title: Evaluation of methods to solubilize and analyze cell-associated ectoenzymes.
abstract: To quantify the functional diversity of microbes that use hydrolytic ectoenzymes, the feasibility of separating cell-associated proteins on polyacrylamide gels and detecting enzyme activity via fluorescent substrate analogs for aminopeptidases, glucosidases, and esterases was determined. More than 87% of aminopeptidase activity was associated with particulate cell material in all of the 10 Gram-negative bacterial strains that were investigated. Although 7-amino-4-methylcoumarin-leucine (AMC-leucine) provided high activities after incubation with eight strains from the Cytophaga-Flexibacter-Bacteroides group, very poor responses were noted with two gamma Proteobacteria that grew well on protein. Therefore, this molecule was not a universal substrate for aminopeptidases. Methods of increasing stringency were evaluated to release enzyme activity from particulate material. Some methods (treatment with 0.1% Triton X-100) gave good results with some but not all strains. Cell lysis by shearing produced the most consistent results. Ectoenzyme activities could be localized on polyacrylamide gels using fluorescent substrate analogs. However, some activities were located in high molecular weight complexes, and methods that disrupted these complexes (such as treatment with sodium dodecyl sulfate at elevated temperature) destroyed enzyme activity. In addition, the enzymes from different strains showed the same electrophoretic mobility. Therefore, the analysis of functional diversity by this approach is limited by the difficulty in solubilizing particulate enzymes under conditions where they retain activity.
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA. akonopka@purdue.edu
authors.forename: Allan
authors.initials: A
authors.lastname: Konopka
authors.forename: Taissiia
authors.initials: T
authors.lastname: Zakharova
mesh_terms: D000626:Aminopeptidases / Q000737:chemistry / Q000378:metabolism; D001704:Biopolymers / Q000737:chemistry / Q000378:metabolism; D019149:Bioreactors; D002458:Cell Fractionation; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005959:Glucosidases / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000145:classification / Q000201:enzymology* / Q000254:growth & development; D006867:Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D012995:Solubility
chemical_list: D001704:Biopolymers; D005456:Fluorescent Dyes; D006867:Hydrolases; D004950:Esterases; D005959:Glucosidases; D000626:Aminopeptidases