Date of Award

Summer 1980

Document Type


Degree Name

Master of Science (MS)


Chemistry & Biochemistry



Committee Director

James H. Yuan

Committee Member

Patricia A. Pleban

Committee Member

Thomas O. Sitz

Call Number for Print

Special Collections LD4331.C45 Y43


Nicotinamide adenine dinucleotide glycohydrolase (NADase) of bull semen is an extracellular, soluble enzyme capable of catalyzing the hydrolysis of the nicotinamide N-ribosidic linkage of NAD+. In search for a rapid, simple procedure for the preparation of this enzyme, it was found that Matrex Gel Red A (Amicon) column packing served very well as an affinity gel for NADase. The enzyme was applied to the Matrex Red A column (21 cm x 1.5 cm) in 10 mM sodium phosphate buffer, pH 6.0. Surprisingly, two fractions of NADase activity were obtained when the column was eluted with a sodium chloride linear gradient from Oto 2.5 M. The two forms, NADase I and NADase II, were eluted at the approximate salt concentrations of 0.25 Mand 1.20 M, respectively.

Investigation of these two forms was continued. Gel filtration chromatography was performed to determine the approximate molecular weights of 55,000 to 60,000 daltons and the Stokes' radii of 31.8 Å for NADase I and 0 31.5 Å for NADase II. Kinetic studies of the active substances showed differences in their abilities to

catalyze the hydrolysis of NAD+. NADase I was irnmediately active in hydrolyzing the substrate, whereas NADase II possessed a characteristic lag phase before catalyzing the reaction. The apparent Michealis constant, Km, for NADase I and for the maximum velocity of the m NADase II reaction was determined to be 1.0 x 10-4 M, which is the same value determined for the homogeneous bull semen NADase. Further studies of the dual forms of the enzyme were made by observing the ability to catalyze the base exchange reaction with 3-acetylpyridine and NAD+ to form free nicotinamide and 3-acetylpyridine adenine inucleotide. Earlier investigation showed that bull semen NADase was unable to catalyze the base exchange. However, both NADase I and NADase II were found to cause the base exchange as the other mammalian NADase's. Thin layer chromatography was used to provide visual evidence ·of the existence of the exchange products.


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