Large-scale purification and characterization of recombinant Pseudomonas ceramidase: Regulation by calcium

Bill X. Wu, Christopher F. Snook, Motohiro Tani, Erika E. Büllesbach, Yusuf A. Hannun

研究成果: ジャーナルへの寄稿学術誌査読

17 被引用数 (Scopus)

抄録

Ceramidases (CDases) hydrolyze ceramide to sphingosine (SPH) and fatty acid. Pseudomonas CDase (pCDase) is a homolog of mammalian neutral ceramidases and may play roles in disease pathogenesis. In this study, pCDase was cloned and expressed in Escherichia coli (E. coli). The expressed recombinant pCDase was solubilized by optimizing several factors, including culture medium, the concentration of isopropyl-β-thiogalactopyranoside (IPTG), temperature, and time of induction, which were identified to be critical for the optimal production of recombinant pCDase. The recombinant pCDase was purified using nickel-nitrilotriacetic acid affinity, phenyl-Sepharose, and Q-Sepharose column chromatography, which gave an overall yield of 0.45 mg/l purified protein of starting culture. The activity of the recombinant pCDase followed classical Michaelis-Menten kinetics, with optimum activity in the neutral pH range. Both the hydrolytic and the reverse activities of CDase were stimulated by calcium with an affinity constant (Ka) of 1.5 μM. Kinetics studies showed that calcium caused a decrease of Km and an increase in V max of pCDase. Calcium and D-erythro-sphingosine caused significant changes in the near ultraviolet circular dichroism (CD) spectra and the changes were inhibited in the presence of EGTA. These results identify important interactions between calcium and pCDase, which may play an essential role in the interaction of pCDase and its substrate.

本文言語英語
ページ(範囲)600-608
ページ数9
ジャーナルJournal of Lipid Research
48
3
DOI
出版ステータス出版済み - 3月 2007
外部発表はい

!!!All Science Journal Classification (ASJC) codes

  • 生化学
  • 内分泌学
  • 細胞生物学

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