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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Purification and Characterization of a Serine Proteinase from Acanthamoeba culbertsoni

  • Park, Ki-Won (Section of DNA Analysis, National Institute of Scientific Investigation) ;
  • Song, Chul-Yong (Department of Biology, Faculty of Natural Science, Chung-Ang University)
  • Received : 1996.05.20
  • Published : 1996.09.30
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Abstract

A serine proteinase was purified from Acanthamoeba culbertsoni by 41~80% ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography and gel filtration chromatography. The molecular weight of the purified enzyme was estimated to be 108.0 kDa by gel filtration chromatography and 54.0 kDa by SDS-PAGE. Therefore, the purified enzyme seemed to be a dimer. Isoelectric point was 4.5. The enzyme activity was highly inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate (OFP) and phenylmethyl sulfonylfluoride (PMSF). It had a narrow pH optimum of 6.5~7.5 with a maximum at pH 7.0. These data suggested that the purified enzyme was a neutral serine proteinase. Optimal temperature was $37^{\circ}C$. It was stable for at least 16 h at $4^{\circ}C$ and $37^{\circ}C$, but it was rapidly inactivated at $65^{\circ}C$ The activity of the purified enzyme was not influenced significantly by $Mg^{2+}$, $Mn^{2+}$, $Zn^{2+}$ or $Ca^{2+}$. However, the enzyme activity was highly inhibited by $Hg^{2+}$ The enzyme degraded type I collagen and fibronectin, but not BSA, hemoglobin, lysozyme, immunoglobulin A or immunoglobulin G.

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