Figure 3. Diffusible pool of μ-calpain rapidly binds in presence of raised cytosolic [Ca²⁺].

A, Western blot of μ-calpain and corresponding Coomassie stain of myosin heavy chain (MHC) for individual skinned fibres that were first exposed to a rigor wash solution (W) with the free [Ca²⁺] buffered at 1 μm. The majority of the μ-calpain remained in the skinned fibre (F) when the wash was for only 3 min (left-hand pair of lanes), but most was lost from a fibre with a 30-min wash (right-hand pair of lanes). B and C, similar experiments with wash solution containing 5 μm and 40 μm free Ca²⁺, respectively. The proportion of μ-calpain lost from the fibre decreased at higher [Ca²⁺], and the extent of autolysis of μ-calpain increased. The total of all μ-calpain in the fibre and wash (normalized to MHC) remained approximately the same as in untreated skinned fibres. D, percentage of μ-calpain remaining in a skinned fibre when the fibre was washed in a rigor solution with indicated free [Ca²⁺] for 1, 3 or 30 min (or 60 min at 50 nm and 200 nm Ca²⁺). Values are based on the total amount of μ-calpain present in the fibre and wash, irrespective of whether it was autolysed. Values are means (± s.e.m.) with number of fibres indicated, except for five cases at 1–8 μm Ca²⁺ where values for individual fibres are shown.