Purification and characterization of alkaline protease and neutral protease from chromatin of rats.
It was previously reported that, in addition to a known chymotrypsin-like protease capable of hydrolyzing histones with an optimum pH of 8 (neutral protease), another protease is bound to the chromatin of various rat tissues and in situ hydrolyzes casein more quickly than histones with an optimum pH of 10 (alkaline protease). In the present study, the alkaline protease was purified 14 000-fold to approx. 75% purity from the chromatin of Rhodamine sarcoma. This tumor contains both proteases at higher levels than normal tissues. For purification, affinity columns of Sepharose with bound soybean trypsin inhibitor, casein and histones were successively used. Also, the neutral protease was purified 920-fold to an apparently homogeneous state by affinity chromatography on a Sepharose column with bound soybean trypsin inhibitor under conditions, in which an excess amount of the enzyme was applied on the column so that part of the enzyme would pass through the column without adsorption and the enzyme thus adsorbed was then eluted. The purified alkaline and neutral proteases had molecular weights of approx. 18 000 and 27 000, respectively, and isoelectric points of approx. 11. The former enzyme hydrolyzed casein (100) in preference to histones (18) with an optimum pH of 9.5, whereas the latter enzyme preferred histones (100) to casein (32) with an optimum pH of 8. Their actions against other proteins and synthetic substrates were also studied.
D000818:Animals; D002364:Caseins / Q000378:metabolism; D002467:Cell Nucleus / Q000032:analysis; D002843:Chromatin / Q000201:enzymology*; D002846:Chromatography, Affinity / Q000379:methods; D006657:Histones / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008297:Male; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification*; D051381:Rats; D012513:Sarcoma, Experimental / Q000201:enzymology*; D013379:Substrate Specificity
D002364:Caseins; D002843:Chromatin; D006657:Histones; D010447:Peptide Hydrolases