Ca(2+)-activated myofibrillar ATPase: transient kinetics and the titration of its active sites.
The transient kinetics of rabbit psoas Ca(2+)-activated myofibrillar Mg(2+)-ATPase 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 products steps were studied by the Pi burst method. The data obtained were interpreted by the simple scheme [formula; see text] represents the myosin heads with or without actin interaction. The constants obtained with myofibrils (where the molecules are highly organized) were compared with those with myosin subfragment 1 (S1) and cross-linked acto-S1 (where the molecules are dispersed in solution). Myofibrils appear to bind ATP as tightly as do S1 and cross-linked acto-S1. This suggests that with them k-2 less than kcat much less than k2, and it is proposed that the ATP chase method can be used to titrate the ATPase sites in myofibrils. The results of titration and single-turnover experiments revealed that myofibrils may contain partially active myosin heads. It is proposed that these heads bind ATP loosely without hydrolysis, as found with S1 [Tesi, C., N. Bachouchi, N., Barman, T., & Travers, F. (1989) Biochimie 71, 363-372]. There were large Pi bursts with the three preparations, showing that with all of them the release of products step (k4) is rate limiting.(ABSTRACT TRUNCATED AT 250 WORDS)
Houadjeto
M
M
INSERM U128, CNRS, Montpellier, France.
Travers
F
F
Barman
T
T
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002021:Buffers; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D003432:Cross-Linking Reagents; D004789:Enzyme Activation / Q000187:drug effects; D006868:Hydrolysis; D007700:Kinetics; D009210:Myofibrils / Q000187:drug effects / Q000201:enzymology*; D015879:Myosin Subfragments / Q000737:chemistry; D009994:Osmolar Concentration; D010710:Phosphates / Q000737:chemistry; D011817:Rabbits
D002021:Buffers; D003432:Cross-Linking Reagents; D015879:Myosin Subfragments; D010710:Phosphates; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium