Purification and characterization of a myosin-cleaving protease from rat heart myofibrils.
A proteolytic enzyme, which causes the limited degradation of cardiac myosin, was purified from rat heart myofibrils. The purified enzyme (a myosin-cleaving protease) was apparently homogeneous by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Autolysis of the purified enzyme was observed at neutral pH without high concentration of CaCl2. The molecular weight was estimated to be 26 000-27 000. The enzyme was active against casein, N-acetyl-L-tyrosine ethyl ester and N-glutaryl-L-phenylalanine-4-nitroanilide (Glu-Phe-NAn), but less active with N-benzoyl-DL-arginine-4-nitroanilide. Optimum pH values for the enzyme were 9.0 for casein and 8.4 for Glu-Phe-NAn. Caseinolytic activity of the enzyme was completely inhibited with phenylmethylsulfonyl fluoride and diisopropylphosphofluoride and partially inhibited with L-1-tosyl-L-phenylalanine chloromethyl ketone (Tos-PheCH2Cl) and soybean trypsin inhibitor. Tos-LysCH2Cl had no effect. Sulfhydryl reagents, metal-chelating agents and metal ions except for Zn2+ had little or no effect on the activity. Degradation of cardiac myosin with the enzyme produced two fragments having molecular weights of 130 000 and 94 000, accompanied by the disappearance of myosin heavy chain and light chain 2. Myosin degradation with the enzyme was more restrictive than with chymotrypsin.
D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007531:Isoflurophate / Q000494:pharmacology; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D009210:Myofibrils / Q000201:enzymology*; D009218:Myosins / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D051381:Rats; D013379:Substrate Specificity; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000494:pharmacology
D002413:Cations, Divalent; D007531:Isoflurophate; D010664:Phenylmethylsulfonyl Fluoride; D014360:Trypsin Inhibitor, Kunitz Soybean; D010447:Peptide Hydrolases; D009218:Myosins