Erythrocytes NADP+from

Pure NADP+ was isolated from red blood cells in 1934 by Otto Warburg and W. Christian, who had been studying the oxidation of glucose 6-phosphate by erythrocytes.13 They demonstrated a requirement for a dialyzable coenzyme which they characterized and named triphosphopyridine nucleotide (TPN+, but now officially NADP+ Fig. 15-1). Thus, even before its recognition as an important vitamin in human nutrition, nicotinamide was identified as a component of NADP+. 

Equations (10) and (11) indicate three intermediates, EHj, EHj-NADP, and EHj-NADPH, which are formed at rates sufficient to require their consideration as reactants with GSSG. Figure 10 hypothesizes a simple binary complex mechanism based on the early kinetic studies with enzyme from erythrocytes (39, 40), peas (191), yeast (99), and P. chrysogenum (193). If this hypothesis is correct then NADP dissociates from EHs prior to reaction with GSSG and only EH and EHj-... 

The sum reaction for the oxidative segment of the PPP is shown by reaction (1). Using insightful planning and remarkable chemistry, Warburg et al. (8) had also isolated and characterized (from 100 L of horse erythrocytes) the new pyridine nucleotide coenzyme NADP+ for the above reactions. He recognized and chemically established that the new coenzyme was functionally... 

Figure 21 -8 Major glycolytic pathways of the erythrocyte. Substrates are in uppercase type, and enzymes are in parentheses. EMP, The Embden-Meyerhof pathway HMP hexose monophosphate pathway or pentose shunt RLC, the Rapoport-Luebering cycle ADP, adenosine diphosphate ATP, adenosine triphosphate NAD, nicotinamide-adenine dinudeotide NADH, reduced nicotinamide-adenine dinucleotide NADP, nicotinamide-adenine dinucleotide phosphate NADPH, reduced nicotinamide-adenine dinucleotide phosphate.The step from ribulose-5-phosphate, which is shown as being catalyzed by transketolase and transaldolase, is an abbreviation of this portion of the HMR...

Dietary NAD and NADP are hydrolyzed by enzymes, such as NAD glycohydrolase, in the intestinal mucosa to release nicotinamide, which together with any nicotinic acid is rapidly absorbed in both the stomach and intestine by an Na -dependent facilitated diffusion at low concentrations and passive diffusion at higher concentrations. Nicotinamide is the main circulating form in the plasma, either postabsorption or by release from hydrolyzed liver NAD, and this can be taken up by most tissue requiring NAD by simple diffusion, though there is evidence of a facilitated transport in the erythrocyte. ... 

Under physiological conditions, G6PD is a dimer of identical subunits of M.W. 55,000. The active enzyme contains a molecule of NADP+, removal of which causes dissociation into inactive monomers. Enzymes from rat, cow, and human are very similar, and active interspecies hybrid enzymes can be formed in vitro. The enzyme is inhibited by NADPH at the concentration normally found in hepatocytes and erythrocytes, by ATP competing with glucose-6-phosphate for binding, and by cyanate. A large reserve capacity of G6PD activity exists in the red blood cell and, presumably, in other tissues. 

The status of niacin in relation to most other vitamins is different as it can be synthesized by humans to some extend from tryptophan. Body status determination has been based on the determination of urinary excretion of niacin metabolites, predominately N-methyl-2-pyridone-5-carboxylamide and N-methyl-nicotinamide. The ratio of these compounds has been used as indicator of niacin status. Recent studies suggest that the determination of the two niacin-derived coenzymes, NAD and NADP, in erythrocytes, and their ratio are more reliable indicators of niacin status. However, a broadly accepted and easy to use determination method does not seem to exist. 

Also of interest, P5C reductase from various tissue or cellular sources are differentially sensitive to inhibition by proline (99,117), NADP (86, 117), and adenine nucleotides (86). The enzyme from cultured fibroblasts is sensitive to inhibition by proline and its inhibition of the NADH-mediated reaction (K, = 2 X 10 M) is much greater than of the NADPH-mediated reaction (K = 2 X 10 M). By contrast, the hepatic and erythrocyte enzymes are insensitive to proline but very sensitive to inhibition by NADP+ (H7). Adenine nucleotides also inhibit the hepatic enzyme (86) but not the erythrocyte enzyme. These differential patterns of inhibition suggest that there are isozymes of P5C reductase found in various tissues, an interpretation further supported by the relative deficiency of the NADH-mediated activity in a leukemia cell line (55). Whether these putative isozymes are products of different genes or are interconvertible forms is an intriguing question which awaits definitive studies on enzyme prepared from different sources. 

Fig. 11. Demonstration of proline - P5C cycling in reconstituted systems. Adapted from data published in refs. (35) and (71). (A) The generation of oxidizing potential to drive the pentose phosphate pathway. Intact rat kidney mitochondria were incubated with erythrocyte cytosol and COj production from [l- C]glucose (1 mAf) was measured. With NADPH (0.1 mM) and ADP (0.2) mM) in the incubation, the amount of glucose oxidized in the presence (A) and absence (O) of 10 mM proline is shown. (B) The transfer of reducing potential to proline. The reconstituted system was similar to that for A except intact rat liver mitochondria were incubated with erythrocyte cytosol. In the presence of [ 1 - H]glu-cose (0.8 mM), ADP (5 mM), and NADP (0.3 rnM), the formation of PH]proline in the presence ( ) and absence (O) of unlabeled proline (5 mM) occurs when P5C produced from proline is the recipient of the from NADPPH] generated from [l- H]glucose 6-phosphate by G-6-P dehydrogenase.

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