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미꾸라지 간으로부터 포스포리파아제 C델타 단백질의 생화학적 특성

Biochemical Characterization of Phospholipase Cδ from liver of Mud loach (Misgurnus mizolepis)

김나영|오현석|이상환|이형호|임상욱|정준기|서정수
수산생명의학회 (Aquatic Life Medical Science)|2005.04|한국어병학회지 (JOURNAL OF FISH PATHOLOGY)|제18권 제1호|pp. 67-80

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초록

미꾸라지 (mudloach, Misgunus mizolepis)의 간으로부터 클로닝한 phosphoinositide-specific phospholipase Cδ (ML-PLCδ)를 대장균 (E. coli)에서 과발현시켜 만든 재조합 ML-PLCδ와 미꾸라지 간 조직으로부터 직접 정제한 ML-PLCδ의 생화학적 특성을 비교분석하였다. 우선, pET28a vector (Novagen)를 이용하여 E. coli BL21(DE3)에서 과발현된 재조합 ML-PLCδ은 Ni2+-NTA affinity 크로마토그래피 및 gel filtration 칼럼에 의해서 정제되었다. 미꾸라지 간 조직으로 ML-PLCδ는 open heparin 칼럼 및 분석용 heparin 칼럼등을 통하여 부분 정제하였다. 두개의 재조합 및 wild ML-PLCδ는 phosphatidylinositol 4,5-bis-phosphate (PIP2)에 대한 농도 의존적 PLC 활성을 보여주었고, 그 활성은 포유류 PLCδ 효소와 유사하게 칼슘 농도에 의존적인 활성을 나타내었다. 재조합 및 wild ML-PLCδ는 각각 pH 7.0 및 7.5에서 가장 큰 PI-가수분해 활성을 나타낸다는 사실을 알 수 있었다. 게다가, 재조합 및 wild ML-PLCδ는 sodium doecylcholate (SDC) 및 phosphatidylethanolamine (PE), phosphatidylcholine (PC)와 같은 지질류에 대하여 농도의존적인 활성을 나타내나, spermine과 같은 polyamine류의 존재하에서는 농도 의존적으로 PLC 활성이 감소됨을 알 수 있었다. 미꾸라지 각 기관들의 ML-PLCδ의 발현양상 및 양등을 측정하여 보았을 때 ML-PLCδ는 포유류 PLCδ와 마찬가지로 다양한 형태의 PLCδ가 존재함을 알 수 있었다. 이와 같은 결과들로 미루어서 미꾸라지로부터 얻은 ML-PLCδ는 포유류의 PLCδ isozymes과 유사한 형태의 생화학적 특성을 가지나, 포유류 PLCδ1과 PLCδ3 isozyme의 생화학적 특성을 함께 가짐을 알 수 있었다.

Abstract

Phosphoinositide-specific phospholipase Cδ (PLCδ) plays an important role in many cellular responses and is involved in the production of second messenger. The present study was conducted to obtain the biochemical characteristics of the expressed recombinant PLCδ in E. coli cloned from Misgurnus mizolepis and partially purified PLCδ enzymes from liver tissues of M. mizolepis (wild ML-PLCδ). The ML-PLCδ gene was cloned and expressed under the previous report (Kim et al., 2004), and purified the recombinant protein by successive chromatography using Ni2+-NTA affinity column and gel filtration FPLC column. The wild ML-PLCδ protein was solublized with 2 M KCl and purified by successive chromatography on open heparin-Sephagel and analytical TSKgel heparin-5PW. Both the recombinant and wild ML-PLCδ form of protein showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bis-phosphate (PIP2) or phosphatidylinositol (PI). Its activity was absolutely Ca2+-dependant, which was similar to mammalian PLCδ isozymes. Maximal PI-hydrolytic activations of recombinant and wild ML-PLCδ was at pH 7.0 and pH 7.5, respectively. In addition, the enzymatic activities of recombinant and wild ML-PLCδ were increased in concentration-dependent manner by detergent, such as sodium deoxycholate (SDC), phosphatidylethanolamine (PE) and phosphatidylcholine (PC). The activities decreased in contrast by a polyamine, such as spermine. Western blotting showed that several types of PLCδ isozymes exist in various organs. Taken together our results, it suggested that the biochemical characteristics of ML-PLCδ are similar with those of mammalian PLCδ1 and δ3 isozymes.

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관련 논문AI추천

Regulation properties of phospholipase Cδ cloned from Misgurnus mizolepisHyung Ho Lee, Ji Hea Sung, Joon-Ki Chung, Ju Eun Je, June Woo Lee, Jung Soo Seo, Moo Sang Kim, Na Young Kim, Sang Hwan Lee, Sang Jung Ahn, Soo Jin Jeon수산생명의학회 · 한국어병학회지2007 · 제20권 제2호넙치 간에 있어 가수분해 효소의 부분정제 및 특성규명김나영, 박성일, 서정수, 엄혜경, 위효진, 이상환, 정준기수산생명의학회 · 한국어병학회지2004 · 제17권 제2호소의 뇌조직이 Phospholipase C의 활성화에 미치는 G-단백질의 역할김정희, 변영주, 이동진영남대학교 의과대학 · Journal of Yeungnam Medical Science1992 · 제9권 제2호소의 자궁 및 고환에서 Phospholipase C의 분리 및 뇌 Isozyme과의 비교 연구김정희, 이기녕, Rhee Sue Goo영남대학교 의과대학 · Journal of Yeungnam Medical Science1988 · 제5권 제2호Characterization of Phosphatidylcholine-Hydrolyzing Phospholipase D in the Scuticociliate Parasite, Uronema marinum김기홍, 김나영, 안상중, 이형호, 정준기, Bo Young Jee, Jin Woo Kim, Jung Soo Seo, Moo-Sang Kim, Sung Hee Jung수산생명의학회 · 한국어병학회지2008 · 제21권 제1호소의 뇌로부터 Inositol Triphosphate Kinase의 정체김정희, 이재태영남대학교 의과대학 · Journal of Yeungnam Medical Science1996 · 제13권 제1호

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