Transient kinetics of adenosine 5'-triphosphate hydrolysis by covalently cross-linked actomyosin complex in water and 40% ethylene glycol by the rapid flow quench method.
The initial steps of the ATPase of covalently cross-linked actomyosin subfragment 1 (acto-SF-1) were studied by the rapid flow quench method, and the results obtained were compared with those with reversible (i.e., non-cross-linked) acto-SF-1 and SF-1 under identical conditions. Cross-linked acto-SF-1 plus [gamma-32P]ATP reaction mixture milliseconds old was quenched either in a large excess of unlabeled ATP (ATP chase) or in acid (Pi burst). The conditions were pH 8 and 15 degrees C at 5 mM or 0.15 M KCl and with or without 40% ethylene glycol. In 40% ethylene glycol (5 mM KCl), as with SF-1 and reversible acto-SF-1, the ATP chase was used to titrate active sites and to study the kinetics of ATP binding. Unlike those with SF-1 or reversible acto-SF-1, saturation kinetics were not obtained. The second-order rate constant for ATP binding was 3.1 X 10(6) M-1 s-1 for cross-linked acto-SF-1, 1.8 X 10(6) M-1 s-1 for reversible acto-SF-1, and 2 X 10(6) M-1 s-1 for SF-1. In Pi burst experiments, a transient phase could not be discerned. Because of a high kcat, cross-linked acto-SF-1 was difficult to study in aqueous solution, but at 5 mM KCl, the ATP chase and Pi burst curves were similar to those obtained in 40% ethylene glycol. At 0.15 M KCl the ATP chase curve was difficult to interpret (small amplitude), and there was a small Pi burst.(ABSTRACT TRUNCATED AT 250 WORDS)
Biosca
J A
JA
Travers
F
F
Barman
T E
TE
Bertrand
R
R
Audemard
E
E
Kassab
R
R
D000205:Actomyosin / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D019855:Ethylene Glycol; D005026:Ethylene Glycols; D007700:Kinetics; D008433:Mathematics; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D013997:Time Factors; D014867:Water
D005026:Ethylene Glycols; D015879:Myosin Subfragments; D010446:Peptide Fragments; D014867:Water; D000255:Adenosine Triphosphate; D000205:Actomyosin; D000251:Adenosine Triphosphatases; D009218:Myosins; D019855:Ethylene Glycol