Effect of pH and temperature on activity of μ‐ and m‐calpain

Inappropriate calpain activity has been implicated in a wide variety of human pathologies from loss of muscle mass in the muscular dystrophies, tissue damage in the ischemic areas surrounding a blood vessel blockage (myocardial infarcts, stroke), multiple sclerosis, proteolytic tissue damage following brain injury, and even Alzheimer’s disease. The calpains have generally assumed to be maximally active at pH values slightly above 7, but the effect of temperature on calpain activity has not been studied carefully. To obtain an accurate estimate of calpain activity at different pH values and temperatures, we have measured the proteolytic activity of μ‐calpain and m‐calpain, the two ubiquitous calpains, at 4 different pH values, 7.5, 7.0, 6.5, and 5.8, and at 3 different temperatures, 4°C, 10°C, and 25°C, by using a sensitive BODIPY microtiter plate assay. At 25°C, pH had little effect on activity of μ‐calpain until it decreased below pH 6.5; at pH 5.8, μ‐calpain activity was ~ 50% of the activity at pH 6.5, 7.0, 7.5. At 4°and 10°C, pH had a slightly greater effect on μ‐calpain activity than at 25°C, but the effect was still less than a 35% decrease. Activity of μ‐calpain at 4°C was ~ 65% of the activity at 25°C, indicating that both pH and temperature have relatively little effect on μ‐calpain activity. pH has a much greater effect on m‐calpain activity; m‐calpain was nearly inactive at pH 5.8 (less than 10% of pH 7.5 activity), and m‐calpain activity at pH 7.0 was ~ 75% as high as at pH 7.5, and was 30–40% as high at pH 6.5 as it was at pH 7.0. Temperature had relatively little effect on m‐calpain activity in the range we studied (~80% as high at 4°C as at 25°C). Supported by NRI 2002‐35206‐11630, 2001‐35503‐10776, 2004‐04338, and the MDA