Rate of ATP synthesis by dynein.
The rates of ATP synthesis and release by the dynein ATPase were determined in order to estimate thermodynamic parameters according to the pathway: (Formula: see text). Dynein was incubated with high concentrations of ADP and Pi to drive the net synthesis of ATP, and the rate of ATP production was monitored fluorometrically by production of NADPH through a coupled assay using hexokinase and glucose-6-phosphate dehydrogenase. The turnover number for the rate of release of ATP from 22S dynein was 0.01 s-1 per site at pH 7.0, 28 degrees C, assuming a molecular weight of 750 000 per site. The same method gave a rate of ATP synthesis by myosin subfragment 1 of 3.4 X 10(-4) s-1 at pH 7.0, 28 degrees C. The rate of ATP synthesis at the active site was estimated from the time dependence of medium phosphate-water oxygen exchange. Dynein was incubated with ADP and [18O] Pi, and the rate of loss of the labeled oxygen to water was monitored by 31P NMR. A partition coefficient of 0.31 was determined, which is equal to k-2/(k-2 + k3). Assuming k3 = 8 s-1 [Johnson, K.A. (1983) J. Biol. Chem. 258, 13825-13832], k-2 = 3.5 s-1. From the rates of ATP binding and hydrolysis measured previously (Johnson, 1983), the equilibrium constants for ATP binding and hydrolysis could be calculated: K1 = 5 X 10(7) M-1 and K2 = 14.(ABSTRACT TRUNCATED AT 250 WORDS)
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols; D002923:Cilia / Q000201:enzymology; D003520:Cyclophosphamide / Q000378:metabolism; D004317:Doxorubicin / Q000378:metabolism; D004398:Dyneins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D009218:Myosins / Q000378:metabolism; D010103:Oxygen Isotopes; D010710:Phosphates / Q000378:metabolism; D011241:Prednisone / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013768:Tetrahymena / Q000201:enzymology
D010103:Oxygen Isotopes; D010710:Phosphates; D004317:Doxorubicin; D000255:Adenosine Triphosphate; D003520:Cyclophosphamide; D000251:Adenosine Triphosphatases; D009218:Myosins; D004398:Dyneins; D011241:Prednisone