Reversible inactivation of myosin subfragment 1 activity by mechanical immobilization.
The Mg-ATPase activity of skeletal muscle myosin subfragment 1 (S1) is reversibly eliminated when it is aggregated by the force of osmotic pressure dehydration using polyethylene glycol (PEG). Several experiments indicate nucleotides bind aggregated S1, but the effects of binding are attenuated. Compared with S1 in solution, epsilonADP binds aggregated S1 with reduced affinity, and the bound epsilonADP fluorescence intensity is more effectively quenched by acrylamide. When ATP binds aggregated S1, the tryptophan intensity increases to only 50% of the solution level. Chemical cross-linking of cys-707 to cys-697 by p-phenylenedimaleimide is less efficient for aggregated S1 x MgADP. The data are consistent with aggregated S1 being able to bind nucleotide but not being able to complete the usual conformation change(s) in response to binding. If S1 is kept from aggregating by increasing the ionic strength at the same osmotic pressure, its Mg-ATPase activity and ATP-induced tryptophan fluorescence intensity increase are normal. The combined data are consistent with an ATP hydrolysis mechanism in which S1 segmental motion is coupled to its enzymatic activity. In this model, segmental motion is mechanically constrained by aggregation; the constrained S1 can bind ATP, but it cannot complete the hydrolysis mechanism.
Highsmith
S
S
Department of Biochemistry, School of Dentistry, University of the Pacific, San Francisco, California 94115-2399, USA. shighsmith@uop.edu
Duignan
K
K
Franks-Skiba
K
K
Polosukhina
K
K
Cooke
R
R
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000327:Adsorption; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D027682:Cation Transport Proteins; D003890:Desiccation; D017886:Durapatite / Q000494:pharmacology; D005456:Fluorescent Dyes; D007700:Kinetics; D018482:Muscle, Skeletal / Q000502:physiology*; D015879:Myosin Subfragments / Q000037:antagonists & inhibitors / Q000502:physiology*; D009994:Osmolar Concentration; D009997:Osmotic Pressure; D010710:Phosphates / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology; D011817:Rabbits
D027682:Cation Transport Proteins; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D010710:Phosphates; C015195:1,N(6)-ethenoadenosine diphosphate; D011092:Polyethylene Glycols; D000244:Adenosine Diphosphate; D017886:Durapatite; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; C022761:potassium transporting ATPase; D000252:Calcium-Transporting ATPases