Regulation Of NAD Research Articles

Nitrate‐induced de novo synthesis and regulation of NAD(P)H nitrate reductase from Sphagnum

The NO−3‐triggered induction of nitrate reductase (NR; EC 1.6.6.2) in the bryophyte Sphagnum magellanicum Brid. has been studied, using in vivo and in vitro assays as well as immunological methods. The time‐course of induction was triphasic with maximal NR activity after 6–8 h. Results obtained from Western blots show that NR is synthesized de novo after NO−3 application. The inhibitory effect of cycloheximide on NR induction corroborated this conclusion. Light enhanced the NO−3‐triggered NR induction. The enzyme activity, measured in vivo, increased more than the in vitro activity. No evidence for phytochrome control of NR was found. Nitrate uptake, in contrast to NR activity, showed no lag period after NO−3 application and, under the experimental conditions used, was not rate limiting for NR induction. Neither light nor a NO−3 pretreatment significantly affected NO−3 uptake.

Regulation of NAD- and NADP-linked isocitrate dehydrogenase by thyroxine in the brain and liver of female rats of various ages

The specific activity of NAD- and NADP-linked isocitrate dehydrogenase and their regulation by thyroxine in the brain and liver of female rats of various ages were studied with the ultimate goal of better understanding the decreased physiological functioning of the brain and liver during old age. Both thyroidectomy and thyroxine treatment have differential age-dependent effects on the activities of these enzymes in both tissues. The activity of NAD-ICDH decreases whereas both cytoplasmic and mitochondrial NADP-ICDH increase simultaneously following thyroidectomy. Thyroxine administration induces NAD-ICDH and depresses NADP-ICDH. The degree of induction and/or repression is lowest in old rats. These effects of thyroxine are actinomycin D sensitive in both the tissues of rats.

Purification, kinetic behavior, and regulation of NAD(P)+ malic enzyme of tumor mitochondria.

The purification and kinetic characterization of an NAD(P)+-malic enzyme from 22aH mouse hepatoma mitochondria are described. The enzyme was purified 328-fold with a final yield of 51% and specific activity of 38.1 units/mg of protein by employing DEAE-cellulose chromatography and an ATP affinity column. Sephadex G-200 chromatography yielded a native Mr = 240,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major subunit with Mr = 61,000, suggesting a tetrameric structure, and also showed that the preparation contained less than 10% polypeptide impurities. Use of the ATP affinity column required the presence of MnCl2 and fumarate (an allosteric activator) in the elution buffers. In the absence of fumarate, the Michaelis constants for malate, NAD+, and NADP+ were 3.6 mM, 55 microM, and 72 microM, respectively; in the presence of fumarate (2 mM), the constants were 0.34 mM, 9 microM, and 13 microM, respectively. ATP was shown to be an allosteric inhibitor, competitive with malate. However, the inhibition by ATP displayed hyperbolic competitive kinetics with a KI (ATP) of 80 microM (minus fumarate) and 0.5 mM (plus 2 mM fumarate). The allosteric properties of the enzyme are integrated into a rationale for its specific role in the pathways of malate and glutamate oxidation in tumor mitochondria.

Regulation of NAD(P)H dehydrogenases in plant mitochondria

Plant mitochondria contain four different NADH dehydrogenases, three of which are located on the inner membrane and feed electrons directly into the respiratory chain. One, a Ca 2+-dependent dehydrogenase, faces the intermembrane space and oxidizes exogenous NADH; the other two enzymes, which have markedly different affinities for NADH, face the matrix and oxidize endogenous NADH. The presence and properties of these dehydrogenases introduce a number of hitherto unknown means of regulating electron transport and mitochondrial-cytoplasmic interactions.

Regulation of NAD+‐ and NADP+‐linked isocitrate dehydrogenase in the obligate methylotrophic bacterium Pseudomonas W6

AbstractCell‐free extracts of the obligate methanol‐utilizing bacterium Pseudomonas W6 catalyze the oxydation of isocitrate to α‐ketoglutarate in the presence of NAD+ and NADP+. After electro‐focusing of the crude extract of Pseudomonas W6 actually two distinct bands each of NAD+‐linked isocitrate dehydrogenase (NAD+‐IDH) and of NADP+‐linked isocitrate dehydrogenase (NADP+‐IDH) could be observed. The NAD+‐IDH was completely separated from the NADP+‐IDH by employing DEAE ion exchange chromatography and further purified by affinity chromatography using Cibacron blue F 3G‐A.The NAD+‐IDH was inhibited by a high energy charge, whereas the NADP+‐IDH was found to be independent of energy charge. Consequently the NAD+‐IDH showed the control behaviour of an enzyme of an energy‐generating sequence which, however, equally fulfils a catabolic and an anabolic function. With respect to the inhibition by reduced pyridine nucleotides and α‐ketoglutarate differences between NAD+‐IDH and NADP+‐IDH were also found. Only the NADP+‐linked enzyme exhibited a feedback inhibition by its reaction products α‐ketoglutarate and NADPH. This control behaviour gives evidence for the biosynthetic function of the NADP+‐IDH. These results confer an amphibolic character to the sequence from citrate to α‐ketoglutarate in the incomplete citric‐acid cycle of Pseudomonas W6.

Regulation of NAD+- and NADP+-linked isocitrate dehydrogenase in the obligate methylotrophic bacteriumPseudomonas W6

Cell-free extracts of the obligate methanol-utilizing bacterium Pseudomonas W6 catalyze the oxydation of isocitrate to alpha-ketoglutarate in the presence of NAD+ and NADP+. After electro-focusing of the crude extract of Pseudomonas W6 actually two distinct bands each of NAD+-linked isocitrate dehydrogenase (NAD+-IDH) and of NADP+-linked isocitrate dehydrogenase (NADP+-IDH) could be observed. The NAD+-IDH was completely separated from the NADP+-IDH by employing DEAE ion exchange chromatography and further purified by affinity chromatography using Cibacron blue F 3G-A. The NAD+-IDH was inhibited by a high energy charge, whereas the NADP+-IDH was found to be independent of energy charge. Consequently the NAD+-IDH showed the control behaviour of an enzyme of an energy-generating sequence which, however, equally fulfils a catabolic and an anabolic function. With respect to the inhibition by reduced pyridine nucleotides and alpha-ketoglutarate differences between NAD+-IDH and NADP+-IDH were also found. Only the NADP+-linked enzyme exhibited a feedback inhibition by its reaction products alpha-ketoglutarate and NADPH. This control behaviour gives evidence for the biosynthetic function of the NADP+-IDH. These results confer an amphibolic character to the sequence from citrate to alpha-ketoglutarate in the incomplete citric-acid cycle of Pseudomonas W6.

Calcium ions and the regulation of NAD+-linked isocitrate dehydrogenase from the mitochondria of rat heart and other tissues

The effects of Ca2+ on the activity of isocitrate dehydrogenase (NAD+) in extracts of rat heart mitochondria were explored in the presence of MgCl2 by using EGTA buffers. In the absence of ADP, Ca2+ (about 30 micrometer) resulted in a slight increase in apparent Km for threo-Ds-isocitrate; in the presence of ADP, Ca2+ (about 25 micrometer) greatly lowered the apparent Km for threo-Ds-isocitrate from 227 micrometer to 53 micrometer without changing the maximum velocity. At 100 micrometer-threo-Ds-isocitrate and 1 mM-ADP, there was an 8-fold activation by Ca2+, with a Km for Ca2+ of 1.2 micrometer. This activation was also observed with Sr2+ (Km 3.1 micrometer), but not with Mn2+ (at concentrations below 2.5 micrometer). Similar effects of Ca2+ were also observed on isocitrate dehydrogenase (NAD+) activity in extracts of mitochondria from liver, kidney, brown adipose tissue and white adipose tissue of the rat. The possible regulatory role of changes in the intramitochondrial concentration of Ca2+ is discussed.

Regulation of NAD and NADP oxidoreductases in oranges

Abstract Dehydrogenase activity of malate: NAD oxidoreductase in extracts of orange juice vesicles was suppressed by NADH at 5 per cent mole fraction of NAD + . Dehydrogenase activity of alcohol: NAD oxidoreductase in the extract was suppressed by NADH at molar concentration equal to NAD + . Suppressing the reoxidation of reduced NAD by O 2 -linked dehydrogenases in intact fruit increased the mole fraction of reduced NAD in the vesicles. The alcohol content of the juice was increased and the citric acid content of the juice was decreased by the treatment. The increase in ethanol and decrease in citric acid indicates a diversion of pyruvate from citric acid formation to ethanol formation in the fruit.