Separation of cathepsin B1 and related enzymes from rat skeletal muscle

• Published in: Biochimica et biophysica acta Vol. 577; no. 2; p. 253
• Main Authors: Hardy, M F, Pennington, R J
• Format: Journal Article
• Language: English
• Published: Netherlands 25-04-1979
• Subjects: Animals; Cathepsins - isolation & purification; Chromatography, Gel; Chromatography, Ion Exchange; Female; Muscle Denervation; Muscles - enzymology; Rats
• DOI: 10.1016/0005-2795(79)90029-1
• ISSN: 0006-3002

Rat muscle was extracted at pH 4 and submitted to gel-filtration on Sephadex G-75 and to chromatography on DEAE-Sephadex. Gel-filtration gave a large peak of activity towards Bz-Arg-NNap with an estimated molecular weight of 25,500. Activity towards Bz-Arg-NH2 was present in this peak and in another peak of molecular weight 45,000. The second peak also hydrolysed benzoyl-glycyl-L-arginine. DEAE-Sephadex gave five peaks of Bz-Arg-NNap hydrolysing activity; all showed thiol dependence. Peaks III, IV and V hydrolysed Z-Ala-Arg-Arg-NNap-OMe rapidly; they also inactivated aldolase and were strongly inhibited by leupeptin. They are probably isoenzymes of cathepsin B1. Peak I showed these properties to a relatively small extent. 7-(N-Benzoyl-DL-argininamide)-4-methylcoumarin appears to be an alternative substrate for cathepsin B1; it was hydrolysed also by peak I, but relatively less rapidly. Peaks I and II were inhibited more than peaks III, IV and V by a muscle extract. Total activity of the Bz-Arg-NH2-hydrolysing enzyme in extensor digitorum longus muscle increased after denervation.