Assay of proteases from Actinomyces pyogenes isolated from pigs and cows by zymography

Extracellular proteases of Actinomyces pyogenes were assayed by zymography on SDS-polyacrylamide gel with the concentrated culture supernatant of 7 isolates from pigs and cows. When gelatin was used as a substrate, three proteases with molecular weights of 69, 59 and 55 kilodalton (kDA) were detected in all the isolates from both the pigs and cows. In addition to these proteases, 108 and 102 kDa proteases were detected in the swine and bovine isolates respectively. No protease bands appeared, however, when the calcium ion was absent from the buffer solution of the zymography. When casein was used as a substrate, the bands of 69, 59 and 55 kDa proteases were present, but 108 and 102kDa proteases were not detected in any of the isolates. The activity of all proteases was completely inhibited by phenylmethyl-sulfonyl fluoride and diisopropyl fluorophosphate but not by other protease inhibitors. From this result, it seems likely that the five proteases produced by A, pyogenes, originating from pigs and cows, are serine proteases.