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The NAD-dependent isocitrate dehydrogenases require the presence of divalent metal cation for activity. In order to elucidate the role of metal in this reaction, Michaelis constants for manganese were determined as a function of pH and isocitrate concentration. At constant pH, the Km values for total manganese as well as free manganous ion decreased as the concentration of isocitrate in the reaction mixture was increased; in contrast, the Michaelis constant in terms of the metal isocitrate complexes was invariant, suggesting that manganese binds as a metal chelate of isocitrate. A previous analysis of the Michaelis constants for isocitrate [Cohen, P. F., and Colman, R. F., Biochemistry (1972) 11, 1501] indicated that in the presence of 1-10mM manganese, the substrate binds as free dibasic isocitrate. A model is proposed which allows for the formation of the active enzyme manganese ˙ isocitrate complex from either of two pathways: in the presence of low concentrations of manganese, the enzyme may combine directly with the metal chelate of dibasic isocitrate; alternatively, in the presence of high concentrations of manganese, the enzyme first binds metal ion to yield an enzyme ˙ manganese complex which subsequently reacts with free dibasic isocitrate. The affinity of the enzyme for the manganese ˙ dibasic-isocitrate complex varies with pH, exhibiting a lower Michaelis constant at pH 8 than 6. It is shown that the catalytic function of the free enzyme is dependent on the ionization of an amino acid residue with pKe = 7.26. The measurable pK of this ionizable group is decreased to 6.39 in the enzyme manganese and enzymes manganese isocitrate complexes. The Km for manganese was determined in the presence of various chelating agents. Although citrate and GDP increase the Michaelis constant in terms of total manganese, the Km for the actual substrate (manganese ˙ dibasic-isocitrate) is not changed. This result can be attributed to the competition between these chelators and isocitrate for manganous ion. In contrast, ADP lowers the Km values for the metal isocitrate complex, as well as that for total manganese, suggesting that a direct interaction with the enzyme is involved. [ABSTRACT FROM AUTHOR]

Lactic dehydrogenase from pig heart (H4) shows pH-dependent reversible deactivation, denaturation and dissociation at pH < 6. The initial and final states in the dissociation-association process are found to be the homogeneous fully inactive monomer (Mr = 37500 %plusmn; 1000) on one hand, and a heterogeneous mixture of denatured subunits, native tetramers and inactive higher aggregates on the other hand. The yield of tetramers and the extent to which renaturation occurs correspond to each other. Using optimum conditions of reactivation [pH 7.9, I ≈ 0.2, 20 °C, in the presence of 1 mM dithiothreitol and 10 mM EDTA, enzyme concentration below 0.05 mg/ml], the correlation of reactivation and reassociation may be analyzed. The second-order kinetics of the reactivation process prove reassociation to be the rate-limiting step in the process of reactivation. From this one may conclude that in the case of the heart isoenzyme of lactic dehydrogenase the subunit does not represent the enzymatically active unit. The coenzyme (NAD+) increases the rate of reactivation but not the overall recovery. This suggests the coenzyme is involved in a nucleation step in the process of refolding and/or reassociation. [ABSTRACT FROM AUTHOR]

1. Nitrate reductase has been partially purified from Aspergillus nidulans. It has a sedimentation coefficient of 7.6 (± 0.1) S and a molecular weight of 190000 (± 10000) from sucrose density gradient centrifugation and gel-filtration studies. There is spectroscopic evidence for the presence of a haemoprotein component. 2. It possesses two associated activities, cytochrome c reductase and reduced viologen dye:nitrate reductase. A study of the effects of heat and inhibitors on the three activities suggests that the molecule consists of two functional parts. One of these is heat labile, contains FAD and a haemoprotein and catalyses the transfer of electrons from NADPH to cytochrome c. The other part is heat stable, contains molybdenum and catalyses the transfer of electrons from reduced viologen dyes to nitrate. 3. The Km values for nitrate, NADPH and cytochrome c are reported. In each case the Km for one substrate is not affected by varying the concentration of the other, suggesting a lack of interaction between their respective binding sites. Product inhibition by NADP+ and nitrate under saturating and non-saturating concentrations of NADPH and nitrate suggests a random order rapid-equilibrium mechanism for the enzyme. [ABSTRACT FROM AUTHOR]

The stoichiometry of the liver microsomal monoxygenase system from phenobarbital-pretreated rabbits has been investigated with cyclohexane, n-hexane and perfluoro-n-hexane as substrates. With cyclohexane an almost 1:1:l stoichiometry for cyclohexanol: NADPH: O2 was obtained. With the uncoupler perfluoro-n-hexane no product was formed and 2 moles of NADPH were oxidised per mole of oxygen. The stoichiometry of n-hexane hydroxylation suggested that partial uncoupling with this substrate had occurred. NADH addition together with NADPH had no significant effect on the hydroxylation of cyclohexane but led to a more than additive hydroxylation in the case of nhexane. Oxygen uptake with perfluoro-n-hexane and NADPH was almost doubled in the presence of NADH. It was shown that during uncoupling an increased oxidation of NADH via cytochrome b5 occurs. It was concluded that active oxygen which is not used for monoxygenations is reduced to water by the NADH-cytochrome b5 system and that this sparing effect could be the main mechanism of the well-known synergistic action of NADH on microsomal hydroxylations. [ABSTRACT FROM AUTHOR]

Modification of the lysine residues of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by 2,4,6-trinitrobenzenesulfonic acid follows a biphasic process in the apoenzyme in which two essential lysyl groups are modified per enzyme tetramer with loss of 50 % of the arsenolytic, phosphorolytic, oxidative and NADH-X activities and 15 % of the esterase activity. Further decrease in the activities was much slower even though a higher number of additional residues were being modified. The trinitrophenylated enzyme with two modified lysine residues and 50 % residual activity at pH 8.0 had the same Km for NAD+, glyceraldehyde 3-phosphate and arsenate as well as the same number of active sites as the native enzyme. By spectrofluorimetric and spectrophotometric techniques it was shown that this modified form of the enzyme still binds four NAD+ per mole enzyme by an anticooperative process. Ultracentrifugation and thermodenaturation studies show that the chemical modification did not affect the quaternary structure or the thermostability as compared with the native enzyme. Fluorescence studies show only a decrease in the fluorescence emission but not displacement in the wavelength of the emission maxima. However, the modified enzyme becomes more sensitive to chymotryptic proteolysis, and the pH optimum of the enzyme activity was displaced by one unit to the acid region. When trinitrophenylation was carried out in the presence of NAD+ two lysyl groups were protected and the rate of modification was identical to that found for the slow-reacting lysine residues of the apoenzyme. Moreover, the loss of the enzyme activities follows a linear pattern. Trinitrophenylation of the apoenzyme in presence of glyceraldehyde 3-phosphate or P1 does not decrease the rate of modification of the lysine residues but the enzyme activity is protected and the inactivation process is also linear. However, by prior modification of the active site sulfhydryl groups of the apoenzyrne with 1fluoro-2,4-dinitrobenzene followed by trinitrophenylation of the lysine residues, no change was observed either in the rate of modification of the lysine residues or in the inactivation rate profile as compared with the native enzyme. In both cases a 50 % loss of the initial activity after modification of two lysine residues was observed. This result indicates that the ligand-induced conformational change in the enzyme active sites does not affect the reactivity of the two lysine residues per enzyme tetramer. From these different experimental data it is concluded that two reactive lysine residues per tetramcr of rabbit muscle apo-glyceraldehyde-3-phosphate dehydrogenase are essential for the different enzyme activities. They do not seem to be directly involved either in the catalytic or the substrate binding site, however, they must play an important role in the stabilisation and control of the enzyme active site. In relationship with the reactivity of the two lysine residues, our results seem to be explained by a pre-existing asymmetry in the structure of the rabbit muscle enzyme, rather than by an induced fit mechanism. [ABSTRACT FROM AUTHOR]

The binding of lanthanide ions to the pyrophosphate portion of NAD and AppA causes conformational changes, monitored by circular dichroism, which involve a perturbation of the population distribution of these flexible molecules generating new conformations. Binding occurs to both stacked and unstacked states; the former probably involves a change in the relative orientation of tile nucleotide chromophores without a fundamental change in overall symmetry ;and the latter, by analogy with weaker binding to mononucleotides, changes in glycosidic angle, X. The implications are discussed for the use of lanthanide probes in high-resolution molecular magnetic resonance studies of small molecules possessing conformational flexibility. [ABSTRACT FROM AUTHOR]

A simple optical method for the assay of Δ4-3β-hydroxysteroid dehydrogenase activity in cell-free preparations of rate liver was designed. This test is based on the fact that under the chosen conditions no reduction of the Δ4-bond occurs. Thus the Δ4-3β-hydroxysteroids dehydrogenase activity can be directly measured by the production of Δ4-3-ketosteroids, the sum of which in turn may be quantitated by their isonicotinic acid hydrazone formation. Δ4-3β-Hydroxysteroid dehydrogenases are localized in both cytoplasmic and microsomal fractions, exhibiting higher activity with NAD than with NADP. Microsomal enzyme activities show a significant sex difference which is more pronounced with NADP as coenzyme than with NAD (male:female activity ratios 5.8 with NADP, 1.9 with NAD). No sex differences were observed in the cytoplasmic enzyme activity. These results indicate the existence of two NAD-dependent enzyme activities of which the microsomal activity shows sexual differences. On the basis of the male:female activity ratios a further separate NADP-dependent microsomal enzyme activity can also be distinguished. [ABSTRACT FROM AUTHOR]

Examines the stereochemistry of the enzymatic process with the coenzyme analogues of nicotinamide adenine dinucleotide. Coenzyme substrate; Horse liver; Yeast alcohol dehydrogenase; Conversion of the thionicotinamide analogue of nicotinamide adenine dinucleotide hydrogenase.

The cytoplasmic serum of Hevea brasiliensis latex contains a 2′-nucleotidase. This monoesterase has been isolated and purified 2100 times. Its molecular weight is estimated to be about 115000–120000. Among the nucleotides, of which it hydrolyses the 2′-phosphate group, NADP+ is utilized mainly and the nucleotides, NADPH, adenosine 2′,5′-diphosphate, guanosine 2′,5′-diphosphate, adenosine 2′-phosphate, in decreasing order, In a few cases (coenzyme A, adenosine 3′,5′-diphosphate, guanosine 3′,5′-diphosphate), it reacts very slowly with the 3′-phosphate group. The enzyme does not hydrolyse adenosine 3′-phosphate, 5′-nucleotides and others phosphorylated substrates. The 5′-phosphate group of the nucleotidic ribose and the type of puric base seem to play an important rôle in the formation of the enzyme-substrate complex. The optimum pH of the 2′-nuceotidase is around 5.5 and its Km value for NADP+ is between 0.7 to 1.0 mM at pH 5.5 and between 1.4 to 1.8 at pH 7.0. The ions Na+, K+, Mn2+, Mg2+, Ca2+, Co2+, Ni2+ have no influence on the enzymatic reaction, inorganic pyrophosphate, orthophosphate, adenosine, ADP and ATP are competitive inhibitors; NaF, Zn2+, Mo4+ and chiefly Cu2+ ions are strong non-competitive inhibitors. The Ki of these effectors has been measured. It appears that these molecules, taking into account the concentration and the Ki-value of ATP and Cu2+ in the latex, exert a physiologic effect on the 2′-nucleotidase reaction. This enzyme of Hevea brasiliensis latex is probably an important factor in the mechanism of regulation of polyisoprene synthesis. [ABSTRACT FROM AUTHOR]

Alkylation of NAD+ with iodoacetic acid followed by alkaline rearrangement gave N6-carboxymethyl-NAD+. Condensation of this analogue with 1,6-diaminohexane gave the analogue NAD+- N6-[N-(6-aminohexyl)-acetamide]. The coenzymic activity of the two derivatives was tested with malate dehydrogenase, alcohol dehydrogenase and lactate dehydrogenase. The efficiency relative to unsubstituted NAD+ was in the range of 50-100%. NAD+-N6-[N-(6-aminohexyl)-acetamide] was attached to Sepharose 4B by the cyanogen bromide method. The immobilized NAD+ analogue thus obtained, exhibited cofactor activity when tested in a recycling three-enzyme system (malate dehydrogenase-citrate synthase-lactate dehydrogenase). The immobilized NAD+ analogue proved to be an effective ligand in affinity chromatography. Thus a mixture of albumin, alcohol dehydrogenase and lactate dehydrogenase was resolved with good recovery. The enzymes were eluted with NAD+ and NADH, respectively. [ABSTRACT FROM AUTHOR]

Ferredoxin isolated from a blue-green alga, Nostoc sp., was purified and crystallized. The absorption spectrum of Nostoc ferredoxin had, in the oxidized state, peaks at 276, 331, 423, and 470 nm, a pattern characteristic of chloroplast-type ferredoxin. The 423 : 276 absorption ratio was 0.7 The midpoint oxidation-reduction potential of Nostoc ferredoxin was found to be -406 mV, at pH 7.5. Nostoc ferredoxin mediated the photoreduction of NADP by isolated Nostoc chromatophores and spinach chloroplast from which the native ferrdoxin was removed. The molar ratio of Nostoc ferredoxin to chlorophyll a was about 1:50, ratio higher than usually. The possible evolutionary significance of the properties of Nostoc ferredoxin compared with those of ferredoxins from other photosynthetic organisms is discussed. [ABSTRACT FROM AUTHOR]

By means of arsenate in optical tests the occurrence of both NAD- and NADP-linked glyceraldehyde-3-phosphate dehydrogenase activity has been found in embryo and endosperm tissue from Scots pine seeds. By exclusion of arsenate also evidences for the activity of a non-phosphorylating NADP-linked form have been demonstrated. The results are sustained by inhibition experiments. The enzyme activities have been followed principally during the first 24 hours of the germination process and in relation to the germination controlling light factor. Homogenates from complete seeds and partly also from separated embryo and endosperm tissues have been studied. The predominant activity in both of the tissues was linked to the NAD-form, expressed either on seed (part) or protein basis. No indications for a light regulation of anyone of these enzymes have been found contrary to findings from other plant materials. [ABSTRACT FROM AUTHOR]

Isolated mitochondria of wheat shoots oxidize α-ketoglutarate, DL-malate succinate and NADH with good relative respiration control and ADP: O ratio. They have high affinity for α-ketoglutarate and NADH as substrates and utilize malate and succinate with a respiration ratio of about one-half of α-ketoglutarate. The average ADP : O ratios approach the expected theoretical values, i.e., 3.6±0.2 for α-ketoglutarate, 1.8±0.2 for succinate, and 2.8±0.2 for malate. The ADP: O ratio with NADH is 1.8±0.2. The maximum coupling of oxidation and phosphorylation is obtained at concentrations of 10 mM, 2 mM, 10 mM and 8mM for α-ketoglutarate, NADH, malate and succinate, respectively. -- Wheat mitochondria have little or no dependence on added cofactors. Mitochondria prepared by our procedure apparently retain sufficient amounts of endogenous cofactors required for NAD-linked systems. FAD+ is found to improve succinate oxidation. Cytochrome c does not have any significant effect on respiratory parameters of wheat mitochondria. -- Wheat mitochondria are some what resistant to DNP at 1.7 × 10-5 M. Malonate seems to improve coupling of α-ketoglutarate oxidation. Other Krebs cycle intermediates have been tested on three major substrates of TCA cycle, i.e., α-ketoglutarate, malate and succinate. [ABSTRACT FROM AUTHOR]

Isolated perfused rat liver incorporates nicotinamide at a concentration of 10 μM into NAD via nicotinamide mononuclcotide. A deamidation of nicotinamide could not be demonstrated. Evidence is presented which indicates that the exchange reaction catalyzed by NAD glycohydrolase is not involved in the uptake of nicotinamide into NAD. At the concentrations used, the rate of the incorporation of nicotinamide into NAD is about 40% of the incorporation rate observed with nicotinic acid. Results obtained after perfusion with [14C]nicotinie acid support the concept of an intracellular pyridine nucleotide cycle. However, in contrast to the general opinion, our results demonstrate that in rat liver nicotinamide generated by NAD breakdown is not deamidated but reutilized via nicotinamidc mononucleotide. After perfusion with 0.1 mM [l4C]nicotinamide for 2.5 h, less than 2% of the total radio-activity is excreted into the bile. The same value is obtained after perfusion with 50 μM [14C]nicotinie acid. From these results it is concluded that the excretion of nicotinamide by the liver and the subsequent deamidation within the intestinal tract which have been considered important in the utilization of nicotinamide by the liver, are without biological relevance at normal nicotinamide concentrations. [ABSTRACT FROM AUTHOR]

1. At pH 7.5, inactivation of NAD kinase by bromoacetyl pyridine occurs in three independent pseudo-first-order stages. 2. Below pH 7.5, only two stages are distinguishable. 3. The final stage alkylation leads to total reactivation, the earlier stages to the formation of an enzyme species with modified properties. 4. Effects of pH on the time courses of inactivation suggest that the first stage may involve a variable number of sulphydryl groups. 5. The final stage probably involves alkylation of a sulphydryl, with abolition of NAD binding. 6. No central role m catalysis can be postulated for any of the groups modified by bromoacetyl pyridine; two groups known to participate in catalysis and the binding of ATP · Mg are apparently not alkylated. 7. Inhibition by L-malate and by free ATP may occur by attachment of these effectors at separate modifier sites. [ABSTRACT FROM AUTHOR]

Studies of the alcohol-acetaldehyde interchange, in the presence of analogues of NAD+ and brought about by yeast and horse liver alcohol dehydrogenases, have not provided any evidence in favour of the direct participation of the enzyme in the hydrogen transfer step. A new preparation of 1,4,5,6-tetrahydro-nicotinamide adenine dinucleotide is described. This analogue has been found to be a good competitive inhibitor for both enzymes, thus demonstrating the importance of fixation of the enzyme by a hydrogen bond to the group present in C-3 of the nicotinamide nucleus. [ABSTRACT FROM AUTHOR]

Structural changes of yeast glyceraldehyde-3-phosphate dehydrogenase as a function of satiation with NAD have been measured with the X-ray small-angle scattering technique. Samples of yeast glyceraldehyde-3-phosphate dehydrogenase were investigated at pH 8.5 and 40° and five different degrees of saturation with the coenzyme NAD (degree of saturation &Yoline; = 0, 0.23, 0.46, 0.72, 0.99). Because of the temperature-sensitivity of the sample, the use of a special flow device became necessary to keep the time of exposure of the samples to 40° and to X-ray irradiation below 1 h. Apo-enzyme revealed a contraction in volume depending upon the degree of saturation. Fully saturated the contraction amounts to 7%. As the observed degree of contraction is not linearly proportional to the degree of saturation, the simple sequential mechanism cannot explain the observations. To the extent that they coincide with the quantitative predictions of the allosteric model for this particular enzyme, based on calculations involving the equilibrium constants obtained from chemical relaxation experiments under identical conditions, they strongly support the latter mechanism. Comparison of the experimental scattering curves with theoretical scattering curves of compact full cylinders did not bring about satisfactory agreement in the range of the first submaximum. Hollow circular cylinders with h:2r1 = 0.6:1 (apo-enzyme) or h:2r1 = 0.58:1 (holo-enzyme) may be regarded as sufficiently equivalent in scattering as far as the form of the main maximum concerned as well as the position and relative intensity of the submaximum. Even if the molecule consists of four subunits they consequently must be arranged in such a way that the entire particle is very similar to a hollow cylinder. [ABSTRACT FROM AUTHOR]

The purification and concentration of NAD-glycohydrolase from group-C streptococci was achieved by zone precipitation, adsorption chromatography and re-chromatography on hydroxylapatite. Subsequently, the enzyme could be isolated as a microcristalline product showing double refraction in the polarization microscope. Analytical gel permeation chromatography, analytical gradient elution chromatography on hydroxylapatite, density gradient centrifugation, sodium dodeeylsulfate—urea electrophoresis and agar gel diffusion have been used to demonstrate the homogeneity of the purified enzyme. The polyacrylamide gel electrophoresis shows the enzyme as an uniformly migrating band. Furthermore the electrophoretic mobility of NAD-glycohydrolase corresponded approximately to that of hemoglobin. This finding provides first evidence for the existence of identical subunits of the enzyme protein. Besides the demonstration of its catalytic function and antibody specificity the crystallized enzyme has been shown to possess hemolytic activity which could be activated by thiol groups. Though a variety of efficient purification procedures were employed they failed to remove Streptolysin-O from the enzyme. Considering analytical data and criteria of homogeneity it may be assumed that a chemical correlation exists between the crystallized enzyme from group-C streptococci and Streptolysin-O previously described as two different substances. [ABSTRACT FROM AUTHOR]

Lobster glyceraldehyde-3-phosphate dehydrogenase was found to contain a reactive lysine residue that is specifically acetylated when the NAD-deficient form of the enzyme is incubated with [1-14C]acetyl phosphate at pH 8.5. The residue was identified by sequence analysis as number 182 in the polypeptide chain of the enzyme. Both the acetylation behaviour and the position of the lysine residue are identical with that found for mammalian glyceraldehyde-3-phosphate dehydrogenases. In addition the amino acid sequence around lysine 182 was found to be identical, except for a few conservative changes, with the corresponding sequence in pig glyceraldehyde-3-phosphate dehydrogenase. [ABSTRACT FROM AUTHOR]

Subcellular fractions of a strain of Saccharomyces cerevisiae have been prepared by enzymic digestion of the cell walls, osmotic lysis of the protoplats and density gradient centrifugation of the lysate. The mitochondrial fraction has both NAD-and NADH- phosphorylating enzymes, whilst the cytoplasm contains a single NAD-specific kinase. These three enzymes differ in their kinetic properties and in their stability to heat and to an alkylating agent, 3 (bromoacetyl) The properties of the cytoplasmic NAD kinase resemble those of NAD kinase partially purified from autolysates of pressed bakers' yeast. [ABSTRACT FROM AUTHOR]

1. When assayed with NAD, the Nine course of inactivation of glutamate dehydrogenase by bromopyruvate is biphasic. This analyses as two simultaneous first order reactions: the faster corresponds to destruction of an NAD activating site and the slower to the destruction of the active site. Both reactions are first order in bromopyruvate and both sites are protected by combinations of coenzymes and regulators. 2. When assayed with NADP, activity loss is essentially a single first order princess. When ADP is included in this assay, the activity loss profile is similar to that observed with NAD. With GTP included in this assay, the activity first increases, corresponding to a partial loss of GTP response, and then falls off in a first order reaction. The loss of response to both regulators is analysed in terms of a model in which approximately 50% of the regulatory sites are lost in a first order reaction. 3. Evidence is presented that the sites binding ADP and GTP are not identical. [ABSTRACT FROM AUTHOR]

Changes induced on addition of the cofactor, NAD, to glyceraldehyde-3-phosphate dehydrogenase from yeast have been investigated by hydrodynamic and spectroscopic techniques. As shown by light-scattering and sedimentation and diffusion studies, the tetrameric structure of the holoenzyme persists under all but strong dissociating conditions: there is thus no change in the quaternary structure on binding. On the other hand, landing is accompanied by an increase of some 40/0 in the sedimentation coefficient, which is too large to he explicable in terms of the increase in particle weight by bound ligand. A conformational change is also observed by optical rotatory dispersion and circular dichroism measurements. The magnitude of the Cotton effect associated with the aromatic chromophores of the protein, near 280 nm, increases, whereas the rotation in the neighbourhood of 233 urn undergoes a small diminution as the cofactor is added. it is shown that these changes are conformational in origin, rather than a consequence of WAD attachment per 89. Increasing the temperature in the range 2O-4O° leads to a transition from non-cooperative to cooperative binding of NAD to the apoenzyrne. At 40°, where NAT) binding is weakly sigmoidal, the change in optical rotation is likewise sigmoidal and parallels the increase in the sedimentation coefficient. At 20° both the uptake of WAD and the structural changes arc hyperbolic, in the cooperative situation the data are shown to be consistent with the two-state "concerted" model for ligand-binding and incompatible with a sequential mechanism. [ABSTRACT FROM AUTHOR]

Correlations between structure of a steroid and inducing effect for 3α-hydroxysteroid: NAD oxidoreductase of Pseudomonas testosteroni, were investigated. For this purpose, 0.35 mMole of steroid was added to a culture in logarithmic phase, in which the medium is mainly composed of glucose and casein hydrolysate. After 6 hours, cells are harvested and enzymic activity tested by measuring the increase in absorbance at 340 mμ. We have controlled that the structure used are fully soluble in the culture medium and their concentrations do not change curing the inducing process: 31 steroids have been tested under these conditions. [This equation can not be represent in ASCII. TXT] . A structure will be considered a good inducer if İ > 0.7 and a week one if <0.3. Out results show that the methyl radical in C19, at the junction between ring A and B is quite indispensable to the inducing effect (İ=0.3 for 19-nor-testosterone, İ=0.0 with 17 β-oestradiol; an accessible oxygen function in C3 position is also required (decrease of inducing effect with sulfur or chloro compounds). Blocking of C17 strongly decreases the inducing process (İ=0.0 for 17α-methyl testosterone but progesterone has a powerful effect (İ=1.2). The most favourable shape and the size of the molecule are the plane form and the freedom of the αface (İ=0.5 for retro-testosterone, İ=0.0 for lumi-androsterone). The lengthening the molecule near the A ring (butyrate, propionate, formation of a new ring with 6 carbons) suppresses the inducing effect. Qualitative analysis by thin-layer chromatography on Kieselge GF 254 of the products made from these steroids. show that all inducing structures are metabolised: we have not found a gratuitous inducer for this enzyme. [ABSTRACT FROM AUTHOR]

At pH 9.0 the activity of crystalline glutamate dehydrogenase was increased by the addition to the incubation mixture of various electrolytes including sodium chloride, potassium phosphate, Tris, 2-amino-2-methyl-1,3-propanedil and methionine. Potassium phosphate was most stimulatory and, at an optimum concentration, increased activity three to four-fold. No stimulation was obtained at pH 7.6. Potassium phosphate and the other electrolytes abolished activation by high concentrations of NAD or NADP and altered the inhibitory effects obtained by compounds which presumably do not interact at the NAD binding site. These electrolytes also overcame the inhibition by GTP, a ligand which binds at the same site as NAD, but they did not suppress the reactivity with ADP. Its is proposed that at pH 9.0 potassium phosphate and other electrolytes alter glutamate dehydrogenase so that the regulatory site which binds NAD or GTP is no longer available to these ligands. [ABSTRACT FROM AUTHOR]

The activity of the enzyme dihydropteridine reductase, which catalyses the reduction of quinoid dihydropteridine cofactor in the presence of NADH or NADPH, was measured in a system containing H2O2 and peroxidase. Peroxidase and H2O2 continually reoxidize the reduced tetrahydropteridine cofactor to the quinoid dihydropteridine cofactor, and thus maintains the concentrations of the latter virtually constant. A method for calculation of the actual concentration of dihydropteridine cofactor and for correction of the dihydopteridine reductase activity to allow for variations in the dihydropteridine cofactor concentration have been derived. Even though dihydropeteridine reductase can utilize either NADH and NADPH as the reductant, the former is 15 to 45-fold more active than the latter. This specificity for the nucleotide cofactor has been found for dihydropteridine reductases prepared from five different mammalian species. [ABSTRACT FROM AUTHOR]

The sulphate of 3β-hydroxypregn-5-en-20-one is now reported to be a substrate for crystalline 20β-hydroxysteroid:NAD oxidoreductase prepared from Streptomyces hydrogenans. Utilization of this steroid sulphate was not independent of activity towards pregn-4-ene-3,20-dione or 3β-acetoxypregn-5-en-20-one, but the total rate of reaction for equimolar mixtures of 3β-hydroxypregn-5-en-20-one sulphate and either pregn-4-ene-3,20-dione or 3β-acetoxypregn-5- en-20-one was less than that calculated for the simplest type of mutual competitive inhibition, particularly at low substrate concentrations. The Michaelis constant for NADH was similar (about 5 μM) with each of these compounds as substrate. 3β-Hydroxypregn-5-en-20-one, and its 17α-hydroxy derivative behaved anomalously, and their behaviour is discussed. 3β,17α,21-Trihydroxypregn-5-en-20-one was a good substrate and had kinetic constants similar to those of 17α,21-dihydroxypregn-4-ene-3,20-dione. [ABSTRACT FROM AUTHOR]

Irradiation of fully developed green leaves as well as irradiation of isolated chloroplasts causes a rapid, reversible increase in the activity of NADP-linked phosphoglyceraldehyde dehydrogenase. This increase is closely connected with the functioning of the non-cyclic electron flow of photosynthesis. It is accompanied by a decrease in the activity of the NAD-linked enzyme. The experiments presented here show that it is not the kinase but the dehydrogenase which is the rate limiting factor in the over-all reaction measured up to date. The activation of the ‘dark-enzyme’ (= basic enzyme) resulting in the ‘light-enzyme’ (= activated enzyme) is not due to an unspecific reduction of S-S-groups in the reactive center. Dithiothreitol only restores the activity lost during the isolation procedure of the chloroplasts. NADPH rather than dithiothreitol causes a specific activation of the basic enzyme. How- ever the basic enzyme must be present in the completely reduced form to undergo maximal activation. Incubation with dithiothreitol and NADPH thus results in an activation rate as high as that caused by light in vivo. Activation of the NADP-linked dehydrogenase could also be attained by NADPH after its partial purification by ammonium sulfate fractionation. The activation rate by incubation with different concentrations of NADPH shows a sigmoidal type of curve. There are characteristic differences in sensitivity between the activity of the basic enzyme and its ability to be activated at different temperatures between 52°–60°. The results indicate that the action of NADPH may be an anosteric one, causing a transition from a NAD- (or possibly a NAD + NADP)-dependent to a NADP-dependent form of the enzyme. A diagram for a feed-forward activation of the NADP-linked dehydrogenase which possibly takes part in regulation of C02-fixation is presented. [ABSTRACT FROM AUTHOR]

1. The specificity of pigeon liver NAD kinase for both substrates and for divalent cations has been investigated. 2. The enzyme is activated by Mg++, Co++, Mn++, and Zn++ only: the ATP- metal complexes have different Km, values but the maximum velocities of the reactions are the same. 3. All nucleoside triphosphates investigated will act as substrates, although the Km values and maximum velocities differ. 4. The only NAP analogue found to act as a substrate was 3-acetyl pyridine-adenrne dinucleotide (APAD). 5. The enzyme is inhibited by free Mg and free ATP. 6. The independence of substrate binding sites confirms that a random-addition, rapid equilibrium mechanism applies to NAD kinase. [ABSTRACT FROM AUTHOR]

The properties of human and rabbit erythrocyte glucose-6-phosphate dehydrogenase have been investigated during aging in vivo. The erythrocytes of different age have been prepared by 2 procedures: (a) by inducing a reticulocytosis by phenylhydrazine administration to rabbits and (b) by separating the young and old red cells of man and rabbit according to the different resistence to osmotic lysis. During aging, human and rabbit glucose-6-phosphate dehydrogenase shows a decrease of the catalytic activity and some marked modifications of its properties. Thermolability and NADPase- dependent inactivation increase: the protective effect of XADP and the affinity for both glucose- 6-phosphate and NADP substrates are decreased: no modifications have been demonstrated as regards pH optimum and electrophoretic patterns in starch gel. It is possible that these modifications begin after the expulsion of ribosome material. According to the literature concerning the behaviour of other enzymes in the physiological state and of glucose-6-phosphate dehydrogenase of some pathological conditions, it is possible that the modified properties of the enzyme during aging in vivo can be related to some structural modifications of the protein. [ABSTRACT FROM AUTHOR]

1. Some properties of NAD : isocitrate dehydrogenase from swede (Brassica napus L.) are described. This enzyme shows simpler regulatory properties than that from animals or fungi. 2. Isocitrate, citrate and tricarballylate are positive effectors but neither ADP nor AMP have any effect. Pyruvate, acetate, α-ketoglutarate, succinate and DL-malate do not stimulate or inhibit at 2 mM. 3. ATP and other nucleoside triphosphates inhibit this enzyme but the inhibition appears to be due to the formation of a complex with the activating cation. 4. The principal control of the enzyme is a potent inhibition by NADH competitive with NAD. Elevation of either isocitrate or activating cation concentration lowers the Km for NAD and in this respect MnSO4 is more effective than MnSO4. In contrast, the Ki for NADH is 0.05 mM with 1mM MgSO4 and 0.16 mM with 1 mM MnSOm 5. The enzyme is inhibited by high concentrations (> 20 mM) of inorganic anions. This inhibition is apparently competitive with isocitrate but anions most likely cause a conformational change. 6. The interactions between isocitrate and activating cation have been examined. Elevation of the concentration of either isocitrate or cation raises the affinity of the enzyme for the other to a limiting value. The nature of the cation has only a slight effect on the minimum Km for isocitrate (0.35 mM with MgSO4, 0.25 mM with MnSO4) but the minimum value of the Km for MgSO4 is 100 μM compared with 7 μM for MnSO4. 7. It is concluded that the enzyme binds one molecule each of isocitrate, activating cation and NAD or NADH at the catalytic site and one molecule of citrate or isocitrate at an effector site. [ABSTRACT FROM AUTHOR]

Crystalline 20β-hydroxysteroid : NAD oxidorecluctase preparations from streptomyces hydrogenans were shown to have 3α-hydroxysteroid : NAD oxidoredictase. Compounds of both the 5α-androstane and 5α-androstane and 5α-pregnane series served as substrates. Among the compounds tested, no evidence was found of 3β-hydroxysteroid : NAD oxidoreductase activity. The 3α-hydroxysteroid : NAD oxidoreductase activity did not extend to compounds having 5Β-H, a 4,5 double bond, a 5,10 double bond, or 1,2 and 4,5 double bonds together. In the &5α-H series, a 1,2 double bond prevented the compound from serving as a substrate. Substituents at C-11, -16 and -17 altered the kinetic constant for the 3α-hydroxysteroid: NAD oxidoreductase reaction, and in the case of an 11β-hydroxy or 11-oxo group the effects were similar to the effects reported for these grups for 20β-hydroxysteriod : NAD oxidareductase activity. Kinetic data were consistent with the view that two types of 3α-hydroxysteriod : NAD oxidoreductase were present which involved different active centres, neither of which was the active centre for the 20β-hydroxisteriod : NAD oxidoreductase activity. Itr is not yet established whether these enzymic activities are associagted with the same or different molecules. The 3α-hydroxysteriod : NAD oxidoreductase activies do not correspond to those of previously charcterised enzymes (3α-hydroxysteriod dehydrogenase and α-hydroxycholanate dyhrogenase. [ABSTRACT FROM AUTHOR]

A procedure for the purification of NAD kinase from pigeon liver acetone powder is described. Although not homogeneous, the preparation is largely free of enzymes which interfere with the assay of NAD kinase. The kinetics with magnesium as the activating ion are described: from interrelationships between the kinetic coefficients, the modes of inhibition by NADH, NADPH and ADP, and the independent variation of Vmax and the two K,ms with pH, it is inferred that NAD kinase acts by a random addition, rapid equilibrium mechanism. [ABSTRACT FROM AUTHOR]

The relationship between oxidative phosphorylation and the energy-linked nicotinamide-adenine dinucleotide transhydrogenase reaction catalyzed by phosphorylating submitochondrial particles from beef heart has been investigated. The efficiency of the particles in exhibiting oxidative phosphorylation (P/O ratio) and NADH-linked NADP+ reduction (NADP+/O ratio) was measured, using succinate as oxidizable substrate and rotenone as inhibitor of NADH oxidase, under conditions where the phosphorylating and transhydrogenating systems were operating one by one or in parallel. When succinate oxidation was limited by increasing concentrations of malonate, the P/O ratio remained unchanged in the absence of an active phosphorylating system, and decreased in its presence. The NADP+/O ratio increased with increasing concentrations of malonate, both in the absence and presence of an active phosphorylating system. The (P + NADP+)/O ratio in the combined system approached 2, i.e., the generally accepted maximal ∼/0 ratio for succinate oxidase. Active transhydrogenation caused an increase in the apparent Km for Pi in the phosphorylating system from 1.7 to 4.5 mM, and active phosphorylation caused an increase in the apparent Km for NADH in the transhydrogenating system from 21 to 40 μM. It. is concluded that oxidative phosphorylation and the energy-linked transhydrogenase reaction are competing for a common nonphosphorylated high-energy intermediate generated by the respiratory chain. The kinetic evidence presented further suggests that the two systems interact with two different moieties of this high-energy intermediate. [ABSTRACT FROM AUTHOR]

Piericidin A and oxaloacetate have been used as inhibitors to investigate the operation and localisation of NAD+-linked dehydrogenases and the possible compartmentation of these enzymes within the matrix of the mitochondrion. 1. Mitochondria isolated from etiolated shoots of wheat (Triticum vulgare) were shown to oxidise a variety of NAD+-linked substrates. Oxaloacetate, when added to rapidly respiring preparations (i.e. state 3) resulted in a large but transient inhibition of the oxidation of both pyruvate and citrate, but had little effect on the rate of oxidation of malate. 2. The products of oxidation of malate, in the absence of added NAD+, were found to be a large quantity of pyruvate together with smaller quantities of oxaloacetate and citrate. Using potassium ferricyanide as a non-penetrating electron acceptor it was possible to show that the malate oxidation occurred in the matrix. This is consistent with the operation of both the malic enzyme and a low level of malate dehydrogenase within the matrix of the mitochondrion. 3. The low level of inhibition of malate oxidation by oxaloacetate was a function of the concentration of malate and appeared to be of a competitive nature. 4. Measurement of the redox state of the endogenous NAD+ pool showed that under phosphorylatig conditions both malate and citrate reduce approximately 15% of the total coenzyme. The addition of 0.2 mM oxaloacetate to phosphorylating aerobic mitochondria failed to oxidise any of the NAD+ reduced by malate but resulted in a partial oxidation of NAD+ reduced by citrate. The transition from the aerobic to anaerobic state in the presence of malate resulted in a biphasic reduction of the endogenous NAD+, whereas in the presence of citrate it was monophasic. 5. The oxidation of NAD+-linked subtrates, including malate, was found to be only partially inhibited by piercidin A. The piericidin-A-resistant pathway was found to be located internally on the basis of experiments using ferricyanide as a non-penetrating electron accepter. The first site of phosphorylation was not coupled to oxidation mediated by the piericidin-A-resistant pathway. 6. The presence of piericidin A altered the shape of the malate saturation curve. In the absence of the inhibitor it was found to be biphasic; saturating at 60 mM and in its presence to be monophasic saturing at 15 mM. At high concentrations of malate (60 mM) piericidin A caused approximately a 30% inhibition, similar to that found with other NAD+-linked substrates, while at lower concentrations of malate (15 mM) piericidin A caused a much higher inhibition (80%), which subsequently recovered to the much higher piericidin-A-resistant rate observed with higher concentrations of malate. 7. In the presence of piericidin A oxaloacetate caused severe but transient inhibition of malate oxidation, similar in magnitude to that observed during pyruvate oxidation in the absence of piericidin A. 8. It was concluded that the data were consistent with the spatial separation of both the internal malic enzyme and malate dehydrogenase together with the internally located piericidin-A-sensitive and resistent electron-transport pathways. [ABSTRACT FROM AUTHOR]

The object of this paper is to analyse the effects of the coenzymes NAD(P)+ and NAD(P)H, and the effectors ADP and GTP, on the polyhexameric structure of pig-liver dehydrogenase. The linear polymerisation model proposed by Eisenberg for the native quaternary structure of this protein is valid with any effector; the observed variations of the degree of polymerization are explained by the modification of the apparent association constant of the hexamers. The appendices I and II define the free and associated areas and give, the theoretical foundations of the variation of the association constant of hexamers in terms of the binding of the ligands to the protemers. The increase in the degree of polymerization of the glutamate dehydrogenase with the binding of NAD(P)H is explained by a higher affinity of the coenzymes for the protomers which have an associated area compared to the protomers which have a free area. No variation is observed with NAD(P)+, ADP, or GTP alone. The formation of the protein · GTP · NAD(P)H ternary complex leads to a complete depolymerization when the two ligands are in saturating concentrations. The systematic study of the variations of polymerization in terms of increasing concentration of NAD(P)H at constant concentration of GTP, or in terms of increasing concentration of GTP at constant concentration of NAD(P)H shows that the interaction between the two opposite protomers of two consecutive hexamers is responsible for the sigmoidal shape of the depolymerization curves. The reversibility of this effect by ADP is assigned to a competition between the binding of ADP and the binding of GTP. [ABSTRACT FROM AUTHOR]

The ability of isolated LS-cell nuclei to incorporate [adenine-³H]-NAD into acid-insoluble products is demonstrated. The incorporation has a pH optimum at 8.5 and an apparent temperature optimum at 25 °C. The optimum Mg2+ concentration is dependent upon the NAP concentration; at 1.5 μM NAD it is 2 mM; at 1.5 mM NAD it is 3 mM; and at 9 mM NAD it is above 20 mM Mg2+. Mg2+ can be replaced by Ca2+. Dithiothreitol or mercaptoethanol, up to 10 mM, enhances the incorporation of NAD. With the optimal conditions incorporation of NAD (V) is 128 nmol NAD × 5 min-1 × mg DNA-1 and the Km is 1.47 ± 0.18 mM. Nicotinamide inhibits the incorporation competitively with a Ki of 14.3 ± 1 μM and thymidine inhibits competitively with a Ki of 32.5 The incorporation is not affected by added DNA or poly(U). It is not affected by ribonuclease but is inhibited by about 50% on treatment with deoxyribonuclease. The enzyme system is unstable and the decay is temperature-dependent; there is little decay at 0 °C but above this temperature the rate of decay follows an Arrhenius relationship. The initial rates of enzyme activity also show a temperature dependence with a linear Arrhenius plot. The temperature optimum at 25 °C is the arithmetic result of these two opposing processes. [ABSTRACT FROM AUTHOR]

Rabbit antiserum was prepared against rat liver microsomal NAD glycohydrolase solubilized by lipase treatment and subsequently purified by successive DEAE-cellulose, Sephadex G-100 chromatography and polyacrylamide gel electrophoresis. The antibody inhibited the purified enzyme 60–80%, but the membrane-bound and Triton X-100 solubilized enzyme up to 20–30% only. A single precipitation line was produced when the antibody reacted with the purified enzyme, and with crude lipase and Triton X-100 solubilized enzyme preparations. NAD glycohydrolase from liver rough-microsomal and plasma-membrane fractions reacted identically. The antibody to rat liver NAD glycohydrolase similarly inhibited the lipase or Triton X-100 solubilized and membrane-bound NAD glycohydrolase from rat spleen and brain as well as from mouse liver but not from bovine liver. [ABSTRACT FROM AUTHOR]

The stereochemistry of hydrogen transfer from NADH during the reduction of the 3-oxo groups of various steroids by cortisone reductase has been investigated for a series of substrates showing interesting structural features or reaction characteristics. It was found that the 4B-hydrogen of the NADH was transferred to the substrate in all cases whether the resulting hydroxyl group was 3α or 3β. The 4B-hydrogen was also transferred when the same enzyme catalysed the reduction of the 20-oxo group of pregn-4-ene-3,20-dione, so, although the enzyme can bind steroids allowing hydrogen transfer in various ways, it binds NADH in such a way that only the 4B-hydrogen is available for catalytic transfer to any considerable extent. No reaction was deleted with NADPH. The reaction rate with NADH was lowered by the presence of NAD+ in the two cases tested and, in the one ease in which the reaction was studied in the reverse direction, NADH lowered the rate of reaction with NAD+. In all three cases, the apparent V values did not change significantly, whereas the apparent Km values for the reacting nucleotide changed considerably ("competitive inhibition"). This was consistent with an ordered reaction sequence in which the nucleotide bound before and left after the steroid, but these findings did not eliminate other possibilities. [ABSTRACT FROM AUTHOR]

1. NAD+ forms two pairs of spectrally distinguishable complexes upon binding to oxidized lipoamide dehydrogenase. The shape and magnitude of the spectra is dependent on temperature and pH. The flavin-absorption band at 450 nm is not shifted uniformly. Intrinsic dissociation constants only could be calculated at 430 nm. In 30 mM phosphate buffer pH 7.2 at 25 °C Kd¹ = 50-60 µM, while at 0 °C Kd ¹ = 40 µM for the high-affinity pair; for the low-affinity pair these values are 200-250 µM and 110-130 µM respectively. The high-affinity pair is also present at pH 5.6 at I = 0.1 and I = 0.2, the values at 25 °C are respectively 50 µM and 35 µM. 2. Computer simulation suggests that the activating effect of NAD+ on the NADH:lipoate reductase activity can be explained by assuming that binding of NAD+ shifts the pK-value of a protonated group at the active centre from 6.2 to 4.9-5.0. This suggestion is supported by the observation that binding of NAD+ at the high-affinity site is accompanied by liberation of 0.9 ± 0.07 equiv. H+/mol flavin. From titration data it was calculated that for NAD+ binding 3. The results indicate that binding of NAD+ at the high-affinity site, even at partial saturation, shifts the monomer-dimer equilibrium towards the dimer. Furthermore the difference in reduction state upon reducing the enzyme with NADH at 25 °C and 0 °C is not due to differences in affinity for NAD+, but to conformational changes of the protein around the flavin moiety. It has to be considered that the two active centres of the dimer are not completely independent. [ABSTRACT FROM AUTHOR]

Semi-purified D-mannonate: NAD-oxydoreductase of Escherichia coli K12, which catalyzes a reversible reaction according to the following reaction: D-Fructuronate + NADH + H+ ↔ D-mannonate + NAD+ has been subjected to kinetic analyses with the method described by Cleland.
Initial velocity kinetics show that the mannonate reductase catalyzed reaction is constant with a sequential mechanism; from reciprocal plots, it is apparent that the Michaelis constant for every substrate of direct and reverse reaction is identical with the corresponding inhibition kinetic consent. The product inhibition studies revealed kinetic patterns consistent with a "rapid-equilibrium random bi-bi + dead-end EBQ complex" enzymic mechanism. The various kinetic parameters arc determined in phosphate buffer at pH 6.3 for the direct reaction and in NaOH-glycylglycine buffer at pH 8.4 for the reverse one.
Alternate product inhibition studies with altronate or tagaturonate, dead-end product inhibition studies with ATP and binding studies in relation to the protection of the enzymic activity against thermal inactivation also suggest that the mannonate oxydoreductase reaction is most likely to proceed through the above described mechanism. [ABSTRACT FROM AUTHOR]

Kinetic studies were carried out in order to investigate the enzymic mechanism of Escherichia coli K12 D-altronate: NAD-oxydoreductase.Initial velocity patterns (reciprocal plots) of the forward and reverse reactions are intersecting, referring to a sequential mechanism. The following numerical values of the Michaelis and dissociation constants are extracted graphically from secondary plots (slopes and intercepts of the reciprocal plots versus the reciprocal concentrations of the non-varied substrate): Kia = 5.8 µM, Ka = 85µM and Kb = 670µM for the forward reaction; Kia = 156 µM, Ka = 53µM and Kb = 75 µM for the reverse reaction. At pH 6.3, in phosphate buffer, product-inhibition patterns of the forward reaction surest an ordered bi-bi mechanism for the altronate oxydoreductase with NADH as the first substrate to add to the enzyme. The only competitive inhibition observed is when NAD+ is the product inhibitor and NADH the variable substrate, all others inhibitions berg non competitive. At pH 8.9, in glycylglycine buffer, product inhibition patterns of the reverse reaction also revealed an ordered bi-bi mechanism with altronate as the first substrate to add to the enzyme. The only competitive inhibition observed is when tagaturonate is the product inhibitor and altronate the variable substrate, all others inhibitions being non competitive. Dead-end product inhibition studies with fructuronate, mannonate and ATP, and binding studies in relation to the protection of the enzyme against thermal inactivation, are consistent with an ordered bi-bi enzymic mechanism for the altronate oxydoreductase. The occurrence of two possible enzyme forms of the altronate oxydoreductase according to the physical properties of the reaction medium is suggested. [ABSTRACT FROM AUTHOR]

1. The structural changes of mammalian skeletal muscle D-glyceraldehyde-3-phosphate de- hydrogenase induced by the binding of the coenzyme, NAD, were studied by small-angle X-ray scattering with the double-monitor technique. 2. As shown by our difference method the radius of gyration of the apoenzyme decreases by 0.08 nm on the binding of four molecules of NAD. The decrease in the radius of gyration is not a monotonous function of saturation with NAD. 3. From the analysis of our data it follows that, in the order of binding, the first and third NADs are positioned at a distance of about 2 nm from the centre of gravity of the molecule, whereas the second and fourth NADs are at a distance of about 4 nm. 4. As indicated by the change in the radius of gyration the protomers of the tetrameric molecule are dislocated by about 0.1-0.2 nm (i.e. 1-4°) relative to each other. [ABSTRACT FROM AUTHOR]

1. Dihydroorotate dehydrogenase from Zymobacterium oroticum is shown to catalyse the anti-elimination of hydrogen from the substrate. 2. In the presence of a catalytic amount of NAD+ the enzyme catalyses the exchange between the abstractable hydrogen atoms of dihydroorotate and solvent protons, the exchange of (5S)-H atom being twice as fast as that of the 4-H atom. 3. Sodium-ethoxide-catalysed exchange affects both diastereotopic protons in the methylene group of dihydroorotate, the (5S)-H atom being exchanged somewhat faster than the (5R)-H atom. 4. These findings are d in terms of stereospecificity and mechanism of flavin dependent dehydrogenase reactions. [ABSTRACT FROM AUTHOR]

1. in this study, the hepatic concentrations of free and acetylated CoA and carnitine, and also hepatic redox state, have been determined in normal lactating cows and in lactating cows to which voren (dexamethasone 21-pyridine-4-carboxylate) was administered 48 h previously. 2, The values for the hepatic [CoA]/[acetyl-CoA] and [carnitine]/[acetylcarnitine] ratios in the normal cow were found to be lower than the corresponding values previously reported for other species. Voren administration brought about an increase in both these ratios Only the increase for the [CoA]/[acetyl-CoA] ratio was statistically significant, however. 3. Using the lactate dehydrogenase and glutamate dehydrogenase systems, respectively, values were obtained for the cytosolic and mitochondrial [NAD+]free/[NADH]free ratios in normal bovine liver that were similar to the corresponding values previously obtained for rat liver. Following voren administration there was a fall in the cytosolic value for this ratio and a rise in the mitochondrial value, so that the equilibrium between the cytosolic and mitochondrial redox states appeared to be disrupted 4. Following voren administration there was an increase in the degree of reduction of total NAD. 5. The results are discussed in relation to the antiketogenic action of voren in the cow. [ABSTRACT FROM AUTHOR]

By studying the glutamate dehydrogenase catalyzed oxidation of glutamic acid under conditions where the concentration of enzyme exceeded that of NAD, it was possible to evaluate directly the first-order rate constant (0.73 sec-1) for the decay of an intermediate associated with the burst in absorbance, which is seen at 340 nm in the pre-steady state phase when the NAD concentration exceeded that of enzyme. A study of the wavelength dependence of the absorbance vs time plots when [enzyme]>[NAD] indicated that the intermediate associated with the burst is isosbestic with the ternary abortive complex E · Glu · NADH. This information made it possible to estimate the total amount of NADH (π) formed in the initial rapid phase when [NAD] > [enzyme]. A study of the dependence of the pre-steady-state kinetics on [NAD] revealed that between 27 and 1000 µM NAD, π was invariant at 0.4 µmol NADH formed in the burst phase per polypeptide chain. However, the velocity of the phase immediately following the burst phase increased between 27 and 1000 µM NAD, and the pseudo, first-order rate constant for the burst phase increased between 30 and 300 µM NAD and decreased between 300 and 1000 µM NAD. These results were taken to mean that all the NAD sites on glutamate dehydrogenase are not catalytically equivalent and that binding of NAD to sites which are not catalytically active causes a decrease in the rate of the burst phase and an increase in the rate of the phase following the burst. Below 1000 µM glutamate, a pronounced lag in the onset of the burst phase is seen. This lag suggests that at low glutamate concentration binding of NAD and/or glutamate to the enzyme becomes slower than reduction of E · Glu · NAD. [ABSTRACT FROM AUTHOR]

3-Hydroxy-3-methyl-[3-14C]glutaryl-CoA was reduced to mevalonic acid in the presence of either [4A-³H]- or [4B-³H]NADPH and rat liver 3-hydroxy-3-methylglutaryl-CoA reductase. The resulting doubly labelled mevalonic acid incorporated tritium exclusively from the 4A (4R) position of NADPH. The distribution of tritium at C-5 of the mevalonic acid was determined by a biological degradation. It was concluded that during the reduction of 3-hydroxy-3-methylglutaryl-CoA to mevalonic acid two hydrogen atoms from the 4A (4R) position of NADPH are transferred directly to mevalonic acid. [ABSTRACT FROM AUTHOR]

D-Mannonate : NAD oxidoreductase of Escherichia coli K12 has been purified. The purification consists of four steps: precipitation of nucleic acids with protamine sulfate; ammonium sulfate precipitation; ion-exchange chromatography on DEAE-cellulose and Sephadex G-200 gel filtration. The overall procedure results in a 53-fold purification with a yield of 70%. Mannonate oxidoreductase is an inducible enzyme: glucuronate and fructuronate are good inducers, galacturonate is not. Optimal activity is obtained at pH 6.3 fro fructuronate reduction, and at pH 8.4 for mannonate oxidation. The apparent Michaelis constants for the purified enzyme have been determined: for fructuronate, the Km is 0.5 mM; for NADH 0.03 mM; for mannonate, 1.8 mM and for NAD+, 0.2 mM. Mannonate oxidoreductase and altronate oxidoreductase have been separated on DEAE-Sephadex in order to study mannonate-oxidoreductase specificity. After separation, mannonate oxidoreductase is found to be active with both fructuronate and tagaturonate as substrates. Kinetic studies, differential inhibition of activities by p-chloromercuribenzoate and thermal inactivation studies confirm this double specificity. Other carbohydrates tested are unsuitable as substrates. Mannonate oxidoreductase is active with NADPH as coenzyme and the apparent Km value for NADPH is 0.7 mM. Kinetic inhibition studies of the enzyme by direct and reverse reactional products suggest a random mechanism for mannonate oxidoreductase. Enzyme inhibition specificity appears to be restricted. The value of the energy of activation is 34 kJ/mol (8 kcal/mol); the Q10 is 1.5. The results of thermal inactivation of mannonate oxidoreductase (53 °C) indicate homogeneous-enzyme activity fructuronate in the crude extract. In the absence of inducer, basal activity is dependent on mannonate oxidoreductase only. Thermal inactivations also indicate that the same enzyme, mannonate oxidoreductase, catalyzes the direct reactions with NADH or NADPH, and the reverse reaction. Enzyme substrates, such as NADH, NADPH, fructuronate, tagaturonate, NAD+, mannonate and altronate protect enzymatic activity from degradation by heat. NADH in particular, is effective as a stabilizer; this ability is proportional to its concentration. p-Chloromercuribenzoate is an active inhibitor: at 5 μM, activity is 100% inhibited. Inhibition is non-competitive, with an apparent Ki of 0.1 μM. The end product of the reaction of mannonate oxidoreductase with fructuronate has been identified by means of paper chromatography as D-mannonate. [ABSTRACT FROM AUTHOR]

The binding of NADH to yeast glyceraldehydes-3 phosphate dehydrogenase has been compared with that of NAD+ under otherwise identical conditions (i.e., pH 8.5 and 20 °C). The binding of NAD+ is weakly sigmoidal. Kinetic data obtained with chemical relaxation and rapid-mixing techniques are consistent with the concreted mechanism for the cooperative binding of NAD+. In particular, the apo-enzyme consists of a mixture of two states (90% with low affinity and 10% with high affinity for NAD+) at equilibrium with each other. In contrast to NAD+, the binding of NADH is hyperbolic. Only one relevant relaxation process was observed in the 0.1 msec range. Since the reciprocal relaxation time increase with increasing concentration of NADH, the reaction can be identified with an elementary binding reaction. NADH and NAD+ compete for the same binding site. Rapid displacement of NADH by NAD+ has been used as a diagnostic tool for the ability of NADH to shift the allosteric pre-equilibrium. This technique and the rapid titration of sulfhydryl groups with 5,5′-dithio-his-(2-nitrobenzoic acid) establish that NADH binds with equal affinity to both allosteric states of yeast glyceraldehydes-3-phosphate dehydrogenase. This finding accounts for the relatively simple relaxation spectrum of NADH binding. These and other data from the literature can be rationalized on the basis of separate subsites for the adenine and nicotinamide moieties of NAD+, the accessibilities of which differ in the two allosteric states. The possibility of a regulation of the fraction of enzymically active yeast glyceraldehydes-3-phosphate dehydrogenase by the NADH/NAD+ ratio is discussed. [ABSTRACT FROM AUTHOR]

D-Altronate: NAD-oxidoreductase of Escherichia coli K12 has been purified. The purification consists of four steps: nucleic acids precipitation with protamine sulfate; ammonium sulfate precipitation; ion exchange chromatography on DEAE-cellulose and Sephadex G-150 gel filtration. The overall procedure results in a 53-fold purification with a yield of 60%. Altronate oxidoreductase is an inducible enzyme: galacturonate, tagaturonate, glucuronate and fructuronate are good inducers. The activity remains stable at 4 °C or at — 20 °C for several months. Optimal activity is obtained at pH 6.3 for tagaturonate reduction, and at pH 8.9 for altronate oxidation. Altronate oxidoreductase does not require any cofactor, except NADH. The apparent Michaelis constants have been determined: for tagaturonate, the Km is 350 μM; for NADH, 60 μM; for altronate, 90 μM, and for NAD+, 110 μM. Moreover, altronate oxidoreductase is active with NADPH as coenzyme and the apparent Km value for NADPH is 400 μM. Other carbohydrates tested are unsuitable as substrates. Many other carbohydrates tested do not prove to be inhibitors. The value for the energy of activation is 38 kJ/mol (9 kcal/mol); the Q10 is 1.5. The results of heat inactivation of altronate oxidoreductase (58.5 °C) indicate homogeneous enzyme activity in crude extract. Thermal inactivations also indicate that the same enzyme, altronate oxidoreductase, is induced by galacturonate and glucuronate, and catalyzes the direct reactions with NADH or NADPH. Finally, it has been confirmed by heat inactivation that there is no interference between specific assays of altronate and mannonate oxidoreduetases. Compounds such as NADH, NADPH, altronate and mannonate protect enzymatic activity from degradation by heat, whereas NAD+, tagaturonate and fructuronate do not. NADH, in particular, is effective as a stabilizer; this ability is proportional to its concentration. Enzyme inhibition with para-chloromercuribenzoate is non-competitive and the apparent Ki is 1 mM. para-Chloromercuribenzoate also inactivates altronate oxidoreductase indicating homogeneous activity and specificity of the assay method in crude extracts. Chromatographic properties of the enzyme are identical whether the inducer is galacturonate or glucuronate. Using paper chromatography, the reaction product of enzyme action on D-tagaturonate is identified as D-altronate. [ABSTRACT FROM AUTHOR]

1. Kinetic studies performed with naturally occurring pyridine nucleotides and their thioanalogues point to the existence of a ping-pong bi-bi mechanism for the NADH → TNAD+ and NADPH → (T) NAD+ reactions. Inhibition studies with NAD+ and NADP+, however, exclude this type of mechanism on the basis of the relations found. 2. A ternary complex mechanism is proposed in which the enzyme is first reduced to the four-equivalent reduced state by two moles of donor, before two moles of acceptor are bound to the reduced enzyme; the possibility of complex formation of the oxidized enzyme with NADP+ is included in the scheme. The kinetic patterns of the NADPH → TNAD+ reaction in the absence and presence of NADP+ can be explained by this mechanism. 3. The explanation of the kinetic patterns of the NADH → TNAD+ reaction can be given by slightly modifying the proposed mechanism in such a way that the sequence of addition of the first acceptor molecule and the second donor molecule is changed and assuming irreversible binding of the first acceptor molecule to the two-equivalent reduced enzyme. The high affinity of NAD+ for the reduced enzyme justifies the latter assumption. 4. The reduction of NADP+ and TNAD(P) + proceeds due to the reversible binding of both substrates according to a rapid-equilibrium-random-bi-bi mechanism. The data of the reaction NADH → NADP+ and the NADP+ inhibition patterns of the reaction NADPH → TNADP+ are consistent with this mechanism. [ABSTRACT FROM AUTHOR]