Purification of Two Myosin -Degrading Proteases From Porcine Skeletal Muscle

Abstract

Myosin is the major skeletal muscle protein. However, the protease system responsible for myosin degradation in vivo has not yet been clearly described. Two myosin degrading proteases, a serine protease and a cysteine protease that might have some function in myosin degradation, were isolated from porcine skeletal muscle. Activity of the serine protease was inhibited by DFP, aprotinin, benzamidine, soybean trypsin inhibitor, antipain, leupeptin and beta-mercaptoethanol, but not altered by E-64, pepstatin, or EDTA. Using radiolabeled DFP, a protein with Mr 32,000 was identified on an SDS gel as a DFP-binding protein, which was either the serine protease or a catalytic subunit thereof. The partial amino acid sequence of the Mr 32,000 protein did not match with known primary sequences, indicating that the serine protease was either a uncharacterized protein or a known protein with unknown primary sequence. The serine protease hydrolyzed myosin heavy chains and light chain 1 and 2 at pH 7.5 and 37°C. The cleavage sites of chicken myosin heavy chain by the serine protease were at the carboxyl side of Lys367, Lys561 and Lys768. The serine protease did not hydrolyze Boc-Leu-Leu-Val-Tyr-AMC, but cleaved Boc-Val-Leu-Lys-AMC. The serine protease was optimally active at neutral pH and had little or no activity at pH below 6. On the other hand, activity of the cysteine protease was inhibited with E-64 but not affected by DFP. In contrast to the serine protease, the cysteine protease was activated by beta-mercaptoethanol although both activities were activated by Ca2+. The cysteine protease hydrolyzed myosin heavy chain and light chain 1 and 3 at pH 7.5 and 50°C and cleaved myosin heavy chain and at the carboxyl side of Glu1182, Lys1249, Asn1276 and His1719. The cysteine protease did not hydrolyze either Boc-Leu-Leu-Val-Tyr-AMC, Boc-Val-Leu-Lys-AMC, or Boc-Leu-Leu-Glu-AMC. The enzymatic property of cysteine protease was different from other cysteine proteases including calpains, cathepsin B and L, indicating the cysteine protease was either an uncharacterized protein or a known protein with unknown myosin specificity.