Purification and separation of calpains and calpastatin are used to determine the individual activities of calpain-1 and calpain-2 and their inhibitor calpastatin. We discuss here a method to purify these enzymes using dialysis followed by separation using anion-exchange chromatography coupled with gradient elution. Swollen DEAE Sephacel is used as the column matrix in this method. Calpastatin and both domains of calpain are weakly basic molecules that effectively bind with the DEAE Sephacel and separate well using a stepwise, increasing gradient of NaCl to elute the proteins. Calpastatin binds most weakly with the column matrix, so it elutes first, followed by calpain-1 and, finally, calpain-2.
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This work is supported by DST (Govt. of India) grant SB/FT/LS-283/2012 and the Indian Council of Agricultural Research, New Delhi (Ministry of Agriculture).
Koohmaraie M, Geesink GH (2006) Contribution of post-mortem muscle biochemistry to the delivery of consistent meat quality with particular focus on the calpain system. Meat Sci 74:34–43CrossRefGoogle Scholar
Voahanginirina R, Ingrid F (2010) The role of calpain in diabetes-associated platelet hyperactivation. Adv Pharmacol 59:235–257CrossRefGoogle Scholar
Kripriyalini L (2015) Identification of calpains and calpastatin enzymes from turkey blood and their influence on post-mortem tenderization of skeletal muscle during refrigeration storage. M.V.Sc. Thesis, Deemed University, IVRI, IndiaGoogle Scholar
DeMartino GN, Wachendorfer R, McGuire M, Croall DE (1988) Proteolysis of protein inhibitor of calcium dependent proteases produces lower molecular weight fragments that retain inhibitory activity. Arch Biochem Biophys 262:189–198CrossRefGoogle Scholar
Wilson K, Walker J (2000) Practical biochemistry-Principles and techniques. 5th edn. (Cambridge low-price edition). Replika Press Pvt. Ltd., New Delhi, IndiaGoogle Scholar
Etherington DJ, Taylor MA, Dransfield E (1987) Conditioning of meat from different species. Relationship between tenderizing and the levels of cathepsin B, cathepsin L, calpain I, calpain II and β-glucuronidase. Meat Sci 20:1–18CrossRefGoogle Scholar
Kretchmar DH, Hathaway MR, Epley RJ, Dayton WR (1989) In vivo effect of a b-adrenergic agonist on activity of calcium-dependent proteinases, their specific inhibitor, and cathepsins B and H in skeletal muscle. Arch Biochem Biophys 275:228–235CrossRefGoogle Scholar
Koohmaraie M (1990) Quantification of Ca2+-dependent protease activities by hydrophobic and ion-exchange chromatography. J Anim Sci 68:659–665CrossRefGoogle Scholar
Geesink GH, Koohmaraie M (1999) Post-mortem proteolysis and calpain/calpastatin activity in callipyge and normal lamb Biceps femoris during extended post-mortem storage. J Anim Sci 77:1490–1501CrossRefGoogle Scholar
Karlsson JO, Gustavsson S, Hall C, Nilsson E (1985) A simple one-step procedure for the separation of calpain I, calpain II and calpastatin. Biochem J 231:201–204CrossRefGoogle Scholar
Biswas AK, Tandon S, Beura CK (2016) Simple extraction method for determination of different domains of calpain and calpastatin from chicken blood and their role in post-mortem ageing of breast and thigh muscles at 4±1 °C. Food Chem 200:315–321CrossRefGoogle Scholar