Purification and characterization of two metalloproteinases from squid mantle muscle, myosinase I and myosinase II

Abstract

Metailoproteinases, myosinase I and myosinase II, that hydrolyze the heavy chain of myosin, were purified from squid mantle muscle. Myosinase I does not hydrolyze other muscle proteins, casein, haemoglobin, or MCA-substrates, while II hydrolyzes tropomyosin. Both myosinase I and myosinase II gave a single protein band on SDS-PAGE with a molecular mass of 16 and 20 kDa, respectively. Their activities were inhibited by EDTA and 1,10-phenanthroline, and II was also inhibited by EGTA. They could be reactivated with some divalent cations, I was especially reactivated with Co 2+ and II especially with Zn 2+. The optimum pH of both activities was 7.0; the optimum temperature for both was 40°C. Myosinase I hydrolyzes myosin heavy chains to produce 130 and 90 kDa fragments. The N-terminal amino-acid sequence of the 90 kDa fragment indicates that myosinase I splits the myosin heavy chain between Ala-ll61 and Thr-l162 in subfragment 2. Myosinase II hydrolyzes myosin heavy chain to produce 158 and 65 kDa fragments, and it splits between Glu-1381 and Thr-1382 in LMM. Myosinases I and II are most likely related to the metabolism of myosin in vivo.