DPNH Oxidase Activity Research Articles

Studies of the development of congenital anomalies in rats. III. Effects of inhibition of mitochondrial energy systems on embryonic development.

Pregnant rats were treated with various inhibitors of mitochondrial oxidative energy metabolism and with lowered oxygen tension, and the embryo fetuses examined for the occurrence of congenital malformations and for changes in enzymatic activities. Treatment with all agents tested resulted in the production of skeletal anomalies. Sodium phenobarbital was the most teratogenic of the drugs tested and produced a high incidence of malformations which included cleft palate, tail anomalies, spinal retroflexion, domed head, and facial hypoplasia. Diphenylhydantoin produced a low incidence of syndactyly and oligodactyly. In addition to its effects on fetal growth and development chloramphenicol appeared to interfere with implantation. Tissue preparations from embryos exposed to sodium phenobarbital and chloramphenicol showed markedly lowered levels of DPNH oxidase activity. Cytochrome oxidase activity was also markedly lowered in the preparations from chloramphenicol-exposed embryos. Enzyme activities in preparations from embryos exposed to malonate and diphenylhydantoin appeared unaffected, although the drugs are strong inhibitors of electron transport in vitro; the lack of apparent effect may be due to the fact that both drugs do not bind to the enzyme preparations and were diluted 100- to 200-fold during preparation and assay of the tissue homogenates.

Studies of mitochondrial energy systems during embryogenesis in the rat

Mitochondria isolated from rat embryos of Day 11 to Day 14 of gestation showed an equal capacity to carry out oxidative phosphorylation with P:O ratios of approximately 3 for pyruvate-malate and 2 for succinate as substrates. Activities of succinic dehydrogenase, cytochrome oxidase, and mitochondrial ATPase remained at nearly constant levels in the preparations during this period of gestation, but DPNH oxidase activity increased over 2-fold. The increase in DPNH oxidase activity could be prematurely induced at Day 11 of gestation of the embryos by prior treatment of the pregnant rats with an atmosphere containing 85% oxygen and 0.4% CO 2, but no changes in the growth and development of the treated embryos were found by gross examination.

Catalytic activity and EPR signals of DPNH dehydrogenase in relation to the acquisition and loss of piericidin sensitivity and of coupling site 1

The events accompanying the development of piericidin sensitivity and of energy coupling site 1 during the transition of Candida utilis cells from the log phase to the late stationary phase of growth have been investigated. There is a large increase in DPNH dehydrogenase (DPNHD) activity, and major increases in the EPR signals of iron-sulfur centers 1, 2, and 3 of the enzyme, as measured at 13°K. The increase in DPNHD activity, however, does not reflect the increased development of the same enzyme as is present in the log phase, for the enzyme being synthesized in the stationary phase is different from that present in the log phase, as judged by juglone reductase and DPNH oxidase activities, which declined during the transition, and by stability, kinetics, and in the type of EPR signal present, which are different in the log and stationary phases. On catabolite repression of stationary phase cells the converse occurs: the specific activities of juglone reductase and DPNH oxidase rise, DPNHD activity and EPR signals corresponding to centers 1 and 2 disappear. This process of catabolite repression by ethanol is prevented by cycloheximide.

Reversibility of 15-OH prostaglandin dehydrogenase from swine lung

15-OH-PGDH oxidizes 15-OH PGs to the 15-keto form. In vitro , with a partially purified PGDH, the reaction can be driven in the reverse direction as indicated by 15-keto PGE 1 dependent DPNH oxidase activity and by the formation of 15-OH PGE 1.

Effect of Silicotungstate on Reduced Diphosphopyridine Nucleotide Oxidation in Submitochondrial Particles

Abstract 1. Silicotungstate inhibits DPNH oxidase, succinoxidase, and 32Pi-ATP exchange activity at relatively low concentrations (150 µg per mg of protein). The inhibition is reversed by bovine serum albumin. 2. When the concentrations of silicotungstate are increased (0.3 to 1.4 mg per mg of protein), DPNH oxidase activity appears which is insensitive to rotenone. Addition of serum albumin restores rotenone sensitivity. 3. Silicotungstate was shown to interact directly with DPNH dehydrogenase. The higher levels of silicotungstate convert both the particulate and soluble DPNH dehydrogenase but not succinate dehydrogenase into an oxidase enzyme.

Rhein, a selective inhibitor of the DPNH-flavin step in mitochondrial electron transport

Previous findings in the literature that rhein inhibits DPNH-linked mitochondrial oxidations by acting in the DPNH dehydrogenase region of the respiratory chain have been confirmed and extended. In the micromolar range rhein inhibits DPNH oxidase and DPNH-ferricyanide activities and the energy-linked reduction of DPN by succinate in membrane preparations from heart, as well as the DPNH dehydrogenase and transhydrogenase activities of the soluble, purified enzyme. The inhibition of the activities of the soluble enzyme are purely competitive with respect to substrate. These facts localize the primary inhibition site of rhein between substrate and FMN. In. heart ETP a second noncompetitive inhibition is also present but is detectable only at very low (<10 μM) rhein concentrations. Rhein also inhibits DPNH dehydrogenase in Candida utilis mitochondria and the purified enzyme from liver. On conversion of the heart enzyme to the low molecular weight DPNH-cytochrome reductase the typical effect of rhein disappears and is replaced by a slight stimulation or inhibition, depending on the electron acceptor used, showing that the substrate-binding site is modified in this form of the enzyme. In beef liver mitochondria DPNH oxidation may appear insensitive to rhein, probably because of the strong binding of rhein to other proteins. To a lesser extent unspecific binding of rhein and resultant interference with the inhibition of DPNH dehydrogenase is also shown by BSA and by proteins in heart ETP. Rhein also inhibits transhydrogenations in mitochondria and at higher concentrations lactate and malate dehydrogenases but has no effect on succinate, alcohol (liver and yeast), and glucose-6-P dehydrogenases or on Neurospora DPN-ase, glucose-6-phosphatase, and amine oxidase.

Studies of the Electron Transport Chain of Extremely Halophilic Bacteria: II. Salt dependence of reduced diphosphopyridine nucleotide oxidase

Abstract A scheme of electron transport in Halobacterium cutirubrum is proposed based on the following findings. (a) The slow rate of reduction of b-type cytochromes by DPNH is not consistent with the participation of these components in the main pathway of the oxidation of DPNH. (b) DPNH oxidation proceeds through a fast, 2-heptyl-4-hydroxyquinoline N-oxide (QO)-sensitive and a slow, QO-insensitive path. Succinate oxidation, which is slow, is not QOsensitive. These results suggest a main branch of electron transport including a flavoprotein, reduced by DPNH, and a second branch consisting of flavoproteins and cytochrome b(559), reduced by DPNH and succinate. The branches join at the c-type cytochromes. Cytochrome b(563), although reduced by the dehydrogenase of the main branch, lies outside the main DPNH oxidase pathway. The relation of DPNH oxidase activity to NaCl concentration and the irreversible inactivation of the enzyme in the absence of salt reflect the behavior of a single component of the system, DPNH dehydrogenase. The place of the b-type cytochromes in the proposed electron transport scheme is confirmed through the determination of salt dependence curves.

Inactivation of lyophilized DPNH dehydrogenase from Escherichia coli by oxygen

Lyophilized Escherichia coli membranes lose 50% of their DPNH oxidase activity when exposed to O 2. The oxidation of succinate or lactate is not affected by this treatment. The enzyme damaged by O 2 is the membrane DPNH dehydrogenase (DPNH-ferricyanide oxidoreductase, EC 1.6.99.3). The K m values for DPNH and the electron acceptor K 3Fe(CN) 6 are the same in the native and the inactivated enzyme. The v max of the latter is decreased by 50% for both substrates. The accessibility of non-heme iron to o-phenanthroline is partly increased. It is suggested that the non-heme iron of DPNH dehydrogenase participates in intramolecular electron conductance between the oxidizing and the reducing sites of the enzyme.

Organic structural specificity and sites of coenzyme Q in succinoxidase and DPNH-oxidase systems

In succinoxidase, CoQ 1 had low activity, and CoQ 2 through CoQ 10 had comparable activities in both the acetone- and pentane-extracted systems. In the DPNH-oxidase system, CoQ 1 had low activity; CoQ 2, CoQ 3, and CoQ 4 had about one-fourth the activity; and CoQ 5 and CoQ 6 had about one-half the activity of CoQ 10. CoQ 7, CoQ 8, and CoQ 9 had activity comparable with that of CoQ 10. Hexahydro- and octahydrocoenzyme Q 4 had activities comparable with that of CoQ 4 and CoQ 10 for succinoxidase, but about 10–15% that of CoQ 10 for DPNH-oxidase. The double bond in the isoprenoid unit adjacent to the nucleus, and possible cyclization, are not necessary for activity. 2,3-Dimethoxy-5-methyl-6-heptadecyl-l,4-benzoquinone shows succinoxidase and DPNH-oxidase activities comparable with that of hexahydrocoeuzyme Q 4. The 5-methyl group is not essential for succinoxidase activity, but has some significance for DPNH-oxidase. A tertiary hydroxyl group in the isoprenoid unit adjacent to the nucleus abolishes activity for succinoxidase but not completely for DPNH-oxidase. Rhodoquinone had no activity in the succinoxidase system and 25% that of CoQ 10 in the DPNH-oxidase system. Representatives of biosynthetic precursors, 2-methoxy-5-methyl-6-phytyl-1,4-benzoquinone and 2-methoxy-3-hydroxy-5-methyl-6-phytyl-1,4-benzoquinone, had no activity in either the succinoxidase or DPNH-oxidase systems. Four compounds having a mechanistic relationship between CoQ and vitamin E had no activity. These data support the concept of two sites for the electron transfer of CoQ. The site for CoQ in succinoxidase is not very specific in the structural and steric requirement for the isoprenoid side chain, and has an electron potentiality that is not met by rhodoquinone. The site for CoQ in DPNH-oxidase has a rather specific requirement for a given length of side chain, and an electron potentiality that is significantly met by rhodoquinone.

Dihydroorotate Dehydrogenase: I. GENERAL PROPERTIES

Abstract An improved method is described for the preparation of crystalline dihydroorotate dehydrogenase which is electrophoretically homogeneous. The enzyme exhibits an s20,w value of 7.45 S at infinite dilution, and its molecular weight is 115,000 ± 7,000. It contains 2 moles of FAD, 2 moles of FMN, 4 g atoms of iron, and 4 moles of acid-labile sulfide per mole of enzyme. Apparent Km values were determined for DPNH, orotate, and dihydroorotate. Orotate and oxidized enzyme yield a complex characterized by its absorption spectrum. Thermodynamic parameters for the dissociation of this complex are presented. Substrate specificity was studied with a series of pyrimidine derivatives. The ring hydroxyl and carboxyl substituents are essential for binding and oxidation of reduced enzyme. Halogen substitution at Position 5 increases both Km and Vmax. Reduction of the enzyme by reducing substrates is evidenced by bleaching at 450 mµ and the appearance of long wave length absorption. Reduction by DPNH proceeds rapidly; reduction by dihydroorotate is slow and proceeds to about one-half the extent found with DPNH. However, reduction of the enzyme by dihydroorotate in the presence of DPN is as complete, and perhaps as rapid, as that by DPNH. A large Cotton effect, observed in the visible region of the spectrum, was markedly altered by dithionite or reducing substrate and was shifted to lower wave lengths by 3.5 m urea. A flavin-free iron-protein was obtained which exhibited an absorption spectrum similar to that of iron-proteins from other iron-flavoproteins and to that of spinach ferredoxin. Three techniques were used to demonstrate the formation of an oxygen free radical when enzyme, reduced by substrate, is reoxidized by oxygen. Treatment with cysteine is required for reactions involving orotate or dihydroorotate, but not for DPNH oxidase activity. Similarly, treatment with mercuribenzoate destroyed orotate reductase and dihydroorotate oxidase activity while inhibiting DPNH oxidase activity only slightly. A tentative scheme for the internal electron transport system of this enzyme is presented.

Studies on the Electron Transfer System: LXVIII. FORMATION OF MEMBRANES AS THE BASIS OF THE RECONSTITUTION OF THE MITOCHONDRIAL ELECTRON TRANSFER SYSTEM

Cytochrome oxidase (Complex IV), reduced diphosphopyridine nucleotide-cytochrome c reductase (a mixture of Complexes I and III), and succinic-cytochrome c reductase (a mixture of Complexes II and III) can be induced to form membranes by reducing the bile salt level of the suspending medium (in concentrated solutions of the complexes as prepared, the level of bile salt is sufficient to achieve molecular dispersion of the repeating units). When the membranes formed separately from the individual complex or sets of complexes are mixed prior to or during assay, the mixture of membranes fails to show DPNH oxidase activity (mixture of I + III and IV) or succinic oxidase activity (mixture of II + HI and IV). In a sucrose gradient these mixtures are separable by centrifugation into two distinct bands corresponding to the component membranes. However, the membranes formed by mixing either I + III or II + III with IV in concentrated solution, and then diluting, will catalyze the oxidation of DPNH or succinate by molecular oxygen (the test of reconstitution). Each of these membranes shows only a single band when centrifuged in a sucrose gradient. On the basis of these observations, reconstitution is interpreted in terms of the incorporation within a single membrane of the complete set of complexes necessary for a reconstituted activity. Reconstitution of electron transfer activity does not require selective interactions between, or alignments of, the complexes of the electron transfer chain. A membrane can be formed by any one complex with or without any of the other complexes; any proportions of two or more complexes can be achieved. These observations exclude the notion of stoichiometry among complexes. Cytochrome c, one of the electron transfer links between complexes, is incapable of moving from one membrane to another. Thus, reconstitution requires that not only the complexes but also the mobile components must be in the same membrane system.

The Influence of Deuterium Oxide and Organic Solvents on the Interaction of Respiratory Chain Components

Abstract 1. The influence of D2O and organic solvents on the rate of oxidation of added substrates by heart muscle particles and tightly coupled rat liver mitochondria has been investigated. Solvents examined include glycerol, sucrose, dimethyl sulfoxide, dimethyl formamide, and ethylene glycol. Each of these solvents was found to induce a marked inhibition of respiratory chain activity. 2. The relative constancy in the steady state reduction level of the respiratory carriers at each increment of solvent concentration prevents the assignment of a single specific site of respiratory inhibition, and favors the hypothesis that the solvent effects are due to a nonspecific inhibition which operates in a similar manner at several sites in the respiratory chain. 3. No correlation was found between the inhibition of respiratory activity by solvents and the viscosity of the solvent reaction mixtures. 4. The organic solvent inhibition of the DPNH oxidase activity of heart muscle particles was a linear function of the water content of the solvent reaction mixtures in which the particles were suspended, and independent of the concentration, tonicity, or chemical nature of the solvent used. 5. It is suggested that several protonolytic reactions may be operative during the transfer of reducing equivalents from substrate to oxygen by way of the respiratory chain. The respiratory chain components mediating these reactions are in some manner accessible to, and markedly influenced by, the aqueous phase surrounding the particles, and therefore do not function entirely in a lipid nonaqueous milieu.

The restoration of DPNH oxidase activity by coenzyme Q (ubiquinone)

Coenzyme Q (ubiquinone) can be efficiently removed from lyophilized mitochondria by extraction with pentane. The extracted mitochondria oxidize neither DPNH nor succinate. Both oxidative activities can be specifically restored, however, by addition of coenzyme Q 10, in the presence of added mitochondrial phospholipids and cytochrome c. The restored DPNH-oxidase activity is fully sensitive to the classical respiratory chain inhibitors, rotenone, antimycin A, and KCN.

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.

Pyridine nucleotide oxidizing enzymes of Loctobacillus casei: I. Diaphorase

A diaphorase was isolated from Lactobacillus casei and purified approximately ninetyfold. It has been shown to be free from DPNH oxidase and DPNH peroxidase activity. The enzyme is specific for DPNH rather than TPNH and requires the addition of FMN for maximal activity. A number of dyes will serve as acceptors, including p benzoquinone, indophenol, and ferricyanide. Oxygen, hydrogen peroxide, methylene blue, lipoic acid, cytochrome c, and substrate amounts of flavin nucleotides are inactive as acceptors. Under the assay conditions used, the diaphorase activity was only slightly inhibited by the presence of hydrogen peroxide, cyanide, or N-ethylmaleimide and not inhibited at all by versene or amytal.

Pyridine nucleotide oxidizing enzymes of Lactobacillus casei: II. Oxidase and peroxidase

The soluble DPNH oxidase and peroxidase activities of Lactobacillus casei were isolated in partially purified form and their properties were examined. The two activities could not be separated by any of the techniques of protein separation that were employed. The peroxidase is an FAD enzyme and retains its flavin tenaciously during purification. It is very specific for hydrogen peroxide as electron acceptor and is not effective with methylene blue, indophenol, benzoquinone, and similar compounds. The enzyme is inhibited strongly by p-hydroxymercuribenzoate and moderately by N-ethylmaleimide, but not by cyanide, amytal, or versene. The oxidase is also a flavoprotein, but loses activity during isolation or on storage and reaches maximal activity only when incubated with a thiol compound and FAD. Oxygen, FAD, or methylene blue will serve as electron acceptor, but FMN and ferri-cyanide are totally inactive. The activity was not affected significantly by any of the inhibitors tested.

Studies on the electron transfer system. LII. Binding of cytochome c by reconstituted DPNH oxidase

Varying amounts of cytochrome c are bound by a reconstituted DPNH oxidase complex. Cytochrome c added above a certain constant level does not result in increased DPNH oxidase activity. Evidence is presented to demonstrate the interaction of the polybasic cytochrome protein with particle-bound phospholipid. The necessity of a bound form of cytochrome c for DPNH oxidase activity is shown, and the implications of this general type of binding are discussed.

DT diaphorase

DT diaphorase, which was first purified from the soluble cytoplasmic fraction of rat liver, has been separated and purified from isolated mitochondria. The mitochondrial enzyme displays the same sensitivity to dicoumarol, reactivity with both DPNH and TPNH, and similar rates of reduction of various electron acceptors as the soluble-cytoplasmic enzyme. The residual mitochondrial fragments, free of DT diaphorase, exhibit a DPNH oxidase activity which resembles that of the respiratory chain of intact mitochondria in its sensitivity to amytal and antimycin A but which lacks phosphorylating capacity. Vitamin K3 (2-methyl-1,4-naphthoquinone) when tested on the DT diaphorase-free submitochondrial particles induces no by-pass of the amytal-sensitive site of the DPNH oxidase, in contrast to the case with intact mitochondria. The addition of DT diaphorase to the particles is required for vitamin K3 in order to accomplish such a by-pass. These and related findings are discussed in view of the various proposed functions of the different diaphorases in the pathways of mitochondrial oxidations.

The isolation and identification of a naphthoquinone from electron-transport particles of mycobacterium phlei

1.1. A naphthoquinone was isolated from electron-transport particles of Mycobacterium phlei.2.2. The naphthoquinone was identified by chromatography as a vitamin K(45) and by infrared spectroscopy as a vitamin K2(45).3.3. This compound was active in restoring DPNH oxidase activity to particles which had been extracted with isooctane. It also reactivated the DPNH oxidase in a crude extract which had been exposed to light at a wavelength of 360 mμ.4.4. The inability to restore DPNH oxidase activity in light-exposed particles, with the isolated compound, has been explained by postulating a second light-sensitive component in the electron-transport pathway.

DT diaphorase. III. Separation of mitochondrial DT diaphorase and respiratory chain.

DT diaphorase, which was first purified from the soluble cytoplasmic fraction of rat liver, has been separated and purified from isolated mitochondria. The mitochondrial enzyme displays the same sensitivity to dicoumarol, reactivity with both DPNH and TPNH, and similar rates of reduction of various electron acceptors as the soluble-cytoplasmic enzyme. The residual mitochondrial fragments, free of DT diaphorase, exhibit a DPNH oxidase activity which resembles that of the respiratory chain of intact mitochondria in its sensitivity to amytal and antimycin A but which lacks phosphorylating capacity. Vitamin K 3 (2-methyl-1,4-naphthoquinone) when tested on the DT diaphorase-free submitochondrial particles induces no by-pass of the amytal-sensitive site of the DPNH oxidase, in contrast to the case with intact mitochondria. The addition of DT diaphorase to the particles is required for vitamin K 3 in order to accomplish such a by-pass. These and related findings are discussed in view of the various proposed functions of the different diaphorases in the pathways of mitochondrial oxidations.