Biochemical Characterization of Phospholipase C$\delta$from liver of Mud loach (Misgurnus mizolepis)

미꾸라지 간으로부터 포스포리파아제 C델타 단백질의 생화학적 특성

  • Seo, Jung-Soo (Department of Aquatic Life Medicine, Pukyong National University) ;
  • Lim, Sang-Uk (Faculty of Food Science and Biotechnology, Pukyong National University) ;
  • Kim, Na-Young (Department of Aquatic Life Medicine, Pukyong National University) ;
  • Lee, Sang-Hwan (Department of Aquatic Life Medicine, Pukyong National University) ;
  • Oh, Hyun-Suk (Department of Aquatic Life Medicine, Pukyong National University) ;
  • Lee, Hyung-Ho (Faculty of Food Science and Biotechnology, Pukyong National University) ;
  • Chung, Joon-Ki (Department of Aquatic Life Medicine, Pukyong National University)
  • 서정수 (부경대학교 수산생명의학과) ;
  • 임상욱 (부경대학교 식품생명과학부) ;
  • 김나영 (부경대학교 수산생명의학과) ;
  • 이상환 (부경대학교 수산생명의학과) ;
  • 오현석 (부경대학교 수산생명의학과) ;
  • 이형호 (부경대학교 식품생명과학부) ;
  • 정준기 (부경대학교 수산생명의학과)
  • Published : 20050400

Abstract

Phosphoinositide-specific phospholipase $C\delta$ $PLC\delta$) 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 $PLC\delta$ in E. coli cloned from Misgurnus mizolepis and partially purified $PLC\delta$ enzymes from liver tissues of M. mizolepis (wild ML-$PLC\delta$). The ML $PLC\delta$ gene was cloned and expressed under the previous report (Kim et al., 2004), and purified the recombinant protein by successive chromatography using $Ni^{2+}$-NTA affinity column and gel iltration FPLC column. The wild ML-$PLC\delta$ protein was solublized with 2 M KCI and purified by successive chromatography on open heparin-Sephagel and analytical TSKgel heparin-5PW. Both the recombinant and wild ML-$PLC\delta$ form of protein showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bis-phosphate (PIP$_2$) or phosphatidylinositol (PI). Its activity was absolutely $Ca^{2+}$- dependant, which was similar to mammalian $PLC\delta$ isozymes. Maximal PI-hydrolytic activations of recombinant and wild ML- TEX>$PLC\delta$ was at pH 7.0 and pH 7.5, respectively. In addition, the enzymatic activities of recombinant and wild ML-$PLC\delta$ 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 $PLC\delta$ isozymes exist in various organs. Taken together our results, it suggested that the biochemical characteristics of ML-$PLC\delta$ are similar with those of mammalian $PLC\delta1$ and ${\delta}3$ isozymes.

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