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A more recent version of this article appeared on March 1, 2007

Papers In Press, published online ahead of print December 12, 2006
J. Lipid Res., doi:10.1194/jlr.M600423-JLR200
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Submitted on September 26, 2006
Revised on November 29, 2006
Accepted on December 12, 2006

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

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

Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425

Corresponding Author: hannun{at}musc.edu

Ceramidases (CDases) hydrolyze ceramide to sphingosine and fatty acid. Pseudomonas CDase (pCDase) is a homologue of mammalian neutral ceramidases and may play roles in disease pathogenesis. In this study, pCDase was cloned and expressed in E. coli. The expressed recombinant pCDase was solubilized by optimizing several factors, including culture medium, the concentration of isopropyl-beta-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 Ni-NTA affinity, phenyl-Sepharose, and Q-Sepharose column chromatography, which gave an overall yield of 0.45 mg purified protein per liter of starting culture. The activity of the recombinant pCDase followed classical Michaelis-Menten kinetics, with an optimum activity in the neutral pH range. Both the hydrolytic and reverse activities of CDase were stimulated by calcium with Ka of 1.5 µM. Kinetics studies showed that calcium caused a decrease of Km and an increase in Vmax of pCDase. Calcium and D-erythro-sphingosine caused significant changes in the near UV 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.


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