Isolation of two forms of the high-molecular-mass serine protease, ingensin, from porcine skeletal muscle.
Two forms of a neutral protease that catalyzed the hydrolysis of succinyl-Leu-Leu-Val-Tyr-MCA were isolated from porcine skeletal muscle cytosol by fractionation on DEAE-cellulose, hydroxyapatite and Sephadex G-100. The native enzyme had a molecular mass of above 1000 kDa. Peak A, which was eluted from hydroxyapatite at 50 mM phosphate, was activated 37-fold by the detergent, SDS, while peak B which was eluted at 150 mM phosphate, was activated only 2-fold. After dialysis against water, the B form showed restored ability to be activated by SDS (9.6-fold with 0.04% SDS). The activated peak B was extremely sensitive to divalent and monovalent cations such as Ca2+, Mg2+, Na+, K+ and NH+4 as well as protease inhibitors such as leupeptin, chymostatin and DFP. These results suggest that these proteases are generally latent in the cells and may be regulated by changes in the concentrations of cations in the cytosol. We call this new type of protease, ingensin.
D000818:Animals; D000976:Antipain / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003374:Coumarins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000302:isolation & purification*; D010450:Endopeptidases / Q000302:isolation & purification*; D007527:Isoenzymes / Q000032:analysis*; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000302:isolation & purification*; D009132:Muscles / Q000201:enzymology*; D009842:Oligopeptides; D010436:Pepstatins / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013552:Swine
D003374:Coumarins; D007527:Isoenzymes; D007976:Leupeptins; D009097:Multienzyme Complexes; D009842:Oligopeptides; D010436:Pepstatins; C014195:Streptomyces pepsin inhibitor; D012967:Sodium Dodecyl Sulfate; D000976:Antipain; C032425:N-(N-(3-carboxyoxirane-2-carbonyl)leucyl)isoamylamine; C046582:succinyl-leucyl-leucyl-valyl-tyrosyl-methylcoumarinamide; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D007930:Leucine; C012211:ubenimex; C032854:leupeptin; C031375:pepstatin