Large-scale purification and characterization of recombinant Pseudomonas ceramidase: regulation by calcium.
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.
Wu
Bill X
BX
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA.
Snook
Christopher F
CF
Tani
Motohiro
M
Büllesbach
Erika E
EE
Hannun
Yusuf A
YA
D000581:Amidohydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D055572:Ceramidases; D002518:Ceramides / Q000378:metabolism; D002847:Chromatography, Agarose; D002942:Circular Dichroism; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013110:Sphingosine / Q000378:metabolism
D002518:Ceramides; D011994:Recombinant Proteins; D000581:Amidohydrolases; D055572:Ceramidases; D013110:Sphingosine; D002118:Calcium