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Binding of ADP and 5'-adenylyl imidodiphosphate to rabbit muscle myofibrils
J A Biosca1, L E Greene, E Eisenberg
1Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.
Abstract:
The binding of [3H]ADP and [3H]adenyl-5'-yl-imidodiphosphate ([3H]AMP-PNP) to rabbit skeletal myofibrils was measured at 25 and 7 degrees C, mu = 0.12 M, using [14C]mannitol as a volume marker. We found that ADP bound to myosin heads in overlap with a binding constant of about 10(4) M-1, similar to the value we previously obtained in vitro with acto.S-1. The binding of AMP-PNP to myosin heads was measured both in and out of overlap. The affinity of AMP-PNP to the heads out of overlap was similar to that obtained in vitro with S-1 alone. The binding of AMP-PNP to the myosin heads in overlap was much weaker. We could fit these data with a binding constant of about 1 x 10(3) M-1, assuming a single population of cross-bridges and 1 mol of AMP-PNP bound per mol of myosin head. This value was reduced by a factor of 2 when we corrected for nonspecific binding. It was also possible to fit the data assuming two equal populations of cross-bridges with one of the populations binding AMP-PNP about 5-fold more strongly than the other population. Therefore, for at least half of the cross-bridges in overlap, the binding of AMP-PNP is almost as weak as the value of 3 x 10(2) M-1 we previously measured for the acto.S-1 complex in vitro (Biosca, J. A., Greene, L. E., and Eisenberg, E. (1986) J. Biol. Chem. 261, 9793-9800).
Insights
Adenosine diphosphate (ADP) binds strongly to rabbit skeletal myofibrils, while adenosine-5
Area of Science:
- Muscle physiology
- Biochemistry
- Molecular biology
Background:
- Skeletal myofibrils are the fundamental contractile units of muscle.
- Understanding myosin head interactions with nucleotides is crucial for muscle function.
- Previous studies explored nucleotide binding to isolated myosin subfragment-1 (S-1) and acto-S-1.
Purpose of the Study:
- To quantify the binding affinity of ADP and AMP-PNP to myosin heads within intact rabbit skeletal myofibrils.
- To investigate the influence of myofibril structure (in overlap vs. out of overlap) on nucleotide binding.
- To compare nucleotide binding in myofibrils with in vitro binding data.
Main Methods:
- Measurement of [3H]ADP and [3H]AMP-PNP binding to rabbit skeletal myofibrils at different temperatures (25°C and 7°C).
- Use of [14C]mannitol as a volume marker to determine bound nucleotide concentrations.
- Analysis of binding data using models assuming single or multiple cross-bridge populations.
Main Results:
- ADP exhibited a strong binding constant (approx. 10^4 M^-1) to myosin heads in the overlap region.
- AMP-PNP binding affinity to myosin heads outside the overlap region was similar to in vitro S-1 binding.
- AMP-PNP binding to myosin heads within the overlap region was significantly weaker (approx. 1 x 10^3 M^-1, corrected for non-specific binding).
- Data suggested that at least half of the cross-bridges in overlap bind AMP-PNP weakly, comparable to acto-S-1 in vitro.
Conclusions:
- Myofibril structure influences nucleotide binding affinity to myosin heads.
- The weak binding of AMP-PNP in the overlap region of myofibrils suggests specific structural constraints or interactions.
- These findings provide insights into the regulatory mechanisms of muscle contraction at the molecular level.