Kinetic mechanism of 1-N6-etheno-2-aza-ATP and 1-N6-etheno-2-aza-ADP binding to bovine ventricular actomyosin-S1 and myofibrils.
The fluorescence emission of 1-N6-etheno-2-aza-ATP (epsilon-aza-ATP) at 410-460 nm is enhanced approximately 8-fold upon mixing substoichiometric concentrations of epsilon-aza-ATP with bovine cardiac actomyosin-S1 or myofibrils. The time course of nucleotide fluorescence measured in a front face stopped flow cell upon mixing epsilon-aza-ATP with bovine cardiac myofibrils ([Ca2+] less than 10(-7) M) is essentially the same as that with bovine cardiac actomyosin subfragment-1. In single turnover experiments, the fluorescence rapidly rises to a maximum value, then decreases with a rate constant of 0.04 s-1 at 0 degree C to a final value that is approximately twice the level of the unbound nucleotide. At concentrations of epsilon-aza-ATP greater than 40 microM the kinetics of epsilon-aza-ATP binding is clearly biphasic for both actomyosin-S1 and myofibrils. At 0 degree C, the rate of the more rapid phase is proportional to nucleotide concentration and has a second order rate constant of 1.7 X 10(5) M-1 s-1; the rate of the slower phase extrapolates to a maximum of 4-5 s-1 at high nucleotide concentration. The rate constants for dissociation of epsilon-aza-ADP from bovine cardiac actomyosin-S1 and myofibrils were measured from the decrease in epsilon-aza-ADP fluorescence enhancement observed upon displacement by ATP to be 20 and 18 s-1, respectively, at 0 degree C. These results indicate that most of the cross-bridges in cardiac myofibrils are bound to actin and that the geometric constraints imposed upon the interaction of actin and myosin by the three-dimensional structure of the myofibril do not modify the kinetics of epsilon-aza-ATP binding or epsilon-aza-ADP dissociation.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D000818:Animals; D002417:Cattle; D004985:Ethenoadenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D005434:Flow Cytometry; D007700:Kinetics; D008433:Mathematics; D009206:Myocardium / Q000201:enzymology*; D009210:Myofibrils / Q000378:metabolism*; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D004985:Ethenoadenosine Triphosphate; D015879:Myosin Subfragments; D010446:Peptide Fragments; C042937:aza-epsilon-ADP; C012758:aza-epsilon-ATP; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009218:Myosins