Activation of actin-cardiac myosin subfragment 1 MgATPase rate by Ca2+ shows cooperativity intrinsic to the thin filament.
The magnesium adenosinetriphosphatase (MgATPase) rate of cardiac myosin subfragment 1 (S-1) was studied in the presence of regulated actin in order to investigate the mechanism by which Ca2+ cooperatively induces cardiac muscle contraction. The MgATPase rate increased cooperatively with Ca2+, exhibiting a Hill coefficient of 1.8 and 50% activation at pCa 5.75. This cooperative response occurred despite an experimental design excluding several potential sources of cooperativity. First, to exclude spurious cooperativity due to erroneous calculation of pCa at low ionic strength, the affinities of Ca2+ and Mg2+ for [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) were measured by a novel method using Quin 2. At pH 7.06, 25 degrees C, and mu = 30 mM, the KD was 140 nM for CaEGTA and 2.7 mM for MgEGTA. Second, the cooperativity was not produced by actin-myosin S-1 binding; myosin S-1 was bound to only 1 of every 300 actin promoters, and earlier work [Tobacman, L. S., & Adelstein, R. S. (1986) Biochemistry 25, 798-802] had shown that cardiac myosin S-1 binds with equal affinity to the thin filament at very low Ca2+ and at saturating Ca2+ concentrations. Furthermore, the adenosine 5'-triphosphate turnover rate of the myosin S-1 was independent of enzyme concentration at low, intermediate, and saturating Ca2+ concentrations. Finally, since cardiac troponin has only one regulatory Ca2+-specific site, cooperative interactions between such sites could not occur. These data suggest that part of the cooperativity conferred by interaction between adjacent troponin-tropomyosin complexes is intrinsic to the thin filament and independent of myosin.
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D002413:Cations, Divalent; D002417:Cattle; D003599:Cytoskeleton / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D007700:Kinetics; D008433:Mathematics; D009132:Muscles / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology*; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011817:Rabbits
D000199:Actins; D000634:Aminoquinolines; D002413:Cations, Divalent; D015879:Myosin Subfragments; D010446:Peptide Fragments; D004533:Egtazic Acid; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins; C035739:Quin2; D002118:Calcium