EARLY STEPS OF THE MG2+-ATPASE OF RELAXED MYOFIBRILS - A COMPARISON WITH CA2+-ACTIVATED MYOFIBRILS AND MYOSIN SUBFRAGMENT-1

被引:25
作者
HERRMANN, C [1 ]
HOUADJETO, M [1 ]
TRAVERS, F [1 ]
BARMAN, T [1 ]
机构
[1] CNRS,INSERM,U128,BP 5051,F-34033 MONTPELLIER,FRANCE
关键词
D O I
10.1021/bi00149a038
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The early steps of the Mg2+-ATPase activity of relaxed rabbit psoas myofibrils were studied in a buffer of near-physiological ionic strength at 4-degrees-C by the rapid flow quench technique. The initial ATP binding steps were studied by the ATP chase, and the cleavage and release of product steps by the P(i) burst method. The data obtained were interpreted by [GRAPHICS] where M represents the myosin heads with or without actin interaction. This work is a continuation of our study on Ca2+-activated myofibrils [Houadjeto, M., Travers, F., & Barman, T. (1992) Biochemistry 31, 1564-1569]. Here the constants obtained with relaxed myofibrils were compared with those with activated myofibrils and myosin subfragment 1 (S1). We find that whereas Ca2+ increases 80X the release of products (k4), it has little effect upon the kinetics of the initial binding and cleavage steps. As with activated myofibrils and S1, the second-order binding constant for ATP (k2/K1) was about 1-mu-M-1 s-1 and the ATP was bound very tightly. With activated myofibrils, it was difficult to obtain an estimate for the k(off) for ATP (k-2) but it is <<k(cat). Here with relaxed myofibrils we estimate k-2 < 8 X 10(-4) s-1, which is considerably smaller than k(cat) (0.019 s-1) and also previous estimates for this constant. The overall K(d) for ATP to relaxed myofibrils is less than 8 X 10(-10) M. With S1 this K(d) is about 10(-11) M. The kinetics of the cleavage step with relaxed and activated myofibrils are probably similar but different from those with S1. From our previous work it appears that K3 is larger with activated myofibrils than with S1 but it was difficult to obtain an estimate for this constant. Here with relaxed myofibrils K3 was estimated at 6, which is considerably larger than the 1.7 found with S1.
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页码:8036 / 8042
页数:7
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