The role of the super-relaxed state of myosin in human metabolism

Figures

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  Fig. 1. Measuring the SRX in human fibers. The intensity of the fiber fluorescence, relative to the pre-chase value, is plotted as a function of time during the chase phase of the single nucleotide turnover measurements in the presence (blue circles) or absence (red squares) of 100 μM piperine. Human fibers were obtained from lean subjects. The fibers were first incubated in the fluorescent ATP analog, mantATP, and then chased with ATP. Fiber fluorescence decreases in two phases, a fast phase that is largely completed in roughly 20–30 s, followed by a slow phase with a lifetime of minutes. The slow phase arises from the slow release of nucleotides by myosin in the SRX. The slower phase has a decreased population in the presence of piperine, showing that the SRX has been partially destabilized. Fits to the data for control defined P2 = 0.31 ± 0.01, T2 = 157 ± 9 s; and with piperine P2 = 0.21 ± 0.01, T2 = 157 ± 12 s. Data averaged over a number of fibers is summarized in Table 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 2. Measuring the fraction of mant nucleotides bound specifically to the fiber. The fiber was first incubated in mantATP and at 100 s chased with ATP similar to the control fiber in Fig. 1, red squares. The fit to the decay curve during the chase defined P2 = 0.29 ± 0.01; T2 = 170 ± 7 s. The fiber was then washed with rigor buffer and incubated again, in mantATP with an additional 4 mM ATP. The presence of the ATP competes off mantATP from sites that bind ATP specifically, blue circles. As can be seen, approximately half of the nucleotides are no longer bound to the fiber. An average of ten experiments showed 42 ± 3% of the nucleotides bound nonspecifically. The fiber was then chased with ATP starting at 100 s. The slow release of nucleotides seen in the absence of competing ATP is much reduced, showing that the slow release of nucleotides comes from ATP specific sites. A fit to the data during the chase defined P2 = 0.05 ± 0.02, T2 = 160 ± 15 s. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 3. Effect of piperine on human cardiac cells in the absence, red squares, and presence of 100 μM piperine, blue circles. The experiment was carried out following the same protocols as described in the legend to Fig. 1, with the exception that the muscle sample was a strip of cardiac cells taken from the left ventricle of a human subject. As can be seen, piperine has little effect on the SRX. Fits to the data showed that the fractions of fluorescence that decayed slowly, P2, were 0.27 ± 0.02 for control and 0.29 ± 0.02 with piperine, with respective lifetimes of 180 ± 18 and 171 ± 14 s. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)