NAD" containing

NADH, containing a tertiary amine functional group, has been readily determined by Ru(bpy)32+ ECL. However the oxidized form, NAD+, containing an aromatic secondary amine group produces virtually no ECL signal. This had led to a variety of indirect enzymic methods of analysis, where the activity of the enzyme results in the conversions between NAD+ and NADH. These are discussed in Sec. 8. 

Some enzymes associate with a nonprotein cofactor that is needed for enzymic activity. Commonly encountered cofactors include metal ions such as Zn2+ or Fe2+, and organic molecules, known as coenzymes, that are often derivatives of vitamins. For example, the coenzyme NAD+contains niacin, FAD contains riboflavin, and coenzyme A contains pantothenic acid. (See pp. 371-379 for the role of vitamins as precursors of coenzymes.) Holoenzyme refers to the enzyme with its cofactor. Apoenzyme refers to the protein portion of the holoenzyme. In the absence of the appropriate cofactor, the apoenzyme typically does not show biologic activity. A prosthetic group is a tightly bound coenzyme that does not dissociate from the enzyme (for example, the biotin bound to carboxylases, see p. 379). 

Note that any NAD" containing solutions must be protected from light, which is easily done by wrapping containers with aluminum foil. Radiolabelled P-NAD" is either synthesized from [a- P]ATP by the method of Cassel and Pfeuffer (1978) or purchased and stored frozen. Although the half-life of the radioactive phosphate is approximately two weeks, we do not use P-NAD" longer than three to four weeks after preparation because of radiolysis. 

ADP-ribosylation of tfie samples is carried out in glass tubes (approximate volume 5 ml) in case the reaction has to be followed by precipitation with organic solvents. Therefore, the markings on the tubes should withstand solvents. Each tube contains 10 (xl of preactivated toxin (100 ng PT/tube) and 20 [il of membrane suspension. The reaction is readily started by addition of 30 1 of the P-NAD" -containing buffer, resulting in a total volume of 60 (il per tube. ADP-ribosylation of the thoroughly vortexed sample is conducted for 30 min at 30°C or tor 20 min at 37°C while the tubes are agitated con-tinously. 

Close inspection of Fig. 2-18(b) will show that the unit cell of Nad contains 8 ions, located as follows ... 

Analogs of NAD containing alkylating functions 169) are also known to inhibit the enzyme and these might react with histidine-195. 

Hans von Euler is generally recognized as the first to establish the chemical structure of NAD (Metzler, p. 468). Von Euler and Arthur Harden shared the 1929 Nobel Prize in physiology or medicine for the discovery of coenzymes (including NAD). Later von Euler showed that NAD contains two units of the sugar ribose, two phosphate groups, one adenine unit, and... 

Definitive eviderice regarding the chemical structure of the photoadduct came from mass spectrometric and nuclear magnetic resonance (NMR) measurements. The labeled tripeptide, Val-X-Tyr (residues 147-149), was isolated from fragment A irradiated in the presence of either unlabeled NAD or NAD containing [ C] or [ N] within the nicotinamide moiety. Mass spectra of these peptides were taken following fast atom... 

Schutzbach suggests a reaction mechanism for the decarboxylation of UDP-glucuronic acid which is outlined in Fig. 16. In Step (1) the NAD-containing enzyme would combine with the substrate in a reversible reaction. Stop (2) involves the extraction of hydrogen from carbon-4, with the participation of NAD, and this step is presumably rate-limiting. The 4-keto intermediate (II) would readily decarboxylate in the essentially irreversible Step (3), and the addition of a proton to the carbanion at carbon-5 would result in the formation of UDP-4-keto-xylose (Step (4)]. The reduction of this intermediate (IV) is probably the final step (5) in the reaction and appears to be irreversible, since UDP-xylose cannot be activated to the 4-keto intermediate by incubation with the enzyme. 

Camphor, dibutyl phthalate [84-74-2], and other Hpidic solvents are common plastici2ers. Nad lacquers requite the presence of a suspending agent because pigments have a tendency to settle. Most tinted lacquers contain a suitable flocculating agent, such as stearalkonium hectorite, a reaction product of hectorite [12173 6-6] and stearalkonium chloride [122-19-0],... 

Oxidation of P-nicotinamide adenine dinucleotide (NADH) to NAD+ has attracted much interest from the viewpoint of its role in biosensors reactions. It has been reported that several quinone derivatives and polymerized redox dyes, such as phenoxazine and phenothiazine derivatives, possess catalytic activities for the oxidation of NADH and have been used for dehydrogenase biosensors development [1, 2]. Flavins (contain in chemical structure isoalloxazine ring) are the prosthetic groups responsible for NAD+/NADH conversion in the active sites of some dehydrogenase enzymes. Upon the electropolymerization of flavin derivatives, the effective catalysts of NAD+/NADH regeneration, which mimic the NADH-dehydrogenase activity, would be synthesized [3]. 

The utility of the Zincke reaction has been extended to the preparation of various NAD and NADH analogs. Holy and co-workers synthesized a series of NAD analogs containing nucleotide bases as a means to study through-space interaction between the pyridinium and base portions. Nicotinamide-derived Zincke salt 8 was used to link with various adenine derivatives via tethers that contained hydroxyl (105 —> 106, Scheme 8.4.35), phosphonate (107—>108, Scheme 8.4.36), and carboxylate "... 

Most foods of animal origin contain nicotinamide in the coenzyme form (high bioavialability). Liver and meat are particularly rich in highly bioavailable niacin. Most of the niacin in plants, however, occurs as nicotinic acid in overall lower concentrations and with a lower bioavailability. The major portion of niacin in cereals is found in the outer layer and its bioavailability is as low as 30% because it is bound to protein (niacytin). If the diet contains a surplus of L-tryptophan (Ttp), e.g., more than is necessary for protein synthesis, the liver can synthesize NAD from Trp. Niacin requirements are therefore declared as niacin equivalents (1 NE = 1 mg niacin = 60 mg Trp). 

Sequences of proteins containing Rieske-type clusters have been deduced from the complete operons of several dioxygenases these dioxygenases require electrons from NAD(P)H to convert aromatic compounds to cis-arene diols. The water-soluble dioxygenase systems consist of a reductase and a terminal dioxygenase many dioxygenases also contain a [2Fe-2S] ferredoxin (20). The terminal oxygenases contain a Rieske-type cluster and the ferredoxins may contain either a Rieske-type or a 4-cysteine coordinated [2Fe-2S] cluster. 

The dioxygenase systems consist of a reductase and a terminal oxygenase many dioxygenases also contain a [2Fe-2S] ferredoxin. The reductase reacts with NAD(P)H it can be any of the following (20) ... 

Subsequently, proteolytic fragments of the rabbit renal 25-kDa amiloride-binding protein were micro-sequenced and found to have high sequence homology with rat and human NAD(P)H quinone oxidoreductase. Indeed, enzymatic assays revealed that renal brush border membrane vesicles contain significant NADPH quinone oxidoreductase activity. Presumably NAD(P)H quinone oxidoreductase coincidentally binds amiloride analogs with the same rank order as the Na /H exchanger [39]. 

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