Pyridine Nucleotide-linked Dehydrogenases Research Articles

The effect of calcium chelators on microsomal pyridine nucleotide-linked dehydrogenases of sugarbeet cells

Cell suspension cultures of Beta vulgaris L., treated with calcium chelators or untreated, were used to characterize pyridine nucleotide-dependent diaphorases of microsomes. The microsomal activity of NADH-dependent duroquinone reductase from cultures treated with 10 mM Na 2 EGTA for 24 h increased by a factor of 1.8 with respect to control microsomes, and was mainly associated with particles of d=1.17 g ml -1 . NADPH-duroquinone reductase and NADH-ferricyanide reductase activities showed smaller increases. Bacterial protein-lipopolysaccharide complexes (prLPS) also promoted the increase of microsomal diaphorases; CaEGTA was ineffective. EGTA effects on enzymes of supernatant and mitochondria were negligible, although Na 2 EGTA treatment induced cell aggragation and strong acidification of the medium

A large-scale preparative electrophoretic method for the purification of pyridine nucleotide-linked dehydrogenases

A large-scale preparative electrophoretic method for the purification of pyridine nucleotide-linked dehydrogenases

A large-scale preparative electrophoretic method for the purification of pyridine nucleotide-linked dehydrogenases

A large-scale preparative polyacrylamide gel electrophoresis (PAGE) method that uses a 1.5- or a 2.0-cm-thick slab gel has been developed for the purification of NAD-dependent dehydrogenases. With the 2.0-cm-thick gel, a maximum volume (up to about 160 ml) of enzyme sample was applied to a gel plate, resulting in the application of a large amount of protein and enzyme. After the electrophoretic run, the enzyme band on the gel was detected by activity staining and recovered from the gel by extraction with a fairly loose-fitting glass-Teflon homogenizer. NAD-dependent alanine dehydrogenase, leucine dehydrogenase, and glycerol dehydrogenase were purified in high yields (more than 80%) by the preparative PAGE method. The method can be carried out using a simple slab gel apparatus, which is modified from the conventional analytical apparatus for the purpose of preparative PAGE under conditions used for routine analytical runs. Thus, the method may be suitable for use in purifying NAD(P)-dependent dehydrogenases and many other enzymes after conventional chromatography such as dye-ligand affinity chromatography or ion-exchange chromatography.

Native gel activity stain and preparative electrophoretic method for the detection and purification of pyridine nucleotide-linked dehydrogenases

An activity stain for the detection of pyridine nucleotide-linked dehydrogenases in polyacrylamide gels is described. Following incubation of the gel with substrate and cofactor, bands are visualized under ultraviolet light, where reduced cofactors fluoresce and oxidized cofactors appear black. The methods described are useful for any NAD- or NADP-linked dehydrogenase; the enzymes can be assayed in either the oxidative or the reductive direction. Also described is a preparative polyacrylamide gel system using the activity stain, which can be used as a general purification method for dehydrogenases. The preparative gels are crosslinked with bisacrylylcystamine. These crosslinks can be broken by the addition of thiols after the bands of interest have been located and excised. The protein of interest is then separated from the solubilized acrylamide by adsorption to a suitable resin.

Vitamin A and biochemical development. II. Effect of postnatal excess vitamin-A administration on the activity of pyridine nucleotide-linked dehydrogenases.

The effect of excess vitamin A, (1,000 IU) administration to rat pups on the 4th, 6th, 8th and 10th days of age was studied on the activities of combined HMP dehydrogenases, malic enzyme and isocitrate dehydrogenase on the 11th, 17th and 23rd days of age. The developmental pattern and the effect of vitamin-A administration differs from enzyme to enzyme and organ to organ.

Fluorimetric assay of tobacco leaf dehydrogenases with resazurin

A versatile fluorimetric assay based on the reduction of resazurin to resorufin demonstrated high specific activities for a number of important pyridine nucleotide-linked dehydrogenases in tobacco leaves. The Michaelis constant for the important photosynthetic enzyme, d-glyceraldehyde-3-phosphate:NADP + oxidoreductase (EC 1.2.1.13), determined by the fluorimetric method, was considerably lower than constants determined by conventional extraction and assay methods reported for the enzyme from other plants. The sensitivity of the fluorimetric method enabled the use of dilute enzyme preparations with resultant low background and high substrate specificity. Inclusion of the anti-oxidant diethyldithiocarbamate in the extraction medium preserved the enzymes during extraction. Primary amines inhibited competitively, and phenazine methosulfate non-competitively each of the eight dehydrogenases tested with the fluorimetric assay. The Mn 2+ dependence of NADP-linked dehydrogenases specific for isocitrate and malate was confirmed. The method is rapid, requires a simple combination of ingredients and should be useful for surveying dehydrogenase activity in leaves.

Metabolism of 6-aminonicotinic acid in Escherichia coli.

A late-log-phase culture of an Escherichia coli nadB pncA double mutant took up 6-[7-14C]aminonicotinic acid and excreted 6-[14C]aminonicotinamide. This mutant also accumulated intracellularly several radioactive compounds which have been tentatively identified as 6-amino analogs of compounds in the pyridine nucleotide cycle. It is concluded that 6-aminonicotinamide and 6-aminonicotinic acid probably exert at least a portion of their bacteriostatic effects by being metabolized, by the enzymes of the pyridine nucleotide cycle, to 6-aminonicotinamide adenine dinucleotide and 6-aminonicotinamide adenine dinucleotide phosphate. These compounds are not electron acceptors and are known inhibitors of some pyridine nucleotide-linked dehydrogenases.

Purification and separation of pyridine nucleotide-linked dehydrogenases by affinity chromatography techniques.

A number of different dehydrogenases have been shown to bind to Sepharose-bound N(6)-(6-aminohexyl)-AMP. These dehydrogenases can be specifically eluted by binary adducts of NAD(+) or with cofactor gradients. In such manner pure enzymes can be obtained from crude extracts, as demonstrated in the purification on a preparative scale of lactate dehydrogenase from dogfish muscle. The data presented indicate the usefulness of general ligands as affinity agents. The techniques are particularly adaptable for the isolation of human mutant enzymes in blood or in the purification and concentration of enzymes present at low levels in fluids or tissues, as shown in the extensive purification of serum lactate dehydrogenase and glucose 6-phosphate dehydrogenase from hemolysate. lsoenzymes with different affinities for co-enzymes can be separated by affinity techniques. Application of affinity techniques may lead to the separation of isoenzymes or mutant enzymes that are not separable by electrophoretic methods.

Sequence homologies among pyridine nucleotide-linked dehydrogenases: Possible partial gene duplications in glyceraldehyde-3-phosphate dehydrogenase

Sequence homologies among pyridine nucleotide-linked dehydrogenases: Possible partial gene duplications in glyceraldehyde-3-phosphate dehydrogenase

Pathway of l-Sorbose Metabolism in Aerobacter aerogenes

Abstract The pathway of l-sorbose metabolism in Aerobacter aerogenes was determined to be: l-sorbose → l-sorbose 1-phosphate → d-glucitol 6-phosphate → d-fructose 6-phosphate. Phosphorylation of l-sorbose at C-1 is mediated by a phosphoenolpyruvate-dependent phosphotransferase system, whereas l-sorbose 1-phosphate reduction and d-glucitol 6-phosphate oxidation are mediated by pyridine-nucleotidelinked dehydrogenases. Mutants deficient in l-sorbose 1-phosphate reductase, d-glucitol 6-phosphate dehydrogenase, or a component (Enzyme I) of the phosphotransferase system did not grow on l-sorbose, whereas revertants which had regained these activities grew normally. Extracts of the Enzyme I-deficient mutant failed to catalyze the phosphorylation of l-sorbose with phosphoenolpyruvate, and adenosine 5'-triphosphate could not replace the phosphoenolpyruvate requirement for l-sorbose phosphorylation in extracts of the wild type strain.

Addition products of diphosphopyridine nucleotides with substrates of pyridine nucleotide-linked dehydrogenases

Preparation of the reduced and oxidized forms of addition compounds from diphosphopyridine nucleotide and a number of carbonyl compounds are described; these include pyruvate, pyruvic ethylester, oxaloacetate, α-ketoglutarate, acetaldehyde, butyraldehyde, and α-ketobutyrate. The adducts are closely related to the DPN-acetone and the DPN-dihydroxyacetone adducts, both in their chemical and spectral properties. The chemical structures of the reduced and oxidized adducts have been determined and information concerning the mechanism of formation of the adducts is presented. The lactate, malate, glutamate, and alcohol dehydrogenases show a unique specificity for the reduced adducts; the specificity was found to be related to the normal oxidized substrate utilized by the various enzymes. The inhibition is competitive with DPNH and noncompetitive with the substrate. Oxidized adducts produce considerably less inhibitory effects than the reduced adducts. Evidence is presented showing that the reduced adducts bind to the enzymes in a manner similar to that of the reduced coenzyme. On the basis of the high selectivity of the dehydrogenases for the adducts, a model is proposed which shows the spatial relationship between the reduced coenzyme and the oxidized substrate on the enzyme in the transition state. From this model, a general reaction mechanism is postulated for the pyridine nucleotide-dependent dehydrogenases, which may explain the stereo-specificity of the hydrogen transfer with respect to the coenzyme as well as the substrate.

Structural similarities within groups of pyridine nucleotide-linked dehydrogenases

If the pyridine nucleotide-linked dehydrogenases are related by evolution from one or a few common ancestral genes, it might be possible to suggest special evolutionary relationships among particular dehydrogenases by a comparison of structural and functional properties, even before extensive comparative primary sequence information is available. The dehydrogenases are compared using their amino acid compositions, isoelectric points, subunit molecular weights, and some functional properties. Amino acid compositions of dehydrogenases are tabulated per thousand total residues. The comparison of compositions is facilitated by using the difference index method developed by Metzger. The methods of comparison are evaluated using cytochromes c, proteases, and glyceraldehyde-3-phosphate dehydrogenases. Glutamate and glucose-6-phosphate dehydrogenases appear to be similar in many respects and mutually distinct from the other dehydrogenases. Similarities also appear to exist among the low molecular weight dehydrogenases: liver alcohol, mitochondrial malate, glycerol-3-phosphate, and NADP-linked isocitrate dehydrogenases. The Metzger difference index suggests that the primary sequence homology between H- and M-type lactic dehydrogenase subunits may be very extensive, perhaps being 75% identical.

The formation of ternary complexes by diphosphopyridine nucleotide-dependent dehydrogenases

Formation of abortive ternary complexes between enzyme, coenzyme, and substrate has been demonstrated for several pyridine nucleotide-dependent dehydrogenases by measuring the quenching of protein fluorescence. Evidence is presented suggesting the participation of a ternary complex consisting of lactate dehydrogenase, DPN +, and pyruvate in the phenomenon of substrate inhibition observed with lactate dehydrogenases. This complex is more readily formed with the H-type LDH than with the M-type enzyme under identical conditions. The ternary complex of LDH-DPN +-pyruvate can be formed at physiological levels of pyruvate. Information concerning the structure of the ternary complex has been derived from its absorption and fluorescence spectra, as well as from its kinetic and chemical properties. The pyridine ring in the complex appears to be in the reduced form, similar to that found in DPNH. The ternary complex can be precipitated with ammonium sulfate. Denaturation of the enzyme results in a product which is easily oxidized. Evidence is presented indicating that this product consists of DPN which is covalently bonded to pyruvate. Similar products can be obtained with analogs of DPN, but the reaction for lactate dehydrogenase is specific for pyruvate. Related ternary complexes have been observed from malate dehydrogenase, glutamic dehydrogenase, and alcohol dehydrogenase. The significance of ternary complex formation is discussed in relation to the reaction mechanism of pyridine nucleotide-linked dehydrogenases.

Activity of isocitric dehydrogenase in the fat bodies of the cockroach, Leucophaea maderae (F).

ISOCITRIC dehydrogenase, an enzyme of the tricarboxylic acid cycle, is responsible for the formation of α-ketoglutarate from isocitrate. During the reaction, oxalosuccinate is formed (as an intermediate) owing to the presence of two pyridine nucleotide-linked dehydrogenases in biological systems. One dehydrogenase is linked with NAD and the other is linked with NADP. They first catalyse the oxidation of isocitrate, and then its decarboxylation in the presence of the manganous ion. If NADP is used in the reaction, it is reduced and, because of the reversibility of the process, carbondioxide is fixed in oxalosuccinate and isocitrate. Isocitric dehydrogenase has been isolated from heart tissue of the pig1, yeasts2, and various animal tissues3. This enzyme was discovered in the supernatant fraction of the homogenates of the fat bodies of the cockroach, Periplaneta americana4,5. Its presence has been reported in the haemolymph of the mealworm, Tenebrio molitor, by Prota (see previous communication). Hence, the purpose of this investigation was twofold: to determine whether this enzyme could be detected in normal and aposymbiotic fat bodies of the cockroach, Leucophaea maderae; and to determine whether the presence of the enzyme in this tissue is due to the action of the tissue, the symbionts or both.

Mitochondrial Isocitrate Dehydrogenases from Yeast

Abstract A polarographic technique suitable for kinetic studies of either isolated or bound pyridine nucleotide-linked dehydrogenases was used to investigate the isocitrate dehydrogenases of yeast mitochondria. Intact mitochondria were found to contain both triphosphopyridine nucleotide- and diphosphopyridine nucleotide-linked isocitrate dehydrogenases with properties very similar to those which have been reported for the isolated dehydrogenases. The DPN-linked enzyme in situ exhibits a nonclassical, sigmoid substrate against activity relationship with respect to isocitrate and is activated by adenosine 5'-monophosphate. The TPN-linked enzyme in situ exhibits a hyperbolic substrate against activity relationship which is unaffected by AMP. The DPN-linked dehydrogenase also shows another non-classical increase in activity that occurs in response to high isocitrate concentrations or to heat treatment. This activation is characteristic only of untreated, intact mitochondria and may be secondary to an alteration of the mitochondria themselves.

Enzyme-coenzyme complexes of pyridine nucleotide-linked dehydrogenases.

Enzyme-reduced coenzyme binary complexes produce previously unreported shifts in the spectrum of the free coenzyme. These shifts give rise to difference spectra which resemble a general environmental change for reduced diphosphopyridine nucleotide (DPNH) in the glutamic dehydrogenase-DPNH complex, and indicate a more specific enzyme-coenzyme interaction for yeast alcohol dehydrogenase-DPNH, isocitrate dehydrogenase-TPNH, and lactic dehydrogenase-DPNH complexes.

An evaluation of the histochemical demonstration of certain pyridine nucleotide-linked dehydrogenases.

Farber, Sternberg and Dunlap1,2 have questioned the validity of histochemical techniques for the demonstration of pyridine nucleotide-linked dehydrogenases. They have argued that since the actual reduction of the tetrazole results from its interaction not with a dehydrogenase but with a diaphorase (tetrazole reductase) specific for NADH or NADPH, strictly speaking the reactions are specific only for NADH- or NADPH-tetrazole reductase. Qualitative evidence was presented to show that of five pyridine nucleotide-linked dehydrogenases studied in rat kidney, only two staining patterns emerged, depending on whether the dehydrogenase required NAD+ or NADP+ in the reaction medium. While admitting that the tetrazole reductase was responsible for the actual reduction of tetrazole at the expense of reduced pyridine nucleotide, other workers3,4 have concluded from similar experiments that each dehydrogenase has its own characteristic pattern of distribution. This point of view has been extensively accepted, but anomalies of formazan localization within the cell5,6 and the observed escape of soluble dehydrogenases into the incubation medium7–12 support the original conclusions of Farber and his colleagues.

Coenzyme binding to glyceraldehyde-3-phosphate dehydrogenase after modification by substrate and coenzyme

Summary 1. The coenzyme-binding characteristics of glyceraldehyde-3-phosphate dehydrogenase ( D -glyceraldehyde-3-phosphate:DPN+ oxidoreductase(phosphorylating), EC 1.2.1.12) are altered when the enzyme is incubated with DPNH or DPNH and glyceraldehyde-3-phosphate. Extensive modification occurs only in the presence of oxygen and parallels the loss of enzymic activity. 2. The modified enzyme binds the same mole-ratio of DPNH as does enzyme which has had the DPN+ removed by mixing with charcoal, i.e. 3 moles per mole of protein. However, this binding does not quench, but rather enhances DPNH fluorescence as is the case for all other pyridine nucleotide-linked dehydrogenases thus far tested.

A quantitative study of the validity of the histochemical demonstration for pyridine nucleotide-linked dehydrogenases.

A quantitative histochemical and biochemical study has been made of the loss of pyridine nucleotide-linked dehydrogenases from frozen histological sections of rat liver. Glucose-6-phosphate, 6-phosphogluconate and lactate dehydrogenases were lost rapidly from the sections during incubation in the histochemical medium, but β-OH-butyrate dehydrogenase was lost at a much slower rate. It was shown that a dehydrogenase reaction can occur in a section lacking that particular dehydrogenase if the section is incubated in the presence of another containing the dehydrogenase. The validity of the tetrazolium reaction for demonstrating pyridine-nucleotide-linked dehydrogenases is considered in the light of these results.

GLUCOSE-6-PHOSPHATE DEHYDROGENASE ACTIVITY IN HYPERPLASTIC AND NEOPLASTIC LESIONS OF THE BREAST. A HISTOCHEMICAL STUDY.

CancerVolume 17, Issue 8 p. 1067-1072 ArticleFree Access Glucose-6-phosphate dehydrogenase activity in hyperplastic and neoplastic lesions of the breast. A histochemical study Richard B. Cohen M.D., Richard B. Cohen M.D. Department of Pathology, Harvard Medical School, and the James Homer Wright Pathology Laboratories of the Massachusetts General Hospital, Boston, Mass. 02114Search for more papers by this author Richard B. Cohen M.D., Richard B. Cohen M.D. Department of Pathology, Harvard Medical School, and the James Homer Wright Pathology Laboratories of the Massachusetts General Hospital, Boston, Mass. 02114Search for more papers by this author First published: August 1964 https://doi.org/10.1002/1097-0142(196408)17:8<1067::AID-CNCR2820170813>3.0.CO;2-ECitations: 17AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 Hershey, F. B.; Lewis, C.; Johnston, G., and Mason, S.: Hormonal effects on glucose-6-phosphate dehydrogenase of mouse mammary gland. J. Histochem. 11: 218– 223, 1963. 2 Hershey, F. C.; Lewis, C., Jr.; Johnston, G., and Murphy, J.: Phosphopyridine nucleotide linked dehydrogenases, aldolase and fumarase in mouse breast. J. Histochem. 11: 224– 231, 1963. 3 Marks, P. A., and Banks, J.: Inhibition of mammalian glucose-6-phosphate dehydrogenase by steroids. Proc. Nat. Acad. Sc. 46: 447– 452, 1960. 4 McLean, P.: Carbohydrate metabolism of mammary tissue; I, pathways of glucose catabolism in mammary gland. Biochem. et biophys. acta 30: 303– 315. 1958. 5 Pearse, A. G. E.: Histochemistry: Theoretical and Applied. 2d ed. Boston. Mass. Little. Brown and Company. 1960. 6 Rees, E. D., and Huggins, C.: Steroid influences on respiration, glycolysis, and levels of pyridine nucleotide-linked dehydrogenases of experimental mammary cancers. Cancer Res. 20: 963– 971, 1960. Citing Literature Volume17, Issue8August 1964Pages 1067-1072 ReferencesRelatedInformation