出版社：American Society for Biochemistry and Molecular Biology
摘要：Ceramidases (CDases) hydrolyze ceramide to sphingosine (SPH)and fatty acid. Pseudomonas CDase (pCDase) is a homolog of mammalianneutral ceramidases and may play roles in disease pathogenesis.In this study, pCDase was cloned and expressed in Escherichiacoli (E. coli). The expressed recombinant pCDase was solubilizedby optimizing several factors, including culture medium, theconcentration of isopropyl-ß-thiogalactopyranoside(IPTG), temperature, and time of induction, which were identifiedto be critical for the optimal production of recombinant pCDase.The recombinant pCDase was purified using nickel-nitrilotriaceticacid affinity, phenyl-Sepharose, and Q-Sepharose column chromatography,which gave an overall yield of 0.45 mg/l purified protein ofstarting culture. The activity of the recombinant pCDase followedclassical Michaelis-Menten kinetics, with optimum activity inthe neutral pH range. Both the hydrolytic and the reverse activitiesof CDase were stimulated by calcium with an affinity constant(Ka) of 1.5 µM. Kinetics studies showed that calcium causeda decrease of Km and an increase in Vmax of pCDase. Calciumand D-erythro-sphingosine caused significant changes in thenear ultraviolet circular dichroism (CD) spectra and the changeswere inhibited in the presence of EGTA. These results identifyimportant interactions between calcium and pCDase, which mayplay an essential role in the interaction of pCDase and itssubstrate.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
