‘Artificial’ Myosin Isozymes; Preparation and Characteristics

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Abstract
Using precisely monitored proteolytic digestion conditions rabbit fast skeletal muscle myosin could be selectively modified in different ways. A myosin isozyme with a 20‐kDa alkali light chain 1 (Al) could be obtained by digesting with papain in the presence of Ca2+. Under these conditions alkali light chain 2 (A2) was cleaved at Lys‐17 and lost a 2.3‐kDa N‐terminal fragment including the strongly basic N terminus and about half of the characteristic (Ala‐Pro) sequence. The Nbs2‐[5,5′ dithiobis(2‐nitrobenzoic acid)‐]light chain and A2 were left unmodified and less than 5% of the myosin heavy chain presented a break in the subfragment‐2 region. EDTA and Ca2+ ATPase activities were unchanged. A myosin isozyme with an 18‐kDa Nbs2‐light chain was obtained by limited digestion with trypsin in the presence of Ca2+. The 18.9→18‐kDa conversion was nearly 100% whereas less than 10% of the heavy chain was fragmented and only about 5% of A1 was converted to A1′. The Nbs2‐light chain was cleaved at Arg‐7 preserving Ser‐15 and consequently a phosphorylated modified myosin could be obtained. A quasi‐elastic light‐scattering study showed that this modified myosin in high‐ionic‐strength solutions exhibited physicochemical characteristics quite similar to those of unmodified myosin.