title: 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.
authors.affiliation: Institute of Cytology, St. Petersburg, Russian Federation.
authors.forename: V V
authors.initials: VV
authors.lastname: Matveyev
authors.forename: A M
authors.initials: AM
authors.lastname: Usmanova
authors.forename: A V
authors.initials: AV
authors.lastname: Morozova
authors.forename: J H
authors.initials: JH
authors.lastname: Collins
authors.forename: S Y
authors.initials: SY
authors.lastname: Khaitlina
mesh_terms: D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange / Q000379:methods; D004492:Edetic Acid / Q000494:pharmacology; D004586:Electrophoresis; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011489:Protein Denaturation; D011506:Proteins / Q000378:metabolism
chemical_list: D000199:Actins; D004791:Enzyme Inhibitors; D011506:Proteins; D004492:Edetic Acid; D010450:Endopeptidases; C097881:proteinase ECP 32