Correlated biochemical and cytochemical studies of nicotinamide adenine dinucleotide phosphatase (NADPase) activity in ameloblasts using structural analogues of NADP.

The effects of altering the molecular structure of nicotinamide adenine dinucleotide phosphate (NADP) on enzymatic hydrolysis of the monoester phosphate group was examined at pH 5.0 in rat incisor ameloblasts using eight analogues of the oxidized form of the beta-isomer of NADP (beta-NADP+) modified in either the nicotinamide or adenine regions, or at the site for attachment of the monoester phosphate group to the molecule. Biochemical studies with whole homogenates of unfixed enamel organs revealed that the Michaelis-Menten constant (KM) and the maximum rate of dephosphorylation (Vmax) were different for these analogues relative to values estimated with beta-NADP+ as substrate. For example, the KM value was 3-fold higher and the Vmax value was lower by about 1/2 with the reduced form of the molecule as substrate. The KM value was about 2-fold higher but the Vmax value was about the same with an analogue lacking the nicotinamide group as substrate, while both the KM and the Vmax values were about 2-fold higher with an analogue containing an ethylene group covalently linked to adenine as substrate. In contrast, the KM value was markedly reduced (1/15) and the Vmax value was elevated (4-fold) using an analogue as substrate which contained the phosphate group attached at the 3'-position (3'-NADP+), rather than the 2'-position as in beta-NADP+ and other analogues. Cytochemical studies with glutaraldehyde-fixed enamel organs revealed that reaction product from hydrolysis of beta-NADP+ and other analogues containing the phosphate group attached at the natural 2'-position was localized within the intermediate saccules of the ameloblast Golgi apparatus. Reaction product from hydrolysis of 3'-NADP+, however, was localized at a different site, that is, within granules and membranous connections of the GERL system in the cell. Hence, 3'-NADP+ was not hydrolyzed by NADPase but by another enzyme tentatively identified as Coenzyme A phosphatase.