22] NAD glycohydrolase from bovine seminal plasma

Abstract

This chapter analyzes nicotinamide adenine dinucleotide (NAD) glycohydrolase from bovine seminal plasma. The bull semen NAD glycohydrolase is a glycoprotein containing 10.4% carbohydrate. The amino acid composition indicates the presence of high concentrations of serine, aspartate, and glutamate residues. The enzyme contains no free sulfhydryl groups as indicated by DTNB— that is, 5,5′-dithiobis (2-nitrobenzoic acid)—titration. The presence of tryptophan is confirmed by fluorescence studies. The bull semen NAD glycohydrolase catalyzes the hydrolysis of a variety of dinucleotides. The relative activities of substrates tested are β-NAD (100), NADP (84), α-NAD (1), nicotinamide hypoxanthine dinucleotide (90), 3-acetylpyridine adenine dinucleotide (9.2), 3-acetylpyridine hypoxanthine dinucleotide (11.0), pyridine-3-aldehyde adenine, dinucleotide (5.8), pyridine-3-aldehyde hypoxanthine dinucleotide (9.7), thionicotinamide adenine dinucleotide (2.5), and nicotinamide mononucleotide (8.4). The 3-aminopyridine adenine dinucleotide, 3-aminopyridine adenine dinucleotide phosphate, NADH, and NADPH did not serve as substrates. The purified NAD glycohydrolase at a concentration of 2 mg/ml in 0.01 M potassium phosphate buffer, pH 7.4, is unstable at 4°. It loses 40% of its activity in four weeks. No loss of activity is observed at -15° for a period of two months. Heating the enzyme in a boiling water bath for 5 min completely destroys the enzyme activity.