Purification and characterization of a Ca2+-activated thiol protease from Drosophila melanogaster.
1992
Pinter M. | Stierandova A. | Friedrich P.
A Ca2+-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 silverstaining on SDS-PAGE: it consists of a single polypeptide chain of Mr,app = 94K and pI = 5.46. The proteolytic activity of the purified enzyme is absolutely Ca2+-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 Mr approximately 30K, common with vertebrate calpains, remained unsuccessful. We suggest that the Drosophila enzyme is a novel calpain II-like protease.
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