Dibucaine interacts differently with membrane and protein in cytochrome c oxidase systems

1993 ◽  
Vol 71 (1-2) ◽  
pp. 14-21
Author(s):  
Jia He ◽  
Peter Nicholls
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Proton Translocation ◽  
Tight Binding ◽  
Respiratory Control ◽  
Soret Band ◽  
Red Shift ◽  
Ph Gradients ◽  
Low Concentrations ◽  
Low Ionic Strength

Dibucaine acts as a weak protonophore in cytochrome c oxidase proteoliposomes. At low concentrations in the presence of permeant anions, it stimulates turnover and collapses enzyme-generated pH gradients. At higher concentrations, dibucaine inhibits activity of cytochrome c oxidase in proteoliposomes and the isolated enzyme. It also induces a red shift in the resting spectrum, indicating a change at the binuclear centre. This spectroscopic effect is kinetically biphasic. Dibucaine inhibits steady-state oxidase activity, but not the rate of the red shift in the cytochrome a3 Soret band during turnover. It reacts faster with the partially reduced state than with resting enzyme. The inhibition is kinetically biphasic with a noncompetitive Ki ≈ 0.5 mM. Excess dibucaine effects a maximal turnover decline of 80%. At low ionic strength only the total Vmax is affected; tight binding of cytochrome c and turnover at the "tight" site are unaffected. Dibucaine may bind to an anionic site in a hydrophobic pocket, modifying electron transfer from cytochrome a and CuA to cytochrome a3 - CuB and the oxidized spectrum of the latter centre. Stimulation of turnover in cytochrome c oxidase in proteoliposomes is due to a separate membrane-dependent proton translocation catalysed by dibucaine in the presence of permeant anions.Key words: dibucaine, cytochrome c oxidase, proteoliposomes, respiratory control, inhibition.

ADP-dependent palmitoykarnitine sensitivity of mitochondria isolated from the perfused rabbit heart

1969 ◽  
Vol 47 (6) ◽  
pp. 611-618 ◽  
Author(s):  
K. J. Kako
Keyword(s):  
Cytochrome C ◽  
Respiratory Rate ◽  
Respiratory Control ◽  
Rabbit Heart ◽  
Nadh Oxidation ◽  
Perfused Heart ◽  
Low Concentrations ◽  
Gradual Loss ◽  
Mitochondrial Suspension ◽  
Perfused Hearts

The function of mitochondria as influenced by the depletion of oxidizable substances in the myocardium was examined during perfusion of the isolated rabbit heart. Mitochondria were isolated using proteinase and KCl–albumin medium, and their respiration was measured polarographically at 25 °C.(i) Mitochondria of nonperfused hearts (control) responded to repeated additions of ADP with repeated and reproducible "state 4 to state 3 transitions" in the presence of a variety of oxidizable substrates (glutamate, oxoglutarate, acetate, pyruvate, β-hydroxybutyrate, succinate, and palmitoyicamitine).(ii) The heart of a fed animal was perfused almost to exhaustion in a substrate-free medium and mitochondria were isolated and tested. The following findings were obtained (iii–viii).(iii) The respiratory control was gradually lost in a response to repeated additions of ADP, when palmitoylcamitine was used as the substrate. A larger quantity of mitochondria in a given assay partially counteracted this gradual loss of the respiratory control.(iv) The respiratory control and P/O ratios of mitochondria obtained from these hearts were slightly depressed as compared to those of the control, when measured using glutamate or oxoglutarate. Malate addition was required for pyruvate oxidation by mitochondria from perfused hearts.(v) The state 3 respiratory rate of the mitochondria obtained from an exhausted heart was about one-quarter that of the control mitochondria regardless of substrates used. The values were independent of the amount of mitochondria present in the assay system.(vi) With paimitoyicamitine as the substrate, the respiratory control ratio calculated from the first state 3 to state 4 transition was influenced by the amount of mitochondria present. The ratio was depressed at low concentrations of mitochondria.(vii) The rate of exogenous NADH oxidation in the absence of cytochrome c and the optical density of the mitochondrial suspension at 520 mμ, were similar with mitochondria prepared from nonperfused and perfused hearts. An addition of cytochrome c accelerated the rate of NADH oxidation by the latter more than the control.(viii) Unstimulated ATPase activity was high in mitochondria of the perfused heart; the enzyme was stimulated by DNP to a lesser degree and by a smaller quantity of paimitoyicarnitine than the control.(ix) The mitochondria prepared from the perfused heart of a fasted animal and from the fed rabbit's heart perfused in the presence of glucose, albumin, or 30 mM KCl (cardiac arrest) showed a normal respiratory function.(x) The mitochondria of the heart in anoxic arrest showed uncoupling of oxidative phosphorylation and a low respiratory rate regardless of the hydrogen donors used.(xi) It is concluded that a prolonged lack of hydrogen sources in perfused exhausted rabbit hearts results in the mitochondrial dysfunction.

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.

Sign in / Sign up

Export Citation Format

Share Document