Trypsin-like enzyme activity of the extracellular membrane vesicles of Bacteroides gingivalis W50.
Trypsin-like enzyme activity in spent culture media from 3-d-old batch cultures of Bacteroides gingivalis W50 was measured by using the hydrolysis of N alpha-benzoyl-L-arginine-p-nitroanilide. The cell-free culture medium was fractionated by differential centrifugation at 10,000 g and 75,000 g, yielding two particulate fractions and a soluble supernatant fraction. About 80% of the total recoverable activity was associated with the particulate fractions, the remainder being in the supernatant. Electron microscopy of ruthenium-red/osmium stained ultrathin sections of the pellet fractions showed them to be composed of vesicular particles (extracellular vesicles), between 50 and 250 nm in diameter. Enzyme activity in all three fractions was enhanced by dithiothreitol. Gel-permeation chromatography of the soluble fraction yielded one peak of activity which contained 64 kDa and 58 kDa polypeptides. Enzyme activity from the vesicular fractions could be solubilized by sonication, giving a similar chromatographic profile to the supernatant fraction. The main peak of activity was composed of 64 kDa and 58 kDa polypeptides. In addition, there was a higher molecular mass enzyme activity peak composed of the 64 kDa and 58 kDa components along with 111 kDa, 93 kDa and 70 kDa polypeptides. We conclude that the trypsin-like enzyme of B. gingivalis is released as a soluble protein and is also associated with extracellular vesicles, in which it may exist as a soluble component and also as a protein complex.
Smalley
J W
JW
Department of Dental Sciences, School of Dental Surgery, University of Liverpool, UK.
Birss
A J
AJ
D001439:Bacteroides / Q000201:enzymology* / Q000648:ultrastructure; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D004229:Dithiothreitol / Q000494:pharmacology; D008854:Microscopy, Electron; D014357:Trypsin / Q000378:metabolism*
D014357:Trypsin; D004229:Dithiothreitol