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1 Zoology, La Trobe University, Victoria, Australia
2 School of Exercise and Nutrition Sciences, Deakin University, Burwood, Victoria, Australia
* To whom correspondence should be addressed. E-mail: r.murphy{at}latrobe.edu.au.
µ-calpain and calpain-3 are Ca2+-dependent proteases found in skeletal muscle. Autolysis of calpains is observed by Western blotting as the cleaving of the full-length proteins to shorter products. Biochemical assays suggest that µ-calpain becomes proteolytically active in the presence of 2-200 µM Ca2+. Although calpain-3 is poorly understood, autolysis is believed to result in its activation and this is widely thought to occur at a lower [Ca2+] (~1 µM) than with µ-calpain. We demonstrate the Ca2+-dependent autolysis of the calpains in human muscle samples and rat extensor digitorum longus (EDL) muscles homogenised in solutions mimicking the intracellular environment at various [Ca2+] (0, 2.5, 10 and 25 µM). Autolysis of calpain-3 was found to occur over a similar [Ca2+] range as that for µ-calpain, and both calpains displayed a seemingly higher Ca2+-sensitivity in human compared to rat muscle homogenates, with ~15 % autolysis observed following 1 min exposure to 2.5 µM Ca2+ in human muscle and almost none following 1-2 min exposure to the same [Ca2+] in rat muscle. During muscle activity, intracellular [Ca2+] may transiently peak in the range found to autolyze µ-calpain and calpain-3 and so we examined the effect of two types of exhaustive cycling exercise (30 s 'all-out', n=8, and 70 % VO2 peak until fatigue, n=3) on the amount of autolyzed µ-calpain or calpain-3 in human muscle. No significant autolysis of µ-calpain or calpain-3 occurred due to the exercise. These findings show that the time- and concentration-dependent changes in cytoplasmic [Ca2+] occurring during concentric exercise fall near but below that necessary to cause autolysis of calpains in vivo.
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