NAD" determination

The conclusion that even with the best substrate, ethanol, dissociation of NAD occurs from the active ternary complex is consistent with the evidence-from isotope exchange experiments 32), mentioned previously, that the dissociation of coenzyme is not greatly suppressed in the ternary complex compared with the binary complex, in contrast to liver alcohol dehydrogenase. This is also indicated by the initial rate data in another way 4>a/4>o for the preferred pathway mechanism approximates to the dissociation constant for NAD from the ternary complex (Table I), and is reasonably constant for the three primary alcohols and approximately equal to the dissociation constant of E-NAD, determined independently 40). 

Fig. 1. NAD content of CF-3 cells incubated in Ca + depleted medium O— O), Ca + supplemented medium ( - ), and Ca + depleted medium supplemented with normal levels of Ca + (1.8 mM) after 2 hr of incubation (x—x). Arrow indicates time of Ca + addition. Values are Mean SEM for three experiments. Ca + depleted medium was McCoy s 5a prepared without nicotinamide, nicotinic acid, and CaCl2. It was supplemented with 10% FBS treated as described (14). NAD determinations were performed as described (14). Reprinted with permission from ref. 14.

Human tumor cells were harvested by trypsinization, washed and sonicated in 50 mM Tris-HCl pH 8.0,0.25 M sucrose, 2 mM MgCl2, 1 mM DTT and 0.1 mM PMSF. Initial velocity (45 sec) assay with p pj AD (final NAD concentration 100 jiM), were performed as described by Chemey et al. (14). Data points are means standard deviation of three separate determinations. NAD determinations as in reference 15. 

Figure 3.8. Variation in the relative surface concentration y of Nad determined by Auger electron spectroscopy) with N2 exposure of different Fe single-crystal planes. 1 L (Langmuir) = 10" torrs is approximately the exposure required to saturate an adlayer if the sticking coefficient is unity.

Glucose [50-99-7] urea [57-13-6] (qv), and cholesterol [57-88-5] (see Steroids) are the substrates most frequentiy measured, although there are many more substrates or metaboUtes that are determined in clinical laboratories using enzymes. Co-enzymes such as adenosine triphosphate [56-65-5] (ATP) and nicotinamide adenine dinucleotide [53-84-9] in its oxidized (NAD" ) or reduced (NADH) [58-68-4] form can be considered substrates. Enzymatic analysis is covered in detail elsewhere (9). 

Biological activity (BA) was chosen as such parameter. The BA determined using a system and a technique for a class of natural polyphenolic bonds nicotinamide adenine dinucleotide restored (NAD H ) - ferricyanide (KjFe(CN)g) in a phosphates buffer solution. 

The physicochemical properties of the reactants in an eiKyme-catalyzed reaction dictate the options for the assay of enzyme activity. Spectrophotometric assays exploit the abihty of a substrate or product to absorb hght. The reduced coenzymes NADH and NADPH, written as NAD(P)H, absorb hght at a wavelength of 340 run, whereas their oxidized forms NAD(P) do not (Figure 7—9). When NAD(P)+ is reduced, the absorbance at 340 run therefore increases in proportion to—and at a rate determined by—the quantity of NAD(P)H produced. Conversely, for a dehydrogenase that catalyzes the oxidation of NAD(P)H, a decrease in absorbance at 340 run will be observed. In each case, the rate of change in optical density at 340 nm will be proportionate to the quantity of enzyme present. 

The use of H-labeled substrates has been used to determine details of the dehydrogenation of ciT-dihydrodiols produced by dioxygenases from aromatic substrates (Morawski et al. 1997), and it was possible to demonstrate the specificity of hydrogen transfer from the dihydrodiol substrates to NAD. 

Both ADH and ALDH use NAD+ as cofactor in the oxidation of ethanol to acetaldehyde. The rate of alcohol metabolism is determined not only by the amount of ADH and ALDH2 enzyme in tissue and by their functional characteristics, but also by the concentrations of the cofactors NAD+ and NADH and of ethanol and acetaldehyde in the cellular compartments (i.e., cytosol and mitochondria). Environmental influences on elimination rate can occur through changes in the redox ratio of NAD+/NADH and through changes in hepatic blood flow. The equilib-... 

Fig. 21.1 The interactions between the bound coenzyme molecule and the amino acids at positions 47 and 369 in the / , / 2, and / 3 polymorphic variants as observed in their respective structures determined by X-ray crystallography. The dashed lines indicate possible hydrogen-bonds between the amino acids and the phosphate oxygens of the bound coenzyme molecule, NAD(H). Arg47 is substituted by a His residue in the f 2 isozyme and Arg369 is substituted by a Cys residue in the / 3 isozyme. In each case, the substitution results in a net loss of hydrogen-bonding interactions and weaker affinity for the coenzyme.

In Franchini et al. (2004) we introduced four Lick/IDS index-index diagrams, i.e. NaD vs Ca4227, NaD vs Mg2, NaD vs Mgb, and NaD vs CaMg, to identify SSA and a-enhanced stars irrespectively of their Teg, log g and [Fe/H]. By applying this method to the 84 normal (i.e. excluding binaries and variable) stars from the S4N web site with [Fe/H] determined by AP04, it results that 8 stars are... 

The bioluminescent determinations of ethanol, sorbitol, L-lactate and oxaloacetate have been performed with coupled enzymatic systems involving the specific suitable enzymes (Figure 5). The ethanol, sorbitol and lactate assays involved the enzymatic oxidation of these substrates with the concomitant reduction of NAD+ in NADH, which is in turn reoxidized by the bioluminescence bacterial system. Thus, the assay of these compounds could be performed in a one-step procedure, in the presence of NAD+ in excess. Conversely, the oxaloacetate measurement involved the simultaneous consumption of NADH by malate dehydrogenase and bacterial oxidoreductase and was therefore conducted in two steps. 

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. 

Enzyme-coupled ECL enables the selective determination of many clinical analytes that are not in themselves directly electrochemiluminescent, but that can act as substrates for a variety of enzymic reactions. There are two general strategies for ECL the use of dehydrogenase enzymes, which convert NAD+ to NADH, and oxidase enzymes, which produce hydrogen peroxide. 

The possibility of isolating the components of the two above-reported coupled reactions offered a new analytical way to determine NADH, FMN, aldehydes, or oxygen. Methods based on NAD(P)H determination have been available for some time and NAD(H)-, NADP(H)-, NAD(P)-dependent enzymes and their substrates were measured by using bioluminescent assays. The high redox potential of the couple NAD+/NADH tended to limit the applications of dehydrogenases in coupled assay, as equilibrium does not favor NADH formation. Moreover, the various reagents are not all perfectly stable in all conditions. Examples of the enzymes and substrates determined by using the bacterial luciferase and the NAD(P)H FMN oxidoreductase, also coupled to other enzymes, are listed in Table 5. 

Ostensibly minor variations of a synthetic procedure sometimes can have significant consequences. For example, substituting KOCMe(CF3)2 for LiOC-Me(CF3)2 is believed [85] to lead to formation of the alkylidyne complex shown in Eq. 16 instead of the known [83] Mo(CH-f-Bu)(NAd)[OCMe(CF3)2]2 (Ad=ad-amantyl). A proton is likely to be transferred before formation of the final product, since it has been known for some time that both W(CH-f-Bu)(NAr)[OC-Me(CF3)2]2 and W(C-f-Bu)(NHAr)[OCMe(CF3)2]2 are stable species that cannot be interconverted in the presence of triethylamine [41]. In such circumstances the nucleophilicity of the alkoxide ion and the nucleophilicity and acidity of the alcohol formed upon deprotonation of the alkylidene will be crucial determinants of whether the imido nitrogen atom is protonated at some stage during the reaction. At this stage few details are known about side reactions in which amido alkylidyne complexes are formed. 

Lee HC, Walseth TF, Bratt GT, Hayes RN, Clapper DL 1989 Structural determination of a cyclic metabolite of NAD+ with intracellular Ca2+-mobilizing activity. J Biol Chem 264 1608-1615... 

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