IMMUNOLOGICAL ANALYSIS OF 2 CALPAIN-LIKE CA2-DEPENDENT PROTEINASES FROM LOBSTER STRIATED MUSCLES - RELATIONSHIP TO MAMMALIAN AND DROSOPHILACALPAINS()

Lobster skeletal muscles contain four Ca2+-dependent cysteine proteina ses (CDPs I, IIa, IIb, and Iii) that degrade myofibrillar proteins. Lo bster CDPs share many properties with calpains from vertebrate tissues , but differ in native mass and subunit composition. Recently, cDNAs e ncoding a calpain-like protein (Dm-calpain; 91.5 or 94 kDa) have been isolated from fruit fly, Drosophila melanogaster. To further clarify t he relationship between invertebrate CDPs and mammalian calpains, anti bodies specific for mu-, m-, p94 (nCL-1), and Dm-calpains and lobster CDP IIb (native M(r) 195,000, subunit M(r) 95,000) were used in immuno blots to test for antigenic cross-reactivity, No common epitopes were found between CDP IIb and vertebrate calpains. However, polyclonal ant ibodies to CDP IIb cross-reacted strongly with a C-terminal 70-kDa por tion of Dm-calpain expressed in Escherichia coli. Conversely, polyclon al antibodies to Dm-calpain recognized CDP IIb. A second CDP, CDP IIa (native M(r) 125,000), was partially purified from lobster muscle; enz yme activity coeluted with a 60-kDa polypeptide using anion-exchange c hromatography. The 60-kDa protein reacted with a polyclonal antibody r aised against a 20-amino acid peptide sequence found around the cataly tic cysteine residue of mu- and m-calpains, but not with antibodies ra ised against other regions of mu- Or m-calpain or with the anti-CDP II b antibody. These results suggest that (1) the CDP IIb is the homolog of Drosophila calpain in crustaceans and (2) the active site regions o f CDP IIa and mu- and m-calpains are similar. (C) 1997 Academic Press.