烟酰胺腺嘌呤二核苷酸激酶研究进展

本文介绍了ＮＡＤ￣＋激酶的来源和分布、测定方法、最适ｐＨ、反应平衡常数、影响酶稳定性的因子、比活力和动力学常数、三磷酸核苷酸特异性、对金属离子的需要、抑制剂和底物类似物、反应机制、反应的感光性、酶固定化、与钙调蛋白的关系、各种效应物等方面研究的进展,并进行了评述。     

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.     

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.     

Specificity for Nicotinamide Adenine Dinucleotide and Nicotinamide Adenine Dinucleotide Phosphate of Nitrate Reductase from the Salt-tolerant Alga Dunaliella parva

Nitrate reductase of the salt-tolerant alga Dunaliella parva could utilize NADPH as well as NADH as an electron donor. The two pyridine nucleotide-dependent activities could not be separated by either ion exchange chromatography on DEAE-cellulose or gel filtration on Sepharose 4B. The NADPH-dependent activity was not inhibited by phosphatase inhibitors. NADPH was not hydrolyzed to NADH and inorganic phosphate in the course of nitrate reduction. Reduction of nitrate in vitro could be coupled to a NADPH-regenerating system of glycerol and NADP-dependent glycerol dehydrogenase. It is concluded that the nitrate reductase of D. parva will function with NADPH as well as NADH. This is a unique characteristic not common to most algae.     

Levels of Nicotinamide Adenine Dinucleotide and Reduced Nicotinamide Adenine Dinucleotide in Facultative Bacteria and the Effect of Oxygen

Nicotinamide adenine dinucleotide (NAD) and reduced NAD (NADH) levels have been measured in bacterial cultures. The cofactors were assayed by using the very sensitive cycling assay described previously by Cartier. Control experiments showed that the level of total NAD(H) falls during harvesting, and so samples were taken quickly from growing cultures and extracted immediately without separating the cells from the medium. Total NAD(H) ranged from 4.0 to 11.7 mumoles/g of dry cells for three facultative organisms, Klebsiella aerogenes, Escherichia coli, and Staphylococcus albus. NADH was remarkably constant in these bacteria; only one out of ten series of determinations was outside the range 1.4 to 1.9 mumoles/g of dry cells. NAD(+) showed much greater variation. An anaerobe (Clostridium welchii) had significantly more total NAD(H) whereas an aerobe Pseudomonas aeruginosa had about as much NAD(H) as the facultative organisms. NAD and NADH were measured during growth: once more NADH was much more constant than NAD. During change-over between aerobiosis and anaerobiosis, NADH showed a temporary increase but then returned to a constant level, whereas NAD changed from high aerobically to low anaerobically. These results are discussed in terms of the control mechanisms that may be involved.     

Production of Nicotinamide Adenine Dinucleotide Glycohydrolases by Bacteria

The distribution of heavy metals (Fe, Mn, Zn, Cu, Pb, Ni and Cd) were investigated in various organs and tissues of striped dolphin, Stenella coeruleoalba. The animals were caught alive at Taiji, on the coast of Kii Peninsula, during the open season in December 1978. Determination of the metals was made by atomic absorption spectrophotometry and significant differences of metal concentration in the positions of the muscle, blubber and skin, respectively, were observed. The front ventral muscle of matured dolphins showed the highest concentrations of Zn and Cd and lowest Fe when compared to other parts of the muscle. Most of the metals recorded relatively low concentrations in melon rather than in the other lipid layers of blubber. In skin tissue, the concentrations of Fe, Mn and Zn were significantly higher in black-colored skin than in white skin. Moreover, a difference in the concentrations of metals according to bone position was observed. In general, high concentrations of most of the metals were found in liver, kidney and bone, with low concentrations in brain and the lipid layer of blubber. Furthermore, relatively high concentrations of Cu, Mn and Zn were found in skin, and for Mn, Zn, Ni and Cd it was likewise in pancreas and the reproductive organs. Based upon these results, the nature of the organ(s) of a dolphin that has to be selected for ecological and hygienic comparison was discussed.     

Production of Nicotinamide Adenine Dinucleotide by Saccharomyces carlsbergensis

The accumulation of NAD was studied by culturing yeast in the presence of NAD precursors, Among the strains tested, Saccharomyces carlsbergensis showed the highest ability for the accumulation of NAD, Additions of pantothenate, inositol, zinc ion and fatty acids were effective for the accumulation of NAD. Under the optimal culture condition, NAD level in Saccharomyces carlsbergensis reached 42 mg per gram dry cells. Surfactants belonging to alkyl sulfate were effective on the leaking of the intracellular NAD, and about 75 mg of NAD per 100 ml was accumulated.     

Purification and Properties of Pantothenate Kinase from Brevibacterium ammoniagenes IFO 12071

Pantothenate kinase (ATP: pantothenate 4′-phosphotransferase, EC 2.7.1.33) was purified about 200-fold from the cell extract of Brevibacterium ammoniagenes IFO 12071 by ammonium sulfate fractionation, DEAE-cellulose chromatography, and Sephadex G-150 gel filtration. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular weight was calculated approximately 45,000. The enzyme catalyzed the formation of pantothenic acid 4′-phosphate and ADP from pantothenate and ATP in the presence of Mg²⁺ ATP could be substituted for, partly, by ITP, GTP, and UTP. The enzyme phosphorylated not only pantothenate, but also pantothenoylcysteine, pantetheine, and pantothenyl alcohol. Apparent Km values were 6.7×10^?5 m for pantothenate, 3.5×10^?5 m for ATP, and 10^?3 m for Mg²⁺. The reaction was inhibited by the intermediates of CoA biosynthesis, of which CoA itself was a most effective inhibitor. Other properties of the enzyme were also investigated.     

Properties of Nicotinamide Adenine Dinucleotide Phosphate-Dependent Formate Dehydrogenase from Clostridium thermoaceticum

Li, Lan-Fun (Western Reserve University School of Medicine, Cleveland, Ohio), Lars Ljungdahl, and Harland G. Wood. Properties of nicotinamide adenine dinucleotide phosphate-dependent formate dehydrogenase from Clostridium thermoaceticum. J. Bacteriol. 92: 405-412. 1966.-A nicotinamide adenine dinucleotide phosphate (NADP)-dependent formate dehydrogenase has been isolated from C. thermoaceticum. The enzyme is very sensitive to oxygen and requires sulfhydryl compounds for activity. The apparent K(m) at 50 C and pH 7.0 for NADP is 5.9 x 10(-5)m and for formate, 2.2 x 10(-4)m. The enzyme is most active at about 60 C and at pH values between 7.0 and 9.0. The enzyme catalyzes an exchange between C(14)O(2) and formate, which requires NADP, but net synthesis of formate from CO(2) and reduced nicotinamide adenine dinucleotide phosphate could not be demonstrated. The reaction does not involve ferredoxin.     

Synthesis of Methylene-Bridged Analogues of Nicotinamide Riboside,Nicotinamide Mononucleotide and Nicotinamide Adenine Dinucleotide

Abstract

5-O-tert-Butyldimethylsilyl-1,2-O-isopropylidene-3(R)-(nicotinamid-2-ylmethyl)-α-D-ribofuranose (11a) and ?3(R)-(nicotinamid-6-ylmethyl)-α-D-ribofuranose (11b) were prepared by condensation of 5-O-tert-butyldimethylsilyl-1,2-O-isopropylidene-α-D-erythro-3-pentulofuranose (10) with lithiated (LDA) 2-methylnicotinamide and 6-methylnicotinamide, respectively, and then deprotected to give 1,2-O-isopropylidene-3-(R)-(nicotinamid-2-ylmethyl)-α-D-ribofuranose(12a) and 1,2-O-isopropylidene-3(R)-(nicotinamid-6-ylmethyl)-α-D-ribofuranose (12b). Benzoylation as well as phosphorylation of compounds 12 afforded the corresponding 5-O-benzoate (13b) and 5-O-monophosphates (14a and 14b). Treatment of 13b with CF₃COOH/H₂O caused 1,2-de-O-isopropylidenation with simultaneous cyclization to the corresponding methylene-bridged cyclic nucleoside - 3′,6-methylene-1-(5-O-benzoyl-β-D-ribofuranose)-3-carboxamidopyridinium trifluoro-acetate (8b) - restricted to the “anti” conformation. In a similar manner compounds 14a and 14b were converted into conformationally restricted 2,3′-methylene-1-(β-D-ribofuranose)-3-carboxamidopyridinium-5′-monophosphate (9a - “syn”) and 3′,6-methylene-1-(β-D-ribofuranose)-3-carboxamido -pyridinium-5′monophosphate (9b - “anti”) respectively. Coupling of derivatives 12a and 12b with the adenosine 5′-methylenediphosphonate (16) afforded the corresponding dinucleotides 17. Upon acidic 1,2-de-O-isopropylidenation of 17b, the conformationally restricted P¹-[6,3′-methylene-1-(β-D-ribofuranos-5-yl)-3-carboxamidopyridinium]-P²-(adenosin-5′-yl)methylenediphosphonate 18b -“anti” was formed. Compound 18b was found to be unstable. Upon addition of water 18b was converted into the anomeric mixture of acyclic dinucleotides, i. e. P¹-[3(R)-nicotinamid-6-ylmethyl-D-ribofuranos-5-yl]-P²-(adenosin-5′-yl)-methylenediphosphonate (19b). In a similar manner, treatment of 17a with CF₃COOH/H₂O and HPLC purification afforded the corresponding dinucleotide 19a.

     

Mode of Nicotinamide Adenine Dinucleotide Utilization by Escherichia coli

Exogenous nicotinamide adenine dinucleotide is not utilized per se by Escherichia coli, but is converted to nicotinamide and thence to nicotinamide adenine dinucleotide via nicotinate.     

The Reduction of Nicotinamide Adenine Dinucleotide by Mitochondria Isolated from Helianthus tuberosus

Intact mitochondria isolated from Jerusalem artichoke tubersoxidize both malate and citrate. Both substrates were equallyeffective in reducing the endogenous pool of pyridine nucleotidesin the presence of respiratory inhibitors. Only malate, andnot citrate, was capable of reducing exogenous NAD⁺ under similarassay conditions. The rate at which malate reduced added NAD⁺was biphasic; the initial rapid phase was inhibited by the accumulationof oxaloacetate while the velocity of the second, slower, phasewas found to be insensitive to accumulated oxaloacetate. Theaddition of the detergent Decon 90 to intact mitochondria stimulatedboth the rapid and slow phases of NAD⁺ reduction and failedto convert the biphasic rate into a constant rate. Decon 90was found to cause inhibition of soluble malate dehydrogenase.Extensive efforts to purify the mitochondria using sucrose densitygradients failed to remove all of the soluble malate dehydrogenasefrom the preparation of mitochondria and approximately 3% ofthe total malate dehyrogenase present in the preparation appearedto behave as if it were outside the inner membrane.     

The Distribution of Enzymes Which Synthesize Nicotinamide Adenine Dinucleotide and Nicotinamide Adenine Dinucleotide Phosphate in Monkey, Rabbit, and Ground Squirrel Retinas

The activity of adenosinetriphosphate:nicotinamide adenylyltransferase (EC 2.7.7.1) was measured in all the layers of monkey, rabbit, and ground squirrel retinas. Nicotinamide adenine dinucleotide (NAD) kinase (EC 2.7.1.23) distribution was measured in monkey and rabbit retinas. An attempt was made to measure NAD synthetase (EC 6.3.5.1), but the activities in the retinal layers were too low to produce a reliable increment in the levels of endogenous NAD. In monkey retina the adenylyl transferase was highest by far in the outer and inner nuclear layers, lower and variable in ganglion cell and fiber layers, and almost absent elsewhere. Rabbit retina differed in that activity was nearly absent in the outer nuclear layer, whereas in the ground squirrel outer nuclear layer activity was double that of the inner nuclear layer. The species differences suggest that adenylyl transferase is almost absent from cone cell nuclei and high in rod cell nuclei. NAD kinase distribution in monkey retina was almost the mirror image of that of adenylyl transferase.     

Nicotinamide Adenine Dinucleotide (NAD+) Repletion Attenuates Bupivacaine-Induced Neurotoxicity

Bupivacaine is one of the most toxic local anesthetics but the mechanisms underlying its neurotoxicity are still unclear. Intracellular nicotinamide adenine dinucleotide (NAD⁺) depletion has been demonstrated to play an essential role in neuronal injury. In the present study, we investigated whether intracellular NAD⁺ depletion contributes to bupivacaine-induced neuronal injury and whether NAD⁺ repletion attenuates the injury in SH-SY5Y cells. First, we evaluated the intracellular NAD⁺ content after bupivacaine exposure. We also examined the cellular NAD⁺ level after pretreatment with exogenous NAD⁺. We next determined cell viability and the apoptosis rate after bupivacaine treatment in the presence or absence of NAD⁺ incubation. Finally, cell injuries such as nuclear injury, reactive oxygen species (ROS) production, and mitochondrial depolarization were detected after bupivacaine treatment with or without NAD⁺ pretreatment. Bupivacaine caused intracellular NAD⁺ depletion in a time- and concentration-dependent manner. Cellular NAD⁺ replenishment prevented cell death and apoptosis induced by bupivacaine. Importantly, exogenous NAD⁺ attenuated bupivacaine-induced nuclear injury, ROS production, and mitochondrial depolarization. Our results suggest that NAD⁺ depletion is necessary for bupivacaine-induced neuronal necrosis and apoptosis, and that NAD⁺ repletion attenuates neurotoxicity resulting from bupivacaine-treatment.     

Electrophoretic Heterogeneity of Bacterial Nicotinamide Adenine Dinucleotide Phosphate-Specific Isocitrate Dehydrogenases

The specific activities of the nicotinamide adenine dinucleotide phosphate-dependent isocitrate dehydrogenase in crude cell-free extracts of 15 different microorganisms, grown aerobically in simple mineral salts media containing glucose as the sole carbon source, ranged from a maximum of 0.820 in Pseudomonas aeruginosa to a minimum of 0.145 in Thiobacillus novellus. Polyacrylamide gel electrophoresis indicated that the bacterial species studied contained electrophoretically distinct proteins exhibiting isocitrate dehydrogenase activity. The electrophoretic mobilities, as well as the differences in stability of the enzyme observed in this study, indicate that the physical and chemical properties of isocitrate dehydrogenase may differ widely between bacterial species.     

Nicotinamide Adenine Dinucleotide Phosphate-Dependent Formate Dehydrogenase from Clostridium thermoaceticum: Purification and Properties

The nicotinamide adenine dinucleotide phosphate (NADP)-dependent formate dehydrogenase in Clostridium thermoaceticum used, in addition to its natural electron acceptor, methyl and benzyl viologen. The enzyme was purified to a specific activity of 34 (micromoles per minute per milligram of protein) with NADP as electron acceptor. Disc gel electrophoresis of the purified enzyme yielded two major and two minor protein bands, and during centrifugation in sucrose gradients two components of apparent molecular weights of 270,000 and 320,000 were obtained, both having formate dehydrogenase activity. The enzyme preparation catalyzed the reduction of riboflavine 5'-phosphate flavine adenine dinucleotide and methyl viologen by using reduced NADP as a source of electrons. It also had reduced NADP oxidase activity. The enzyme was strongly inhibited by cyanide and ethylenediaminetetraacetic acid. It was also inhibited by hypophosphite, an inhibition that was reversed by formate. Sulfite inhibited the activity with NADP but not with methyl viologen as acceptor. The apparent K(m) at 55 C and pH 7.5 for formate was 2.27 x 10(-4) M with NADP and 0.83 x 10(-4) with methyl viologen as acceptor. The apparent K(m) for NADP was 1.09 x 10(-4) M and for methyl viologen was 2.35 x 10(-3) M. NADP showed substrate inhibition at 5 x 10(-3) M and higher concentrations. With NADP as electron acceptor, the enzyme had a broad pH optimum between 7 and 9.5. The apparent temperature optimum was 85 C. In the absence of substrates, the enzyme was stable at 70 C but was rapidly inactivated at temperatures above 73 C. The enzyme was very sensitive to oxygen but was stabilized by thiol-iron complexes and formate.     

The Effect of Exogenous Nicotinamide Adenine Dinucleotide on the Oxidation of Nicotinamide Adenine Dinucleotide-linked Substrates by Isolated Plant Mitochondria

The oxidation of malate, citrate, and α-ketoglutarate by cauliflower (Brassica oleacea L.) bud mitochondria was inhibited by rotenone. This inhibition was relieved upon addition of NAD⁺ to the medium, and ADP/O values were lowered to less than 2 when both rotenone and NAD⁺ were present. Dinitrophenol did not affect the relief of rotenone inhibition by exogenous NAD⁺.     

Metabolic Changes in Nicotinamide Adenine Dinucleotide in Response to Anthrax Toxin

Bacillus anthracis produces a toxin both in vitro and in vivo which, when injected intravenously into rats, brings about the death of the animals accompanied by gross pulmonary edema. Lung tissue removed prior to death showed, in vitro, a 30% reduction in overall oxidative metabolism (Q(o2)), whereas the nicotinamide adenine dinucleotide (NAD)-independent succinic dehydrogenase remained unaffected. The NAD concentration in the lungs of injected animals was reduced by 50%. Upon addition of NAD, the Q(o2) of lung tissue from injected animals rose to control values. At 45 min after toxin injection, the serum lactate concentration began to rise, showing about a 3.5-fold increase over controls after 75 min. No changes occurred in the pyruvate concentration. These changes may be explained by increased use of the pyruvate for glycolytic energy production with further loss of NAD. Additional experiments with liver, spleen, kidney, and brain tissues showed that the toxin-induced reduction of Q(o2) is an effect specific for lung tissue. Brain tissue showed a significant increase in oxidative metabolism upon the addition of the toxin, whereas the other tissues remained unaffected. It is suggested that a principal effect of the toxin is to inhibit, in lung tissue, the regeneration of NAD in the respiratory chain.