Isolation and characterization of a novel 45 kDa calponin-like protein from anterior byssus retractor muscle of the mussel Mytilus galloprovincialis

Abstract

A calponin-like protein of 45 kDa was isolated from mussel anterior byssus retractor muscle (ABRM) and its inhibitory effects on actomyosin Mg2+-ATPase was demonstrated. The 2-D electrophoresis for ABRM myofibrils gave a spot of 45 kDa protein in addition to myofibrillar proteins such as myosin and actin. The 45 kDa protein, which was more basic and showed a slightly higher molecular weight than actin, was isolated by ion-exchange chromatography and subjected to chymotryptic digestion. N-terminal amino acid sequencing of polypeptide fragments produced gave two sequences, ASQKGMTSFGAVRHH and GMDRALISKMGSKYDSGL, both of which showed a high homology to those of vertebrate calponins and invertebrate calponin-related proteins. Furthermore, the 45 kDa protein strongly reacted with commercially available antibody raised against chicken smooth muscle calponin, demonstrating that the mussel ABRM 45 kDa protein is a new member of the calponin family. Then, actomyosin Mg2+-ATPase activity of ABRM was measured in the presence and absence of the 45 kDa protein. The 45 kDa protein clearly inhibited actomyosin Mg2+-ATPase activity in a dose-dependent manner as in the case of other vertebrate calponins. These results indicate that the 45 kDa calponin-like protein is involved in the thin filament-associated regulation of molluscan smooth muscle contraction, possibly of a unique contraction called catch.