Circadian control of meal timing during temporal isolation.
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Circadian Rhythm↗
Biomedical subjects
R Wever
Publications and source records attributed to R Wever.
Myeloperoxidase is more efficient than eosinophil peroxidase in the in vitro killing of newborn larvae of Trichinella spiralis.
Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) catalyse the formation of hypochlorite (OCl-) from chloride ions (OCl-) and hydrogen peroxide (H2O2). OCl- proved to be highly toxic for Trichinella spiralis newborn larvae (NBL) in in vitro assays. Using purified human MPO and EPO it was found that even at neutral pH both enzymes under appropriate conditions are able to kill NBL. The rate at which OCl- is produced is much lower in the EPO- than in the MPO-mediated reaction. This difference in enzymic activity may explain why in the MPO-mediated reaction half the amount of OCl- was sufficient to kill 50% of the NBL, as compared to the EPO-mediated reaction. Purified human eosinophil major basic protein showed excellent OCl- scavenging properties, resulting in a significant decrease in the EPO-mediated NBL killing. Addition of ammonium ions [(NH4)2SO4] to the EPO-mediated reaction increased the NBL killing remarkably. It was concluded that in vitro MPO is more efficient than EPO in killing NBL. Furthermore, it was suggested that although eosinophils show marked parasiticidal effects in various in vitro systems, their primary biological role might be the regulation of the inflammatory reactions.
Animals↗
The effect of D-penicillamine on human myeloperoxidase, a mechanism for the efficacy of the drug in rheumatoid arthritis.
We investigated the effect of D-penicillamine on the ability of myeloperoxidase, purified from human leukocytes, to catalyse the oxidation of chloride ions to hypochlorite (HOCl) in the presence of H2O2. It is shown that, due to the interaction of D-penicillamine with both myeloperoxidase itself and HOCl, the chlorinating activity of myeloperoxidase in the presence of H2O2 and chloride ions is prevented. A concentration of 100 microM D-penicillamine inhibits the chlorinating activity of myeloperoxidase completely, which Is due to the stabilization of Compound II, an inactive form of the enzyme. In addition, HOCl reacts directly with D-penicillamine. Analysis of the reaction products of D-penicillamine and HOCl showed that D-penicillamine was oxidized to penicillamine disulphide and penicillamine sulphinic acid, and eventually deaminated (indicated by the release of ammonia). Lower concentrations of D-penicillamine (10 microM) inhibited myeloperoxidase less, but still acted as effective scavengers of HOCl. In very low concentrations (1 microM), D-penicillamine did not scavenge HOCl effectively, but rather stimulated the chlorinating activity of myeloperoxidase. However, when instead of D-penicillamine a comparable amount of ascorbate was added, a similar but even larger stimulation was observed. Since the concentration of free D-penicillamine in serum from rheumatoid patients treated with this drug is about 20 microM (Saetre, R. and Rabenstein, D.L. (1978) Anal. Chem. 50, 276-280), the therapeutic effect of D-penicillamine may be due to the protection of tissues against the reactive HOCl released by activated granulocytes at inflammation sites.
Amino Acids↗
An EPR study of the photodissociation reactions of oxidised cytochrome c oxidase-nitric oxide complexes.
Complexes of oxidised cytochrome c oxidase with NO in the absence and presence of ligands such as formate, fluoride and cyanide are photodissociable. After photodissociation at 10 K the EPR spectrum of the high-spin cytochrome a3+3 in the absence of ligands or in the presence of fluoride or formate disappears - as does the EPR spectrum of the low-spin cytochrome a3+3 in the presence of cyanide. The action spectra of the photodissociation reaction of these complexes show slight differences but all have maxima at 640-660 nm and below 400 nm, and are assigned to a diamagnetic Cu+B-NO+ complex. The differences in the action spectra in the presence of various ligands are due to binding of these anions to the cytochrome (a3-CuB) couple. The disappearance of the cytochrome a3 signal upon photodissociation of the Cu+B-NO+ complex is explained by a magnetic interaction between cytochrome a3+3 and Cu2+B in the photodissociated complex. The temperature at which NO recombines with Cu2+B is about 30 K and slightly affected by the presence of added ligands. It is suggested that in the oxidised ligand-cytochrome c oxidase complexes the coupling ligand between cytochrome a3+3 and Cu2+B is cyanide, fluoride and formate. The observation that two ligands may bind simultaneously to the cytochrome a3-CuB couple leads to further support for the notion that during turnover of cytochrome c oxidase both metal ions are involved in binding and reduction of oxygen.
Animals↗
The peroxidation of thiocyanate catalysed by myeloperoxidase and lactoperoxidase.
Peroxidation of SCN- to OSCN-, catalysed by myeloperoxidase and lactoperoxidase, was studied. The rate of this reaction showed sharp optima between pH 5 and 7.5, the position of which is determined by the concentrations of both SCN- and H2O2. At low pH values, both SCN- and H+ inhibited myeloperoxidase and lactoperoxidase competitively with respect to H2O2. The inhibition constants of SCN- for myeloperoxidase and lactoperoxidase (2 and 6 mM, respectively) are independent of pH. For these enzymes a Ki for H+ of 1 microM was found that corresponded to an ionisable group on the enzymes (pKa = 6) which controls the enzymic activity. A kinetic expression is proposed that explains most of the data. The physiological consequences of the corresponding mechanism are discussed.
Animals↗
Electron transfer after flash photolysis of mixed-valence carboxycytochrome c oxidase.
The light-induced difference spectra of the fully reduced (a2+ a23+-CO) complex and the mixed-valence carboxycytochrome c oxidase (a3+ a23+-CO) during steady-state illumination and after flash photolysis showed marked differences. The differences appear to be due to electron transfer between the redox centres in the enzyme. The product of the absorbance coefficient and the quantum yield was found to be equal in both enzyme species, both when determined from the rates of photolysis and from the values of the dissociation constants of the cytochrome a23+-CO complex. This would confirm that the spectral properties of cytochrome a3 are not affected by the redox state of cytochrome a and CuA. When the absorbance changes after photolysis of cytochrome a23+-CO with a laser flash were followed on a time scale from 1 mus to 1 s in the fully reduced carboxycytochrome c oxidase, only the CO recombination reaction was observed. However, in the mixed-valence enzyme an additional fast absorbance change (k = 7 X 10(3) s-1) was detected. The kinetic difference spectrum of this fast change showed a peak at 415 nm and a trough at 445 nm, corresponding to oxidation of cytochrome a3. Concomitantly, a decrease of the 830 nm band was observed due to reduction of CuA. This demonstrates that in the partially reduced enzyme a pathway is present between CuA and the cytochrome a3-CuB pair, via which electrons are transferred rapidly.
Animals↗
EPR studies of the photodissociation reactions of cytochrome c oxidase-nitric oxide complexes.
Three complexes of NO with cytochrome c oxidase are described which are all photodissociable at low temperatures as measured by EPR. The EPR parameters of the cytochrome a2+(3)-NO complex are the same both in the fully reduced enzyme and in the mixed-valence enzyme. The kinetics of photodissociation of cytochrome a2+(3)-NO and recombination of NO with cytochrome a2+(3) (in the 30-70 K region) revealed no differences in structure between cytochrome a2+(3) in the fully reduced and the mixed-valence states. The action spectrum of the photodissociation of cytochrome a2+(3)-NO as measured by EPR has maxima at 595, 560 and 430 nm, and corresponds to the absorbance spectrum of cytochrome a2+(3)-NO. Photodissociation of cytochrome a2+(3)-NO in the mixed-valence enzyme changes the EPR intensity at g 3.03, due to electron transfer from cytochrome a2+(3) to cytochrome a3+. The extent of electron transfer was found to be temperature dependent. This suggests that a conformational change is coupled to this electron transfer. The complex of NO with oxidized cytochrome c oxidase shows a photodissociation reaction and recombination of NO (in the 20-40 K region) which differ completely from those observed in cytochrome a2+(3)-NO. The observed recombination occurs at a temperature 15 K lower than that found for the cytochrome a2+(3)-NO complex. The action spectrum of the oxidized complex shows a novel spectrum with maxima at 640 and below 400 nm; it is assigned to a Cu2+B-NO compound. The triplet species with delta ms = 2 EPR signals at g 4 and delta ms = 1 signals at g 2.69 and 1.67, that is observed in partially reduced cytochrome c oxidase treated with azide and NO, can also be photodissociated.
Electron Spin Resonance Spectroscopy↗
Characterization of the peroxidase in human eosinophils.
Human eosinophil peroxidase is a cationic protein with a higher content of arginine, the enzyme being poorly soluble in water. The purified enzyme is able to carry out the peroxidative chlorination of monochlorodimedon. Like myeloperoxidase the position of the pH optimum of this reaction depends on the ration of the concentrations of chloride and H2O2. Compared to myeloperoxidase the pH optimum is shifted by 0.8 pH unit to more acid pH values. The physiological consequences of the properties of the eosinophil peroxidase are discussed.
Eosinophils↗
Characterization of hereditary partial myeloperoxidase deficiency.
We studied a family with a partial myeloperoxidase deficiency. The myeloperoxidase in the neutrophils and monocytes of the parents and their two sons had normal spectral properties (determined optically and by EPR). Enzymic characteristics (oxidation of iodide) were indistinguishable from those of normal myeloperoxidase; moreover, immunological identity between the myeloperoxidase in the leukocytes of the family members and normal myeloperoxidase was found. No differences in heat stability were observed. The neutrophils and monocytes of the sons contained 9% to 18% of the myeloperoxidase content of normal cells; the neutrophils and monocytes of the parents contained 45% to 58%. These data suggest either that the parents are heterozygous and the sons homozygous for hereditary partial myeloperoxidase deficiency or that each parent is heterozygous for a different type of myeloperoxidase deficiency and the sons combine both deficiencies. The oxidative metabolism of the neutrophils during phagocytosis was not affected by the myeloperoxidase deficiency. The killing of Staphylococcus aureus was apparently normal. The perforation of Escherichia coli by the neutrophils of the sons, however, was retarded in comparison with normal neutrophils.
Adult↗
Spectral properties of myeloperoxidase and its ligand complexes.
The effects of ligands with various field strengths on the optical absorption spectrum of myeloperoxidase have been investigated. As is the case with other hemoproteins, the Soret peak in the optical absorption spectra at 77 K moves to longer wavelengths when strong-field ligands are present, whereas binding of such ligands as chloride and fluoride, which stabilize the high-spin state, shows the opposite effect. With a ligand of intermediate field strength, such as azide, the optical spectrum is not affected at room temperature, but lowering of the temperature results in the formation of the low-spin form of the enzyme. Similarly, in native myeloperoxidase a spin state equilibrium is found in which the low-spin state is favoured at high ionic strength and displays corresponding changes in the optical spectra. From the ligand- and the temperature-induced changes in the optical spectra of the ferric enzyme it is concluded that the band at 620-630 nm is an alpha band of the low-spin heme iron species, whereas the bands at 500 and 690 nm are probably 'charge-transfer' bands of the heme with the iron in the high-spin state.
Azides↗
Human eosinophil peroxidase: a novel isolation procedure, spectral properties and chlorinating activity.
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Chlorides↗
The killing of newborn larvae of Trichinella spiralis by eosinophil peroxidase in vitro.
Helminth infections in mammals are characterized by a high level of eosinophils in parasitized tissues and blood, and it has recently been suggested that these cells have a direct parasiticidal effect. Newborn larvae of Trichinella spiralis can be killed within 20 min by incubation at room temperature in a cell-free system, including purified human eosinophil peroxidase (EPO), H2O2 and chloride at pH 5.5. Killing was measured by microscopic observation of the larvae. The larvicidal effect was dependent on each component of the EPO/H2O2/Cl- system and could be prevented by using SO4(2-) instead of Cl-. Killing was totally inhibited by sodium azide and catalase, and substantially by bovine serum albumin, a protein that is an effective scavenger for HOCl. Since larvae could also be killed directly by HOCl under these conditions and EPO is able to oxidize Cl- to hypochlorous acid, it is very likely that the larvicidal effect of the EPO system is due to formation of hypochlorous acid. It is proposed that in vivo, the combination of EPO, which is exocytosed onto the surface of the parasite, and H2O2, which is generated by stimulated eosinophils, is responsible for the larvicidal effect.
Animals↗
The dependence of onset and duration of sleep on th circadian rhythm of rectal temperature.
The sleep-wake cycle and the circadian rhythm of rectal temperature were recorded in subjects who lived singly in an isolation unit. In 10 subjects, the freerunning rhythms remained internally synchronized, 10 other subjects showed internal desynchronization. Times of onset and end of bedrest ("sleep") were determined in each cycle and referred to the phase of the temperature rhythm. In the synchronized subjects, onset of sleep occurred, on the average, 1.34 h before the minimum of temperature, and end of sleep 6.94 h thereafter, with narrow distributions. The desynchronized subjects had a broad bimodal distribution of sleep onsets (peaks 6.3 and 1.3 h before the minimum); the duration of sleep varied between more than 15 h when sleep began about 10 h before the temperature minimum, and less than 4 h when sleep began several hours after the minimum. The dependence of sleep duration on body temperature is interpreted as a continuing action of the coupling forces between the two rhythms after mutual synchronization is lost.
Adult↗
Some enzymatic characteristics of eosinophil peroxidase from patients with eosinophilia and from healthy donors.
Some enzymatic characteristics of human eosinophil peroxidase were compared with those of human myeloperoxidase. Both enzymes catalyzed the oxidation of iodide by hydrogen peroxide. This assay proved to be very sensitive; the activity of 100 eosinophils/ml could be measured. The position of the pH optimum of this reaction was linearly dependent on the logarithm of the iodide/H2O2 ratio. At the same substrate ratio, this optimum was about 1 pH unit higher for eosinophil peroxidase than for myeloperoxidase. This difference may be related to the action of myeloperoxidase inside an acidified phagolysosome as opposed to the extracellular action of eosinophil peroxidase on the surface of certain parasites. Under defined conditions (KI, 1.4 mM; H2O2, 0.18 mM; cetyltrimethylammonium bromide, 0.008% [wt/vol]; pH 6), the activity of eosinophil peroxidase could be measured in a mixed granulocyte suspension independently of myeloperoxidase. Eosinophils from patients with eosinophilia were found to contain as much peroxidase activity as did eosinophils from healthy donors. No enzymatic differences in eosinophil peroxidase were found between the two types of donors.
Cetrimonium↗
Extracellular proton release by stimulated neutrophils.
We have tried to elucidate the mechanism of phagosome acidification in human neutrophils. Assuming that phenomena occurring at the plasma membrane reflect reactions in the phagocytic vacuoles, we have stimulated human neutrophils with agents that induce a "respiratory burst," and we have measured the release of protons into the extracellular medium. Phorbol myristate acetate, N-formyl-methionyl-leucyl-phenylalanine and serum-opsonized zymosan particles each caused a rapid release of protons, concomitant with the increase in oxygen consumption. The stimulated release of protons was strictly coupled to the increase respiration of the cells, because inhibition of the respiration of either anaerobiosis, chlorpromazine, or glycolytic inhibitors also inhibited the release of protons. Also, in the presence of the above-mentioned stimulating agents, neutrophils from three patients with chronic granulomatous disease enhanced neither respiration not proton release. In normal cells, the ratio of deltaH+/-deltaO2 was 1.04 +/- 0.19 (mean +/ SD, n = 13). The mechanism of this proton release is not clear. The amount of lactic and carbonic acid produced by stimulated neutrophils was inadequate to explain the amount of protons released. Perhydroxyl radicals were also ruled out as the source of the protons. Because the cells did not release measurable amounts of phosphate ions, a phosphate-hydroxyl-ion antiport was also excluded. Finally, the lack of any effect of uncouplers renders it unlikely that a respiration-driven proton gradient is built up across the plasma membrane.
Granulomatous Disease, Chronic↗
Thiocyanate as a cofactor in myeloperoxidase activity against Streptococcus mutans.
Inhibition of glycolysis of some oral bacteria was established in vitro by an antibacterial system, consisting of myeloperoxidase, H2O2, and a cofactor. When thiocyanate was used in physiological concentration as a cofactor, the system acted (at low pH) bactericidally on Streptococcus mutans, as indicated by the determination of viable counts. However, at neutral pH, the glycolysis of Streptococcus mutans was inhibited, while its viability remained unaffected. The possible role of such a pH-dependent antibacterial system in the oral cavity is discussed.
Androstenedione↗
Redox reactions in mixed-valence cytochrome c oxidase.
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Anaerobiosis↗
The halide complexes of myeloperoxidase and the mechanism of the halogenation reactions.
The spectral changes caused by the addition of halides to myeloperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) have been investigated and the dissociation constants of the enzyme-halide complexes have been determined. The pH dependence of the dissociation constants suggests that halide binding is associated with a protonation step in myeloperoxidase. Myeloperoxidase catalyzes the peroxidative chlorination and bromination of monochlorodimedone. It is shown that at low pH, chloride acts as a competitive inhibitor with respect to H2O2, whereas at higher pH, H2O2 inhibits the chlorination reaction. The dissociation constant (Kd) of the spectroscopically detectable complex and the Km for chloride are considerably smaller than the inhibition constant (Ki) for chloride. These halogenation reactions are strongly pH dependent, the logarithm of the Km for chloride varies linearly with pH. The position of the pH optimum of the chlorination and bromination reaction is a linear function of the logarithm of the [halide]/[H2O2] ratio. A mechanism of the chlorination and bromination reaction is suggested with substrate inhibition for both hydrogen peroxide and the halide.
Catalysis↗