Purification and characterization of the proteinase ECP 32 from Escherichia coli A2 strain

Abstract

The proteinase previously described as an unidentified component of E. coli A2 extracts which hydrolyses actin at a new cleavage site (Khaitlina et al. (1991) FEBS Lett. 279, 49) was isolated and further characterized. A chromatographic method of proteinase purification was developed by which a purity of more than 80% was attained. The enzyme was identified as a single, 32 kDa polypeptide (ECP 32) by SDS-PAGE and non-denaturing electrophoresis as well as by ion-exchange chromatography and gel filtration. The N-terminal sequence of ECP 32 was determined to be: AKTSSAGVVIRDIFL. The activity of ECP 32 is inhibited by o-phenanthroline, EDTA, EGTA and zincone. The EDTA-inactivated enzyme can be reactivated by cobalt, nickel and zinc ions. Based on these properties ECP 32 was classified as a metalloproteinase (EC 3.4.24). Limited proteolysis of skeletal muscle actin between Gly-42 and Val-43 was observed at enzyme substrate mass ratios of 1:25 to 1:3000. Two more sites between Ala-29 and Val-30, and between Ser-33 and Ile-34 were cleaved by ECP 32 in heat- or EDTA-inactivated actin. Besides actin, only histones and DNA-binding protein HU were found to be substrates of the proteinase, confirming its high substrate specificity. Its molecular mass, N-terminal sequence and enzymatic properties distinguish ECP 32 from any known metalloproteinases of E. coli, and we therefore conclude that it is a new enzyme.