Immunological analysis of two calpain-like Ca2+-dependent proteinases from lobster striated muscles: relationship to mammalian and Drosophila calpains.
Lobster skeletal muscles contain four Ca2+-dependent cysteine proteinases (CDPs I, IIa, IIb, and III) that degrade myofibrillar proteins. Lobster CDPs share many properties with calpains from vertebrate tissues, but differ in native mass and subunit composition. Recently, cDNAs encoding a calpain-like protein (Dm-calpain; 91.5 or 94 kDa) have been isolated from fruit fly, Drosophila melanogaster. To further clarify the relationship between invertebrate CDPs and mammalian calpains, antibodies 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 immunoblots to test for antigenic cross-reactivity. No common epitopes were found between CDP IIb and vertebrate calpains. However, polyclonal antibodies to CDP IIb cross-reacted strongly with a C-terminal 70-kDa portion of Dm-calpain expressed in Escherichia coli. Conversely, polyclonal antibodies to Dm-calpain recognized CDP IIb. A second CDP, CDP IIa (native M(r) 125,000), was partially purified from lobster muscle; enzyme activity coeluted with a 60-kDa polypeptide using anion-exchange chromatography. The 60-kDa protein reacted with a polyclonal antibody raised against a 20-amino acid peptide sequence found around the catalytic cysteine residue of mu- and m-calpains, but not with antibodies raised against other regions of mu- or m-calpain or with the anti-CDP IIb antibody. These results suggest that (1) the CDP IIb is the homolog of Drosophila calpain in crustaceans and (2) the active site regions of CDP IIa and mu- and m-calpains are similar.
Beyette
J R
JR
Department of Biology, Colorado State University, Fort Collins 80523, USA.
Emori
Y
Y
Mykles
D L
DL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002154:Calpain / Q000737:chemistry / Q000276:immunology*; D003429:Cross Reactions; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000276:immunology*; D004331:Drosophila melanogaster / Q000201:enzymology*; D000939:Epitopes; D006801:Humans; D007106:Immune Sera; D015151:Immunoblotting; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology*; D008121:Nephropidae / Q000201:enzymology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology
D000939:Epitopes; D007106:Immune Sera; D011994:Recombinant Proteins; D002154:Calpain; D003546:Cysteine Endopeptidases; D002118:Calcium