A partially purified fraction of bovine muscle cathepsins prepared from a crude extract by precipitation with ammonium sulfate between 45 and 55% saturation was assayed for activity on 18 peptides and other synthetic substrates, and on preparations of four natural substrates isolated from beef. Three of the synthetic substrates were partially hydrolyzed by the enzyme preparation. The hydrolysis of carbobenzoxy-L-glutamyl-L-tyrosine indicated endopeptidase activity. Dipeptidase and/or endopeptidase activity was evidenced by the hydrolysis of glycyl-L-tyrosine and L-leucyl-L-tyrosine. When using either of these two peptides as the substrate, a chromatographically distinct compound was detected which was absent in the enzyme and substrate blanks.
The fraction had no detectable enzymatic action on crude preparations of actin, myosin, and actomyosin. Commercially purified serum albumin was hydrolyzed, but at a much slower rate than denatured hemoglobin, which has been widely used for assaying catheptic activity. Sarcoplasmic proteins indigenous to the crude extract appeared to be readily hydrolyzed by the cathepsins.