Purification and characterization of a Serratia marcescens metalloprotease.
An extracellular, nonelastolytic, neutral metalloprotease of Serratia marcescens was purified by sequential ammonium sulfate precipitation, hydroxyapatite adsorption chromatography, flat-bed isoelectric focusing, and Sephadex G-100 gel filtration. The protease preparation had a 280/260 nm absorbance ratio of 1.8, was free of detectable amounts of endotoxin, carbohydrate, phosphorus, and other known extracellular enzymes of S. marcescens, and was homogeneous by Ouchterlony double immunodiffusion and Grabar-Williams immunoelectrophoresis. Crossed immunoelectrophoresis, thin-layer electrofocusing in polyacrylamide gel, and polyacrylamide disc gel electrophoresis showed three to four closely migrating, Coomassie blue-staining components in the protease preparation. However, zymogram analyses of the patterns showed that protease activity was associated with each component and that the protease was, therefore, microheterogeneous. The isoelectric point and sedimentation coefficient of the protease were approximately 5.3 to 5.4 and 4.2S, respectively, and the molecular weight estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration was approximately 52,500 and 44,000, respectively. The pH optimum range, with azocasein as the substrate, was 5.5 to 7.5. The enzyme contained a high percentage of acidic amino acids, no cysteine, and 1 g-atom of Zn(2+) and 7 g-atoms of Ca(2+) per mol. Various heavy metal ions and chelating agents and heating at 60 degrees C for 15 min inactivated the enzyme. Intracorneal, intratracheal, and intradermal administration of the protease into rabbits elicited rapid and extensive tissue damage. The minimum lethal intravenous dose for mice was approximately 17 mg/kg of body weight.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D005110:Extracellular Space / Q000201:enzymology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008667:Metalloproteins / Q000302:isolation & purification; D008670:Metals / Q000032:analysis; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification* / Q000378:metabolism / Q000633:toxicity; D011480:Protease Inhibitors / Q000494:pharmacology; D011817:Rabbits; D012706:Serratia marcescens / Q000201:enzymology*; D014461:Ultracentrifugation
D000596:Amino Acids; D008667:Metalloproteins; D008670:Metals; D011480:Protease Inhibitors; D010447:Peptide Hydrolases