Characterization of 73 kDa thiol protease from Serratia marcescens and its effect on plasma proteins.
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 sulfhydryl 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 sulfhydryl 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 presence of cysteine or dithiothreitol. The protease was not inhibited by high molecular weight kininogen but was inhibited by alpha 2-macroglobulin. The protease bound stoichiometrically to alpha 2-macroglobulin with 1:1 molar ratio and 25% activity remained constant even after the addition of 4 molar excess of alpha 2-macroglobulin. The protease extensively degraded IgG, IgA, fibronectin, fibrinogen, and alpha 1-protease inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)
Molla
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A
Department of Microbiology, Kumamoto University Medical School.
Yamamoto
T
T
Maeda
H
H
D000596:Amino Acids / Q000032:analysis; D001798:Blood Proteins / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012706:Serratia marcescens / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D000596:Amino Acids; D001798:Blood Proteins; D013438:Sulfhydryl Compounds; D003546:Cysteine Endopeptidases