EPR and fluorescence depolarization studies on bovine cardiac myosin.
To test for possible differences in local conformation and S1 flexibility, bovine cardiac and rabbit skeletal myosins were labeled with a fluorophore (1,5-IAEDANS) and a spin label having iodoacetamide reactivity. The marked activation of the Ca2+-ATPase (6- to 8-fold) and inhibition of the K+ (EDTA)-ATPase (80-90%) by both labels indicated specific labeling of the fast-reacting thiols (SH1) of both myosins. Fluorescence depolarization studies of 1,5-IAEDANS-labeled cardiac myosin indicated that, like skeletal myosin, the SI moieties of cardiac myosin exhibit considerable segmental flexibility with respect to the rod portion of the molecule. This indicates that segmental flexibility may be a property of all myosins. Cardiac and skeletal myosins immobilized spin labels to approximately the same extent, indicating a similarity in steric restraints around the SH1 thiol of the two myosins. The magnitude of the changes in spin label mobility accompanying binding of MgADP and hydrolysis of MgATP was reduced in cardiac myosin relative to skeletal myosin. This suggests that the lower catalytic center activity of cardiac myosin is associated with more restricted conformational changes accompanying formation of M.ADP and M.ADP.Pi. From measurements of spin label mobility, the affinity of cardiac and skeletal myosin for ADP were similar: Kd (ADP) = 7 microM, n = 1.6. The EPR spectrum of spin labels attached to cardiac and skeletal myosin showed similar saturation effects upon actin binding indicating immobilization of myosin heads occurs with both proteins.
D000199:Actins / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001842:Bone and Bones / Q000032:analysis; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D005454:Fluorescence Polarization; D007460:Iodoacetamide / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism; D009206:Myocardium / Q000032:analysis*; D009218:Myosins / Q000032:analysis*; D009711:Nucleotides / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism
D000199:Actins; D009711:Nucleotides; D000251:Adenosine Triphosphatases; D009218:Myosins; D008274:Magnesium; D011188:Potassium; D002118:Calcium; D007460:Iodoacetamide