Purification and characterization of squid brain myosin.
Myosin was extracted from frozen squid brain and purified by a modification of the procedure of Pollard et al. (Pollard, T.D., Thomas, S.M., and Niederman, R. (1974) Anal. Biochem. 60, 258-266). Myosin was eluted from Bio-Gel A-15m column as a single peak of (K+-EDTA)-activated ATPase ((K+-EDTA)-ATPase) activity with an average partition coefficient (Kav) of 0.22. In sodium dodecyl sulfate-acrylamide gel electrophoresis, the purified myosin showed a predominant band with similar electrophoretic mobility as the heavy chain of rabbit skeletal muscle myosin, and two less intense bands near the bottom of the gel. No actin band was seen. The properties of the (K+-EDTA)-ATPase activity were: (a) the time course of the reaction was biphasic at 25 degrees but linear at 32 degrees; (b) the optimum rate of reaction was obtained between 0.3 and 0.8 M KCl; (c) the pH optimum was between 8.0 and 9.0; (d) the reaction was specific for ATP with an apparent Km of 0.19 mM. ATPase activity in 0.06 M KCl and 5 mM MgCl2 was increased about 1.5 times by a 10-fold excess of rabbit skeletal muscle F-actin and about 5 times by a 40-fold excess. The actin activation was inhibited slightly by the addition of 0.2 mM CaCl2 and completely by the addition of 10 mM CaCl2. Myosin formed arrowhead patterns with rabbit skeletal muscle F-actin as observed by electron microscopy of negatively stained samples. It also aggregated in bipolar filaments which attached to decorated actin filaments at different angles, as well as formed cross-connections and ladder-like patterns between actin filaments. These two forms of interactions between myosin and actin were abolished by treatment with MgATP.
D000199:Actins / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D049832:Decapodiformes / Q000032:analysis*; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008854:Microscopy, Electron; D009218:Myosins / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012964:Sodium / Q000494:pharmacology; D013696:Temperature
D000199:Actins; D046911:Macromolecular Substances; D004492:Edetic Acid; D012964:Sodium; D000251:Adenosine Triphosphatases; D009218:Myosins; D008274:Magnesium; D002118:Calcium