detection of membrane-bound proteases of FRANCISEllA TUlARENSIS

Sterile preparations of membrane fractions were prepared by processing of live Francisella tularensis cells of different subspecies with 4.5M urea solution and differential centrifugation. For the first time, proteolytic activity was detected and studied by tests of radial enzyme diffusion and substrate polyacrylamide gel electrophoresis using gelatin as a substrate. Spectrum of gelatinases in the resulted preparation were detected. Quantitative inter-strain differences in the protease activities and their qualitative composition in membrane preparations of various virulent F.tularensis strains was analyzed. Avirulent F.tularensis 21/400 subsp. holarctica (I-214) strain demonstrated the greatest gelatinase activity in enzyme diffusion method and the lowest hydrolytic activity was seen in F.tularensis B-399 A-Cole subsp. tularensis (I-386) and F.tularensis Utah 112 subsp. novicida (I-384), other preparations showed intermediate activity. Enzyme electrophoresis in the protease spectra determined the presence of proteins with proteases activity 50–100kDa, and in the spectrum preparations of F. tularensis I-386 and I-384 were detected additional bands of proteases.