Further characterization of the structural and functional properties of the cross‐linked complex between F‐actin and myosin S‐1

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Abstract
Several structural and functional properties of the covalent complex, formed upon cross‐linking of the myosin heads (S‐1) to F‐actin with 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide, were characterized. The elevated Mg2+‐ATPase activity was measured during a 1‐month storage of the complex under various conditions. In aqueous medium it showed a rapid time‐dependent decrease but it was significantly more stable in the presence of 50% ethylene glycol at ‐20°C. The ATPase loss most likely reflects a progressive conformational change within the S‐1 ATPase site resulting from its greater exposure to the medium, induced by the permanently bound F‐actin. The covalent acto‐S1 complex was submitted to depolymerization‐repolymerization experiments using different depolymerizing agents (0.6 M KI; 4.7 M NH4Cl; low‐ionic‐strength solution). The depolymerization led to an immediate loss of the enhanced Mg2+‐ATPase activity; this activity was almost entirely recovered upon repolymerization of the complex. The protein material formed upon depolymerization of the covalent acto‐S1 was analyzed by gel chromatography, gel electrophoresis, analytical ultracentrifugation and electron microscopy. It comprised mainly small‐sized actin oligomers associated with the covalently bound S‐1 and only a limited amount of free G‐actin. The results illustrate the relationships between the filamentous state of actin and its ability to stimulate the Mg2+ ‐ATPase activity of S‐1. They also indicate that the binding of S‐1 to F‐actin is transmitted to several neighbouring actin subunits and strengthens the interactions between actin monomers. Acto‐S1 cross‐linked complexes were prepared in the presence of tropomyosin and the tropomyosin‐troponin system. Under the conditions employed, the regulatory proteins were not cross‐linked to actin or S‐1 and did not affect the extent or the pattern of S‐1 cross‐linking to F‐actin. Measurements of the elevated Mg2+ ‐ATPase activity of the cross‐linked preparations revealed that tropomyosin and the tropomyosin‐troponin complex, in the absence of Ca2+, inhibit ATP hydrolysis; the extent of ATPase inhibition (up to 50%) was dependent on the amount of covalently bound S‐1, being larger at low level of S‐1 cross‐linking; the addition of Ca2+ restored the ATPase activity to the control value. The data provide direct evidence that the regulatory proteins can modulate directly the kinetics of ATP hydrolysis by the covalent acto‐S1 complex as has earlier been suggested for the reversible complex [Chalovich, J. M. and Eisenberg, E. (1982) J. Biol. Chem. 257, 2432‐2437]. Finally the selective limited digestion of tropomyosin with trypsin in the absence and presence of acto‐S1 was employed as a probe of the binding of the regulatory protein to the covalent acto‐S1. It indicated that the preliminary cross‐linking of S‐1 leads to an increase of the amount of tropomyosin bound to F‐actin; however the extra binding of tropomyosin did not change its ability to regulate the activity of the covalent acto‐S1 complex.