Active N-Acylsphingosine Amidohydrolase 2 (ASAH2)

The human ASAH2 gene encodes N-acylsphingosine amidohydrolase-2, also known as neutral or non-lysosomal ceramidase. ASAH2 is a type II integral membrane protein that can be cleaved to produce a soluble secreted protein. The enzyme is abundant in the brush border membranes of the intestine, but is also expressed in tissues such as kidney, brain and liver. An N-terminally truncated form of human ASAH2 has been shown to localize to mitochondria. A major physiological function of ASAH2 is the metabolism of dietary sphingolipids, but the enzyme may also be involved in the generation of messenger molecules such as sphingosine and sphingosine 1-phosphate. The activity of recombinant human ASAH2 is measured by its ability to hydrolyze the substrate C12:0 ceramide into sphingosine and dodecanoic acid in the assay buffer 25 mM MES, 150 mM NaCl, 1% (w/v) Sodium Cholate, pH 6.5. The rhASAH2 is diluted to 0.15 ug/ml in assay buffer and 50 ul diluted rhASAH2 was loaded into a black well plate and start the reaction by adding 200 µL of 250 µM substrate, with a substrate blank containing 50 µL assay buffer and 200 µL substrate and a protein blank containing 50 ul diluted rhASAH2 and 200 ul assay buffer. Incubated at 37 ℃ for 1h, then stop reactions by heating them at 95-100 °C for 5 minutes. Add 250 µL of 2 mg/ml o-PA mixture to all reaction vials, including controls and incubate at room temperature for 10 minutes. Load 200 µL (in duplicate) of reaction mixtures and controls in a plate and read at excitation and emission wavelengths of 330 nm and 450 nm (top read), respectively, in endpoint mode. The specific activity of recombinant human ASAH2 is > 9700 pmol/min/µg.