PRIMARY PEPTIDE SEQUENCES FROM SQUID MUSCLE AND OPTIC LOBE MYOSIN IIs: A STRATEGY TO IDENTIFY AN ORGANELLE MYOSIN

Abstract

The squid giant axon provides an excellent model system for the study of actin-based organelle transport likely to be mediated by myosins, but the identification of these motors has proven to be difficult. Here the authors purified and obtained primary peptide sequence of squid muscle myosin as a first step in a strategy designed to identify myosins in the squid nervous system. Limited digestion yielded fourteen peptides derived from the muscle myosin which possess high amino acid sequence identities to myosin II from scallop (60-95%) and chick pectoralis muscle (31-83%). Antibodies generated to this purified muscle myosin were used to isolate a potential myosin from squid optic lobe which yielded 11 peptide fragments. Sequences from six of these fragments identified this protein as a myosin II. The other five sequences matched myosin II (50-60%, identities), and some also matched unconventional myosins (33-50%). A single band that has a molecular weight similar to the myosin purified from optic lobe copurifies with axoplasmic organelles, and, like the optic lobe myosin, this band is also recognized by the antibodies raised against squid muscle myosin II. Hence, this strategy provides an approach to the identification of a myosin associated with motile axoplasmic organelles.

VASP protects actin filaments from gelsolin: An in vitro study with implications for platelet actin reorganizations

“…We also attempted to avoid urea by eluting VASP with DMSO from the poly-L-proline columns as has been successful for the purification of profilin [Lindberg et al, 1988], but this did not apply for VASP. VASP, profilin, and Grb2 were simultaneously eluted with 6 M Urea in 50 mM Tris-HCl (pH 7.4) and identified by microsequencing of each band performed as we described [Medeiros et al, 1998;Bearer, 2000]. At least two phosphorylated forms of VASP (50 and 46 kDa) [Halbrugge et al, 1990] were present.…”

2E4 (Kaptin): A novel actin-associated protein from human blood platelets found in lamellipodia and the tips of the stereocilia of the inner ear

“…Blots were performed as described. The Coomassie-stained band corresponding to the band recognized by MAb 2E4 was excised from the gel and subjected to in-gel proteolysis followed by HPLC separation and automated Edman degradative sequencing as we described [20].…”

Muscular myosin isoforms of Taenia solium (Cestoda)

“…Pre-stained broad range molecular weight markers (MW) were used. separation of these proteins is difficult because the interaction of the hydrophobic region of myosin with paramyosin is necessary for the solubility and assemblage of thick filaments (Medeiros et al, 1998;Cohen and Parry, 1998). Paramyosin is an important molecule in T. solium and has been profusely described (Laclette et al, 1991;Guerra et al, 1982;Flisser et al, 1980).…”