scholarly journals Dynamics of proteoliposome formation. Intermediate states during detergent dialysis

1987 ◽  
Vol 246 (3) ◽  
pp. 737-744 ◽  
Author(s):  
J M Wrigglesworth ◽  
M S Wooster ◽  
J Elsden ◽  
H J Danneel
Keyword(s):  
Electron Microscopy ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Protein Conformation ◽  
Respiratory Control ◽  
Molar Ratio ◽  
Gel Chromatography ◽  
Protein Orientation ◽  
Intermediate States ◽  
Small Unilamellar Vesicles

1. The intermediate structures formed during dialysis of mixtures of cholate, phospholipid and cytochrome c oxidase were analysed by gel chromatography and electron microscopy. Measurements of trapped phosphate and the degree of respiratory control were used to assess the integrity of the vesicular structures formed. Protein orientation in the bilayer was monitored by the accessibility of cytochrome c to cytochrome c oxidase. 2. The results indicate that proteoliposome formation by the detergent-dialysis procedure takes place in three distinct stages. In the first stage, cholate/phospholipid and cholate/phospholipid/protein micelles coexist in solution and grow in size as the detergent is slowly removed. At a detergent/phospholipid molar ratio of about 0.2, micelle fusion results in the formation of large bilayer aggregates permeable to both phosphate and cytochrome c. It is at this stage that cytochrome c oxidase is incorporated into the bilayer. In the final stage of dialysis the bilayer sheets fragment into small unilamellar vesicles. 3. The orientation of membrane protein in the final vesicles appears to be determined by the effect of protein conformation on the initial curvature of the bilayer sheets during the fragmentation process.

scholarly journals Electron microscopy of cytochrome c oxidase-containing proteoliposomes: imaging analysis of protein orientation and monomer-dimer behaviour

1993 ◽  
Vol 292 (3) ◽  
pp. 933-946 ◽  
Author(s):  
M Tihova ◽  
B Tattrie ◽  
P Nicholls
Keyword(s):  
Electron Microscopy ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Respiratory Control ◽  
Fracture Plane ◽  
Aggregation State ◽  
Protein Orientation ◽  
Small Areas ◽  
Probable Presence ◽  
Two Sides

1. Cytochrome c oxidase-containing vesicles were prepared by cholate dialysis using bovine heart cytochrome c oxidase with egg and dioleoylphosphatidylcholine/dioleoylphosphatidylethanolamines (1:1, w/w) at two ratios of phospholipid to protein (25 mg/mg and 10 mg/mg). With each mixture, one or two (FII, FIII) fractions with mostly outward-facing cytochrome aa3 were separated from a fraction (FI) containing mostly inward-facing enzyme and protein-free liposomes by DEAE-Sephacel chromatography. 2. FII and FIII fractions from egg phospholipid mixtures had 60-80% outward-facing enzyme; FII and FIII fractions from dioleoyl phospholipids showed 50-70% outward-facing enzyme. Egg and dioleoyl phospholipid mixtures maintained good respiratory control ratios (8-13) only at the higher lipid/protein ratios. 3. Platinum/carbon replicas of freeze-fractured vesicle surfaces were subjected to image analysis. The results showed two types of membrane projection with average heights of 7.5 nm and 3.5 nm from the fracture plane. The former were more numerous on the convex faces. Calculated areas of the projections indicated the probable presence of both enzyme dimers and higher aggregates. Oxidase dimers may have membrane areas of 70-80 nm2 at the high (7.5 nm) side and 40-50 nm2 on the low (3.5 nm) side. 4. Proteoliposomes prepared with enzyme depleted of subunit III contained predominantly much smaller projecting areas. These probably represent monomers with high side areas of 35-40 nm2 and low side areas of 20-25 nm2. Electron microscopy thus directly confirms the predicted change of aggregation state resulting from subunit depletion. 5. The results are compared with those from two-dimensional crystals. Assuming that the high and low projections are two sides of one family of transmembrane molecules, a total length of 11 nm matches 11-12 nm lengths obtained by crystallography. Our membrane areas match the areas obtained in earlier ‘crystal’ studies better than the small areas obtained recently by electron cryomicroscopy.

scholarly journals Reconstitution of cytochrome c oxidase in phospholipid vesicles containing polyvinylic polymers

1989 ◽  
Vol 257 (3) ◽  
pp. 783-787 ◽  
Author(s):  
P Sarti ◽  
G Antonini ◽  
F Malatesta ◽  
B Vallone ◽  
S Villaschi ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Functional Characterization ◽  
Respiratory Control ◽  
Phospholipid Vesicles ◽  
Lipid Phase ◽  
Protein Orientation ◽  
Highly Hydrophobic ◽  
Polymer Interaction

Cytochrome c oxidase was reconstituted in phospholipid vesicles in the presence of highly hydrophobic poly(vinyl alkanoate) polymers. Electron-microscopy observations demonstrated that polymer interaction with the lipid phase induces vesicles to adopt smaller diameters than those typical of standard proteoliposomes. Functional characterization of these polymer-proteoliposome structures indicates that the reconstitution of the enzyme proceeds efficiently without causing either scrambling of the protein orientation in the membrane or loss of respiratory control. A clear dependence of respiratory control ratio on vesicle size was also demonstrated, which is in agreement with a previous model proposed for control of activity of cytochrome c oxidase vesicles [Brunori, Sarti, Colosimo, Antonini, Malatesta, Jones & Wilson (1985) EMBO J. 4, 2365-2368].

scholarly journals 1616. Mechanism of Thrombocytopenia Induced by Oxazolidinones Antibiotics (Linezolid, Tedizolid): Demonstration of Impairment of Megakaryocyte Differentiation From Human Hematopoietic Stem Cells associated with Mitochondrial Toxicity

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S801-S801
Author(s):  
Paul M Tulkens ◽  
Tamara V Milosevic ◽  
Gaëlle Vertenoeil ◽  
William Vainchenker ◽  
Stefan N Constantinescu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Stem Cells ◽  
Cytochrome C ◽  
Hematopoietic Stem Cells ◽  
Cytochrome C Oxidase ◽  
Panel Member ◽  
Human Model ◽  
Mitochondrial Toxicity ◽  
Review Panel ◽  
Hematopoietic Stem

Abstract Background Linezolid causes thrombocytopenia, which limits its use. In cell culture and in tissues from treated patients, linezolid impairs mitochondrial protein synthesis (due to structural similarities and common binding sites between bacterial and mitochondrial ribosomes). Recent studies have shown that mitochondria act as a key relay in the process leading from activation of the thrombopoietin receptor to megakaryocytes differentiation. Methods Validated ex-vivo human model of hematopoietic stem cells (HSC) differentiation for (i) measuring megakaryocytes, granulocyte-monocytes, and burst-forming unit-erythroids colony formation; (ii) differentiation into megakaryocytes (conversion of CD34+ into CD41+/CD42+ cells; morphology) and proplatelets formation, (iii) mitochondrial toxicity (electron microscopy; cytochrome c-oxidase activity [partly encoded by the mitochondrial genome]). Results We show that linezolid (and the recently approved tedizolid), both at concentrations corresponding to their human serum concentrations) inhibit the maturation of HSC into fully differentiated megakaryocytes (CD41 and CD42-positive cells) and the formation of proplatelets. Optic and Electron microscopy) showed an impairment of the formation of typical megakaryocytes (lack of large polylobulated nuclei and of intracellular demarcation membrane system [required for platelet formation]), together with disappearance of the internal structure of mitochondria. Biochemical studies showed a complete suppression of the activity of cytochrome c-oxidase (a key enzyme of the mitochondrial respiratory chain). Conclusion Our study provides for the first time insights in the mechanism of thrombocytopenia induced by linezolid and tedizolid, identifying mitochondria as their target and showing that the drugs will impair the differentiation of hematopoietic stem cells into mature platelets-releasing megakaryocytes. It illustrates how mitochondria dysfunction may play a key role in toxicology and diseases, while paving the way for rational approaches for the design and screening of less toxic derivatives for the benefit of future patients. Disclosures Paul M. Tulkens, MD, PhD, Bayer (Consultant, Advisor or Review Panel member, Speaker’s Bureau)Menarini (Speaker’s Bureau)Merck (Advisor or Review Panel member, Speaker’s Bureau)Trius (now part of Merck) (Advisor or Review Panel member, Research Grant or Support) Françoise Van Bambeke, PharmD, PhD, Bayer (Speaker’s Bureau)

scholarly journals Cytochrome c-mediated electron transfer between ubiquinol-cytochrome c reductase and cytochrome c oxidase. Kinetic evidence for a mobile cytochrome c pool

1984 ◽  
Vol 217 (2) ◽  
pp. 551-560 ◽  
Author(s):  
R J Froud ◽  
C I Ragan
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
State Level ◽  
Complex Iii ◽  
Molar Ratio ◽  
Cytochrome C Reductase ◽  
Molar Ratios ◽  
Ubiquinol Oxidase ◽  
Mediated Electron Transfer ◽  
Pool Model

Ubiquinol oxidase has been reconstituted from ubiquinol-cytochrome c reductase (Complex III), cytochrome c and cytochrome c oxidase (Complex IV). The steady-state level of reduction of cytochrome c by ubiquinol-2 varies with the molar ratios of the complexes and with the presence of antimycin in a way that can be quantitatively accounted for by a model in which cytochrome c acts as a freely diffusible pool on the membrane. This model was based on that of Kröger & Klingenberg [(1973) Eur. J. Biochem. 34, 358-368] for ubiquinone-pool behaviour. Further confirmation of the pool model was provided by analysis of ubiquinol oxidase activity as a function of the molar ratio of the complexes and prediction of the degree of inhibition by antimycin.

Sulphide oxidation and oxidative phosphorylation in the mitochondria of the lugworm

1997 ◽  
Vol 200 (1) ◽  
pp. 83-92 ◽  
Author(s):  
S Vökel ◽  
M K Grieshaber
Keyword(s):  
Oxygen Consumption ◽  
Cytochrome C ◽  
Oxidative Phosphorylation ◽  
Cytochrome C Oxidase ◽  
Electron Flow ◽  
Respiratory Control ◽  
Sulphide Oxidation ◽  
Atp Production ◽  
Sulphide Concentration ◽  
Flow Through

Oxygen consumption, ATP production and cytochrome c oxidase activity of isolated mitochondria from body-wall tissue of Arenicola marina were measured as a function of sulphide concentration, and the effect of inhibitors of the respiratory complexes on these processes was determined. Concentrations of sulphide between 6 and 9 µmol l-1 induced oxygen consumption with a respiratory control ratio of 1.7. Production of ATP was stimulated by the addition of sulphide, reaching a maximal value of 67 nmol min-1 mg-1 protein at a sulphide concentration of 8 µmol l-1. Under these conditions, 1 mole of ATP was formed per mole of sulphide consumed. Higher concentrations of sulphide led to a decrease in ATP production until complete inhibition occurred at approximately 50 µmol l-1. The production of ATP with malate and succinate was stimulated by approximately 15 % in the presence of 4 µmol l-1 sulphide, but decreased at sulphide concentrations higher than 15­20 µmol l-1. Cytochrome c oxidase was also inhibited by sulphide, showing half-maximal inhibition at 1.5 µmol l-1 sulphide. Sulphide-induced ATP production was inhibited by antimycin, cyanide and oligomycin but not by rotenone or salicylhydroxamic acid. The present data indicate that sulphide oxidation is coupled to oxidative phosphorylation solely by electron flow through cytochrome c oxidase, whereas the alternative oxidase does not serve as a coupling site. At sulphide concentrations higher than 20 µmol l-1, oxidation of sulphide serves mainly as a detoxification process rather than as a source of energy.

scholarly journals Subcellular localization of ferritin and iron taken up by rat hepatocytes

1989 ◽  
Vol 262 (2) ◽  
pp. 685-688 ◽  
Author(s):  
J C Sibille ◽  
M Ciriolo ◽  
H Kondo ◽  
R R Crichton ◽  
P Aisen
Keyword(s):  
Acid Phosphatase ◽  
Cytochrome C ◽  
Subcellular Localization ◽  
Cytochrome C Oxidase ◽  
Density Gradient ◽  
Rat Hepatocytes ◽  
Sucrose Density Gradient ◽  
Gel Chromatography ◽  
Density Gradient Ultracentrifugation

The subcellular localization of ferritin and its iron taken up by rat hepatocytes was investigated by sucrose-density-gradient ultracentrifugation of cell homogenates. After incubation of hepatocytes with 125I-labelled [59Fe]ferritin, cells incorporate most of the labels into structures equilibrating at densities where acid phosphatase and cytochrome c oxidase are found, suggesting association of ferritin and its iron with lysosomes or mitochondria. Specific solubilization of lysosomes by digitonin treatment indicates that, after 8 h incubation, most of the 125I is recovered in lysosomes, whereas 59Fe is found in mitochondria as well as in lysosomes. As evidenced by gel chromatography of supernatant fractions, 59Fe accumulates with time in cytosolic ferritin. To account for these results a model is proposed in which ferritin, after being endocytosed by hepatocytes, is degraded in lysosomes, and its iron is released and re-incorporated into cytosolic ferritin and, to a lesser extent, into mitochondria.

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