Application of autolysin and deoxyribonuclease profiles generated by renaturing SDS-PAGE in the comparison of selected Proteobacteria.
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.
Charnock
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Department of Microbiology, Institute of Pharmacy, University of Oslo, Postbox 1068, Blindern, 0316 Oslo, Norway.
D001419:Bacteria / Q000145:classification* / Q000201:enzymology*; D003851:Deoxyribonucleases / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000032:analysis*; D011489:Protein Denaturation; D011549:Pseudomonas / Q000145:classification / Q000201:enzymology; D012967:Sodium Dodecyl Sulfate; D013379:Substrate Specificity
D012967:Sodium Dodecyl Sulfate; D003851:Deoxyribonucleases; D009238:N-Acetylmuramoyl-L-alanine Amidase