Artemia franciscana cyst의 세포내 산성 Protease

Characterization of Cytosol Acid Protease from Dormant Cyst of Artemia franciscana

  • 최희선 (부산대학교 자연과학대학 생물학과) ;
  • 최경희 (부산대학교 자연과학대학 생물학과) ;
  • 류태형 (부산대학교 자연과학대학 생물학과)
  • 발행 : 1997.09.01

초록

Artemia franciscana의 cyst에서 protease를 정제하가 위해 세포를 파쇄하여 원심 분리한 후 상등액을 회수하였다. 그상등액에서 40-60% 황산암모늄을 첨가하여 침전시켜서 염을 제가한 후, Sphadex G-200 및 DEAE-Sephadex A-50 column chromatography를 하여 정제한 후 본 효소의 특성을 조사한바 그 특성은 아래와 같았다. 정제 과정을 거치는 동안, 비활성은 13.64배 증가하였고, 수율은 7%였다. 그리고 이효소는 pH 6에서는 안정하였으나, pH 8이상에서는 대부분의 활성이 실활 되었고, 최적 pH는 3.0이었다. 따라서 이 효소는 전형적인 산성 pro-tease로 판정되었다. 본 효소는 60$^{\circ}$C이상의 온도에서 대부분 실활 되었고, 최적 활성 반응 온도는 35$^{\circ}$C였다. 금속 이온의 영향을 알아본 결과, $Cu^{++}$, Z$Zn^{++}$, $Fe^{++}$가 효소의 활성을 저해하였고, 중금속 chelator인 EDTA는 반대로 효소 활성을 증가시켰다. 단백질분해효소에 특이적인저해제를 첨가하여 실험한 결과 thiol protease ingibitor인 antipain, chymostatin, leupeptin, E-64, iodoacetate등에 효과적으로 효소활성이 저해받으므로 본 효소는 acid thiol protease라 판정했다.

A thiol protease has been isolated and partially purified from encysted brine shrimp Artemia franciscana using a four-step procedure(filtration, salting out, gel filtration and ion exchange chromatography). The optimum pH of the enzyme for caseinolytic activity was appeared to be 3.0, and the enzymematic activity was stable up to pH 6.0 but lost completely at the pH higher than 8.0. The optimal temperature of the enzyme was appeared to be 35$^{\circ}$C, and ninety percent of the enzyme activity was lost at 45$^{\circ}$C. Various metal ions, e.g., zinc, copper, iron, inhibited the enzyme activity; however, heavy metal chelator, e.g., EDTA, stimulated the enzyme activity. The protease was concluded to be a member of the thiol group protease, since it was inhibited by thiol protease inhibitors and iodoacetate. The protease was also concluded to be a acid protease based on optimum pH.

키워드

참고문헌

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