Quantitation of tissue calpain activity after isolation by hydrophobic chromatography |
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| Authors: | R Gopalakrishna S H Barsky |
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| Affiliation: | 1. Department of Electrical and Electronics Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 Serdang, Malaysia;2. Advanced Lightning, Power and Energy Research (ALPER), Faculty of Engineering, Universiti Putra Malaysia, 43400 Serdang, Malaysia;3. Department of computer Engineering, University of Al-Mustansiriyah, 10001, Baghdad, Iraq;4. Centre for Artificial Intelligence Research and Optimization, Torrens University Australia, Fortitude Valley, Brisbane, 4006 QLD, Australia;5. Yonsei Frontier Lab, Yonsei University, Seoul, Korea;6. Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, Serdang, 43400, Selangor, Malaysia;7. School of Electrical Engineering, College of Engineering, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia;8. University Research and Innovation Center, Obuda University, 1034 Budapest, Hungary |
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| Abstract: | A rapid and reliable method for quantitating tissue calpains (Ca2+-activated, neutral, thiol proteases) was developed using hydrophobic chromatography with phenyl-Sepharose. Calpains I and II isolated by this method are free of endogenous inhibitor(s) (calpastatin), activator(s), and nonspecific proteases. These calpains expose hydrophobic regions in the presence of Ca2+ and bind tightly to phenyl-Sepharose. Inactivation of bound calpain is prevented by the addition of leupeptin (20 microM). Calpains I and II bound initially by phenyl-Sepharose in a Ca2+-dependent manner are then eluted successively on the basis of their Ca2+-independent binding to phenyl-Sepharose. Because calpastatin may prevent binding of calpain to phenyl-Sepharose by forming a protease-inhibitor complex in the presence of Ca2+, preadsorbing the protease to a suspension of phenyl-Sepharose beads initially in the absence of Ca2+ separates most of the calpain present in tissue extracts from calpastatin. The isolated calpains obtained are assayed by casein digestion. This quantitation procedure is suitable for measuring calpain activity in various tissues and cells including erythrocytes. |
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