Application of autolysin and deoxyribonuclease profiles generated by renaturing SDS-PAGE in the comparison of selected P
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© Springer-Verlag 1997
O R I G I N A L PA P E R
Colin Charnock
Application of autolysin and deoxyribonuclease profiles generated by renaturing SDS-PAGE in the comparison of selected Proteobacteria
Received: 16 December 1996 / Accepted: 20 January 1997
Abstract SDS-PAGE of cell-free extracts in gels containing bacterial murein or DNA allowed, after enzyme renaturation and staining of nonhydrolysed substrate, the detection of multiple autolysin or deoxyribonuclease activities directly in the gel as zones of clearing. Enzyme profiles of Proteobacteria which are, or were at one time, classified in the genus Pseudomonas were compared. For each species, a relatively large number of autolysin and deoxyribonuclease activities were detected. The distribution, numbers and intensities of zones of clearing in the gel provided complex species-specific patterns. Extensive data from two fundamental, and presumably evolutionarily distinct classes of enzymes were thus generated for purposes of comparison. Neither analysis suggested that these bacteria could represent a single natural cluster of species, lending support to their present multigeneric status. Ethidium-bromide-stained gels could be subsequently stained with Coomassie blue. This allowed the mapping of many deoxyribonuclease activities to particular peptides in the cell-free extract. In addition, modification of the substrate or renaturation buffer enabled a preliminary characterisation of several deoxyribonucleases in terms of their stability, substrate specificity, and other parameters expected to affect enzyme activity. Individual deoxyribonucleases could be located and screened for desired properties without prior purification. Key words Proteobacteria · Renaturing SDS-PAGE · Autolysin · Deoxyribonuclease
Introduction Renaturation of enzymes in substrate-containing SDSpolyacrylamide gels provides a means of directly investigating the cellular complement of a class of enzymes with
Colin Charnock Department of Microbiology, Institute of Pharmacy, University of Oslo, Postbox 1068, Blindern, 0316 Oslo, Norway Tel. +47-2285-4566; Fax +47-2285-4605
related activities. Information obtained includes the number of individual enzyme types that regain activity against the included substrate and, for monomeric enzymes, their molecular mass. Bands of activity obtained after renaturing SDS-PAGE are unlikely to represent every individual enzyme type. The methodology requires that the enzyme can be restored to an active form and, therefore, must not be irreversibly denatured by heat/SDS treatment or contain subunits of different size. A requirement for cofactors, activators (such as processing by the action of a protease), and other factors influencing enzyme activity will also affect the results obtained, as will the degree to which a particular enzyme, governed by its specificity, hydrolyses the incorporated substrate. This study concerns itself with two physiologically central, and presumably evolutionarily distinct classes of eubacterial enzymes – autolysins and deoxyribon
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