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.     

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.     

An enzyme degrading reduced nicotinamide-adenine dinucleotide in Proteus vulgaris.

Cell-free extracts of a strain of Proteus vulgaris degrade NADH to reduced nicotinamide riboside, adenosine and two molecules of phosphate. The system is weakly active in fresh cell extracts, but activity is increased about 10-fold on rapid heating to 70-100 degrees C. On returning to room temperature, the activity returns rapidly to its initial low value but can be re-activated by again heating to 70-100 degrees C. Reversible activation can also be effected by extremes of pH or by teatment with 8M-urea. Activation appears to be due to reversible changes in conformation of the protein of the enzyme rather than to combination of the enzyme with a heat-labile inhibitor. The active form can be stabilized by addition of PPi. The system, which also possesses 5'-nucleotidase activity not separable from the NADH pyrophosphatase, requires Co2+ (0.4mM) for maximum activity. Although activated at relatively high temperatures, it is not enzymically active until cooled to 50-60 degrees C. It may be purified by affinity chromatography (with NAD+ as ligand) to an activity over 400 times that of the crude cell extract, and yields only one major band on polyacrylamide-gel electrophoresis.