TPNH Oxidase Research Articles

Glutamine-binding subunit of glutamate synthase and partial reactions catalyzed by this glutamine amidotransferase.

In the course of studies on glutamine-dependent carbamyl phosphate synthetase from Aerobacter aerogenes, we purified another protein which was found to be glutamate synthase (EC 2.6.1.53). The enzyme, obtained in apparently homogeneous form (monomer molecular weight about 227,000; s(20,omega) = 17.6 S), was found to be a typical glutamine amidotransferase in that it exhibits glutaminase activity and can utilize ammonia in place of glutamine as a nitrogen donor. The enzyme also catalyzes at low rates the oxidative deamination of glutamate in the presence of TPN, and it exhibits TPNH oxidase activity. The enzyme is similar to the glutamate synthase found in Escherichia coli in that it is an iron-sulfide flavoprotein. Treatment of the enzyme with sodium dodecyl sulfate or potassium thiocyanate dissociates it into nonidentical subunits exhibiting molecular weights of about 175,000 and 51,500. The glutamine-dependent activity of the enzyme is inhibited by L-2-amino-4-oxo-5-chloropentanoic acid, but this chloroketone analog of glutamine does not affect the ammonia-dependent glutamate synthase activity. Studies with [(14)C]chloroketone show that the reagent binds to the heavy subunit only. Inhibition by the chloroketone and its binding to the heavy subunit are markedly reduced in the presence of L-glutamine. Sedimentation velocity studies carried out in potassium thiocyanate indicate that iron-sulfide and flavin sites are also located on the heavy subunit. While these studies show that glutamate synthase resembles other glutamine amidotransferases in certain of its catalytic properties, the findings indicate that the light subunit of this enzyme, in contrast to that of several other glutamine amidotransferases, does not function to bind glutamine. It is of interest that the enzyme exhibits an unusually high affinity for ammonia as compared to a number of other glutamine amidotransferases. Glutamate synthase is inhibited (competitively with respect to glutamine) by low concentrations of methionine sulfone, methionine sulfoximine, and methionine sulfoxide.

P-Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens: REACTIVITY WITH OXYGEN

Abstract p-Hydroxybenzoate hydroxylase, a flavoprotein isolated from Pseudomonas fluorescens, was anaerobically reduced and its reoxidation reactions with O2 studied with a stopped flow spectrophotometer. All effectors of this enzyme which stimulate its TPNH oxidase activity were found also to affect its reactivity with O2. Both the rates of reaction and the binding constant for O2 were enhanced by the effectors. During the reoxidation of the enzyme complexed with the substrate effector, p-hydroxybenzoate, a rapidly formed oxygenated-intermediate is observed. This intermediate is analogous to the oxygenated intermediate previously seen in the presence of 2,4-dihydroxybenzoate, another substrate effector (Spector, T., and Massey, V. (1972) J. Biol. Chem. 247, 5632–5636). It is believed that these intermediates are a hydroperoxide form of the flavin prosthetic group and are catalytic species that are produced prior to hydroxylation. When the reduced enzyme was reoxidized in its uncomplexed form, or complexed with a nonsubstrate effector (i.e. a compound which stimulates the TPNH oxidase activity but is not hydroxylated), no spectral intermediates were detected.

A function for α‐tocopherol: Stabilization of the microsomal membrane from radical attack during TPNH‐dependent oxidations

AbstractEvents accompanying electron transport in the membrane fraction of liver and other tissues have led us to propose a specific function for α‐tocopherol based on a sequence of biochemical changes we observed to occur in these membranes and on pertinent information from other laboratories. The activity of a membrane‐bound enzyme system (TPNH oxidase) which involves transport of electrons from substrate to oxygen, has been shown to promote simultaneous formation of peroxide functions on the β position polyunsaturated fatty acids (PUFA) of phospholipids in the membrane. The phospholipid peroxides then undergo a chain cleavage reaction producing phospholipids containing a variety of carbonyl moieties in the β position. The process results in marked alteration of the membrane structure. During the overall reaction α‐tocopherol present in the membrane is converted to a compound more polar than tocopheryl quinone and the conversion is dependent on the same enzymic factors promoting the phospholipid alterations. The membrane alteration process is enhanced in microsomes from animals fed diets containing relatively high levels of PUFA or diets low in α‐tocopherol, and is diminished by low levels of dietary PUFA or relatively high levels of α‐tocopherol. The experimental data indicate that enzymic electron transport associated with TPNH oxidation by the microsomal membrane involves free radical functions. The latter apparently can promote extensive peroxidative alterations of phospholipids that result in structural changes in the membrane unless adequate α‐tocopherol is present in this organelle. This system appears to be part of the microsomal drug metabolizing system.

Metabolic and bactericidal activities of human eosinophils.

Summary. Eosinophils obtained from three patients with transient eosinophilia and polymorphonuclear neutrophils(PMN) obtained from young adults with acute suppurative infections were compared with regard to their metabolic and bactericidal responses to phagocytosis. Eosinophils ingested zymosan particles and staphylococci more slowly and to a lesser extent than did PMN, and displayed a more potent oxidative response during phagocytosis which led to a greater production of intracellular hydrogen peroxide. This enhanced oxidative response was primarily due to increased activity of TPNH oxidase, a granule enzyme, rather than the soluble DPNH oxidase which mediates hydrogen peroxide production in neutrophils. Despite the enhanced peroxide production, eosinophils were less bactericidal than PMN.

Ethanol oxidation by liver microsomes: Evidence against a separate and distinct enzyme system

The TPNH dependent oxidation of ethanol by rat liver microsomes has been studied. Microsomes isolated by the usual centrifugation techniques contain TPNH oxidase, catalase and alcohol dehydrogenase (ADH). The latter two enzymes, known to be involved in ethanol metabolism, appear to be present largely as contaminants and their activity can be removed or decreased by washing. No evidence was obtained for the presence of a separate and distinct membrane bound ethanol oxidase. It is concluded that the TPNH and O 2 dependent metabolism of ethanol by microsomes can readily be explained by the coupled and combined activities of TPNH oxidase, catalase and ADH.

SOME OBSERVATIONS ON THE PROPERTIES OF CRYSTALLINE BACTERIAL LUCIFERASE*

Abstract— We describe a number of properties of our crystalline bacterial luciferase preparations, such as absorption spectra and fluorescence characteristics which are not typical of flavoproteins. Acid extraction of the enzyme or digestion with proteolytic enzymes do not lead to the liberation of flavin‐like compounds, as judged by fluorescence techniques. Other dyes such as reduced neutral red will replace FMNH2 in this system, and irradiation of the enzyme with u.v. light does not change the ratio of light production with FMNH2 and reduced neutral red. Thus, all efforts to detect flavin in our enzyme have failed, suggesting that FMN is not necessarily involved in the emitting complex. It is of interest that addition of hydrosulfite to luciferase solutions shifts the fluorescence emission to the blue and thus close to that of bioluminescence emission. Separations of two different FMN‐dependent DPNH oxidases are described, one of which is closely associated with luciferase and exhibits both DPNH and TPNH oxidase activity.

ON THE MECHANISM OF DRUG HYDROXYLATION IN RAT LIVER MICROSOMES

The TPNH- and O(2)-dependent drug hydroxylation system of liver microsomes has been studied using normal rats and rats in which the drug-hydroxylating activity has been enhanced by repeated injections of phenobarbital. The oxidative demethylation of aminopyrine is employed as an assay. Optimal conditions for the assay with regard to the concentrations of TPNH and aminopyrine are established. TPN inhibits the reaction in a competitive manner, similarly to its effect on the microsomal TPNH-cytochrome c reductase. Drug hydroxylation, but not the "TPNH oxidase," TPNH-cytochrome c, -2,6-dichlorophenolindophenol, or -neotetrazolium reductase reaction, or the TPNH-dependent lipid peroxidation, is blocked by carbon monoxide. Microsomes from phenobarbital-treated rats exhibit increased activities of the various TPNH-linked reductase reactions, parallel to the increased drug hydroxylation activity, whereas the "TPNH oxidase" activity does not change appreciably. Measurements with microsomes from drug-treated animals reveal a 1:1:1 stoichiometry of aminopyrine-dependent oxygen uptake, TPNH oxidation, and formaldehyde formation. Attempts to solubilize the drug-hydroxylating enzyme system are also presented. It is concluded that the drug-hydroxylating enzyme system involves the microsomal TPNH-cytochrome c reductase and CO-binding pigment, and a hypothetic reaction scheme accounting for the data presented is proposed.

Variation in the activity of liver microsomal drug-metabolizing enzymes in rats in relation to the age

The variation in the activity of the microsomal drug-metabolizing enzymes in rats in relation to age was studied. The systems used were the in-vitro metabolism of hexobarbital, pentobarbital, meprobamate, carisoprodol and strychnine, the in-vivo metabolism of pentobarbital, meprobamate and carisoprodol and the duration of pentobarbital hypnosis and carisoprodol paralysis and the toxicity of strychnine and OMPA (Octamethylpyrophosphoramide). New-born rats showed only a very low metabolic activity, but this increased progressively until 30 days after which it gradually decreased with aging. In the metabolisms in vitro, 30-day-old rats showed about 2.7, 2.2, 1.7 and 1.5 times higher activity than those of 250, 150, 100 and 60 days old, respectively. Similar, though smaller, differences were observed in vivo. The duration of pentobarbital hypnosis, carisoprodol paralysis and strychnine toxicity was almost the same in immature (30 days old) and adult rats (60–150 days old). Possibly, the immature have a higher sensitivity to the drugs than the adult rats, but this is overshadowed by the higher metabolic activity in the immature rats. The possible mechanism for the marked difference in the activity in vitro of the drug-metabolizing enzymes among the differently aged rats is discussed in connection with the variation of in-vivo metabolism and activity of microsomal TPNH oxidase.

In vitro effect of adrenaline and other amines on glucose metabolism in sheep thyroid, heart, liver, kidney and testicular slices

Adrenaline, noradrenaline, and serotonine stimulate the oxidation of glucose carbon 1 and glucose carbon 6 by the thyroid tissue of various species in vitro. This effect has been related to the activation by these hormones of a DPNH and a TPNH oxidase system. The effect of adrenaline depends on its prior oxidation to adrenochrome. A similar but weaker effect of adrenaline and adrenochrome on glucose carbon 1 oxidation by liver and kidney cortex slices has been observed. A stimulation of glucose carbon 1 oxidation by substrates of monoamine oxidase has been observed in thyroid and testis slices. While this mechanism accounts for the effect of adrenaline on testis slices it does not account for the effect of this hormone on thyroid slices. The glycogenolytic effect of adrenaline on liver and heart tissue has been demonstrated by an increased glucose output by liver slices and a decreased glucose uptake by heart slices in the presence of this hormone. The biological significance of these three in vitro actions of adrenaline on glucose catabolism is discussed and it is concluded that only the glycogenolytic effect is physiological.

ADP-activated lipid peroxidation coupled to the TPNH oxidase system of microsomes

The presence of lipid peroxides, detected by the formation of malonaldehyde (MA), has been observed in tissues isolated from vitamin E-deficient animals (Zalkin and Tappel, 1960). Lipid peroxidation has also been found to occur in isolated subcellular fractions when these were incubated aerobically in the presence of hematin compounds (Tappel and Zalkin, 1960), ferrous ions (Ottolenghi, 1959; Hunter et al., 1963), and ascorbate (Ottolenghi, 1959; Thiele and Huff, 1960). In this paper, we wish to report the occurrence of a TPNH-induced peroxidation of lipids in rat-liver microsomes. The reaction requires activation by ADP or other pyrophosphates, and appears to be coupled to the TPNH oxidase system of the microsomes. The latter system was first described by Gilette et al. (1956) in connection with studies of TPNH-linked hydroxylations. Some properties of the TPNH-induced lipid peroxidation are described below and compared with those of the non-enzymically induced lipid peroxidation which occurs in the presence of ascorbate.

Enzymes in Candida albicans. II. Tricarboxylic acid cycle and related enzymes.

Rao, G. Ramananda (Indian Institute of Science, Bangalore, India), M. Sirsi, and T. Ramakrishnan. Enzymes in Candida albicans. II. Tricarboxylic acid cycle and related enzymes. J. Bacteriol. 84:778-783. 1962.-Evidence is presented to show the operation of the tricarboxylic acid cycle in Candida albicans, by studies with whole cells, cell-free preparations, and by the demonstration of most of the enzymes involved in the cycle. Cell-free extracts contained the following enzymes: condensing enzyme; aconitase; isocitric, alpha-ketoglutaric, succinic, and malic dehydrogenases; malic enzyme; fumarase; reduced diphosphopyridine nucleotide (DPNH) oxidase; DPNH-cytochrome c reductase; reduced triphosphopyridine nucleotide (TPNH) cytochrome c reductase; and diaphorase. Pyruvic dehydrogenase, TPNH oxidase, and transhydrogenase activities could not be detected under the test conditions.

Studies on the mechanism of action of thyroid stimulating hormone on glucose oxidation

Incubation of thyroid slices with added thyroid stimulating hormone results in an increase in the concentration of oxidized triphosphopyridine nucleotide. Since thyroid stimulating hormone also stimulates acetate and pyruvate oxidation, it is suggested that the previously reported increase in glucose oxidation is not a primary effect of thyroid stimulating hormone but results secondarily from the ability of this hormone to increase the TPN concentration in the thyroid. Attempts to demonstrate an effect of thyroid stimulating hormone on TPNH oxidase were unsuccessful, and it was not possible to stimulate glucose oxidation in thyroid homogenates fortified with TPN or TPNH. The amount of glucose oxidized by thyroid homogenates appeared to be a function of the concentration of TPN added. An effect of 3·4·1 o −11 M TSH was obtained when dog thyroid slices were used. This concentration is considered to be in the physiological range. Monoiodotyrosine and diiodotyrosine also stimulated glucose oxidation in thyroid slices. Acetylated thyroid stimulating hormone neither stimulated glucose oxidation in thyroid slices nor did it inhibit the action of one-tenth as much thyroid stimulating hormone. Thyroid glands obtained from guinea pigs injected with thyroid stimulating hormone for two days prior to sacrifice oxidized more glucose than did glands from control animals.

On the mechanism of glucose metabolism in the plant tumor-inducing organism Agrobacterium tumefaciens.

Extracts of the plant tumor-inducing organism Agrobacterium tumefaciens do not oxidize glucose directly to gluconic or to 2-ketogluconic acid. Such extracts contain an extremely active, cyanide-sensitive DPNH oxidase. TPNH oxidase shows little activity and pyridine nucleotide transhydrogenase appears to be absent. Hexokinase is active, glucose-6-phosphate dehydrogenase functions either as a DPN- or as a TPN-linked system, 6-phosphogluconic acid dehydrogenase is TPN-specific, and glyceraldehyde-3-phosphate dehydrogenase is DPN-specific.Evidence is presented to show that the pentose cycle operates in such preparations and also that the extent to which it functions is determined by the rate-limiting dehydrogenation and decarboxylation of 6-phosphogluconate. Thus, heptulose phosphate is formed readily from ribose-5-phosphate and much less effectively from 6-phosphogluconate. The net formation of hexose as a product of the cycle is also shown. It was possible to demonstrate under certain conditions that an effective competition exists between glyceraldehyde-3-phosphate dehydrogenase and transaldolase for available triose phosphate.From reaction rates and other data it is clear that the Entner–Doudoroff 6-phosphogluconate-splitting pathway is the major avenue of hexose phosphate utilization in cell-free extracts. Pyruvate thus formed is oxidized via the tricarboxylic acid cycle while glyceraldehyde-3-phosphate can be changed slowly into pyruvate or be recycled into hexose phosphate. The latter pathway is facilitated by the existence of a highly active fructose-1,6-diphosphatase.Whereas phosphohexoseisomerase is found to be highly active, phosphofructokinase shows very little activity, aldolase does not appear to be very effective, and the conversion of phosphoglyceric acid to pyruvate is extremely slow. For these reasons, glycolysis is considered to play a very minor role in glucose metabolism in these extracts.Data on the effects of some carbohydrate inhibitors on glucose-6-phosphate dehydrogenase and on phosphohexoseisomerase are also given.

Some haem protein-linked pyridine nucleotide oxidation systems in Rhodospirillium rubrum

Ammonium sulfate fractionation of a crude citrate extract obtained from lyophilized cells of R. rubrum, a facultative photoheterotrophe, yields a soluble enzyme preparation which contains “bound” bacterial cytochrome c ( “ cytochrome c 2” ) and variant heme protein (“RHP”). The following light-insensitive reactions, enzymically-catalyzed by this preparation, have been noted: (a) TPNH oxidase (b) DPNH oxidase (c) TPNH-cytochrome c 2 reductase (d) DPNH-cytochrome c 2 reductase (e) TPNH-RHP reductase (f) DPNH-RHP reductase (g) TPNH-2,6-DCPI reductase and (h) DPNH-2,6-DCPI reductase. The reaction between DPNH and 2,6-DCPI indicates 1o-fold purification of the enzyme with respect to the initial crude extract. Using this preparation, some characteristics of these catalyzed reactions have been determined. The evidence obtained from studies of heat inhibition and variations with pH suggests that reactions (g) and (h) are catalyzed by different enzymes, the latter being much more reactive. The rates of oxidase reactions (a) and (b) are very small, being 1 300 and 1 50 respectively of those found for DPNH and TPNH using 2,6-DCPI as H-acceptor. The bound RHP is rapidly oxidized, whereas cytochrome c 2 is only partially oxidized when the enzyme preparation is aerted. When DPNH is added in air, only cytochrome c 2 is reduced. Under anaerobic conditions, both endoegenous heme proteins are reduced to the maximum extent, but at different rates. These preliminary studies are in accord with an oxidation scheme in which dehydrogenation of the substrate DPNH proceeds via RHP to both cytochrome c 2 and oxygen as hydrogen acceptors.

TPNH-cytochrome c reductase of chloroplasts and its role in photosynthetic phosphorylation

1. 1. The extract from acetone-dried powder of washed chloroplasts shows a marked DPNH and TPNH-cytochrome c reductase activity. TPNH-cytochrome c reductase has been further purified to a specific activity nine times greater than in the extract. The enzyme requires FMN or FAD for full activity, and is severely inhibited by p-chloro-mercuribenzoic acid, while practically insensitive to KCN, NaN 3, iodoacetate and o-iodosobenzoate. A marked TPNH oxidase activity seems associated with the reductase activity of the preparation. 2. 2. Isolated whole chloroplasts actively reduce cytochrome c in the dark in the presence of TPNH or DPNH. The rate of reduction of cytochrome is greatly enhanced by FMN, particularly with TPNH as electron donor. Participation of the TPNH-cytochrome c reductase system in the photosynthetic activity of the chloroplasts is indicated by the fact that FMN and TPN + significantly increase the rate of reduction of cytochrome c by illuminated chloroplast suspensions. 3. 3. The addition of cytochrome c to chloroplast suspensions almost doubles their photosynthetic phosphorylation activity. When TPN + is added as well, phosphorylation is further increased, but TPN + alone has little effect. The presence of FMN, a powerful activator of photosynthetic phosphorylation, augments, on an absolute basis, the effects of either cytochrome c or cytochrome c plus TPN +. 4. 4. These results, together with those of other authors, are interpreted as indicating that the role of TPN in photosynthesis is not only that of a reducing agent for phosphoglycerate, but also that of an intermediary electron carrier in photosynthetic phosphorylation.

The metabolism of Acetobacter peroxidans: I. Oxidative enzymes

Several oxidative enzymes of the catalase-negative bacterium Acetobacter peroxidans have been investigated. Alcohol and acetaldehyde dehydrogenases are TPN-specific. TPNH oxidase, TPNH-linked cytochrome c reductase, diaphorase, peroxidase, and the H 2O 2 peroxidations of reduced TPN and or reduced cytochrome can be demonstrated in cell-free extracts. The entire electron transport chain is antimycin A insensitive. The conversion of acetaldehyde to acetate is not CoA dependent. It appears as if the usual tricarboxylic acid cycle is inoperative during acetate oxidation by this microorganism.