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Ceramidase catalyzes the hydrolysis of the N-acyl linkage between the fatty acid and sphingosine base in ceramide to produce sphingosine and a free fatty acid. Three isoforms of ceramidases (acid, neutral, and alkaline) have been characterized based on differences in their catalytic pH optimum., C12 NBD dihydro ceramide is a fluorescent ceramide analog that contains a saturated bond in the C-4/C-5 position of the sphingosine backbone. C-12 NBD ceramide, which contains the C-4/C-5 double bond, is a fluorescent ceramidase substrate that can be used for the measurement of alkaline and neutral ceramidase activity from a variety of sources. C12 NBD dihydro ceramide may exhibit reduced activity in ceramidase assays compared to C-12 NBD ceramide. This is based on the observation that saturation of the C-4/C-5 sphingosine bond in C-16-ceramide results in approximately a 10-fold reduction in efficiency as a substrate for rat brain ceramidase compared to the unsaturated substrate. However, experimental characterization of C12 NBD dihydro ceramide as a ceramidase substrate needs to be performed.
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