PURIFICATION AND CHARACTERIZATION OF THE PROTEINASE ECP-32 FROM ESCHERICHIA-COLI A2 STRAIN

The proteinase previously described as an unidentified component of E. coli A2 extracts which hydrolyses actin at a new cleavage site (Khait lina et al. (1991) FEBS Lett. 279, 49) was isolated and further charac terized. A chromatographic method of proteinase purification was devel oped by which a purity of more than 80% was attained. The enzyme was i dentified 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 t o be: AKTSSAGVVIRDIFL. The activity of ECP 32 is inhibited by o-phenan throline, EDTA, EGTA and zincone. The EDTA-inactivated enzyme can be r eactivated by cobalt, nickel and zinc ions. Based on these properties ECP 32 was classified as a metalloproteinase (EC 3.4.24). Limited prot eolysis of skeletal muscle actin between Gly-42 and Val-43 was observe d at enzyme substrate mass ratios of 1:25 to 1:3000. Two more sites be tween 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 histon es and DNA-binding protein HU were found to be substrates of the prote inase, confirming its high substrate specificity. Its molecular mass, N-terminal sequence and enzymatic properties distinguish ECP 32 from a ny known metalloproteinases of E. coli, and we therefore conclude that it is a new enzyme.