Purification and characterization of a Ca(2+)-activated thiol protease from Drosophila melanogaster.
A Ca(2+)-activated thiol protease was purified from Drosophila melanogaster. The procedure involves Phenyl-Sepharose, Reactive Red-Agarose and Q-Sepharose fast flow (or MonoQ) chromatographic steps. The enzyme eluting from Q-Sepharose fast flow seems to be homogeneous as judged by silver staining on SDS-PAGE: it consists of a single polypeptide chain of M(r),app = 94K and pI = 5.46. The proteolytic activity of the purified enzyme is absolutely Ca(2+)-dependent, characterized by 0.6 mM free Ca2+ at half-maximal activity. Ca2+ ions cannot be replaced effectively by the divalent cations Mg2+, Mn2+, Zn2+, Ba2+, and Cd2+. The enzyme shows the inhibitor pattern of thiol proteases. Human recombinant calpastatin (domain I) completely inhibits the enzyme at a nearly 1:1 molar ratio. Several of these properties resemble those of vertebrate calpain II. However, various attempts to detect a small subunit of M(r) approximately 30K, common with vertebrate calpains, remained unsuccessful. We suggest that the Drosophila enzyme is a novel calpain II-like protease.
Pintér
M
M
Institute of Enzymology, Hungarian Academy of Sciences, Budapest.
Stierandova
A
A
Friedrich
P
P
D000818:Animals; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000494:pharmacology; D002413:Cations, Divalent; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003546:Cysteine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology; D013552:Swine
D002135:Calcium-Binding Proteins; D002413:Cations, Divalent; D046911:Macromolecular Substances; D011480:Protease Inhibitors; D011994:Recombinant Proteins; C033668:calpastatin; D003546:Cysteine Endopeptidases; D002118:Calcium