Abstract
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-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 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 microM. 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.
Highlights
Ceramidases (CDases) hydrolyze ceramide to sphingosine (SPH) and fatty acid
The Pseudomonas CDase (pCDase)-BL21 E. coli cells were first grown in LB medium and induced with 1 mM IPTG for 6 h
Because the pCDase has been purified from medium as a secreted protein [32], we tested for the recombinant pCDase in the medium
Summary
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. This study provides knowledge for the regulation and biochemical properties of pCDase and demonstrates that CD spectroscopy is a useful tool for the analysis of interactions between the enzyme, its substrates, and calcium activator. Enzyme activity was measured using detergent/lipid mixed micelles with D-erythro-C12-NBD-ceramide as a substrate at a concentration of 50 mM or 1.08 mol% in a 50 mM (pH 7.1) Tris buffer contained 1 mM CaCl2, 0.3% (w/v) Triton X-100 final concentration, with a total volume of 100 ml.
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