[123] Nicotinamide deamidase

Publisher Summary This chapter describes the various assay methods, purification procedure, and properties of the enzyme nicotinamide deamidase. It is stable at all stages of purification. The purified enzyme can be stored indefinitely at -15°C. Under the standard assay conditions, the reaction is linear for at least 6 hours. A variety of compounds cause inhibition of the deamidase such as small amounts of surface active agents, thyroxine and thyroxine analogs, ammonium sulfate, and diisopropylfluorophosphate. Inhibition by detergents brings about a shift in pH optimum to high values. Sulfhydryl reagents inhibit the deamidase only at high concentrations. The deamidase has such a high K m for its substrate that assays based on disappearance of substrate are impractical. Several assay procedures based on the determination of products are used, namely—nicotinic acid-7- 14 C, ammonia (NH 3 ) by Nessler's method, NH 3 by the indophenol method, and by coupling to glutamic dehydrogenase. In crude extracts where release of ammonia from a variety of sources is possible, estimation of nicotinic acid is the preferred method. The use of the more rapid and simpler methods for the determination of ammonia is recommended for assaying the purified enzyme.