Calf-Spleen Nicotinamide - Adenine Dinucleotide Glycohydrolase Solubilization Purification and Properties of the Enzyme

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Abstract
NAD glycohydrolase [EC 3.2.2.6] of calf spleen was solubilized with [hog] pancreatic lipase [EC 3.1.1.3] and purified approximatively 800-fold to a specific activity of 7 units/mg of protein by successive DEAE-cellulose and carboxymethyl-cellulose chromatography. The purified enzyme has a MW of 24,000 and is characterized by a double band on disc gel electrophoresis. Some kinetic properties of the NAD-glycohydrolase-catalyzed hydrolysis of NAD were examined using a titrimetric assay for enzyme activity. The reaction is subject to inhibition by excess of substrate, which disappears at high ionic strength and low pH. At a pH below 5 the kinetics display an apparent activation by substrate. The effects of pH (4.5-9.0) on the kinetic parameters do not reveal an essential ionizable group in the catalytic process.