Hamdan Medical Journal (previously the Journal of Medical Sciences)

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Biochemical Characterization of Reverse Activity of Human Recombinant Neutral Ceramidase

Sehamuddin Galadari, Faisal Thayyullathil, Shahanas Chathoth, Mahendra Patel, Abdulkader Hago
Published in : Journal of Medical Sciences ; Vol 2, No 3 (2009)
DOI : 10.2174/1996327000902030128

Abstract


Ceramidases are enzymes that cleave the Nacyl linkage of ceramide and as a result generate sphingosine and a free fatty acid. We have purified a recombinant human neutral ceramidase from Hela cells, which is able to catalyze the reverse reaction of ceramidase i.e. generating ceramide. In order to understand the biochemistry of the human recombinant neutral ceramidase (nCDase), we have used sphingosine and C12-NBD-fatty acid as a substrate. Interestingly, this enzyme has a narrow pH optimum in the neutral range, and a very low enzyme activity is detected in the acid and alkaline range. The human nCDase is activated by Ca2+ at low concentrations, and it is inhibited by Cu2+ and Zn2+. The human recombinant nCDase is also inhibited by phospholipids such as: phosphatidic acid (PA), phosphatidyl ethanolamine (PE), phosphatidyl serine (PS), phosphatidyl choline (PC) and phosphatidyl glycerol (PG) by about 70-90% at 0.5 mM concentration. However, phosphatidyl inositol (PI) significantly stimulated the reverse activity at a very low concentration. Human nCDase was also inhibited by pyrimidine mono nucleotides such as TMP and UMP. This finding is significant as it demonstrates for the first time an effect by nucleotides on CDase activity. The enzyme is also inhibited by both oxidized and reduced GSH.

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