Partial purification and characterization of phosphatidylcholine hydrolyzing enzyme from liver membrane of flounder , Paralichtys olivervaceus

넙치 간에 있어 가수분해 효소의 부분정제 및 특성규명

  • Published : 2004.08.30

Abstract

In the present study, phosphatidylcholine (PC) hydrolyzing enzyme had been isolated from membrane of flounder liver. PC hydrolyzing enzyme solubilized in 1% Triton X -100 from membrane was partially purified by sequential chromatography on Heparin Sepharose CL-6B and Heparin-5PW columns. The products by membrane-bound hydrolyzing enzyme were identified as phosphatidic acid and choline, but in the presence of primary alcohol, phosphatidylethanol was produced at the expense of phosphatidic acid. These data suggest that membrane-bound enzyme may be a PC-phosphoipase D (PLD) type. The enzyme had pH optimum at below 6.0 and temperature optimum at $37^\circ{C}$. The activity of PC-PLD was dose-dependently increased by $Ca^{2+}$ but not $Mg^{2+}$. The activity of PC-PLD was stimulate by PC, PIP2 and PE.

본 실험은 넙치 (Paralichthys olivaceus) 간으로부터 membrane부분에 존재하는 PC 가수분해 효소의 특성에 대해 조사하였다. 먼저 간 조직을 초고속 원심 분리기와 nonion-detergent인 1% triton X - 100을 이용하여 membrane 부분을 분리하였으며, Heprain-Sepharose CL-6B 칼럼과 Heparin-5PW 칼럼을 이용하여 분리정제 하였다. 얻어진 PC 가수분해효소에 대한 반응 ․ 생성물을 확인하기 위해 autoradiography를 실시하였다. 지용성 부분의 결과에서 transphosphatidylation 반응의 결과물인 PEtOH을 형성하는 것으로 보아 PC-PLD임을 알 수 있었다. 얻어진 PC 가수분해효소에 대한 생화학적 특성을 조사한 결과 적정 pH가 6.5이하인 산성 조건 및 $37^\circ{C}$의 배양온도에서 최고 활성을 나타내었으며, 이가 이온들에 대한 영향의 경우 칼슘은 1.67mM 농도에서 최고 활성을 나타냈으나, 마그네슘은 활성에 영향을 미치지 않았다. 각종 세포막 기질에 대한 영향을 조사한 결과 PC는 $0.75\mu{M}$, PIP2는 $2.35\mu{M}$, PE는 $26.8\mu{M}$ 농도에서 최고 활성을 나타내었다. 이상의 결과부터 넙치 간조직의 막층부분에 존재하는 PC를 가수분해효소는 PC-PLD가 존재함을 알 수 있었다.

Keywords

References

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