J. Biochem, 1988, Vol. 104, No. 4 616-621
© 1988 Japanese Biochemical Society
research-article |
Characterization of 73 kDa Thiol Protease from Serratia marcescens and Its Effect on Plasma Proteins1
*Department of Microbiology Kumamoto University Medical School, Kumamoto, Kumamoto 860
**Department of Allergy, Institute for Medical Immunology Kumamoto University Medical School, Kumamoto, Kumamoto 860
2To whom correspondence should be addressed
The 73-kDa protease (73K protease) was purified from a clinical isolate of Serratia marcescens kums 3958. The purified protease appeared homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol. The protease is active in a broad pH range with maximum activity at pH 7.5–8.0. The protease appeared to be a thiol protease, since it was inhibited by suithydryl reactive compounds such as p-chloromercuribenzoic acid, fluorescein mercuric acetate (FMA), iodoacetamide, and N-ethylmaleimide, and the protease activity was enhanced by various reducing agents such as cysteine, glutathione, 2-mercaptoethanol, and dithiothreitol. The protease contained 2 mol of free suiflaydryl residues per mol of protease. When the protease was reacted with FMA, a maximum of 2 mol of FMA per mol of enzyme was found reacted, based on fluorescence quenching in which the enzyme inactivation was paralleled linearly with the loss of both SH groups. This indicates possible equal involvement of the two thiol groups for the enzyme activity. The inactivation of the protease by FMA was partially restored by a dialysis in the presene of cysteine or dithiothreitol. The protease was not inhibited by high molecular weight kininogen but was inhibited by 
2 The protease bound stoichiometrically to 2 with 1: 1 molar ratio and 25% activity remained constant even after the addition of 4 molar excess of 1-protease inhibitor.The protease extensively degraded IgG, IgA, fibronectin, fibrinogen, and a inhibitor. The protease completely inactivated the 1-protease inhibitor within 30–45 min, even at a very low enzyme concentration. Transferrin, lysozyme, and albumin were almost resistant to proteolysis. The high susceptibility of these defense-oriented humoral proteins to the 73K protease may become additively augmented in the presence of 56- and 60-kDa (56K and 60K) proteases secreted by S. marcescens at the site of invasion and thus enhance the pathogenic potentials.
1This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, 1987 and in part by a Grant from Yakult Honsha Co., Tokyo, 1988.