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...

Full description

Saved in:
Bibliographic Details
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
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0006-2960
DOI:10.1021/bi00150a012