CHARACTERIZATION OF A BASIC SERINE PROTEINASE (PL-SIMILAR-TO-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 similar to 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 chromatogra phy on sulphopropyl-Sephadex C-25 at pH 8.6. D. nodosus strains that c ause benign foot-rot do not secrete a corresponding basic proteinase w ith a pI of similar to 9.5. Benign strains secrete a closely related, but distinct, proteinase which has the same molecular mass and N-termi nal sequences as the 'virulent' basic proteinase, but a lower pI of si milar to 8.6. The basic proteinases from both strains appear to intera ct with other proteins present in the culture medium, which results in anomalous behaviour on gel filtration. Pure D. nodosus 'virulent' bas ic proteinase has a molecular mass of 36 kDa and showed a low solubili ty at I < 0.05 precipitating quantitatively from solution as microcrys tals. 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 activi ty is rapidly lost. Bivalent-metal ions (e.g. Ca2+) are required to ma intain the structural integrity and stability of the proteinase; in th e presence of EDTA or conditions that cause protein unfolding, the pro teinase undergoes rapid and complete autolysis. Cleavage of oxidized i nsulin A- and B-chain showed that the basic proteinase has a broad spe cificity, including cleavage at lysine and arginine bonds.