Purification of cytochrome c peroxidase for monitoring H2O2 production.

One of the most precise methods of determining hydrogen peroxide (H2O2) formation by biological systems is based on measuring the rate of enzyme-substrate complex formation between H2O2 and cytochrome c peroxidase (CCP). The main problem with this method is that CCP is not commercially available and has to be prepared in the laboratory. We have modified some currently available methods for purifying a highly active preparation of CCP in about 4 d. It includes a batch extraction of protein using DEAE-sepharose followed by concentration either by lyophilization or by passing the extract through a small DEAE-sepharose column instead of by ultrafiltration. The concentrated preparation is passed through a Sephadex G-75 column and the final CCP crystallized against water. The final preparations had a purity index (PI, ratio of absorbance at 408 nm/280 nm, equivalent to heme/protein ratio) above 1.2. These changes make the overall procedure very simple, preserving enzyme activity and spectral properties. In addition, we point out that special care has to be taken to eliminate cytochrome c from crude CCP extracts. Cytochrome c not only introduces an artifact when determining PI, but is also may act as a hydrogen donor for CCP when monitoring H2O2 formation, thus decreasing the sensitivity of this method.

Extramitochondrial release of hydrogen peroxide from insect and mouse liver mitochondria using the respiratory inhibitors phosphine, myxothiazol, and antimycin and spectral analysis of inhibited cytochromes.

The fumigant insecticide phosphine (PH3) is known to inhibit cytochrome c oxidase in vitro. Inhibition of the respiratory chain at this site has been shown to stimulate the generation of superoxide radicals (O2-), which dismutate to form hydrogen peroxide (H2O2). This study was performed in order to investigate the production of H2O2 by mitochondria isolated from granary weevil (Sitophilus granarius) and mouse liver on exposure to PH3. Other respiratory inhibitors, antimycin, myxothiazol, and rotenone were used with insect mitochondria. Hydrogen peroxide was measured spectrophotometrically using yeast cytochrome c peroxidase as an indicator. Insect and mouse liver mitochondria, utilizing endogenous substrate, both produced H2O2 after inhibition by PH3. Insect organelles released threefold more H2O2 than did mouse organelles, when exposed to PH3. Production of H2O2 by PH3-treated insect mitochondria was increased significantly on addition of the substrate alpha-glycerophosphate. Succinate did not enhance H2O2 production, however, indicating that the H2O2 did not result from the autoxidation of ubiquinone. NAD(+)-linked substrates, malate and pyruvate also had no effect on H2O2 production, suggesting that NADH-dehydrogenase was not the source of H2O2. Data obtained using antimycin and myxothiazol, both of which stimulated the release of H2O2 from insect mitochondria, lead to the conclusion that glycerophosphate dehydrogenase is a source of H2O2. The effect of combining PH3, antimycin, and myxothiazol on cytochrome spectra in insect mitochondria was also recorded. It was observed that PH3 reduces cytochrome c oxidase but none of the other cytochromes in the electron transport chain. There was no movement of electrons to cytochrome b when insect mitochondria are inhibited with PH3. The spectral data show that the inhibitors interact with the respiratory chain in a way that would allow the production of H2O2 from the sites proposed previously.

Binding of [14C] DDT by submitochondrial particles.

1. Binding of [14C] DDT by submitochondrial particles and by liposomes prepared from lipids extracted from the particles was studied by the discontinuous sucrose gradient method. 2. Binding of the insecticide was a biphasic linear function of the biomembrane- and liposome-concentration with a break in the binding curve occurring at identical concentrations of phospholipid for both the biomembrane and vesicle. The biphasic binding curve is interpreted in terms of decreased availability of binding sites as a result of particle-particle interaction. 3. [14C] DDT was bound mainly by the membrane lipids and only negligible binding was detected for the delipidated membrane. 4. A 100-200-fold excess of unlabeled DDT had no effect on the binding of [14C] DDT and a 600-fold excess of unlabeled DDT reduced the binding by 20% suggesting that binding of [14C] DDT by lipids was nonspecific. 5. These results are discussed in relation to the strong inhibition by DDT of mitochondrial bioenergetics.

Perturbation of phospholipid bilayers by DDT.

The localization of the effects of DDT (5-50 mol%) addition on the acyl chain dynamics in unilamellar vesicles of two phosphatidylcholines (DPPC and egg PC) has been investigated by steady-state fluorescence polarization of a series of n-(9-anthroyloxy) fatty acids (n = 2, 6, 9, 12 and 16) whose fluorophore is located at a graded series of depths from the surface to the centre of the bilayer. The results show that DDT is a fluidizer of DPPC and egg PC bilayers. The increase in microviscosity of DPPC bilayers at 23 degrees C begins at the centre of the bilayer (5 mol% DDT) and proceeds outward to the surface with increasing concentration of DDT (17 mol%). This pattern of effects is not evident in fluid bilayers of DPPC at 54 degrees C or egg PC at 23 degrees C. DDT (33 mol%) also lowers the phase transition temperature of DPPC bilayers by approximately 2 Cdeg. DDT (17 mol%) had no effect on the mean excited fluorescence life-time of 2-AP and 12-AS in DPPC, DOPC and egg PC bilayers. No quenching of 2-AP fluorescence was evident.

The effect of DDT and related insecticides on the mitochondrial ATPase of houseflies.

1. DDT is a weak inhibitor of the ATPase of housefly muscle mitochondria in the absence of Mg2+ but an activator in the presence of Mg2+. 2. By contrast, DDT and several p,p'-substituted alpha-trichlomethylbenzylanilines were strong inhibitors of the ATPase activity in the presence of the uncoupler, dinitrophenol. 3. Maximum inhibition of the DNP-ATPase was achieved when the ATPase complex was dissociated from its endogenous protein inhibitor. 4. The inhibition by DDT was noncompetitive, maximum at acid pH and independent of temperature. The inhibition was counteracted by exogenous phosphatidylcholine and phosphatidylethanolamine. 5. The ATPase was also activated by NH+4 in the presence of valinomycin. This activation was reversed by K+ and strongly inhibited by DDT. 6. The possible mechanisms involved in the inhibition by DDT are discussed.

Role of phospholipids in the DDT-induced efflux of potassium in human erythrocytes.

Incubation of human erythrocytes for 1-2 h at 37 degrees C in a suspension of dipalmitoylphosphatidylcholine (DDPC) liposomes results in a phospholipid enrichment of erythrocyte membranes by 45-55% and a depletion of cholesterol by 19-24%. The enrichment by DPPC was time and concentration dependent. By contrast, dioleoylphosphatidylcholine (DOPC) liposomes were less effective in enriching the membranes with phospholipid and in depleting the membranes of cholesterol. Concomitantly, the DDT-induced efflux of K+ was reduced in the case of DPPC-enriched erythrocytes but enhanced in DOPC-enriched erythrocytes. These results suggest that DDT partitions more readily into the unsaturated than the saturated phospholipids of the erythrocyte membrane. It is concluded that the extent to which DDT affects the flux of K+ across the membrane is dependent on the fluidity of the lipid phase. We also report here a rapid method for cholesterol depletion of red blood cells in comparison to previously reported methods.

Use of the fluorescent probe, 1-anilino-8-naphthalene sulfonate, to monitor the interaction of pesticide chemicals with mitochondrial membranes.

1. The interaction of mitochondrial and submitochondrial membranes with DDT, methoxychlor and kelthane as monitored by fluorescence changes of ANS was studied. 2. The three organochlorine pesticide chemicals reduced the succinate- and ATP-dependent quenching of ANS fluorescence of mitochondria in a concentration-dependent manner. The results suggest that they blocked the supply of energy to the inner membrane. 3. The reversal of the substrate-induced quenching of ANS by uncouplers was abolished by DDT and kelthane probably as a consequence of the inhibition of protonophoric activity of the uncoupler. 4. Both DDT and kelthane were relatively ineffective inhibitors of substrate-induced quenching of ANS fluorescence of submitochondrial particles. The results suggest an asymmetry of the inner membrane with respect to DDT-inhibition of substrate-induced energization. 5. The possible basis of this asymmetry as well as of inhibition of energy supply to the membrane is discussed.

The effect of DDT on K+ transport in mouse liver mitochondria.

This study describes DDT-induced changes in membrane permeability of mitochondria and erythrocytes to K+ as monitored by a K+-selective electrode. DDT is a strong inhibitor of valinomycin-mediated K+ uptake and the corresponding H+ efflux and an inducer of K+ leakage out of mitochondria but not to any significant extent out of erythrocytes. The inhibition of K+ uptake and H+ efflux was a function of (a) preincubation time between mitochondria and DDT, (b) mitochondrial concentration, (c) the nature of the carrier solvent and (d) temperature. The kinetics of inhibition of K+ uptake showed that DDT is an uncompetitive inhibitor with respect to valinomycin and a competitive inhibitor with respect to K+. The efflux of endogenous K+ showed a sigmoid dependency on DDT concentration and was reduced to endogenous rates when the temperature was lowered below the gel-liquid crystalline phase transition of the lipids. It is suggested that the DDT-induced changes in membrane permeability are due to perturbation of the lipid phase and that its toxicity may be due in part to hyperpolarization of subcellular membranes.