Effect Of NAD Research Articles

Exogenous NAD+ Effects on Plant Mitochondria

Addition of NAD(+) to purified potato (Solanum tuberosum L.) mitochondria respiring alpha-ketoglutarate and malate in the presence of the electron transport inhibitor rotenone, stimulated O(2) uptake. This stimulation was prevented by incubating mitochondria with N-4-azido-2-nitrophenyl-aminobutyryl-NAD(+) (NAP(4)-NAD(+)), an inhibitor of NAD(+) uptake, but not by 1 mm EGTA, an inhibitor of external NADH oxidation. NAD(+)-stimulated malate-cytochrome c reductase activity, and reduction of added NAD(+) by intact mitochondria, could be duplicated by rupturing the mitochondria and adding a small quantity to the cuvette. The extent of external NAD(+) reduction was correlated with the amount of extra mitochondrial malate dehydrogenase present. Malate oxidation by potato mitochondria depleted of endogenous NAD(+) by storing on ice for 72 hours, was completely dependent on added NAD(+), and the effect of NAD(+) on these mitochondria was prevented by incubating them with NAP(4)-NAD(+). External NAD(+) reduction by these mitochondria was not affected by NAP(4)-NAD(+). We conclude that all effects of exogenous NAD(+) on plant mitochondrial respiration can be attributed to net uptake of the NAD(+) into the matrix space.

魚類筋肉のグリセルアルデヒド―リン酸脱水素酵素に関する研究 ＩＩ サケ・タラ科魚類筋肉のグリセルアルデヒド―リン酸脱水素酵素のＮＡＤ含量とその添加効果

For the crystallization of purified glyceraldehyde-phosphate dehydrogenases (EC. 1.2.1.12) from fish muscles, the requirement of each enzyme for added NAD is different from species to species. Thus, we attempted to determine the bound NAD content in the enzymes. The results obtained by the titration with NAD and the ratio of E280:E260 indicated that each fish enzyme contained varying amounts of bound NAD and less than one mol of NAD per mol of enzyme. The exogenous NAD-added fish enzymes can be resolved into multiple forms having different amounts of NAD by ion exchange chromatography. The effect of NAD on the conformation of the enzyme was investigated by comaring the loss of enzyme activity in the presence of urea and the regain of enzyme activity on dilution of urea. In contrast to the rabbit enzyme, which reversed the high extent of its activity in the renaturation, the fish enzymes regained little or no activity. In the presence of NAD, the activities of fish enzymes were recoverable in both the denaturation and the renaturation. The fish enzymes supplied with added NAD attained to the same stability for urea as that of the rabbit enzyme.

Kinetic studies with rat-brain succinic-semialdehyde dehydrogenase.

A simple procedure is described that gives an approximately 100-fold purification of rat brain succinic-semialdehyde dehydrogenase with a high yield. The enzyme exhibits a relatively low Km value for succinic semialdehyde (2.5 microM) and is inhibited by high concentrations of that substrate in an uncompetitive manner with respect to NAD+ (Ki = 150 microM). p-Hydroxybenzaldehyde was shown to give competitive inhibition with respect to succinic semialdehyde and uncompetitive inhibition with respect to NAD+. Initial rate studies in the presence of a fixed concentration of this inhibitor allowed a more accurate estimation of the kinetic parameters for the uninhibited reaction. The results of these studies, together with analysis of the dead-end inhibition by AMP and the effects of NAD+ and 3-acetylpyridine--adenine dinucleotide as alternative acceptors in the reaction, were consistent with the enzyme-catalysed reaction obeying a compulsory-order mechanism in which NAD+ was the first substrate to bind to the enzyme and NADH was the last product to dissociate from it.

Radioprotection on nucleated and anucleated erythrocytes by oxide-reduction coenzymes

Abstract The protective effects of NAD, FAD and Quinone and mixtures of these compounds were studied on gamma irradiated rabbit and chicken erythrocytes. The dose relative factor (DRF 37) was evaluated by visible absorbancy measurements of liberated hemoglobin. The DRF 37 obtained on rabbit erythrocytes were : NAD+FAD+Quinone mixture : 11,1; NAD+Quinone mixture : 6,1; FAD+Quinone mixture : 6,1; NAD: 1,6; FAD: 5,5; Quinone: 5,1. The DRF 37 obtained with the mixture NAD+FAD+Quinone on chicken erythrocytes was 3,9. The high efficiency of the radioprotective mixture NAD+FAD+Quinone is discussed.

Effect of NAD+ on Malate Oxidation in Intact Plant Mitochondria

Potato tuber mitochondria oxidizing malate respond to NAD(+) addition with increased oxidation rates, whereas mung bean hypocotyl mitochondria do not. This is traced to a low endogenous content of NAD(+) in potato mitochondria, which prove to take up added NAD(+). This mechanism concentrates NAD(+) in the matrix space. Analyses for oxaloacetate and pyruvate (with pyruvate dehydrogenase blocked) are consistent with regulation of malate oxidation by the internal NAD(+)/NADH ratio.

Stimulation and inhibition of the protein synthetic process by NAD+ in lysed rabbit reticulocytes.

NAD+ at 0.16 mM stimulates the initiation step of the protein synthetic process in lysed rabbit reticulocytes. This conclusion is based on the stimulation of (i) the transfer of formylmethionine from f[35S]Met-tRNAfMet into polypeptide, (ii) the accumulation of the initial dipeptide, methionylvaline, in the presence of pactamycin, and (iii) the formation of the 40 S initiation complex. The effect of NAD+ changes from a stimulatory role on protein synthesis to an inhibitory role at concentrations greater than 0.16 mM. At 4.0 mM NAD+, protein synthesis is inhibited. This has been demonstrated experimentally by using the same three assays described above. In addition, 4.0 mM NAD+ inhibits MettRNAfMet.initiation factor.GTP ternary complex formation. The elongation and termination steps of polypeptide synthesis are not affected by 0.16 to 4.0 mM NAD+. The data presented clearly show that the stimulatory activity of 0.16 mM NAD+ and the inhibitory activity of 4.0 mM NAD+ affects the initiation step of the protein synthetic process in lysed rabbit reticulocytes.

The role of arginine residues in the function of d-glyceraldehyde-3-phosphate dehydrogenase

Inactivation of apo-glyceraldehyde-3-phosphate dehydrogenase from rat skeletal muscle in the presence of butanedione is the result of modification of one arginyl residue per subunit of the tetrameric enzyme molecule. The loss of activity follows pseudo-first-order kinetics. NAD + increases the apparent first-order rate constant of inactivation. The effect of NAD + on the enzyme inactivation is cooperative (HIll coefficient = 2.3–3.2). Glyceraldehyde 3-phosphate protected the holoenzyme against inactivation, decreasing the rate constant of the reaction. At saturating concentrations of substrate the protection was complete. The Hill plot demonstrates that the effect is cooperative. This suggests that subunit interactions in the tetrameric holoenzyme molecule may affect the reactivity of the essential arginyl residues. In contrast, glyceraldehyde 3-phosphate had no effect on the rate of inactivation of the apoenzyme in the presence of butanedione. 100 mM inorganic phosphate protected both the apoenzyme and holoenzyme against inactivation. The involvement of the microenvironment of the arginyl residues in the functionally important conformational changes of the enzyme is discussed.

Alcohol dehydrogenase polymorphism in Apis mellifera.

A polymorphic system of ADH isozymes is described in the honeybee Apis mellifera. Three and six different electrophoretic patterns were found, respectively, in drone and worker pupae analysis. The data indicate that the ADH isozymes are controlled by three alleles, Adh-1(1), Adh-1(2), and Adh-1(3). The frequency of the Adh-1 alleles is different in two analyzed subspecies, Apis mellifera adansonii (African bees) and Apis mellifera ligustica (Italian bees). In the African bees, the frequencies are 0.256 and 0.697 for Adh-1(1) and Adh-1(2), respectively. In the Italian bees, these values are shown to be 0.902 and 0.098, respectively. The allele Adh-1(3) was not detected in the Italian bee population. The effect of NAD on the resolution of this system was investigated, and only one region of ADH activity was obtained in drone pupae analysis when NAD was used in the gels. However, two different regions of activity were observed in the same samples, in the absence of the coenzyme. ADH activity was not detected in young larvae, but it increased to a maximum in prepupal and white-eyed pupal phases. It then declined progressively to total absence in the emerging bees.

The effect of NAD + and NAD + analogs on protein synthesis in rabbit reticulocytes: Replacement of the energy regenerating system

The addition of NAD+ to a cell-free protein synthesizing system derived from lysed rabbit reticulocytes replaces the requirement for exogenously added energy regenerating systems. NAD+ is effective at a concentration of 5 μM and maximal stimulation is achieved above 25 μM NAD+. NADH can completely replace NAD+. NAD+ analogs substituted in the adenosine moiety, i.e., with 8-bromoadenosine, tubercidin, 3′-deoxyadenosine, 2′-deoxyadenosine, formycin, 1,N6-ethenoadenosine, and inosine, show the following activities compared to NAD+: 110, 82, 27, 18, 14, 9, and 5%. NADP+, NADPH, and analogs with substitutions in the nicotinamide moiety can not replace NAD+; thionicotinamide NAD+ has about 34% activity relative to NAD+. The lysate contains less than 1 μM NAD+. Evidence for an enzyme system capable of rapidly converting NAD+ to ATP is presented.

Study of yeast D-glyceraldehyde-3-phosphate3 dehydrogenase with the use of a fluorescent probe, 1-anilino-8-naphthalene sulfonate

1. 1. The effect of NAD + and its fragments, adenosine and 5'-AMP on the interaction of yeast d-glyceraldehyde-3-phosphate dehydrogenase with l-anilino-8-naphthalene sulfonate has been studied. 2. 2. A competitive relationship between NAD + and the dye has been demonstrated using fluorimetric technique; in the case of adenosine and 5'-AMP a direct method of analytical ultracentrifugation was employed. 3. 3. The results obtained suggest the dye binding at the adenine subsite of the dehydrogenase. 4. 4. The fluorescence of 1-anilino-8-naphthalene sulfonate bound to d-glyceraldehyde-3-phosphate dehydrogenase increases and the emission maximum shifts to shorter wavelengths on addition of nicotinamide mononucleotide. 5. 5. This suggests some conformational changes to occur in the micro-environment of the bound dye in response to the interaction with the ligand in the nicotinamide subsite. 6. 6. A quantitative relationship between the nicotinamide mononucleotide concentration on one hand, and the fluorescence change on the other, was used to determine the dissociation constant of the ligand, which was found to be 13.0 mM (0.1 M glycine buffer pH 8.5 at 20°C).

The investigation of substrate-induced changes in subunit interactions in glyceraldehyde 3-phosphate dehydrogenases by measurement of the kinetics and thermodynamics of subunit exchange

An investigation was made of changes in subunit interactions in glyceraldehyde 3-phosphate dehydrogenase on binding NAD+, NADH and other substrates by using the previously developed method of measurement of rates and extent of subunit exchange between the rabbit enzyme (R4), yeast enzyme (Y4) and rabbit-yeast hybrid (R2Y2) [Osborne & Hollaway (1974) Biochem. J. 143, 651-662]. The free energy of activation for the conversion of tetramer into dimer for the rabbit enzyme (R4 leads to 2R2) is increased by at least 12kJ/mol in the presence of NAD+. This increase is interpreted in terms of an NAD+-induced 'tightening' of the tetrameric structure probably involving increased interaction at the subunit interfaces across the QR plane of the molecule [see Buehner et al. (1974) J. Mol. Biol. 82, 563-585]. This tightening of the structure only occurs on binding the third NAD+ molecule to a given enzyme molecule. Conversely, binding of NADH causes a decrease in the free energy of activation for the R4 leads to 2R2 and Y4 leads to 2Y2 conversions by at least 10kJ/mol. This is interpreted as a NADH-induced 'loosening' of the structures arising from decreased interactions across the subunit interfaces involving the QR dissociation plane. In the presence of NADH the increase in the rate of subunit exchange is such that it is not possible to separate the hybrid from the other species if electrophoresis is carried out with NADH in the separation media. In the presence of a mixture of NADH and NAD+ the effect of NAD+ on subunit exchange is dominant. The results are discussed in terms of the known co-operativty between binding sites in glyceraldehyde 3-phosphate dehydrogenases.

Effect of NAD on flounder muscle glyceraldehyde 3-phosphate dehydrogenase

Flounder muscle (Pseudopleuronectes americanus) glyceraldehyde-3-phosphate dehydrogenase was characterized as to its stability towards various inactivating treatments in the presence and absence of the enzyme cofactor, NAD. Incubation of a partially purified enzyme preparation at urea concentrations greater than 2 M produced a very rapid inactivation. NAD greatly reduced the rate of inactivation at all the urea concentrations tested. Incubation of each of the three major muscle enzyme forms in 0.1 percent trypsin or chymotrypsin for forty-five minutes decreased the activity of each form by 65 percent and 55 percent, respectively. NAD (5mM) afforded complete protection to each enzyme form from proteolytic digestion by these two enzymes. Exposure of each form to 50 degrees or 20 mM ATP also led to gross inactivation which could be greatly reduced if the respective incubations were performed in the presence of 5mM NAD. NAD was also found to be required for the renaturation of the unfolded urea-denatured subunits to form the active tetramer.

Evidence for the occurrence in submitochondrial particles of a dual respiratory chain containing different forms of cytochrome b

1. Addition of ascorbate + N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD) to beef-heart submitochondrial particles (Mg-ATP particles) in the presence of KCN causes extensive reduction of cytochromes c 1 + c and a + a 3, and a partial reduction of cytochrome b. Addition of ATP results in an increased cytochrome b reduction, with a slight decrease in the level of reduced cytochrome a + a 3. Subsequent addition of NAD + causes a further substantial reduction of cytochrome b and a partial oxidation of cytochrome a + a 3. The total amount of cytochrome b reduced at this stage is close to that obtained by adding NADH or succinate to the particles in the presence of KCN. 2. Cytochrome b reduced upon the addition of TMPD and NAD + has an absorption maximum at 562 nm, and that reduced upon the addition of ATP at 565 nm, with a shoulder at 558 nm. All three phases of cytochrome b reduction are inhibited by antimycin, and the ATP- and NAD +-induced phases are also inhibited by FCCP and oligomycin. The NAD +-induced cytochrome b 562 reduction is ATP-dependent, and is abolished by rotenone and by pyruvate + lactate dehydrogenase, indicating that it proceeds by way of NADH generated through reverse electron transport via cytrochrome b 565 (+ b 558). The effect of NAD + in inducing cytochrome b 562 reduction is duplicated by fumarate in a non-additive fashion. 3. The three phases of cytochrome b reduction are accompanied by roughly proportional extents of reduction of ubiquinone and flavorprotein. The amounts of cytochrome b, ubiquinone and flavoprotein, reduced upon the addition of ascorbate + TMPD, ATP and NAD +, are close to the total enzymically (NADH and succinate) reducible contents of these components in the particles. 4. The results are interpreted to indicate that submitochondrial particles contain a dual respiratory chain, one including cytochrome b 565 (+ b 558) and a functional Coupling Site II, and another, including cytochrome b 562 and no functional Coupling Site II. The possible significance of these results for the functional organization of mitochondria is discussed.

The effect of pH on the characteristics of the binding of nicotinamide-adenine dinucleotide by nicotinamide-adenine dinucleotide-specific isocitrate dehydrogenase from pea mitochondria.

1. The effect of pH on the V(max.) and concentration of NAD(+) at half-maximum velocity at a constant isocitrate concentration was examined, and the results were related to the requirements for binding of H(+) ions to the enzyme. 2. The effect of varying the NAD(+) concentration on the pH optimum with constant isocitrate concentration was studied. 3. A comparison has been made between the effect of isocitrate concentration on the characteristics of binding of NAD(+) and the effect of NAD(+) concentration on the characteristics of isocitrate binding at three different pH values. 4. The mechanistic and metabolic significance of these studies is considered.

The effect of NAD + on the catalytic efficiency of glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle

The catalytic center activity for the oxidation of glyceraldehyde, acetaldehyde and propionaldehyde by glyceraldehyde-3-phosphate dehydrogenase was not observed to be appreciably affected by the degree of saturation of the enzyme with NAD +. A small increased in intrinsic rate on going from E( NAD +) I to E( NAD +) 4 was noted for glyceraldehyde (1.1 fold) and a small decrease in intrinsic rate for acetaldehyde (1.4 fold) and propionaldehyde (1.2 fold). These small changes, contrasted with the approx. 1·10 7-fold decrease in ligand affinity induced by the binding of NAD +, are consistent with the induced fit model of allosterism. The initial rate of oxidation of glyceraldehyde-3-phosphate by glyceraldehyde-3-phosphate dehydrogenase, in the presence of arsenate, decreased rapidly within the first few percent of the reaction. Investigation of the end product inhibition of this reaction suggests that the accumulation of 1-arseno-3-phosphoglycerate is partly responsible for the observed reduction in rate, and thus that the spontaneous hydrolysis of this mixed anhydride may not be rapid relative to the rate of catalysis.

Steoid delta-isomerase of the bovine adrenal gland: kinetics, activation by NAD and attempted solubilization.

Treatment of miorosomes of the bovine adrenal gland with deoxycholate yields a preparation which is not sedimented at 100,000 Xg. Molecular sieve chromatography on Sepharose 2B showed that the steroid Δ-isomerase activities of such preparations reside in particles having a wide range of size. While the kinetic properties of an acetone powder and a deoxycholate treated preparation of the microsomes suggest the existence of a single isomerase capable of acting upon C10 and C21-Δ5-3-ketosteroids, gel filtration resulted in a dissociation of isomerase activity toward androst-5-ene-3,17-dione and pregn-5-ene-3,20-dione. Kinetic analysis showed that NAD stimulates the steroid Δ-isomerase of an acetone powder preparation by accelerating the breakdown of the enzyme-substrate complex; this effect of NAD is not obtained after treatment of the microsomes with deoxycholate. (Endocrinology 83: 864, 1968)

Factors influencing the utilization of reduced nicotinamide adenine dinucleotide by pigeon heart mitochondria.

Pigeon heart mitochondria that were studied exhibited ADP:O and respiratory control ratios of 2.3–3.1 and 6–20 respectively with glutamate as substrate. This coupled respiration of glutamate was reversibly inhibited by ATP. In polarographic and photometric experiments at 28° in a medium containing 0.23 M mannitol, 0.07 M sucrose, 0.02 M Tris, and 0.02 mM EDTA at pH 7.2 such mitochondria oxidized 15–150 μM NADH with essentially zero order kinetics and an apparent Kmof approximately 4 μM. NADH oxidation, which was not coupled to phosphorylation, was increased 50% by the addition of 5 mM orthophosphate (Pi) but was inhibited by albumin, NAD, ATP, and ADP. The effect of NAD became marked only at concentrations of 2–5 mM. However, ATP in concentrations (5–10 mM) similar to those found in heart, inhibited by 50–75% the oxidation of physiological levels (~ 10 μM) of NADH. These and other findings are compatible with the view that the oxidation of cytoplasmic NADH is a normal property of heart mitochondria, and that the control of this process by changes in the intracellular levels of ATP and Pimay influence myocardial carbohydrate utilization.

Nicotinamide Adenine Dinucleotide in Potato Tuber Slices in Relation to Respiratory Changes with Age

The respiration of slices of a variety of bulky storage tissues rises gradually three to fourfold within a day of cutting (7, 8,9, 10). The respiration rate of fresh tissue can be sharply raised with 2,4 dinitrophenol, however, as demonstrated in chicory and potato slices. Both the age-induced and DNPinduced respiration increments are inhibited by malonate. It has been shown that whereas malate restores the respiration of malonate inhibited aged chicory slices to the normal level, malate is virtually without effect upon the respiration rate of 2,4 dinitrophenol-treated, malonate-inhibited fresh tissue (9). The foregoing observation suggested that the respiration of fresh storage organ slices may be limited by the levels of endogenous nucleotides, particularly NAD. However NAD concentrations were not ascertainable by the methods used in the foregoing study. In the present study NAD levels of fresh and aged potato slices were determined and the effect of NAD on the oxidation of organic acids by mitochondria isolated from fresh and aged tissue was studied.