Further characterization of the structural and functional properties of the cross-linked complex between F-actin and myosin S-1.
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 degrees 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].(ABSTRACT TRUNCATED AT 400 WORDS)
Rouayrenc
J F
JF
Bertrand
R
R
Kassab
R
R
Walzthöny
D
D
Bähler
M
M
Wallimann
T
T
D000199:Actins; D000251:Adenosine Triphosphatases / Q000378:metabolism; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase; D002845:Chromatography / Q000379:methods; D004355:Drug Stability; D004586:Electrophoresis / Q000379:methods; D004789:Enzyme Activation / Q000187:drug effects; D066298:In Vitro Techniques; D015879:Myosin Subfragments; D009218:Myosins; D010446:Peptide Fragments; D011485:Protein Binding; D013048:Specimen Handling; D013329:Structure-Activity Relationship; D014335:Tropomyosin / Q000378:metabolism
D000199:Actins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D014335:Tropomyosin; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins