TY - JOUR
T1 - Large-scale purification and characterization of recombinant Pseudomonas ceramidase
T2 - Regulation by calcium
AU - Wu, Bill X.
AU - Snook, Christopher F.
AU - Tani, Motohiro
AU - Büllesbach, Erika E.
AU - Hannun, Yusuf A.
PY - 2007/3
Y1 - 2007/3
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=33947149638&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33947149638&partnerID=8YFLogxK
U2 - 10.1194/jlr.M600423-JLR200
DO - 10.1194/jlr.M600423-JLR200
M3 - Article
C2 - 17164222
AN - SCOPUS:33947149638
SN - 0022-2275
VL - 48
SP - 600
EP - 608
JO - Journal of Lipid Research
JF - Journal of Lipid Research
IS - 3
ER -