Reduced Diphosphopyridine Nucleotide Research Articles

An enzymatic fluorometric micromethod for the determination of glycerol

A simple and rapid method was developed for determining glycerol in plasma or whole blood in a sample of 0.1 ml. The method was a fluorometric adaptation of existing spectrophotometric methods with enzymatic conversion of glycerol when reduced diphosphopyridine nucleotide is formed in proportion to the glycerol concentration in the sample. The method requires only capillary samples and is therefore suitable for repeated registration of glycerol during experimental conditions.

Isolation and Properties of Aromatic α-Keto Acid Reductase

Abstract An enzyme which catalyzes the reduction of aromatic α-keto acids to phenyllactic acids has been isolated from beef and dog heart. This enzyme has been designated aromatic α-keto acid reductase. Reduced diphosphopyridine nucleotide is a required cofactor for aromatic α-keto acid reductase activity. Substrates for aromatic α-keto acid reductase include 3,5-diiodo-p-hydroxyphenylpyruvic acid, p-hydroxyphenylpyruvic acid, and phenylpyruvic acid. 3,5-Diiodo-p-hydroxyphenylpyruvic acid has a high affinity for the enzyme (Km of 4.3 x 10-5 m). The enzyme is present in many mammalian tissues. These include heart, skeletal muscle, kidney, adrenal, liver, thyroid, and blood.

Partial Resolution of the Enzymes Catalyzing Oxidative Phosphorylation: VII. OXIDATIVE PHOSPHORYLATION IN THE DIPHOSPHOPYRIDINE NUCLEOTIDE-CYTOCHROME b SEGMENT OF THE RESPIRATORY CHAIN: ASSAY AND PROPERTIES IN SUBMITOCHONDRIAL PARTICLES

Abstract 1. A spectrophotometric procedure is described which specifically measures oxidative phosphorylation in submitochondrial particles in the diphosphopyridine nucleotide-cytochrome b segment of the respiratory chain. It is based on the reduction of exogenous coenzyme Q1 by reduced diphosphopyridine nucleotide catalyzed by phosphorylating submitochondrial particles from beef heart and rat liver. 2. Previously described methods for assaying formation of adenosine triphosphate at the first site of oxidative phosphorylation were found to be unsatisfactory. 3. Both the reduction of coenzyme Q1 and the accompanying formation of adenosine triphosphate in this system are unaffected by antimycin and KCN but are almost completely abolished by rotenone or Amytal. 4. The reduction of coenzyme Q1 by succinate is not accompanied by phosphorylation. 5. A survey of various electron acceptors revealed that oxidative phosphorylation at the first phosphorylation site was only observed if electrons passed through the rotenone-sensitive site. 6. In submitochondrial particles from beef heart, octylguanidine, phenethylbiguanidine, and dicumarol failed to exhibit a clear-cut site specificity of action. 7. Addition of the cold-labile, soluble adenosine triphosphatase (F1) to deficient submitochondrial particles stimulated oxidative phosphorylation at all three sites in the respiratory chain.

1-Phosphofructokinase from an Anaerobe

Abstract A soluble 1-phosphofructokinase has been extracted from anaerobic bacteria (Bacteroides symbiosus) and sufficiently purified to reveal its mode of action. The enzyme requires a nucleoside 5'-triphosphate (adenosine, inosine, guanosine, or uridine triphosphate) and magnesium or manganous ions. It has been obtained free from hexokinase, d-glyceraldehyde kinase, aldolase, and reduced diphosphopyridine nucleotide oxidase. Sorbose-1-P and fructose-6-P are neither substrates nor inhibitors for the enzyme. A spectrophotometric method for the estimation of fructose 1-phosphate is described. It is recommended that hereafter the phosphofructokinases be clearly identified as to their substrate specificities. The previously reported finding of 6-phosphofructokinase in B. symbiosus was not confirmed.

Beef Heart Malic Dehydrogenases: IV. FLUORESCENT BINARY AND TERNARY COMPLEXES OF THE SUPERNATANT ENZYME

Abstract The stoichiometry of reduced diphosphopyridine nucleotide binding to supernatant malic dehydrogenase (S-MDH) was determined by fluorometric techniques and a Sephadex gel filtration procedure. Both types of measurement yielded a stoichiometry of 1 mole of DPNH bound per mole of enzyme. This stoichiometry was unaffected by the presence of d-malate, a compound which is known to form a ternary enzyme complex having distinctive fluorescent properties. The dissociation constants for the binary enzyme-DPNH and ternary enzyme-DPNH-d-malate complexes with respect to DPNH were determined. Several criteria were used to determine the effectiveness of a number of compounds in forming ternary complexes with S-MDH and DPNH. A requisite for ternary complex formation was a dicarboxylic acid with chain length of 3 to 4 carbon atoms. Although an α-hydroxyl group was not necessary for ternary complex formation, the introduction of such a group in a specific steric configuration resulted in a loss of capacity to form such a complex. Formation of strong fluorescing complexes by a number of dicarboxylic acids is not necessarily related to their effectiveness as inhibitors of S-MDH activity. The presence of an α-hydroxyl group appears to be required for inhibition, although added steric considerations involving the β carbon atom may obliterate such inhibitory capacity.

Electron-transport systems of yeast. II. Purification and properties of a soluble reduced diphosphopyridine nucleotide dehydrogenase.

Electron-transport systems of yeast. II. Purification and properties of a soluble reduced diphosphopyridine nucleotide dehydrogenase.

Transhydrogenation in Root Tissue: Mediation by Carbon Dioxide

The C(14)O(2) incorporated into organic acids by excised corn roots is released with an estimated halftime of 3 hours. Homogenates of root tissue can mediate these reactions by coupling, in series, phosphoenolpyruvate carboxylase, diphosphopyridine nucleotide malic dehydrogenase, phosphopyridine nucleotide malic dehydrogenase. An important consequence is the transfer of hydrogen from reduced diphosphopyridine nucleotide to triphosphopyridine nucleotide which is then reduced.

An experimental evaluation of a histochemical diagnosis of burn depth

<h2>Summary</h2> Preliminary findings are reported on the effects of linear burns on rat skin, using neotetrazolium chloride and reduced diphosphopyridine nucleotide as histochemical criteria for the detection of DPNH dehydrogenase activity in burned tissues. The appearance of a red band of cells in the injured dermis, immediately following thermal injury, as detected by this technique, may be of particular relevance to the rapid early diagnosis of burn depth. Above this band the dermal and epithelial tissue has been irreparably damaged and is destined to be sloughed; below the band, the dermal tissue unless further insulted will undergo repair. If epidermal elements (pilosebaceous units) are present below the band, resurfacing is greatly facilitated.

Mental Retardation in Methemoglobinemia Due to Diaphorase Deficiency

SEVERAL specific genetic lesions may result in congenital methemoglobinemia. Abnormal hemoglobins with mutations affecting alpha or beta globin near the site of heme attachment are found in most cases with a dominant mode of transmission.1 The biochemical defect in most cases of recessively inherited congenital methemoglobinemia is an almost complete deficiency of erythrocyte dihydronicotinamide adenine dinucleotide (NADH) or reduced diphosphopyridine nucleotide (DPNH), diaphorase activity.2 , 3 This enzyme functions with NADH as the principal agent reducing methemoglobin, which is constantly produced physiologically by the oxidation of hemoglobin. Gibson4 established the site of the metabolic defect and suggested that the reductase was a . . .

Electrochemical Reduction of Diphosphopyridine Nucleotide*

Electrochemical Reduction of Diphosphopyridine Nucleotide^*

Comparative carbohydrate catabolism and methemoglobin reduction in pig and human erythrocytes

AbstractPig red cells were characterized structurally and chemically, the rates of certain of their carbohydrate metabolic pathways were determined, the capacity to utilize these pathways for methemoglobin reduction was measured, and the results were compared with similar studies in human erythrocytes. Pig cells are smaller, contain less hemoglobin but are similar to human cells in mean cellular hemoglobin concentration. Unlike most other mammalian red cells, including human erythrocytes, pig cells are glucose “free” or nearly so; plasma concentrations of glucose are similar in both species. On a per cell basis, the content of adenosine triphosphate and reduced glutathione is similar in both species. Pig red cells catabolize glucose at about one‐tenth the human rate; for each mole of glucose consumed, two moles of lactate are formed. In both species, a similar proportion (5 to 10%) of the total glucose catabolized passes via the phosphogluconate oxidative pathway. Both species form lactate from inosine at similar rates; the pathways involved appear similar. Pig hemolysates prepared in water form lactate from glucose, glucose‐6‐phosphate, or inosine; freeze‐thawing destroys this potential. Methemoglobin reduction rates of red cells suspended in plasma are similar in both species. With human cells the plasma glucose concentration accounts for the rate; with pig cells, the lactate level appears responsible. Cells of both species apparently link methemoglobin reduction to reduced diphosphopyridine nucleotide generated via Embden‐Meyerhof glycolysis but can couple reduced triphosphopyridine nucleotide, generated via the phosphogluconate oxidative pathway, to methemoglobin reduction as well.

CONTENT AND SYNTHESIS OF HEPATIC PYRIDINE NUCLEOTIDES IN FETAL AND NEONATAL RABBITS.

The concentration and the synthesis of pyridine nucleotides in the liver and placenta of the rabbit have been studied as a function of developmental age. The concentration of total hepatic pyridine nucleotides increased progressively during fetal and neonatal development, reaching a maximal level in the adult period. This increase was due primarily to elevation in diphosphopyridine nucleotide (DPN) and reduced triphosphopyridine nucleotide (TPNH). In the postmature state, hepatic reduced diphosphopyridine nucleotide (DPNH) and triphosphopyridine nucleotide (TPN) levels were less than in term or 5-day-old rabbits. The concentration of total pyridine nucleotides in the placenta diminished during gestation. Following parenteral administration of nicotinamide to the pregnant rabbit, the concentration of nicotinamide increased to the same extent in maternal and fetal blood plasma and the total pyridine nucleotide concentration increased in both maternal and fetal liver. The latter increment was greater in maternal than in fetal liver, and in fetal liver was greatest at term. The increase was due largely to increase in DPN in the developing fetus and neonate, and in the adult rabbit to increase in all pyridine nucleotides.

Metabolic Control Phenomena Involved in Damped Sinusoidal Oscillations of Reduced Diphosphopyridine Nucleotide in a Cell-free Extract of Saccharomyces carlsbergensis

Metabolic Control Phenomena Involved in Damped Sinusoidal Oscillations of Reduced Diphosphopyridine Nucleotide in a Cell-free Extract of Saccharomyces carlsbergensis

Phase relationship of glycolytic intermediates in yeast cells with oscillatory metabolic control

The phase relations of reduced diphosphopyridine nucleotide (DPNH) and other oscillating intermediates of yeast cells in the aerobic-anaerobic transition are analyzed by the phase plane plot. Adenosine-5′-diphosphate and -monophosphate are roughly in phase with DPNH, insofar as they precede it somewhat. Fructose-1,6-diphosphate, dihydroxyacetone phosphate, and glyceraldehyde-3-phosphate also follow the changes in DPNH, but their action is slightly retarded Pyruvate, which reacts with FDP at the beginning, distinctly comes into phase with DPNH in the second period. These data indicate that neither feedback mechanisms nor the kinetics of phosphofructokinase (PFK) alone can explain the observed fluctuations of glycolytic intermediates. A hypothesis is discussed, which takes into account the limited turnover at the pyruvate decarboxylase step, the susceptibility of GAPDH against product as well as substrate inhibition, together with the well-known kinetics of PFK.

RED-CELL ENZYMES AND CO-ENZYMES IN NON-SPHEROCYTIC CONGENITAL HAEMOLYTIC ANAEMIAS.

The non‐spherocytic congenital haemolytic anaemias were classified on haematological grounds by Selwyn and Dacie (1954) into two main types—Type I and Type II. It now seems that Type I cases probably form a heterogeneous group while the Type II cases are probably a homogeneous group (de Gruchy, Santamaria, Parsons and Crawford, 1960). Robinson, Loder and de Gruchy (1961) presented evidence of impairment of cell metabolism in both Type I and Type II cases and suggested that in Type II cases there was an enzymatic defect in glycolysis beyond the stage of 2,3‐diphosphoglycerate (2,3‐DPG) synthesis. This was confirmed by the demonstration by Tanaka, Valentine and Miwa (1962) of a pyruvate kinase (PK) deficiency in Type II cases.This paper reports observations in a number of patients with Type I or Type II non‐spherocytic haemolytic anaemia on (a) the activities of the enzymes of the Embden–Meyerhof glycolytic pathway, (b) the activities of the enzymes glucose‐6‐phosphate dehydrogenase and glutathione reductase, (c) the values of the co‐enzymes diphosphopyridine nucleotide (DPN) and reduced diphosphopyridine nucleotide (DPNH) and (d) the values of reduced glutathione. The significance of the findings is discussed together with their relevance to the classification of these anaemias.

Kinetic Studies on Reduced Diphosphopyridine Nucleotide Dehydrogenase by Electron Paramagnetic Resonance Spectroscopy

Kinetic Studies on Reduced Diphosphopyridine Nucleotide Dehydrogenase by Electron Paramagnetic Resonance Spectroscopy

Oxidation of Reduced Diphosphopyridine Nucleotide by Tetrahymena pyriformis Preparations

Oxidation of Reduced Diphosphopyridine Nucleotide by Tetrahymena pyriformis Preparations

OXIDATION OF EXTRAMITOCHONDRIAL DIPHOSPHOPYRIDINE NUCLEOTIDE BY VARIOUS TISSUES OF THE MOUSE.

Exogenous reduced diphosphopyridine nucleotide was not oxidized by mitochondria but by cytoplasmic hydrogen-accepting systems catalyzed by lactic, alpha-glycerophosphate, and malic dehydrogenases. The three enzymes were found in all tissues of the mouse; the amount of activity of each enzyme differed in the various tissues, however. It is suggested that each tissue has a unique pattern for oxidation of extramitochondrial diphosphopyridine nucleotide.

The Mechanism of Stimulation of the Reduced Diphosphopyridine Nucleotide Oxidase of Heart Muscle Particles and Mitochondria by Soluble Cytochrome c

The Mechanism of Stimulation of the Reduced Diphosphopyridine Nucleotide Oxidase of Heart Muscle Particles and Mitochondria by Soluble Cytochrome c

Hydrogen Transfer between Reduced Diphosphopyridine Nucleotide Dehydrogenase and the Respiratory Chain: II. AN INITIAL LAG IN THE OXIDATION OF REDUCED DIPHOSPHOPYRIDINE NUCLEOTIDE

Hydrogen Transfer between Reduced Diphosphopyridine Nucleotide Dehydrogenase and the Respiratory Chain: II. AN INITIAL LAG IN THE OXIDATION OF REDUCED DIPHOSPHOPYRIDINE NUCLEOTIDE