Purification and characterization of four proteases from a clinical isolate of Serratia marcescens kums 3958

Abstract

Four distinct proteases were purified to homogeneity fromn culture filtrates of Serratia marcescens kums 3958, a fresh isolate from a patient with a severe corneal ulcer. Purification was achieved by ammonium sulfate precipitation, DEAE-cellulose ion-exchange chromatography, and Sephadex gel filtration chromatography. The proteases were differentiated from each other by polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate and by immunodiffusion in agarose gels. The molecular weights of these purified proteases were estimated to be 56 x 103, 60 x 103, and 73 x i03 (hereafter designated 56K, 60K, and 73K proteases, respectively). The 73K protease was separated into 73Ka and 73Kb upon isoelectricfocusing. The isoelectric points of the 56K (major) and 60K, 73Ka, and 73Kb proteases (minors) were approximately 5.3, 4.4, 5.8, and 7.3, respectively. Both 56K and 60K enzymes were completely inactivated by EDTA at pH 5.0 and were reactivated by zinc ion; thus, they are mnetalloenzymes, whereas 73K (73Ka and 73Kb) enzymes appear to be thiol proteases. Carbohydrate, cysteine, and cystine were not detected in the 56K and 60K proteases. Amino acid compositions, partial amino acid sequence, and enzymological and immunological properties revealed that these four enzymes are distinct from each other.

Plasmin in tear fluid of patients with corneal ulcers: basis for new therapy

“…These inhibitors of collagenase had also very little effect on the proteolytic activity of tear fluid of' patients 1, 2, 8 and 9 in vitro. In keratitis caused by the opportunistic pathogen Serratia marcescens the Mr 56 000 bacterial metalloproteinase is thought to be a major pathogenic factor (Lyerly 8r Kreger 1979;Matsumoto 1984;Kamata et al 1985). On the basis of our results it seems possible that therapeutic intervention with proteinase inhibitor could also be beneficial in these patients.…”

Enhanced Vascular Permeability in Solid Tumor Involving Peroxynitrite and Matrix Metalloproteinases

“…proMMP-9 from human leukocytes and Serratia marcescens 56-kDa metalloproteinase were purified as reported previously. 34,35) Ovomacroglobulin (ovoM), a proteinase inhibitor from chicken egg white (720 kDa), was from JIMRO (Takasaki), and soybean trypsin inhibitor (SBTI, Kunitz type, 20 kDa) was from Fujimoto Pharmaceutical Co., Osaka. α-Gelatin monomer was a product of Serva Feinbiochemica, GmbH, Heidelberg, Germany.…”

Culture conditions affect cytotoxin production by Serratia marcescens

“…It is primarily a nosocomial pathogen that causes bacteremia (Volkow-Femandez et al, 1976;, cell-bound hemolysin (Ruan and Braun, 1990;, the presence of fimbriae and adherence to uroepithelial cells (Yamamoto et al, 1985;Parment et al, 19921, and production of four extracellular proteinases (Matsumoto et al, 1984). Among them, the 56 kDa proteinase has been the most extensively studied because it has been obtained from clinical isolates and is produced in the largest amounts (Maeda and Morihara, 1995).…”