Measurement of ADP-ribosyl cyclase activity

Purified proteins (mBST-1, mCD38) were incubated with 0.5 mM β-NAD at 37 °C for 1 h in 20 mM Tris–HCl buffer (pH 7.2) with 0.1% Triton-X 100. Samples were treated with 0.6 M perchloric acid (PCA), and precipitates were removed by centrifugation. PCA was removed by mixing the aqueous sample with 3 parts 2 M KHCO₃. After centrifugation at 15,000×g for 10 min, the aqueous layer was collected and neutralized with 20 mM sodium phosphate (pH 8.0). The enzyme product, cADPR, was measured by modification of the cycling method described previously [68]. To remove all contaminating nucleotides, the samples were incubated overnight with the following hydrolytic enzymes at 37 °C: 0.44 units/ml nucleotide pyrophosphatase, 12.5 units/ml alkaline phosphatase, 0.0625 units/ml NAD glycohydrolase, and 2.5 mM MgCl₂ in 20 mM sodium phosphate buffer (pH 8.0). Enzymes were removed by filtration using Centricon 3 filters. To convert cADPR to β-NAD⁺, the samples (0.1 ml/tube) were incubated with 50 μl of a cycling reagent containing 0.3 μg/ml Aplysia ARC, 30 mM nicotinamide, and 100 mM sodium phosphate (pH 8.0) at room temperature for 30 min. The samples were further incubated with the cycling reagent (0.1 ml) containing 2% ethanol, 100 μg/ml alcohol dehydrogenase, 20 μM resazurin, 10 μg/ml diaphorase, 10 μM riboflavin 5′-phosphate, 10 mM nicotinamide, 0.1 mg/ml bovine serum albumin (BSA), and 100 mM sodium phosphate (pH 8.0) at room temperature for 2 h. An increase in resorufin fluorescence was measured at 544 nm excitation and 590 nm emission wavelengths using a fluorescence plate reader (Molecular Devices Corp., Spectra-Max GEMINI). Various known concentrations of cADPR were also included in the cycling reaction to generate a standard curve.