Protease-susceptible sites and properties of fragments of aortic smooth-muscle myosin

Abstract

We have examined the protease susceptibility of aortic myosin, the thermal unfolding profiles of myosin rod and light meromyosin (LMM) and the solubility properties of the LMM fragments. Two major protease-susceptible sites were found, located at the head-rod junction and the heavy meromyosin (HMM)-LMM junction. Both tryptic and chymotryptic digestion of aortic myosin rod produced the LMM (80-85 kDa) and short subfragment 2 (S-2) (40-45 kDa) segments, which were similar to those of gizzard myosin rod and differed from the short LMM (70 kDa) and long S-2 (58 kDa) segments produced from skeletal-muscle rod. The thermal unfolding profile of aortic myosin rods exhibited three helix-unfolding transitions, at 47.5, 51 and 54 degrees C, similar to those of gizzard rods yet different from those of skeletal-muscle rods. There was a dramatic difference in the solubility of aortic LMM fragments of various molecular mass, as for gizzard smooth-muscle LMM and rabbit skeletal-muscle LMM. LMM fragments of molecular mass 77 kDa or more were completely insoluble in low-ionic-strength buffer, whereas LMM fragments of molecular mass 73 kDa or less were completely soluble in low-ionic-strength buffer. Proteolytic digestion patterns of LMM showed two additional protease-susceptible sites located 13 and 30 kDa from the ends of the LMM molecule. This suggests the existence of flexible regions within the LMM molecule, which may be responsible for the folded form of aortic myosin.

Muscular myosin isoforms of Taenia solium (Cestoda)

“…This information, together with the finding that peptides between 40 and 30 kDa had different concentrations in cysticerci and tapeworms, points to differences in amino acid sequences and therefore suggests that they correspond to isoforms. We can also suggest that digestion performed in the gel was a successful procedure since similar peptides to those described in previous reports were found (King et al, 1995;Eddinger and Murphy, 1988;Tashiro et al, 1985). Furthermore, previous findings (King et al 1995) show that the flexible region in the tail of smooth muscle myosin is found in peptides that have molecular weights similar to those found in T. solium myosin (30e32 kDa).…”

“…The expression of myosin depends on the developmental stage, on the type and degree of contractile activity and tissue, therefore several myosin isoforms are found in the same organism (Arner et al, 2003;Berg et al, 2001;Babu et al, 2000). Myosin ATPase activities, limited proteolysis, electrophoretic separation and immunochemical assays are successful strategies for demonstrating the presence of isoforms (De la Cruz and Ostap, 2004;Blough et al, 1997;King et al, 1995;Kimura et al, 1991). Type II myosin and several actin isoforms were found in muscle fibers of Taenia solium Ambrosio et al, 1997).…”

Comparison of biochemical and immunochemical properties of myosin II in taeniid parasites

“…The chymotrypsin subunit is at 0.9. related but different genes. The peptides common to all the digested MHCs probably correspond to conserved myosin regions, as described in previous reports (King et al, 1995;Eddinger and Murphy, 1988;Tashiro et al, 1985); the others are taeniid-specific. Our data show that metacestodes and adults of various taeniid parasites have different myosin II isoforms as judged by enzyme activity, V max , antigenic specificity and MHC peptide maps, as found previously for T. solium myosin and actin (Ambrosio et al, 1997Gonzalez-Malerva et al, 2004).…”

The Tip of the Coiled-coil Rod Determines the Filament Formation of Smooth Muscle and Nonmuscle Myosin

“…2). Interestingly, the antibody binding sites were nearly identical to the flexible regions of the rod that are predicted according to the amino acid sequence and electron microscopic observation (13,(35)(36)(37)(38)(39)(40), suggesting that the flexible regions have high antigenicity (Fig. 2).…”