Figure 2. Diffusible and bound pools of μ-calpain in single muscle fibres at rest.
A, Western blots for calpain-3 and μ-calpain, and corresponding Coomassie stain of myosin heavy chain (MHC), in an intact section of each of two extensor digitorum longus (EDL) fibres (I) and in the total skinned segments (S1 and S2) and the associated sarcolemmal cuffs (C1 and C2) of two other EDL fibres. Calpain-3 and μ-calpain were present predominantly in their unautolysed full-length forms (94 and 80 kDa, respectively). B, μ-calpain and corresponding MHC remaining in the skinned fibre (F) or lost to the wash solution (W), after washing single skinned EDL fibre segments for the indicated time. (Each W on left side of corresponding F). Wash solution was a K+-based physiological solution with free [Ca2+] buffered at 20 nm. C, similar experiment with calpain-3 in skinned fibres exposed to 20 nm Ca2+ wash solution for 3 or 30 min. Calpain-3 is not lost from the fibre even with extensive washing. D, time course of washout of μ-calpain from single skinned EDL fibres; values are the mean (±s.e.m.) μ-calpain remaining in the skinned segment (expressed as a percentage of the total present in the skinned segment and wash solution) in indicated number of fibre segments.