Extracellular vesicle-associated and soluble trypsin-like enzyme fractions of Porphyromonas gingivalis W50.
Soluble vesicle-associated trypsin-like enzyme fractions (VSF) were prepared by sonication from extracellular vesicles (ECV) from strains W50 and W50/BE1. High-(H), intermediate-(I) and low-(L) molecular-weight VSF enzyme subfractions were identified by non-dissociative gel filtration chromatography with Mr 160, 95 and 60 kDa respectively. The chromatographic profiles of W50 VSF from 48-h and 72-h cultures were identical. W50/BE1 VSF displayed a higher ratio of the 160 to 60 kDa components. This ratio was reduced in VSF from 72-h cultures. Extracellular soluble protein (EP) trypsin profiles were similar to their respective VSF, but the 60 kDa component predominated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a loss of soluble extracellular polypeptides with culture age. A polyclonal antiserum to EP subfraction L reacted in immunoblots with a 50 kDa peptide of subfraction L of W50. Whole EP and its subfraction H displayed a 50 kDa immunoreactive peptide but no peptides of higher molecular weight. This antiserum reacted with a similar sized peptide, and with lower-molecular-weight components in whole ECV. Gelatin substrate zymography of whole EP following non-reducing SDS-PAGE revealed a major 80 kDa protease that increased with culture age. Minor protease bands of 70 and 50 kDa were also detected.
Smalley
J W
JW
Department of Clinical Dental Sciences, University of Liverpool.
Birss
A J
AJ
D001425:Bacterial Outer Membrane Proteins / Q000032:analysis*; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D016966:Porphyromonas gingivalis / Q000201:enzymology*; D012697:Serine Endopeptidases / Q000032:analysis / Q000378:metabolism; D014357:Trypsin / Q000032:analysis* / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D012697:Serine Endopeptidases; D014357:Trypsin