Fig. S4.

The single-nucleotide turnover experiments used to measure the SRX in skinned skeletal muscle fibers. (A) Control fiber in the presence (solid circles) and absence (open squares) of 100 µM piperine. (B) Fiber exchanged with RLC5-MDCC also in the presence and absence of piperine. (C) The raw data for the control fiber in the absence of piperine showing the fiber fluorescence (open squares) and the background (solid circles). For the control fiber in the absence of piperine the value of P2 is 0.34 and that of T2 is 185. In the presence of piperine, these are inhibited by 41 and 42%, respectively. For the exchanged fiber in the absence of piperine the value of P2 is 0.25 and that of T2 is 194. In the presence of piperine, these are inhibited by 40 and 41%, respectively. The P2 of the exchanged fiber is inhibited relative to the control fiber by 27%, with little change in T2. A similar small change in P2, 0.26 ± 0.02, vs. control, P2 = 0.31, was also seen after exchange with unlabeled RLC-5 by Nogara et al. (18), suggesting that the mutation has a small effect but the addition of the probe causes no further change. In C the fiber starts in rigor, where the fiber fluorescence is indistinguishable from background. The autofluorescence of the fiber was not changed in the presence of ATP or piperine. Upon the addition of 250 μM mantATP at 80 s, both the background and fiber fluorescence increase dramatically. The chase phase begins at 220 s with the wash by 4 mM ATP. The background drops immediately, and fiber fluorescence decreases slowly as mant nucleotides are released and diffuse out of the fiber. In the analysis of the data the background is subtracted from the fiber signal to give net fiber intensity. The net fiber intensity during the chase is then normalized to the value before the chase, providing the curve shown for the control fiber in A.