binding protein with a molecular mass of 66 kDa, found in chromatin, was likely to be converted to the alkaline protease of 25 kDa when chromatin was incubated at pH 10 or in 6 M urea/0.1 M phosphoric acid at the extraction. The distribution of proteolytic activities and iPr2P-F-binding proteins were compared among chromatins from different strains and from cells in different growth phases and it was found that these three proteases were present in all of them but with different proportions. Considering that rat liver chromatin contains equivalents to these proteases [Tsurugi, K. and Ogata, K. (1982) J. Biochem. (Tokyo) 92, 1369-1381], the results suggested that they play some important roles in the function of eukaryotic chromatin.
D002352:Carrier Proteins / Q000032:analysis; D002843:Chromatin / Q000201:enzymology*; D010450:Endopeptidases / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007531:Isoflurophate / Q000378:metabolism; D010447:Peptide Hydrolases / Q000032:analysis* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development; D012697:Serine Endopeptidases
D002352:Carrier Proteins; D002843:Chromatin; D007531:Isoflurophate; D010450:Endopeptidases; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases