Localization and Regulation of Two NAD Nucleosidases in Ehrlich Ascites Cells

Abstract

After differential centrifugation of Ehrlich ascites cells by the de Duve method, NAD nuclosidase activities were found in the nuclear and the microsomal fractions, but not in the lysosomal fraction. The nuclear NAD nucleosidase is inhibited 50% by 0.2 mM nicotinamide and is highly specific for β-NAD. Its activity is markedly decreased after DNase treatment; it is associated with chromatin and is very probably identical with an enzyme that polymerizes the adenosine diphosphate ribose moiety of NAD to an acid-insoluble polymer first described in rat liver nuclei by Chambon, Weill, and Mandel. The microsomal NAD nucleosidase is inhibited 50% by 1.2 mM nicotinamide, and splits NAD, NADP and NMN at a ratio of 1:0.5:0.2. NAD and NADP nucleosidase activities are reciprocally inhibited by NADP and NAD. Evidence is presented that the microsomal enzyme is active mainly at the surface of the cell. Nicotinamide concentration may be one factor regulating the nuclear but not the microsomal NAD nucleosidase. Within the cell, NAD is protected by membrane structure from degradation by the microsomal NAD nucleosidase.