A nicotinamide-adenine dinucleotide assay utilizing liver alcohol dehydrogenase

An assay for the determination of NAD has been developed utilizing the coupled oxidoreductase activity of liver alcohol dehydrogenase. The coupled reaction between ethanol and lactaldehyde is driven by the removal of one of the products, acetaldehyde, into a semicarbazide solution. Under the stated conditions, a linear relationship exists between the absorbance of acetaldehyde semicarbazone and NAD concentration in the reaction mixture. The principal advantages of this method are speed and simplicity. NAD⁺ and NADH are assayed by the same procedure, which is also used to measure NADP⁺ and NADPH after these nucleotides have been converted to NAD⁺ and NADH, respectively.     

Calf-spleen nicotinamide-adenine dinucleotide glycohydrolase. Properties of the active site.

The interaction between the nicotinamide adenine dinucleotide binding domain of calf spleen NAD glycohydrolase and its ligands has been studied. The use of competitive inhibitors, structurally related to different portions of the NAD molecule (i.e. adenosine and nicotinamide moieties), revealed the considerable importance of the binding between the pyrophosphate linkage and probably an arginyl residue of the active site. This interaction allows the positioning of the substrate in a conformation which permits catalysis to occur. The binding between the 2'-hydroxyl of the adenosine moiety and a residue of the active site, which exists in NAD-linked dehydrogenases, is probably missing in the calf spleen NAD glycohydrolase, based on the inhibition by salicylates, 2'-deoxyadenosine 5'-monophosphate and the hydrolysis of the 2'-deoxyadenosine analogue of NAD. The NAD glycohydrolase could be completely inactivated by 2,3-butanedione, an arginyl-modifying reagent. The reaction followed pseudo-first-order kinetics and the modification was found to be reversible. Woodward's reagent K, a reagent for carboxyl residues, partially inactivated the enzyme, which resulted in a change of the NAD glycohydrolase kinetic parameters Km and V. The inactivation rate was complicated by a parallel decomposition of the reagent.     

An improved enzymatic cycle for nicotinamide-adenine dinucleotide phosphate.

We describe the use of column chromatography on the nonpolar adsorbent. Amberlite XAD-2, and on silanized silica gel in the desalting and partial purification of cobalamins. These techniques are both simpler and more versatile than phenol extraction, without sacrificing efficiency. In addition, a solvent system for thin-layer chromatography on silanized silica gel is described which rapidly separates naturally occurring cobalamins.     

Erythrocytic nicotinamide-adenine dinucleotide phosphate levels and the genetic regulation of erythrocytic glucose 6-phosphate dehydrogenase activity in the inbred mouse

NADP measurements in erythrocytes of eight inbred strains of mice showed a correlation between low NADP and low erythrocytic G6PD levels. In vitro heat inactivation tests indicated that the NADP differences found could explain the previously demonstrated lability of G6PD in the erythrocytes of the low G6PD activity strain, C57L/J. Breeding experiments suggested a common genetic control of erythrocytic NADP levels and G6PD activity in crosses between intermediate and low strains, but an effect of sex and other variables, as yet unidentified, precluded a strong demonstration of unitary genetic control.