Biochemical properties of membrane-associated proteases of Brachyspira pilosicoli isolated from humans with intestinal disorders.
A membrane-associated, subtilisin-like, serine protease activity was found in both pathogenic and non-pathogenic strains of Brachyspira species in a previous study, but the biochemical properties of the enzyme were not investigated. The purpose of the present study was to characterize further the biochemical properties, including substrate specificity, of the membrane-associated protease of Brachyspira pilosicoli isolated from humans with intestinal disorders. Protease activity of detergent-enriched membrane protein extracts of B. pilosicoli was assessed using fluorescent dye-labelled synthetic peptides as substrates and determination of electrophoretic mobility of cleavage products in agarose gels. Each activity was further confirmed with class-specific protease inhibitors and thermal denaturation. The presence of a hydrophilic membrane-associated thermolabile serine endopeptidase with specificity for Leu was confirmed. Two additional hydrophilic membrane-associated thermostable proteolytic activities were identified, one with a putative Ala specificity, and one a carboxypeptidase. Taken together, these data suggest that, in addition to a previously described membrane-associated subtilisin-like serine protease, the membrane of B. pilosicoli contains proteins with at least two other proteolytic activities.
Dassanayake
Rohana P
RP
Department of Veterinary and Biomedical Sciences1, Center for Biotechnology2 and Department of Biochemistry3, University of Nebraska-Lincoln, Lincoln, NE 68583-0905, USA.
Caceres
Nancy E
NE
Department of Veterinary and Biomedical Sciences1, Center for Biotechnology2 and Department of Biochemistry3, University of Nebraska-Lincoln, Lincoln, NE 68583-0905, USA.
Sarath
Gautam
G
Department of Veterinary and Biomedical Sciences1, Center for Biotechnology2 and Department of Biochemistry3, University of Nebraska-Lincoln, Lincoln, NE 68583-0905, USA.
Duhamel
Gerald E
GE
Department of Veterinary and Biomedical Sciences1, Center for Biotechnology2 and Department of Biochemistry3, University of Nebraska-Lincoln, Lincoln, NE 68583-0905, USA.
D017400:Brachyspira / Q000201:enzymology* / Q000302:isolation & purification; D002614:Chelating Agents / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004795:Enzyme Stability; D006801:Humans; D007410:Intestinal Diseases / Q000382:microbiology*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013145:Spirochaetales Infections / Q000382:microbiology*; D013379:Substrate Specificity
D002614:Chelating Agents; D008565:Membrane Proteins; D010455:Peptides; D015842:Serine Proteinase Inhibitors; D004492:Edetic Acid; D012697:Serine Endopeptidases