NAD Kinase Activity Research Articles

Distribution of calmodulin within wheat leaf cells

Subcellular distribution of calmodulin within wheat leaf cells was measured by a enzymatic method (activation of calmodulin-deficient NAD kinase from pea seedlings) or a radioimmunoassay. Most of cellular calmodulin was localized in cytosol fraction (89–93%), and remainders were in the mitochondrial (5–9%), the chloroplast (1–2%) and the microsomal (< 1%) fractions. Calmodulin concentration in the chloroplast was sufficient to saturate NAD kinase which was localized mostly in the chloroplast. Therefore, the light-induced Ca 2+ transport in intact wheat chloroplasts (Muto, S. FEBS Lett. (1982) 139, 250–254] may play an important role in regulation of chloroplast NAD kinase activity.

Calmodulin-dependent and independent NAD kinase activities from cytoplasmic and chloroplastic fractions of spinach (Spinacia oleracea L.)

NAD kinase activity has been found in a soluble, cytoplasmic fraction and in the chloroplasts prepared from green spinach leaves. A small amount of both the cytoplasmic and the chloroplastic NAD kinase activities was retained on a calmodulin-Sepharose affinity column. The cytoplasmic NAD kinase eluted from the affinity column was found to be enhanced by calmodulin in a Ca(2+)-dependent manner. The chloroplastic enzyme which is located exclusively in the stroma and not in the envelope and thylakoid fractions was not affected by Ca(2+) and calmodulin. The stromal fraction of purified chloroplasts contained only a negligible amount of calmodulin, most probably due to cytoplasmic contamination. Based on these data, two different mechanisms for the light-dependent modulation of spinach NAD kinase activity are suggested.

Production of NADP by immobilized cells with NAD kinase

By the radiation-copolymerization method with polyethylene glycoldimethacrylate (PGD) as a main polymerizable reagent, microbial cells of Brevibacterium ammoniagenes were immobilized with high specific activity of NAD kinase and high mechanical strength. The reagents used for the immobilization such as PGD, polyvinyl alcohol (PVA), and N,N'-methylenebisacrylamide (Bis) did not inversely affect the enzyme activity. Freezing and irradiation treatment of the cell-reagent solution did not inactivate the enzyme either, but longer freezing time or a lower irradiation dose (less than 400 krad) resulted in the unsatisfactory mechanical strength of the immobilized cells. Almost all of NAD and ATP consumed were converted into NADP within three hours reaction time. The drum reactor was found to be ideal for the reaction of immobilized cells, since it gave little mechanical stress to the immobilized cells for the effective mixing of the cells and the substrates. The immobilized cells were subjected to three hours reaction repeatedly for 30 times without any activity loss.

Gonadotropin releasing hormone stimulates calmodulin redistribution in rat pituitary.

Calcium (Ca2+) seems to have an informational role in many tissues. In particular, it fulfills the requirements of a second messenger for gonadotropin releasing hormone (GnRH)-stimulated luteinizing hormone (LH) release from the pituitary gonadotrope (see ref. 1 for review). Very little is known about the effect of this ion on intracellular targets or the mechanism by which Ca2+ mobilization stimulates LH release. One intracellular target for Ca2+ is calmodulin, a ubiquitous intracellular Ca2+ receptor that has been shown to modulate many cellular functions, including cyclic nucleotide and glycogen metabolism, protein phosphorylation, microtubule assembly and disassembly, a Ca2+ flux, and the activities of NAD kinase, tryptophan 5' monooxidase and phospholipase A2 (see refs 2-5 for reviews). We have now used a specific and sensitive radioimmunoassay to determine the quantity and distribution of calmodulin in the gonadotrope before and during GnRH-stimulated LH release. The data indicate that GnRH stimulates redistribution of calmodulin from the cytosol to the plasma membrane and suggest that the molecule may have a role in the mechanism of stimulus-secretion coupling.

Glycolytic pathway as an ATP generation system and its application to the production of glutathione and NADP

Efficient ATP generation is required to produce glutathione and NADP. Hence, the generation of ATP was investigated using the glycolytic pathway of yeast. Saccharomyces cerevisiae cells immobilized using polyacrylamide gel generated ATP from adenosine, consuming glucose and converting it to ethanol and carbon dioxide. Under optimal conditions, the ATP-generating activity of immobilized yeast cells was 7.0 μmol h −1 ml −1 gel. A column packed with these immobilized yeast cells was used for continuous ATP generation. The half-life of the column was 19 days at a space velocity of (SV) 0.3 h −1 at 30°C. The properties of glutathione- and NADP-producing reactions coupled with the ATP-generating reaction were investigated. Escherichia coli cells with glutathione synthesizing activity and Brevibacterium ammoniagenes cells with NAD kinase activity were immobilized in a polyacrylamide gel lattice. Under optimal conditions, the immobilized E. coli cells and immobilized B. ammoniagenes cells produced glutathione and NADP at the rates of 2.1 and 0.65 μmol h −1 ml −1 gel, respectively, adding ATP to the reaction mixture. In order to produce glutathione and NADP economically and efficiently, the glutathione- and NADP-producing reactions were finally coupled with the ATP-generating reaction catalysed by immobilized S. cerevisiae cells. To compare the productivities of glutathione and NADP, and to compare the efficiency of ATP utilization for the production of these two compounds, the two reactor systems, co-immobilized cell system and mixed immobilized cell system, were designed. As a result, these two compounds were also found to be produced by these two kinds of reactor systems. Using the data obtained, the feasibility and properties of ATP generation by immobilized yeast cells are discussed in terms of the production of glutathione and NADP.

Immobilization of microbial cells containing NAD-kinase.

Microbial cells having NAD-kinase activity, Brevibacterium ammoniagenes, were immobilized by the radiation-copolymerization method under low temperature with the activity recovery of more than 80%. Compared to the native microbial cells the immobilized cells were more stable against heat and pH change. The immobilized cells were subjected to the 5 hr reaction repeatedly 20 times without any activity loss.

Continuous production of NADP by immobilized Brevibacterium ammoniagenes cells.

Whole cells of Brevibacterium ammoniagenes IAM 1645 having the polyphosphate NAD-kinase were successfully immobilized in a polyacrylamide gel lattice. The immobilized cells were activated by treatment with organic solvents or detergents. The pH optimum of the immobilized cells for the production of NADP was 7.0, and divalent metal ions were required to maintain the elevated activity of polyphosphate NAD-kinase. Highly pure NADP was continuously produced in high yield by the immobilized cell column. The half-life of this column was about eight days.

Calcium-dependent regulator of NAD kinase in higher plants

An activator protein of NAD kinase from the pea, Pisum satavum L., has been shown to be Ca 2+-dependent. This plant activator protein also stimulates the activity of modulator protein dependent-cyclic nucleotide phosphodiesterase from porcine brain. This stimulation is similar to that observed with modulator protein isolated from animal sources. Furthermore, Ca 2+-dependent modulator proteins isolated from porcine brain, bovine brain, and the coelenterate, Renilla , will regulate the NAD kinase activity of peas. Other common properties of the plant activator protein and animal modulator proteins are their acidic nature, heat stabilities, similar Stokes' radii, and their interactions with troponin I.

Continuous production of NADP by immobilized Achromobacter aceris cells.

Several microorganisms having higher nicotinamide adenine dinucleotide kinase (NAD kinase, EC 2.7.1.23) activity were immobilized into polyacrylamide gel lattices. The enzyme activity field by immobilization was highest in Achromobacter aceris AKU 0120. By the incubation of the immobilized A. aceris cells at pH 4.0, the NAD kinase activity increased and the adenosine triphosphate (ATP)-degradation activity disappeared completely. Enzymatic properties of the immobilized A. aceris cells were investigated and compared with those of intact cells. The optimal pH and the optimal temperature of immobilized cells were the same as those of intact cells. Immobilized cell NAD kinase was more stable than that of intact cells. The operational half-life of immobilized cells was 20 days when the substrate solution was passed through a column packed with immobilized cells at a flow rate which gives a space velocity (SV) of 0.1 hr-1 at 37 degrees C. On the other hand, the half-life of the intact cells was only 6 hr.

Spontaneous fluctuations in NAD-kinase activity of rabbit skeletal muscles

Spontaneous fluctuations in the time of activity of a 280–300-times purified preparation of NAD-kinase from rabbit skeletal muscles are described after its dilution. No flucturations of activity were found in an unfrozen but undiluted preparation. After preincubation of the diluted enzyme with substrates (NAD and ATP) its activity did not fluctuate.

Cyclic nucleotides, thioldisulfide status of proteins, and cellular control processes

It is shown that cyclic nucleotides can have a variety of effects on cell division, cell shape, cell adhesion, and cell movement, depending on the cells selected and the conditions under which they are used. For example, while CHO cells elongate under the influence of exogenous dibutyryl CAMP, Y-1 adrenal tumor cells round up and polyoma-transformed 3T3 cells show no change in shape. The totality of experience with cyclic nucleotides suggests that where they have been used by cells as control elements involving the four processes listed above, they are superimposed on basic cellular processes that progress in their absence--that is, they must be acting indirectly. In attempting to understand the inhibitory action of methyl xanthines on egg development, we were forced to abandon the idea that they acted through cyclic nucleotides. We found that methyl xanthines inhibited the activation of glutathione reductase and that glutathione oxidizing agents act as mitotic inhibitors. Further, we found that tubulin polymerizability, NAD-kinase activity, and a mitotic apparatus associated Ca+2-ATP-ase were all inhibited by oxidation of some of their sulfhydryls and were activated by reduction of the resulting disulfides. These results are discussed in terms of reported cycles and activations of glutathione reductase (GR) in cells and reports that mixed disulfides of glutathione and proteins can act as substrates for GR. Using the fact that a CAMP-dependent protein kinase has been reported to be activated by glutathione, we have suggested potential sites where sulfhydryl control processes and cyclic nucleotide control processes and cyclic nucleotide control processes may interact in certain restricted cases.

Photoactivation of NAD Kinase through Phytochrome

The specificities of phosphate donors and the effects of metal chelating agents and divalent metal ions on NAD kinase activation by phytochrome-far red-absorbing form (Pfr) were examined. ATP was the most efficient phosphorylating agent. Uridine 5'-triphosphate, cytidine 5'-triphosphate (CTP), inosine 5'-triphosphate, and guanosine 5'-triphosphate in this order caused significant phosphorylation in the dark. Under red light, striking photoactivation of NAD kinase was obtained with ATP and subsequently CTP.In the presence of exogenous Mg(2+), which is required for NAD kinase activity, alpha-nitroso-beta-naphthol, cyanide, and dimethylglyoxime, strongly inhibited the activation by red light without affecting the level of NAD kinase in the dark.Of the divalent cations tested with the KCN-treated phytochrome preparation, only Co(2+) was effective for photoactivation of NAD kinase. Even when Mg(2+), an essential component of NAD kinase, was added to the assay system, the further addition of Co(2+) was required for the activation of NAD kinase by Pfr.

Pigeon-liver NAD kinase. The structural and kinetic basis of regulation of NADPH.

NAD kinase was purified from pigeon liver by an improved procedure which included chromatography on phosphocellulose. The resultant preparation was homogeneous as judged by gel electrophoresis, but electrofocusing gave indications of heterogeneity. The enzyme appeared to be of molecular weight 270000, and to consist of subunits of molecular weight 34000; it may therefore be an octomer. Kinetic studies over a wide range of substrate concentrations revealed departures from Michaelis-Menten behaviour with the substrate NAD+; these were interpreted tentatively in terms of negative homotropic interactions between identical binding sites, since thermal and chemical inactivation studies revealed no evidence for more than one type of catalytic site. The significance of the kinetics and of the type of inhibition produced by NADPH is discussed in terms of the regulation of NAD kinase activity in vivo.

Kinetics of Activation of Nicotinamide Adenine Dinucleotide Kinase by Phytochrome—Far Red-absorbing Form

The effects of exogenous redox cofactors and purine analogues on the activation of the NAD kinase by Pfr were examined. Addition of phenazine methosulfate, flavin mononucleotide, or methylene blue increased the activation of NAD kinase by red light in a partially purified preparation of phytochrome. Phenazine methosulfate and flavin mononucleotide do not absorb light in the red or far red region, so they do not act as light receptors in this activation. Thus they probably intervene in electron transfer between phytochrome and NAD kinase. Addition of kinetin with these compounds increased photopotentiation further. In the presence of phenazine methosulfate and kinetin, the activation of NAD kinase by red light was counteracted by illumination with far red light immediately after red light.The relation between the dose of illumination with red light and the degree of activation of NAD kinase was measured. Illumination for 5 minutes with 800 ergs cm(-2) sec(-1) of red light was sufficient to obtain maximal activation. In the dark, the activated state of NAD kinase was maintained for about 10 min and then rapidly lost. The time of preservation of the activated state was greatly shortened by illumination with far red light, indicating the participation of phytochrome.

Photoregulation of Nicotinamide Adenine Dinucleotide Kinase Activity in Cell-free Extracts

This article gives evidence that NAD kinase activity is controlled by the action of phytochrome. The NADP level rapidly increased in the cotyledons of seedlings of Pharbitis nil strain Violet (a short day plant), when the inductive dark for flowering was interrupted with a 5-minute illumination of red light. Illumination with far red light immediately after illumination with red light counteracted partly the effect of the latter.A partially purified phytochrome preparation from the uppermost internodes including the hook of Pisum sativum var. Alaska contained NAD kinase activity, and this increased greatly on illumination with red light. The effects of red and far red light on NAD kinase activity were reversible. The Km value of NAD kinase in the phytochrome preparation for NAD was 1.84 mm in the dark and 0.90 mm under red light. A broad optimum of the NAD kinase reaction and its photopotentiation appeared in the region of pH 6.6 to 8.6.The NAD kinase activity in a phytochrome preparation from the coleoptiles of Avena sativa was also activated by red light.It was suggested that the Pfr type of phytochrome may contribute to the decrease of the Km value of NAD kinase reaction by causing a conformational alteration of NAD kinase.

Distribution and Properties of NAD Phosphorylating Reaction

Distribution of NAD phosphorylating reactions, phosphorylation through NAD kinase and phosphotransferase, was investigated. NAD kinase activity was distributed rather widely in bacteria, whereas phosphotransferase activity with p-NPP and NAD was limited to a few genera. Proteus mirabilis showed strong activity of phosphotransferase besides NAD kinase activity.Partial purification of the phosphotransferase was attempted. The enzyme preparation possessed phosphatase activity as well as phosphotransferase activity. Phosphorylation of NAD proceeded maximally under the conditions below pH 4.0. Cu2+ showed stimulating effect on the activity. Besides p-NPP and phenylphosphate, various nucleotides, especially 2′ (or 3′) isomers, served as excellent phosphoryl donors, and various kinds of nucleosides and nucleotides were phosphorylated to form nucleoside monophosphates and nucleoside diphosphates.

Does ADP regulate respiration following fertilization of sea urchin eggs?

The hypothesis that the ADP level regulates the respiration rate following fertilization of sea urchin eggs has been critically examined in the eggs of Strongylocentrotus purpuratus. Simultaneous measurements were made of (1) respiration rate, (2) adenine nucleotide levels and (3) energy utilization resulting from NAD kinase activity. Both respiration and energy utilization increase, but no changes in ATP, ADP, or AMP level were observed at the times of maximum respiratory activity. These findings do not support the hypothesis that respiration is limited by ADP. Alternate factors possibly controlling respiration include enzyme availability and Ca 2+-uptake by mitochondria.

Reversible inactivation of nicotinamide adenine dinucleotide kinase in extracts of unfertilized sea urchin eggs

NAD kinase activity in extracts of unfertilized sea urchin eggs is lost on dialysis against dilute buffer and can be recovered by incubation with NAD and dithiothreitol.

Changes in the activities of enzymes of the biosynthetic pathway of the nicotinamide nucleotides in rat mammary gland during the lactation cycle.

1. The activities of NMN pyrophosphorylase, NMN adenylyltransferase and NAD kinase in the mammary glands of rats at different stages of pregnancy, lactation and involution were measured. 2. NMN pyrophosphorylase has a low activity early in pregnancy, but its activity increases at parturition and in early lactation to reach a maximum at the tenth day of lactation, after which it remains constant until it declines abruptly in involution. 3. NMN adenylyltransferase is already quite active by the tenth day of pregnancy and its activity does not rise further in the second half of gestation. After a sharp rise in activity at parturition, the activity of the enzyme declines slowly throughout the period of lactation and, more sharply, in involution. 4. NAD kinase has a low activity for most of pregnancy, but its activity rises at parturition to a value at 2 days of lactation that is maintained until the tenth day. Between the tenth and fifteenth days of lactation the activity almost doubles, but falls sharply in mammary involution. 5. The relation of the activities of these enzymes to the rates of synthesis of NAD and NADP is discussed.

Inhibition of Rat Liver Nicotinamide Adenine Dinucleotide Kinase by Reduced Nicotinamide Adenine Dinucleotide Phosphate

Abstract Rat liver NAD kinase (ATP:NAD 2'-phosphotransferase, EC 2.7.1.23) was purified about 70-fold. The Michaelis-Menten constants (Km) for NAD and ATP were 8 x 10-4 m and 2 x 10-3 m, respectively. NAD kinase activity was markedly inhibited by NADH and also NADPH. The Ki of NADH was approximately 1 x 10-4 m, and that of NADPH was approximately 5 x 10-5 m. Both inhibitions were competitive with NAD, and the inhibition of NADH and NADPH was partially additive. NADP had little or no inhibitory effect on the enzyme activity. p-Hydroxymercuribenzoate (2 x 10-5 m) inhibited NAD kinase activity. This inhibition was reversed by 1-cysteine or 2-mercaptoethanol. The possible role of the reduced pyridine nucleotides in the control of NADP synthesis has been considered.