Early steps of the Mg(2+)-ATPase of relaxed myofibrils. A comparison with Ca(2+)-activated myofibrils and myosin subfragment 1.
The early steps of the Mg(2+)-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 Pi burst method. The data obtained were interpreted by [formula: see text] where M represents the myosin heads with or without actin interaction. This work is a continuation of our study on Ca(2+)-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 microM-1 s-1 and the ATP was bound very tightly. With activated myofibrils, it was difficult to obtain an estimate for the koff for ATP(k-2) but it is much less than kcat. Here with relaxed myofibrils we estimate k-2 less than 8 x 10(-4) s-1, which is considerably smaller than kcat (0.019 s-1) and also previous estimates for this constant. The overall Kd for ATP to relaxed myofibrils is less than 8 x 10(-10) M. With S1 this Kd is about 10(-11) M.(ABSTRACT TRUNCATED AT 250 WORDS)
Herrmann
C
C
INSERM U128, CNRS, Montpellier, France.
Houadjeto
M
M
Travers
F
F
Barman
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D004533:Egtazic Acid / Q000494:pharmacology; D007700:Kinetics; D009126:Muscle Relaxation; D009210:Myofibrils / Q000187:drug effects / Q000201:enzymology*; D015879:Myosin Subfragments / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D011817:Rabbits; D013696:Temperature; D013816:Thermodynamics
D015879:Myosin Subfragments; D010710:Phosphates; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium