A high-molecular-weight cysteine endopeptidase from rat skeletal muscle.
A cytosolic enzyme of high molecular weight (about 500 000), which attacks native or denatured proteins (inter alia, casein, globin and hexokinase) was purified about 1000-fold from mixed rat skeletal muscles, including muscles freed of mast cells by prior treatment of the animals with the degranulator, compound 48/80. Peptides of varying size were generated from radioactively labelled globin, but no free amino acids were formed; free tyrosine was also not released from azocasein. The pH optimum was 7.5 and the presence of an essential cysteine group was suggested because dithiothreitol (1 mM) stimulated the activity and N-ethylmaleimide (5 mM) and p-chloromercuriphenylsulphonic acid (1 mM) were inhibitors. The activity was markedly inhibited by Zn2+ but not by leupeptin, chymostatin or pepstatin. The enzyme was stabilized by ATP, at concentrations as low as 0.1 mM, against inactivation at 42 degrees C. The endopeptidase was clearly separated on gel chromatography from another large protease, also sensitive to Zn2+, but with marked aminopeptidase activity and the properties of hydrolase H. The activity levels of the protease, assayed after chromatography on Sepharose 6B of high-speed supernatant fractions, did not vary significantly in skeletal muscle samples which were derived from denervated, starved, diabetic or hyperthyroid animals, in all of which the abnormal physiological states expressed themselves as enhanced rates of tyrosine released by incubated soleus and extensor digitorum longus muscles. Nevertheless, the enzyme described here may be part of an ATP-dependent, multi-component proteolytic system similar to that already known to be present in reticulocytes.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002712:Chlorides; D003546:Cysteine Endopeptidases; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D006980:Hyperthyroidism / Q000201:enzymology; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D009121:Muscle Denervation; D009132:Muscles / Q000201:enzymology*; D051381:Rats; D013217:Starvation; D013379:Substrate Specificity; D015032:Zinc / Q000494:pharmacology; D017967:Zinc Compounds; D003189:p-Methoxy-N-methylphenethylamine / Q000494:pharmacology
D002712:Chlorides; D017967:Zinc Compounds; D003189:p-Methoxy-N-methylphenethylamine; C016837:zinc chloride; D000255:Adenosine Triphosphate; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D015032:Zinc