Date of Award

Spring 1983

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry & Biochemistry

Program/Concentration

Chemistry

Committee Director

James H. Yuan

Committee Member

Frank E. Scully, Jr.

Committee Member

Robert L. Ake

Call Number for Print

Special Collections LD4331.C45C43

Abstract

Nicotinamide adenine dinucleotide glycohydrolase (NADase) (E.C. 3.2.2.5) from human seminal plasma (HSP) was partially purified through a multistep purification procedure, including affinity chromatography on Amicon Red Matrex Gel A. The NADase activity was assayed by a fluorometric method with nicotinamide 1,N6 -ethenoadenine dinucleotide as the substrate. The enzymatic cleavage of etheno-NAD by NADase was accompanied by an eight-fold increase in fluorescence. This large increase in fluorescence provided a sensitive way to follow the rate of etheno-NAD hydrolysis.

NADase was found to be less specific for the purine moiety of NAD, since etheno-NAD was accepted as a substrate. The enzyme exhibited an optimum pH for hydrolysis at pH 7.0 and a K of 0.54 pM.

The properties of the substrate binding site of NADase were studied with several inhibitors. The adenosine derivatives, adenosine, adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), adenosine 5'-triphosphate (ATP), and adenosine diphosphoribose (ADP-ribose), were found to competitively inhibit the NADase-catalyzed hydrolysis of etheno-NAD. In addition, NADase was shown to be competitively inhibited by a homologous series of n-alkyl phosphates from n-butyl phosphate to n-dodecyl phosphate. A small positive chain length effect was observed in the binding of the n-alkyl phosphates to the enzyme. Nicotinamide, on the other hand, was shown to be a noncompetitive inhibitor of NADase.

The results of inhibition study of the enzyme suggest that the substrate binding site of the HSP NADase contains a pyrophosphate binding region and a hydrophobic region.

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DOI

10.25777/436k-7z41

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