Expansion of the inner membrane compartment and its relation to mitochondrial volume and ion transport.

Glutaraldehyde has been used to fix mitochondria undergoing rapid volume changes associated with energized ion transport under oscillatory state conditions and valinomycin-induced potassium uptake. Fixation was found to prevent structural changes which normally occur during ion accumulation or loss. By correlating packed volume measurements with electron microscopy, it is shown that changes in volume associated with ion movements reflect changes in the inner membrane compartment and that this compartment can be related to the sucrose inaccessible space. The method can therefore be used to accurately determine volume changes that arise from ion translocation.

Studies on epithelial cells isolated from guinea pig small intestine.

Sheets of mucosal epithelial cells were released from guinea pig small intestine after incubation with ethylenediaminetetraacetate. Cells in sheets retained their columnar shape for 24 hr at room temperature, and exclusion of nigrosine suggested they had intact plasma membranes. When sheets were disaggregated individual cells had normal morphology for at least 4 hr. During isolation 16% of the total protein and 24% of the total lactic dehydrogenase were lost from the cells, but subsequent enzyme leakage was low. Leakage increased with shaking, incubation at 37 degrees C, or increasing the oxygen tension of the suspending medium, but was minimal when the Na(+):K(+) ratio in the medium was 8:1 and the osmolarity was high. Losses of particulate enzyme activities were negligible. Respiration was constant for up to 4 hr and was insensitive to calcium, bicarbonate, oxygen tension, and pH. It was inhibited by cyanide and iodoacetate and varied with the Na(+):K(+) ratio of the extracellular fluid and the structural integrity of the cells. All preparations concentrated potassium and excluded sodium, but lost this ability if ouabain was added or cells were broken. Potassium-42 uptake was also sensitive to temperature, ouabain, and structural integrity. The preparations are being used to study cell metabolism in the intestinal epithelium.

Turnover and vectorial properties of cytochrome c oxidase in reconstituted vesicles.

1. Proteoliposomes containing cytochrome c oxidase and phospholipid have been made by sonication and by the cholate dialysis procedure. In both methods of preparation, only about 50% of the enzyme molecules are oriented in the membrane with their cytochrome c reaction sites exposed to the outside of the vesicle. 2. The activity of cytochrome c oxidase in the reconstituted vesicles in not increased by incubation in 1% Tween 80. Experiments on reconstituted vesicles containing internal (entrapped) cytochrome c indicate that turnover of enzyme oxidising entrapped cytochrome c in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethyl-p-phenylendediamine is at a very much lower rate than enzyme oxidising external ferrocytochrome c. 3. Oxidation of ascorbate by externally added cytochrome c results in an electrogenic production of OH- inside the vesicles, which can be monitored using entrapped phenol red. Polylysine inhibits, but does not abolish, the internal alkalinity change in reconstituted vesicles oxidising internal (entrapped) cytochrome c using externably added ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine. When 2,3,5,6-tetramethyl-p-phenylenediamine is used as the permeable redox mediator, an increase in internal acidity can be monitored under the same conditions.

Slow ('resting') forms of mitochondrial cytochrome c oxidase consist of two kinetically distinct conformations of the binuclear CuB/a3 centre--relevance to the mechanism of proton translocation.

We have purified slow ('resting') cytochrome oxidase from bovine heart, free of contamination with fast ('pulsed') enzyme. This form of the enzyme shows two kinetic phases of reduction of haem a3 by dithionite (k = 0.020 +/- 0.005 s-1 and k = 0.005 +/- 0.002 s-1). The presence of ligands that bind to the oxidized or reduced binuclear centre (formate or carbon monoxide respectively) has no effect on these rates. Varying the dithionite concentration also has no effect on either phase, although at low dithionite concentrations a lag phase is observed as the rate of haem a reduction is slower. The results are consistent with a model for reduction of the slow enzyme where the rate of electron transfer to the binuclear centre is the limiting step, rather than an equilibrium model where the haem a3 redox potential is low. Increasing the pH decreases the rate of the slower phase of dithionite reduction, but has no effect on the faster phase. EPR studies show that the slow phase (only) correlates with the disappearance of the g' = 12/g' = 2.95 signals, with the same pH dependence; again the presence of formate has no effect on these results. Deconvolution of the oxidized optical spectra shows that the enzyme reduced in the slow phase has a blue-shifted Soret band, relative to that reduced in the faster phase. Incubation of the oxidized enzyme at high pH causes a line-broadening of both the g' = 12 and g' = 2.95 EPR signals with no obvious effect on the amount of signal. The results are interpreted in a model where the presence of a carboxylate bridge between haem a3 and CuB defines the slow enzyme. It is suggested that the two rates of dithionite reduction are the result of different ligation to CuB--where water is the ligand the binuclear centre is FeIV/CuI (EPR-silent) and where hydroxide is the ligand the binuclear centre is FeIII/CuII (g' = 12/g' = 2.95 EPR signals).

Activation by reduction of the resting form of cytochrome c oxidase: tests of different models and evidence for the involvement of CuB.

(1) The reaction of the resting form of oxidised cytochrome c oxidase from ox heart with dithionite has been studied in the presence and absence of cyanide. In both cases, cytochrome a reduction in 0.1 M phosphate (pH 7) occurs at a rate of 8.2.10(4) M-1.s-1. In the absence of cyanide, ferrocytochrome a3 appears at a rate (kobs) of 0.016 s-1. Ferricytochrome a3 maintains its 418 nm Soret maximum until reduced. The rate of a3 reduction is independent of dithionite concentration over a range 0.9 mM-131 mM. In the presence or cyanide, visible and EPR spectral changes indicate the formation of a ferric a3/cyanide complex occurs at the same rate as a3 reduction in the absence of cyanide. A g = 3.6 signal appears at the same time as the decay of a g = 6 signal. No EPR signals which could be attributed to copper in any significant amounts could be detected after dithionite addition, either in the presence or absence of cyanide. (2) Addition of dithionite to cytochrome oxidase at various times following induction of turnover with ascorbate/TMPD, results in a biphasic reduction of cytochrome a3 with an increasing proportion of the fast phase of reduction occurring after longer turnover times. At the same time, the predominant steady state species of ferri-cytochrome a3 shifts from high to low spin and the steady-state level of reduction of cytochrome a drops indicating a shift in population of the enzyme molecules to a species with fast turnover. In the final activated form, oxygen is not required for fast internal electron transfer to cytochrome a3. In addition, oxygen does not induce further electron uptake in samples of resting cytochrome oxidase reduced under anaerobic conditions in the presence of cyanide. Both findings are contrary to predictions of certain O-loop types of mechanism for proton translocation. (3) A measurement of electron entry into the resting form of cytochrome oxidase in the presence of cyanide, using TMPD or cytochrome c under anaerobic conditions, shows that three electrons per oxidase enter below a redox potential of around +200 mV. An initial fast entry of two electrons is followed by a slow (kobs approximately 0.02 s) entry of a third electron.(ABSTRACT TRUNCATED AT 400 WORDS)

Water permeability in human erythrocytes: identification of membrane proteins involved in water transport.

The water permeability of human erythrocytes has been monitored by nuclear magnetic resonance (NMR) before and after treatment of the cells with various sulfhydryl reagents. Preincubation of the cells with N-ethylmaleimide (NEM), a non-inhibitory sulfhydryl reagent, results in a faster and more sensitive inhibition of water exchange by mercurials. The inhibition of water exchange by p-chloromercuribenzene sulfonate (PCMBS) was maximal at a binding of approximately 10 nmol PCMBS per mg protein when non-specific sulfhydryl groups are blocked by NEM. Inhibition by PCMBS has been correlated with the binding of 203Hg to erythrocyte membrane proteins. A significant binding of label to band 3 and the polypeptides in band 4.5 occurs, with approximately 1 mol of mercurial bound per mol of protein. Inhibition of water transport by sulfhydryl reagents does not induce major morphological changes in the cells as assessed by freeze-fracture and scanning electron microscopy.

Antimycin inhibition of the cytochrome bd complex from Azotobacter vinelandii indicates the presence of a branched electron transfer pathway for the oxidation of ubiquinol.

Antimycin A and UHBDT inhibit the activity of the purified cytochrome bd complex from Azotobacter vinelandii. Inhibition of activity is non-competitive and antimycin A binding induces a shift to the red in the spectrum of a b-type haem. No inhibitory effects were seen with myxothiazol. Steady-state experiments indicate that the site of inhibition for antimycin A lies on the low-potential side of haem b558. In the presence of antimycin A at concentrations sufficient to inhibit respiration, some direct electron transfer from ubiquinol-1 to haem b595 and haem d still occurs. The results are consistent with a branched electron transfer pathway from ubiquinol to the oxygen reduction site.

Chemiosmotic coupling in cytochrome oxidase. Possible protonmotive O loop and O cycle mechanisms.

Using the principle of specific vectorial ligand conduction, we outline directly coupled protonmotive O loop and O cycle mechanisms of cytochrome oxidase action that are analogous to protonmotive Q loop and Q cycle mechanisms of QH2 dehydrogenase action. We discuss these directly coupled mechanisms in the light of available experimental knowledge, and suggest that they may stimulate useful new research initiatives designed to elucidate the osmochemistry of protonmotive oxygen reduction in cytochrome oxidase.

Uncoupling of intestinal mitochondrial oxidative phosphorylation and inhibition of cyclooxygenase are required for the development of NSAID-enteropathy in the rat.

BACKGROUND: The pathogenesis of NSAID-induced gastrointestinal damage is believed to involve a nonprostaglandin dependent effect as well as prostaglandin dependent effects. One suggestion is that the nonprostaglandin mechanism involves uncoupling of mitochondrial oxidative phosphorylation. AIMS: To assess the role of uncoupling of mitochondrial oxidative phosphorylation in the pathogenesis of small intestinal damage in the rat. METHODS: We compared key pathophysiologic events in the small bowel following (i) dinitrophenol, an uncoupling agent (ii) parenteral aspirin, to inhibit cyclooxygenase without causing a 'topical' effect and (iii) the two together, using (iv) indomethacin as a positive control. RESULTS: Dinitrophenol altered intestinal mitochondrial morphology, increased intestinal permeability and caused inflammation without affecting gastric permeability or intestinal prostanoid levels. Parenteral aspirin decreased mucosal prostanoids without affecting intestinal mitochondria in vivo, gastric or intestinal permeability. Aspirin caused no inflammation or ulcers. When dinitrophenol and aspirin were given together the changes in intestinal mitochondrial morphology, permeability, inflammation and prostanoid levels and the macro- and microscopic appearances of intestinal ulcers were similar to indomethacin. CONCLUSIONS: These studies allow dissociation of the contribution and consequences of uncoupling of mitochondrial oxidative phosphorylation and cyclooxygenase inhibition in the pathophysiology of NSAID enteropathy. While uncoupling of enterocyte mitochondrial oxidative phosphorylation leads to increased intestinal permeability and low grade inflammation, concurrent decreases in mucosal prostanoids appear to be important in the development of ulcers.

Routes of cytochrome a3 reduction. The neoclassical model revisited.

A model for cytochrome oxidase is presented in which cytochrome a, cytochrome a3, and CuB are mutally interacting centers. Cytochrome a3, at equilibrium, is always reduced after CuB. The redox potential of cytochrome a declines progressively as the a3 CuB center is reduced. Dithionite reduction involves up to five steps: (i) reduction of cytochrome a and CuA; (ii) reduction of CuB; (iii) dissociation of ligands (exogenous and endogenous) from cytochrome a3; (iv) spin state changes (high to low) in cyt. a3 and (v) reduction of cytochrome a3. Any of (ii), (iii), and (iv) may be implicated as part of the slow step in this process, which is seen in the resting enzyme.

Spectrophotometric characterization of intermediate redox states of cytochrome oxidase.

The spectrophotometric characteristics of hemes a and a3 in cytochrome oxidase have been examined over the range 380 nm to 900 nm. Difference spectra (relative to the oxidized form) are presented for ferrous, high-spin oxidized, low-spin oxidized, early "pulsed," late "pulsed," and two-peroxide-treated states of the enzyme. Comparisons indicate that the decay product of the initial peroxide complex of the enzyme is identical to a low-spin pulsed form of the enzyme. A high-spin pulsed form of the enzyme persists for several hours to days after preparation.

A hydrogen-donating monohydroxamate scavenges ferryl myoglobin radicals.

The addition of 25 microM hydrogen peroxide to 20 microM metmyoglobin produces ferryl (FeIV = O) myoglobin. Optical spectroscopy shows that the ferryl species reaches a maximum concentration (60-70% of total haem) after 10 minutes and decays slowly (hours). Low temperature EPR spectroscopy of the high spin metmyoglobin (g = 6) signal is consistent with these findings. At this low peroxide concentration there is no evidence for iron release from the haem. At least two free radicals are detectable by EPR immediately after H2O2 addition, but decay completely after ten minutes. However, a longer-lived radical is observed at lower concentrations that is still present after 90 minutes. The monohydroxamate N-methylbutyro-hydroxamic acid (NMBH) increases the rate of decay of the fenyl species. In the presence of NMBH, none of the protein-bound free radicals are detectable; instead nitroxide radicals produced by oxidation of the hydroxamate group are observed. Similar results are observed with the trihydroxamate, desferrioxamine. "Ferryl myoglobin" is still able to initiate lipid peroxidation even after the short-lived protein free radicals are no longer detectable (E.S.R. Newman, C.A. Rice-Evans and M.J. Davies (1991) Biochemical and Biophysical Research Communications 179, 1414-1419). It is suggested that the longer-lived protein radicals described here may be partly responsible for this effect. The mechanism of inhibition of initiation of lipid peroxidation by hydroxyamate drugs, such as NMBH, may therefore be due to reduction of the protein-derived radicals, rather than reduction of ferryl haem.

Quantization of membrane potential generation by cytochrome c oxidase in small vesicles.

Proteoliposomes incorporating cytochrome c oxidase have been prepared by the cholate dialysis method and by sonication. Sonication produces multilamellar vesicles heterogeneous in size in contrast to a more uniform preparation of unilamellar vesicles produced by the dialysis procedure. Respiratory control in both preparations ranges between 4 and 8. From an electron microscopic analysis of proteoliposome size, the average electrical capacitance/vesicle for the dialyzed and sonicated preparations is calculated as 15 X 10(-18) F and 130 X 10(-18) F, respectively. These capacitance values would lead to a quantization of membrane potential generation by the enzyme at 77 mV/turnover for the dialyzed preparation and 9 mV/turnover for sonicated vesicles. It is argued that these differences can explain the dependence of H+ translocation on the number of turnovers of cytochrome c oxidase in dialyzed preparations in contrast to the lack of dependence on number of turnovers in sonicated preparations.

Electrostatic interactions at the plasma membrane.

Hydrogen-ion titration has been used to detect the presence of charged groups on the human red-cell plasma membrane. The findings are discussed in terms of the effect of the local environment on electrostatic interactions between the charged groups.

Formation and reduction of a 'peroxy' intermediate of cytochrome c oxidase by hydrogen peroxide.

In the presence of micromolar concentrations of H2O2, ferric cytochrome c oxidase forms a stable complex characterized by an increased absorption intensity at 606-607 nm with a weaker absorption band in the 560-580 nm region. Higher (millimolar) concentrations of H2O2 result in an enzyme exhibiting a Soret band at 427 nm and an alpha-band of increased intensity in the 589-610 nm region. Addition of H2O2 to ferric cytochrome c oxidase in the presence of cyanide results in absorbance increases at 444nm and 605nm. These changes are not seen if H2O2 is added to the cyanide complex of the ferric enzyme. The results support the idea that direct reaction of H2O2 with ferric cytochrome a 3 produces a 'peroxy' intermediate that is susceptible to further reduction by H2O2 at higher peroxide concentrations. Electron flow through cytochrome a is not involved, and the final product of the reaction is the so-called 'pulsed' or 'oxygenated' ferric form of the enzyme.