Effect of an ADP analog on isometric force and ATPase activity of active muscle fibers.
The role played by ADP in modulating cross-bridge function has been difficult to study, because it is hard to buffer ADP concentration in skinned muscle preparations. To solve this, we used an analog of ADP, spin-labeled ADP (SL-ADP). SL-ADP binds tightly to myosin but is a very poor substrate for creatine kinase or pyruvate kinase. Thus ATP can be regenerated, allowing well-defined concentrations of both ATP and SL-ADP. We measured isometric ATPase rate and isometric tension as a function of both [SL-ADP], 0.1-2 mM, and [ATP], 0.05-0.5 mM, in skinned rabbit psoas muscle, simulating fresh or fatigued states. Saturating levels of SL-ADP increased isometric tension (by P'), the absolute value of P' being nearly constant, approximately 0.04 N/mm(2), in variable ATP levels, pH 7. Tension decreased (50-60%) at pH 6, but upon addition of SL-ADP, P' was still approximately 0.04 N/mm(2). The ATPase was inhibited competitively by SL-ADP with an inhibition constant, K(i), of approximately 240 and 280 microM at pH 7 and 6, respectively. Isometric force and ATPase activity could both be fit by a simple model of cross-bridge kinetics.
Karatzaferi
Christina
C
Department of Biochemistry & Biophysics, Cardiovascular Research Institute, University of California, San Francisco, California 94143, USA.
Myburgh
Kathryn H
KH
Chinn
Marc K
MK
Franks-Skiba
Kathleen
K
Cooke
Roger
R
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D007537:Isometric Contraction / Q000187:drug effects*; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000502:physiology*; D018482:Muscle, Skeletal / Q000187:drug effects / Q000502:physiology*; D009994:Osmolar Concentration; D011817:Rabbits; D013113:Spin Labels
D013113:Spin Labels; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases