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Papers In Press, published online ahead of print March 1, 2007
J. Lipid Res., doi:10.1194/jlr.M600423-JLR200

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Journal of Lipid Research, Vol. 48, 600-608, March 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

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. Hannun¹

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

The online version of this article (available at http://www.jlr.org) contains an additional figure.

Published, JLR Papers in Press, December 12, 2006.

¹ To whom correspondence should be addressed. e-mail: hannun{at}musc.edu

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-ß-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 (K_a) of 1.5 µM. Kinetics studies showed that calcium caused a decrease of K_m 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.

Supplementary key words circular dichroism • shphingolipid • ceramide

Abbreviations: CD, circular dichroism; CDase, ceramidase; IPTG, isopropyl-beta-thiogalactopyranoside; LB, Luria-Bertani; nCDase, neutral ceramidase; NOE, N-oleoylethanolamine; P. aeruginosa, Pseudomonas aeruginosa; pCDase, Pseudomonas ceramidase; S1P, sphingosine-1-phosphate; SPH, sphingosine

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