Purification and characterization of a Ca(2+)-activated thiol protease from Drosophila melanogaster

• Published in: Biochemistry (Easton) 1992-09, Vol.31 (35), p.8201-8206
• Main Authors: Pintér, M, Stierandova, A, Friedrich, P
• Format: Article
• Language: English
• Subjects: Animals; Brain - enzymology; Calcium - pharmacology; Calcium-Binding Proteins - pharmacology; Cations, Divalent; Chromatography, Affinity; Chromatography, Ion Exchange; Cysteine Endopeptidases - isolation & purification; Cysteine Endopeptidases - metabolism; Drosophila melanogaster - enzymology; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Kinetics; Macromolecular Substances; Molecular Weight; Protease Inhibitors - pharmacology; Recombinant Proteins - pharmacology; Swine
• ISSN: 0006-2960
• DOI: 10.1021/bi00150a012

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.