American
Journal of Physiology, November 27, 2002
The Effect of an ADP Analog on Isometric Force and ATPase Activity of Active
Muscle Fibers
CHRISTINA KARATZAFERI1*,
KATHRYN H MYBURGH2,
MARC K CHINN3,
KATHLEEN FRANKS-SKIBA1,
and ROGER COOKE1
1 Biochemistry and Biophysics, UCSF,
San Francisco, California, USA
2 Physiological Sciences, University of Stellenbosch, Stellenbosch,
Matieland, South Africa
3 Biochemistry and Biophysics, UCSF, San Francisco, California, USA;
Physiological Sciences, University of Stellenbosch, Stellenbosch, Matieland,
South Africa
The role played by ADP in modulating
cross-bridge function has been difficult to study, as 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, ~ 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 ~ 0.04 N.mm-2. The ATPase
was inhibited competitively by SL-ADP with an inhibition constant,
Ki, of approx. 240 and 280 µM at pH 7 and 6 respectively.
Isometric force and ATPase activity could both be fit by a simple
model of cross-bridge kinetics.
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