Ron Wever

Tetrahydrobiopterin restores endothelial function in hypercholesterolemia.

In hypercholesterolemia, impaired nitric oxide activity has been associated with increased nitric oxide degradation by oxygen radicals. Deficiency of tetrahydrobiopterin, an essential cofactor of nitric oxide synthase, causes both impaired nitric oxide activity and increased oxygen radical formation. In this study we tested whether tetrahydrobiopterin deficiency contributes to the decreased nitric oxide activity observed in hypercholesterolemic patients. Therefore, L-mono-methyl-arginine to inhibit basal nitric oxide activity, serotonin to stimulate nitric oxide activity, and nitroprusside as endothelium-independent vasodilator were infused in the brachial artery of 13 patients with familial hypercholesterolemia and 13 matched controls. The infusions were repeated during coinfusion of L-arginine (200 microg/kg/min), tetrahydrobiopterin (500 microg/min), or the combination of both compounds. Forearm vasomotion was assessed using forearm venous occlusion plethysmography and expressed as ratio of blood flow between measurement and control arm (M/C ratio). Tetrahydrobiopterin infusion alone did not alter M/C ratio. Both the attenuated L-mono-methyl-arginine-induced vasoconstriction as well as the impaired serotonin-induced vasodilation were restored in patients during tetrahydrobiopterin infusion. Tetrahydrobiopterin had no effect in controls. In conclusion, this study demonstrates restoration of endothelial dysfunction by tetrahydrobiopterin suppletion in hypercholesterolemic patients.

Demonstration by EPR spectroscopy of the functional role of iron in soybean lipoxygenase-1

1. The EPR spectrum at 15 degrees K of soybean lipoxygenase-1 in borate buffer pH 9.0 has been studied in relation to the presence of substrate (linoleic acid), product (13-L-hydroperoxylinoleic acid) and oxygen. 2. The addition of 13-L-hydroperoxylinoleic acid to lipoxygenase-1 at pH 9.0 gives rise to the appearance of EPR lines at g equals 7.5, 6.2, 5.9 and 2.0, and an increased signal at g equals 4.3. 3. In view of the effect of the end product on both the kinetic lag period of the aerobic reaction and the fluorescence of the enzyme, it is concluded that 13-L-hydroperoxylinoleic acid is required for the activation of soybean lipoxygenase-1. Thus it is proposed that the enzyme with iron in the ferric state is the active species. 4. A reaction scheme is presented in which the enzyme alternatingly exists in the ferric and ferrous states for both the aerobic and anaerobic reaction.

Implications for the catalytic mechanism of the vanadium-containing enzyme chloroperoxidase from the fungus Curvularia inaequalis by X-ray structures of the native and peroxide form.

Implications for the catalytic mechanism of the vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis have been obtained from the crystal structures of the native and peroxide forms of the enzyme. The X-ray structures have been solved by difference Fourier techniques using the atomic model of the azide chloroperoxidase complex. The 2.03 A crystal structure (R = 19.7%) of the native enzyme reveals the geometry of the intact catalytic vanadium center. The vanadium is coordinated by four non-protein oxygen atoms and one nitrogen (NE2) atom from histidine 496 in a trigonal bipyramidal fashion. Three oxygens are in the equatorial plane and the fourth oxygen and the nitrogen are at the apexes of the bipyramid. In the 2.24 A crystal structure (R = 17.7%) of the peroxide derivate the peroxide is bound to the vanadium in an eta2-fashion after the release of the apical oxygen ligand. The vanadium is coordinated also by 4 non-protein oxygen atoms and one nitrogen (NE2) from histidine 496. The coordination geometry around the vanadium is that of a distorted tetragonal pyramid with the two peroxide oxygens, one oxygen and the nitrogen in the basal plane and one oxygen in the apical position. A mechanism for the catalytic cycle has been proposed based on these X-ray structures and kinetic data.

Bromoperoxidase and iodoperoxidase enzymes and production of halogenated methanes in marine diatom cultures

Halogenated methanes produced in the oceans are important as carriers of chlorine, bromine, and iodine into the atmosphere. There they play roles in the regulation of ozone in the stratosphere and perhaps in the Arctic troposphere at polar sunrise. While the mechanisms for the production of some polyhalogenated compounds by marine macrophytes have previously been substantially elucidated, the same has not been true in the case of marine phytoplankton. We describe laboratory experiments on the production of various brominated and iodinated compounds in cultures of marine diatoms, obtained from the Provasoli-Guillard Center for Culture of Marine Phytoplankton collections (Bigelow Laboratory for Ocean Sciences, Maine, USA ; CCMP). Species examined included Nitzschia sp. (CCMP 580), Nitzschia arctica, Porosira glacialis, and two Navicula sp. (CCMP 545 and 546). A suite of brominated compounds, notably bromoform and dibromomethane, is produced by the Nitzschia and Porosira species. Nitzschia sp.(CCMP 580) was grown in sufficient quantities to allow the identification of a bromoperoxidase enzyme, which is assumed to be responsible not only for the CHBr 3 and CH 2 Br 2 production but also for CH 2 I 2 which was measured in those cultures. Chloroiodomethane was produced, either directly by the algae or by a photochemical reaction of CH 2 I 2 . One Navicula species (CCMP 545), found to produce CH 2 I 2 and CH 2 CII, was shown to possess an iodoperoxidase. Bromoform and dibromomethane were not detected in cultures of this species. Other compounds produced in certain of these non axenic cultures included methyl and ethyl iodide, and bromoiodomethane.

Characterization and quantification of the peroxidase in human monocytes

1. 1. The properties of the peroxidase in human moncytes have been compared with those of myeloperoxidase in human granulocytes and with those of isolated human mycloperoxidase. 2. 2. The optical difference spectra of the reduced minus the oxidized states of monocyte and granulocyte homogenates show peaks at 472 and 635 nm that are identical with those observed in the spectrum of reduced isolate myeloperoxidase. 3. 3. Electron paramagnetic resonance spectra of monocytes, granulocytes and isolated myeloperoxidase show a rhombic high-spin haem iron signal with gx = 6.90 and gy = 5.07. 4. 4. Monocyte and granulocyte lystates show a peroxidative reaction with ortho-dianisidine and require the same optimal substrate concentrations. 5. 5. Monocyte and granulocyte homogenates, as well as isolated myeloperoxidase, catalyse the oxidation of iodide by H2O2. This reaction has an acid pH optimum of 4.5–5.5. 6. 6. Ingested zymosan particles are iodinated by monocytes as well as by granulocytes. 7. 7. On the basis of these observations we conclude that human monocytes contain myeloperoxidase. 8. 8. The concentration of myelperoxidase in monocytes is about 1.1 · 10−17 mol per cell. Granulocytes contain three times this amount of myeloperoxidase per cell.

Generation of superoxide radicals during the autoxidation of mammalian oxyhemoglobin

Abstract It is shown that during autoxidation of mammalian oxyhemoglobin to methemoglobin superoxide anions (O2- are generated. We suggest that the physiological role of superoxide dismutase in erythrocytes is to prevent the deleterious action of the superoxide anion.

Bromoperoxidase from Ascophyllum nodosum: a novel class of enzymes containing vanadium as a prosthetic group?

Abstract Reconstitution experiments showed that the transition metal vanadium is essential for enzymic activity of bromoperoxidase from the marine brown alga Ascophyllum nodosum. A linear relationship existed between brominating activity and the amount of vanadium incorporated in the enzyme. Maximal activity was found when the enzyme contained 5.1 nmol vanadium per mg bromoperoxidase. Based on gel-exclusion high-performance liquid chromatography, the molecular mass of bromoperoxidase was estimated as 90 000. Chemical analysis and electron paramagnetic resonance (EPR) demonstrated that bromoperoxidase contains vanadium (4.4 nmol per mg bromoperoxidase). This value corresponds to a ratio of 0.4 mol vanadium per mol bromoperoxidase. The EPR experiments suggest that in bromoperoxidase the vanadium ion is present in the 5 + redox state. These experiments demonstrate for the first time the existence of an enzyme containing vanadium at the active site.

Isolation procedure and some properties of myeloperoxidase from human leucocytes

1. A rapid isolation procedure with a high yield for pure myeloperoxidase (donor:H2O2 oxidoreductase, EC 1.11.1.7) from normal human leucocytes is described. The enzyme was solubilized from leucocytes with the detergent, cetyltrimethylammonium bromide, and purified to apparent homogeneity. The yield of the enzyme was 17% with an absorbance ratio A430nm/A280nm = 0.85. 2. The purified enzyme showed three isoenzyme bands after polyacrylamide gel electrophoresis; ultracentrifuge studies indicated one homogeneous band with a molecular weight of 144 000. After reduction of myeloperoxidase, sodium dodecyl sulfate gel electrophoresis resolved an intense band (63 000 daltons) and a weak band (81 000 daltons). 3. The carbohydrate content of the enzyme was at least 2.5%. Mannose, glucose and N-acetylglucosamine were present. The amino acid composition is reported. 4. The EPR spectrum exhibited a high-spin heme signal with rhombic symmetry (gx = 6.92, gy = 5.07 and gz = 1.95). Upon acidification this signal was converted into a signal with more axial symmetry (g perpendicular = 5.89). At high pH (9.5) the EPR spectrum of the enzyme only shows low-spin ferric heme resonances. The circular dichroism spectra of ferric and ferrous myeloperoxidase in the visible and ultraviolet region show maxima and minima in ellipticity.

Vanadium(V) as an essential element for haloperoxidase activity in marine brown algae: purification and characterization of a vanadium(V)-containing bromoperoxidase from Laminaria saccharina

Abstract The purification and characterization of bromoperoxidase from the marine brown alga Laminaria saccharina is described. The purified enzyme is homogeneous, as verified by polyacrylamide gel electrophoresis and size-exclusion high-performance liquid chromatography; it has a dimeric structure with a relative molecular mass of 108 kDa. Reconstitution experiments showed that the transition metal vanadium is essential for the brominating activity of this enzyme. Analysis of bromoperoxidase with electron paramagnetic resonance in combination with activity measurements indicated that the ratio of vanadium ligated to the protein at the active site increased from 0.3 to 2.0 when the purified enzyme was incubated with an excess of the metal. Properties of bromoperoxidase from L. saccharina are compared with bromoperoxidase from Ascophyllum nodosum (De Boer, E., Van Kooyk, Y., Tromp, M.G.M., Plat, H. and Wever, R. (1986) Biochim. Biophys. Acta 869, 48–53). The presence of ‘vanadium(V)-dependent’ haloperoxidase activity in crude extracts of Fucus spiralis, F. serratus, F. vesiculosus, Pelvetia canaliculata and Chorda filum is described. These experiments demonstrate that vanadium-containing haloperoxidases are more widely spread in nature

The chloroperoxidase from the fungus Curvularia inaequalis; a novel vanadium enzyme.

The presence of vanadium-containing bromoperoxidases in various types of seaweed is well-documented. We now report that the terrestrial fungus Curvularia inaequalis excretes a novel chloroperoxidase which also contains vanadium as a prosthetic group. The chloroperoxidase is excreted in the medium as the only protein and is, therefore, almost purely obtained. Atomic absorption spectroscopy measurements showed that the chloroperoxidase contained vanadium, which was essential for enzymatic activity, in a stoichiometry of 1 mol vanadium per mol of enzyme. When the fungus was grown in media containing low concentrations of vanadate (VO4(3-)) or when vanadate was absent, the enzyme was excreted in an apoform. Addition of vanadate to the apoenzyme purified from the medium, dialyzed holo-enzyme or growth medium led to incorporation of the metal and to a subsequent increase in specific activity from 0.7 to about 7.5 units/mg. The reduced enzyme showed an axially symmetric EPR spectrum (g(o) = 1.971, Ao = 91.7 x 10(-4) cm-1) with 16 hyperfine lines that is essentially the same as the EPR spectrum of the vanadium-containing bromoperoxidase of the seaweed Ascophyllum nodosum. This demonstrates that the active sites in the two enzymes are very similar. The chlorinating and brominating activities of the chloroperoxidase from C. inaequalis were also studied and compared to those of the vanadium bromoperoxidase from A. nodosum. The chlorinating reaction catalyzed by the chloroperoxidase had a pH optimum around 5.5 and the Km for Cl- was small (0.25 mM at pH 4.5), but the logarithm of its value increased linearly with increasing pH. At high bromide concentrations, the pH optima of chloroperoxidase and bromoperoxidase in the brominating reaction were about the same (5.5). However, at low bromide concentrations the pH optimum of the chloroperoxidase was at higher pH values than that of the bromoperoxidase.