Characterization of a basic serine proteinase (pI approximately 9.5) secreted by virulent strains of Dichelobacter nodosus and identification of a distinct, but closely related, proteinase secreted by benign strains.
An extracellular serine proteinase with a PI approximately 9.5 (referred to as 'basic proteinase') was purified to homogeneity, from strains of Dichelobacter nodosus that cause virulent foot-rot, by gel filtration of concentrated culture supernatant on Sephadex G-100 and chromatography on sulphopropyl-Sephadex C-25 at pH 8.6 D. nodosus strains that cause benign foot-rot do not secrete a corresponding basic proteinase with a pI of approximately 9.5. Benign strains secrete a closely related, but distinct, proteinase which has the same molecular mass and N-terminal sequences as the 'virulent' basic proteinase, but a lower pI of approximately 8.6. The basic proteinases from both strains appear to interact with other proteins present in the culture medium, which results in anomalous behavior on gel filtration. Pure D. nodosus 'virulent' basic proteinase has a molecular mass of 36 kDa and showed a low solubility at I < 0.05 precipitating quantitatively from solution as microcrystals. The proteinase shows optimal activity at pH 8.0 and is stable to heating to 55 degrees C for 30 min, but at higher temperatures activity is rapidly lost. Bivalent-metal ions (e.g. Ca2+) are required to maintain the structural integrity and stability of the proteinase; in the presence of EDTA or conditions that cause protein unfolding, the proteinase undergoes rapid and complete autolysis. Cleavage of oxidized insulin A- and B-chain showed that the basic proteinase has a broad specificity, including cleavage at lysine and arginine bonds.
Kortt
A A
AA
Commonwealth Scientific and Industrial Research Organisation, Division of Biomolecular Engineering, Parkville, Victoria, Australia.
Caldwell
J B
JB
Lilley
G G
GG
Edwards
R
R
Vaughan
J
J
Stewart
D J
DJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001439:Bacteroides / Q000201:enzymology* / Q000472:pathogenicity; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics; D014774:Virulence
D000596:Amino Acids; D007328:Insulin; D011480:Protease Inhibitors; D012697:Serine Endopeptidases