Purification and Characterization of Crustacean Calpain-like Proteinases

Calcium-dependent cysteine proteinases (CDPs or calpain-like proteinases) constitute a large family of related proteins in the tissues of invertebrate species. They vary in native mass from 59 kDa for lobster muscle CDP III to 520 kDa for octopus muscle CDP, and vary also in subunit composition (1 ,2 ). Although quite diverse, invertebrate enzymes share essential biochemical properties with mammalian calpains. The invertebrate proteinases require Ca²⁺ for full activity, and have a much greater affinity for Ca²⁺ than for other divalent cations (3 –6 ). Invertebrate calpain-like enzymes are strongly inhibited by cysteine proteinase inhibitors, such as E-64 and related epoxysuccinyl peptides, peptide aldehydes (e.g., MDL 28170 and calpeptin), iodoacetate, iodoacetamide, Hg²⁺ , pCMB. and JV-ethylmaleimide (NEM) (1,2 ,7 ). The serine proteinase inhibitors tosyllysyl chloromethyl ketone (TLCK), tosylphenylalanine chloromethyl ketone (TPCK), leupeptin, and antipain are effective only at higher concentrations. Moreover, cDNA sequences and immunological analysis show significant homologies between mammalian calpains and invertebrate enzymes (1,2 ,7 ).