Stimulation by paraquat of microsomal and cytochrome P-450-dependent oxidation of glycerol to formaldehyde.

Glycerol can be oxidized to formaldehyde by microsomes in a reaction that is dependent on cytochrome P-450. An oxidant derived from the interaction of H2O2 with iron was responsible for oxidizing the glycerol, with P-450 suggested to be necessary to produce H2O2 and reduce non-haem iron. The effect of paraquat on formaldehyde production from glycerol and whether paraquat could replace P-450 in supporting this reaction were studied. Paraquat increased NADPH-dependent microsomal oxidation of glycerol; the stimulation was inhibited by glutathione, catalase, EDTA and desferrioxamine, but not by superoxide dismutase or hydroxyl-radical scavengers. The paraquat stimulation was also inhibited by inhibitors, substrate and ligand for P-4502E1 (pyrazole-induced P-450 isozyme), as well as by anti-(P-4502E1) IgG. These results suggest that P-450 still played an important role in glycerol oxidation, even in the presence of paraquat. Purified NADPH-cytochrome P-450 reductase did not oxidize glycerol to formaldehyde; some oxidation, however, did occur in the presence of paraquat. Reductase plus P-4502E1 oxidized glycerol, and a large stimulation was observed in the presence of paraquat. Rates in the presence of P-450, reductase and paraquat were more than additive than the sums from the reductase plus P-450 and reductase plus paraquat rates, suggesting synergistic interactions between paraquat and P-450. These results indicate that paraquat increases oxidation of glycerol to formaldehyde by microsomes and reconstituted systems, that H2O2 and iron play a role in the overall reaction, and that paraquat can substitute, in part, for P-450 in supporting oxidation of glycerol. However, cytochrome P-450 is required for elevated rates of formaldehyde production even in the presence of paraquat.

Structural determinants for alcohol substrates to be oxidized to formaldehyde by rat liver microsomes.

Glycerol can be oxidized to formaldehyde by rat liver microsomes and by cytochrome P450. The ability of other alcohols to be oxidized to formaldehyde was determined to evaluate the structural determinants of the alcohol which eventually lead to this production of formaldehyde. Monohydroxylated alcohols such as 1- or 2-propanol did not produce formaldehyde when incubated with NADPH and microsomes. Geminal diols such as 1,3-propanediol, 1,3-butanediol, or 1,4-butanediol also did not yield formaldehyde. However, vicinal diols such as 1,2-propanediol or 1,2-butanediol produced formaldehyde. With 1,2-propanediol, the residual two-carbon fragment was found to be acetaldehyde, while with 1,2-butanediol, the residual three-carbon fragment was propionaldehyde. Oxidation of 1,2-propanediol to formaldehyde plus acetaldehyde involved interaction with an oxidant derived from H2O2 plus nonheme iron, since production of the two aldehydic products was completely prevented by catalase or glutathione plus glutathione peroxidase and by chelators such as desferrioxamine or EDTA. The oxidant was not superoxide or hydroxyl radical. Product formation was fivefold lower when NADH replaced NADPH, and was inhibited by substrates, ligands, and inhibitors of cytochrome P450. A charged glycol such as alpha-glycerophosphate (but not the geminal beta-glycerophosphate) was readily oxidized to formaldehyde, suggesting that interaction of the glycol with the oxidant was occurring in solution and not in a hydrophobic environment. These results indicate that the carbon-carbon bond between 1,2-glycols can be cleaved by an oxidant derived from microsomal generated H2O2 and reduction of non-heme iron, with the subsequent production of formaldehyde plus an aldehyde with one less carbon than the initial glycol substrate.

Role of cytochrome P450 in the oxidation of glycerol by reconstituted systems and microsomes.

Glycerol can be oxidized by rat liver microsomes to formaldehyde in a reaction that requires the production of reactive oxygen intermediates. Studies with inhibitors, antibodies, and reconstituted systems with purified cytochrome P4502E1 were carried out to evaluate whether P450 was required for glycerol oxidation. A purified system containing phospholipid, NADPH-cytochrome P450 reductase, P4502E1, and NADPH oxidized glycerol to formaldehyde. Formaldehyde production was dependent on NADPH, reductase, and P450, but not phospholipid. Formaldehyde production was inhibited by substrates and ligands for P4502E1, as well as by anti-pyrazole P4502E1 IgG. The oxidation of glycerol by the reconstituted system was sensitive to catalase, desferrioxamine, and EDTA but not to superoxide dismutase or mannitol, indicating a role for H2O2 plus non-heme iron, but not superoxide or hydroxyl radical in the overall glycerol oxidation pathway. The requirement for reactive oxygen intermediates for glycerol oxidation is in contrast to the oxidation of typical substrates for P450. In microsomes from pyrazole-treated, but not phenobarbital-treated rats, glycerol oxidation was inhibited by anti-pyrazole P450 IgG, anti-hamster ethanol-induced P450 IgG, and monoclonal antibody to ethanol-induced P450, although to a lesser extent than inhibition of dimethylnitrosamine oxidation. Anti-rabbit P4503a IgG did not inhibit glycerol oxidation at concentrations that inhibited oxidation of dimethylnitrosamine. Inhibition of glycerol oxidation by antibodies and by aminotriazole and miconazole was closely associated with inhibition of H2O2 production. These results indicate that P450 is required for glycerol oxidation to formaldehyde; however, glycerol is not a direct substrate for oxidation to formaldehyde by P450 but is a substrate for an oxidant derived from interaction of iron with H2O2 generated by cytochrome P450.

Role of iron, hydrogen peroxide and reactive oxygen species in microsomal oxidation of glycerol to formaldehyde.

Rat liver microsomes can oxidize glycerol to formaldehyde. This oxidation is sensitive to catalase and glutathione plus glutathione peroxidase, suggesting a requirement for H2O2 in the overall pathway of glycerol oxidation. Hydrogen peroxide can not replace NADPH in supporting glycerol oxidation; however, added H2O2 increased the NADPH-dependent rate. Ferric chloride or ferric-ATP had no effect on glycerol oxidation, whereas ferric-EDTA was inhibitory. Certain iron chelators such as desferrioxamine, EDTA or diethylenetriaminepentaacetic acid, but not others such as ADP or citrate, inhibited glycerol oxidation. The inhibition by desferrioxamine could be overcome by added iron. Neither superoxide dismutase nor hydroxyl radical scavengers had any effect on glycerol oxidation. With the exception of propyl gallate, several antioxidants which inhibit lipid peroxidation had no effect on formaldehyde production from glycerol. The inhibition by propyl gallate could be overcome by added iron. In contrast to glycerol, formaldehyde production from dimethylnitrosamine was not sensitive to catalase or iron chelators, thus disassociating the overall pathway of glycerol oxidation from typical mixed-function oxidase activity of cytochrome P450. These studies indicate that H2O2 and nonheme iron are required for glycerol oxidation to formaldehyde. The responsible oxidant is not superoxide, H2O2, or hydroxyl radical. Cytochrome P450 may function to generate the H2O2 and reduce the nonheme iron. There may be additional roles for P450 since rates of formaldehyde production by microsomes exceed rates found with model chemical systems. Elevated rates of H2O2 production by certain P450 isozymes, e.g., P450 IIE1, may contribute to enhanced rates of glycerol oxidation.

Rapid decrease of cytochrome P-450IIE1 in primary hepatocyte culture and its maintenance by added 4-methylpyrazole.

Studies were conducted to evaluate the possible induction or the maintenance of cytochrome P-450IIE1 in primary hepatocyte cultures by the inducing agent 4-methylpyrazole. Hepatocytes were isolated from control (noninduced) rats and from rats treated in vivo with either pyrazole or 4-methylpyrazole to induce P-450IIE1. The content of P-450IIE1 was determined by Western blots with antipyrazole P-450 IgG, and catalytic activity was assessed by assays of dimethylnitrosamine demethylase activity. The treatment with 4-methylpyrazole in vivo increased the content of P-450IIE1 and dimethylnitrosamine demethylase activity sevenfold and fourfold, respectively. In cultures prepared from noninduced hepatocytes, P-450IIE1 levels fell to values of 76%, 65%, 31% and 1% of freshly isolated hepatocytes after 1, 3, 6 and 9 days in culture. A similar decrease in dimethylnitrosamine demethylase was observed during this time. In cultures prepared from induced hepatocytes, the decline in P-450IIE1 was more rapid as levels fell to 77%, 31%, 3% and 3% of initial values after 1, 3, 6 and 9 days in culture. Again, the fall in dimethylnitrosamine demethylase activity paralleled the decline in content of P-450IIE1 and was more rapid with the induced hepatocytes. With cultures prepared from noninduced or induced hepatocytes, the addition of 4-methylpyrazole in vitro did not increase the content of P-450IIE1 or the activity of dimethylnitrosamine demethylase over the initial values. However, 4-methylpyrazole appeared to stabilize the P-450IIE1 and to decrease its rate of decline in culture. In noninduced cultures, the percent remaining content of P-450IIE1 after 6 days was 31% in the absence of and 52% in the presence of 5 mol/L 4-methylpyrazole. In cultures from 4-methylpyrazole-induced hepatocytes, the percent remaining P-450IIE1 after 3 days was 31% in the absence of inducer and 59% with 4-methylpyrazole added in vitro. Similarly 4-methylpyrazole helped to prevent the rapid decline of dimethylnitrosamine demethylase activity in induced and noninduced cultures. Viability of the induced and noninduced cultures in the absence or presence of added 4-methylpyrazole was similar. Levels of mRNA for P-450IIE1 were similar for livers from control rats and from rats treated in vivo with 4-methylpyrazole. The mRNA levels rapidly declined in induced and noninduced cultures, and this decline, unlike the fall in P-450IIE1 or dimethylnitrosamine demethylase activity, could not be prevented by the addition of 4-methylpyrazole in vitro to the cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of the interaction of pyrazole and its metabolite 4-hydroxypyrazole with rat liver microsomes.

Pyrazole is known to interact with and to induce cytochrome P-450 IIE1. Since pyrazole is oxidized by rat liver microsomes to 4-hydroxypyrazole, and several of the actions of pyrazole have been ascribed to its metabolite, experiments were conducted to evaluate the interactions of 4-hydroxypyrazole with microsomes, and to compare these to pyrazole itself. Rats were injected with doses of 4-hydroxypyrazole ranging from 2 to 100 mg/kg body weight/day for 2 days. A slight increase of total cytochrome P-450 was observed at low doses, followed by a decrease at higher concentrations. NADPH-cytochrome P-450 reductase activity was not affected. The oxidation of aniline or dimethylnitrosamine was increased about 50% by the 4-hydroxypyrazole treatment; however, this extent of increase was much less than that produced by pyrazole treatment. In vitro, 4-hydroxypyrazole produced a type II binding spectrum with microsomes, with a peak at about 425 nm and a trough at about 395 nm. The affinity for 4-hydroxypyrazole was increased from a value of about 0.60 mM in control microsomes to a value of about 0.40 mM in microsomes from pyrazole-treated rats. These values are 2-fold greater than those observed with pyrazole as the ligand. 4-Hydroxypyrazole inhibited the microsomal oxidation of ethanol; kinetics of inhibition were mixed. The apparent KI for 4-hydroxypyrazole inhibition of ethanol oxidation by microsomes was about 4 mM, which is about an order of magnitude greater than that for pyrazole. The in vivo and in vitro interactions of 4-hydroxypyrazole with microsomes appear to be similar to those described for pyrazole; however, these interactions are considerably less effective than those of the parent drug, pyrazole. Thus, although some actions of pyrazole may be due to the metabolite 4-hydroxypyrazole, it appears that the induction of P-450 IIE1 and the in vitro interactions of pyrazole with microsomes is not likely to be mediated by prior metabolism of pyrazole to 4-hydroxypyrazole.

Oxidation of pyrazole by reconstituted systems containing cytochrome P-450 IIE1.

Rat liver microsomes oxidize pyrazole to 4-hydroxypyrazole and this oxidation is increased in microsomes isolated from rats treated with inducers of cytochrome P-450 IIE1, such as pyrazole or ethanol. A reconstituted system containing the P-450 IIE1, purified from pyrazole-treated rats, oxidized pyrazole to 4-hydroxypyrazole in a time- and P-450-dependent manner. Oxidation of pyrazole was dependent on the concentration of pyrazole over the range of 0.15 mM to 1.0 mM. In isolated microsomes, glycerol inhibited pyrazole oxidation by about 50% under concentration conditions which occur in the reconstituted system; hence, the values for pyrazole oxidation by the reconstituted systems are underestimated because of the presence of glycerol. Oxidation of pyrazole was inhibited by competitive substrates for P-450 IIE1, such as 4-methylpyrazole, aniline and ethanol, as well as by an antibody raised against the pyrazole-induced P-450 IIE1. Thus, pyrazole is an effective substrate for oxidation by purified P-450 IIE1, extending the substrate specificity of this isozyme to potent inhibitors of alcohol dehydrogenase.

Sensitivity of the rat liver microsomal oxidation of pyrazole to antibody raised against the ethanol-inducible rabbit liver cytochrome P-450 isozyme.

Pyrazole is oxidized to 4-hydroxypyrazole by rat liver microsomes in a cytochrome P-450-dependent reaction and this oxidation can be increased by prior treatment of rats with pyrazole, 4-methylpyrazole, or chronic ethanol feeding. The induction pattern suggests that pyrazole may be an effective substrate for oxidation by P-450 IIE.1. This P-450 isozyme is recognized by antibody (anti-3a IgG) raised against the ethanol-inducible P-450 in rabbits. Experiments were carried out to evaluate the ability of anti-3a IgG to inhibit pyrazole oxidation by microsomes from controls and from rats treated with inducers of P-450 IIE.1. Immunoblots with anti-3a IgG or with the anti-pyrazole P-450 IgG were identical and indicated increased staining of the pyrazole P-450 with microsomes from rats treated with pyrazole, 4-methylpyrazole, or ethanol, relative to saline controls; very little staining occurred with microsomes from pair-fed controls or phenobarbital-treated rats. Rates of pyrazole oxidation were highest with microsomes from rats treated with the inducers of P-450 IIE.1 and lowest with pair-fed controls or rats treated with phenobarbital. Anti-3a IgG produced about a 60% decrease of pyrazole oxidation in microsomes from rats treated with inducers of P-450 IIE.1 and about a 25% decrease with the saline controls; no inhibition was found with microsomes from the phenobarbital-treated rats. The anti-3a IgG-resistant rate of pyrazole oxidation was similar with all the microsomal preparations, and was not due to interaction of pyrazole with hydroxyl radicals.(ABSTRACT TRUNCATED AT 250 WORDS)

Synergistic interactions between NADPH-cytochrome P-450 reductase, paraquat, and iron in the generation of active oxygen radicals.

The toxicity associated with paraquat is believed to involve the generation of active oxygen radicals and the production of oxidative stress. Paraquat can be reduced by NADPH-cytochrome P-450 reductase to the paraquat radical; this results in consumption of NADPH. A variety of ferric complexes, including ferric-ATP, -citrate, -EDTA, ferric diethylenetriamine pentaacetic acid and ferric ammonium sulfate, produced a synergistic increase in the paraquat-mediated oxidation of NADPH. This synergism could be observed with very low concentrations of iron, e.g. 0.25 microM ferric-ATP. Very low rates of hydroxyl radical were generated by the reductase with paraquat alone, or with ferric-citrate or -ATP or ferric ammonium sulfate in the absence of paraquat; however, synergistic increases in the rate of hydroxyl radical generation occurred when these ferric complexes were added together with paraquat. Ferric-EDTA and -DTPA catalyzed some production of hydroxyl radicals, which was also synergistically elevated in the presence of paraquat. Ferric desferrioxamine was essentially inert in the absence or presence of paraquat. This enhancement of hydroxyl radical generation was sensitive to catalase and competitive scavengers but not to superoxide dismutase. The interaction of paraquat with NADPH-cytochrome P-450 reductase and ferric complexes resulted in an increase in oxygen radical generation, and various ferric complexes increased the catalytic effectiveness and potentiated significantly the toxicity of paraquat via this synergistic increase in oxygen radical generation by the reductase.

Oxidation of glycerol to formaldehyde by rat liver microsomes.

Rat liver microsomes catalyzed the oxidation of glycerol to a Nash-reactive material in a time- and protein-dependent manner. Omission of the glycerol or the microsomes or any of the components of the NADPH-generating system resulted in almost a complete loss of product formation. Apparent Km and Vmax values for glycerol oxidation were about 18 mM and 2.5 nmol formaldehyde per min per mg microsomal protein. Carbon monoxide inhibited glycerol oxidation indicating a requirement for cytochrome P-450. That the Nash-reactive material was formaldehyde was validated by a glutathione-dependent formaldehyde dehydrogenase positive reaction. These studies indicate that glycerol is not inert when utilized with microsomes or reconstituted mixed function oxidase systems, and that the production of formaldehyde from glycerol may interfere with assays of other substrates which generate formaldehyde as product.

Characterization and identification of a pyrazole-inducible form of cytochrome P-450.

In vivo administration of the alcohol dehydrogenase inhibitor pyrazole induces a cytochrome P-450 isozyme. The pyrazole-inducible cytochrome P-450 has been purified from rat livers to electrophoretic homogeneity and its biochemical, spectral, and immunological properties characterized. The final preparation had a specific content of 11 nmol of cytochrome P-450/mg of protein. A single band with an apparent molecular weight of 52,000 was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The absolute spectrum of the isolated pyrazole cytochrome P-450 displayed peaks at 648 and 396 nm, suggestive of a high spin cytochrome. The ethylisocyanide difference spectrum exhibited two maxima, one at 457 nm, the other at 428 nm. Pyrazole and dimethyl sulfoxide produced binding spectra with the purified P-450, with peaks at 425 or 419 nm and troughs at 390 or 386 nm, respectively. K8 values for dimethyl sulfoxide and pyrazole were 21 and 0.04 mM, respectively. The catalytic activity of the pyrazole cytochrome P-450 was elevated with aniline and dimethylnitrosamine (low Km) but not with aminopyrine, benzphetamine, ethoxycoumarin, or ethoxyresorufin as substrates. An antibody against pyrazole cytochrome P-450 recognized a 52,000 molecular weight protein upon reaction with saline microsomes. The intensity of the immunoblot was increased when microsomes isolated from pyrazole, 4-methylpyrazole-, acetone-, or chronic ethanol-treated rats were utilized, but not after phenobarbital or 3-methylcholanthrene treatment. Homology at the amino terminus of 19 amino acids was observed between pyrazole P-450 and the isoniazid-inducible P-450j. Based upon the above catalytic, spectral, and immunological properties, it appears that pyrazole induces a form of cytochrome P-450 which is identical to that induced by ethanol and isoniazid.

Reduction of cytochrome b in mitochondria from yeast lacking coenzyme Q.

Mitochondria isolated from coenzyme Q deficient yeast cells had no detectable NADH:cytochrome c reductase or succinate:cytochrome c reductase but had comparable amounts of cytochromes b and c1 as wild-type mitochondria. Addition of succinate to the mutant mitochondria resulted in a slight reduction of cytochrome b; however, the subsequent addition of antimycin resulted in a biphasic reduction of cytochrome b, leading to reduction of 68% of the total dithionite-reducible cytochrome b. No "red" shift in the absorption maximum was observed, and no cytochrome c1 was reduced. The addition of either myxothiazol or alkylhydroxynaphthoquinone blocked the reduction of cytochrome b observed with succinate and antimycin, suggesting that the reduction of cytochrome b-562 in the mitochondria lacking coenzyme Q may proceed by a pathway involving cytochrome b at center o where these inhibitors block. Cyanide did not prevent the reduction of cytochrome b by succinate and antimycin the the mutant mitochondria. These results suggest that the succinate dehydrogenase complex can transfer electrons directly to cytochrome b in the absence of coenzyme Q in a reaction that is enhanced by antimycin. Reduced dichlorophenolindophenol (DCIP) acted as an effective bypass of the antimycin block in complex III, resulting in oxygen uptake with succinate in antimycin-treated mitochondria. By contrast, reduced DCIP did not restore oxygen uptake in the mutant mitochondria, suggesting that coenzyme Q is necessary for the bypass. The addition of low concentrations of DCIP to both wild-type and mutant mitochondria reduced with succinate in the presence of antimycin resulted in a rapid oxidation of cytochrome b perhaps by the pathway involving center o, which does not require coenzyme Q.

Coenzyme Q analogues reconstitute electron transport and proton ejection but not the antimycin-induced "red shift" in mitochondria from coenzyme Q deficient mutants of the yeast Saccharomyces cerevisiae.

Mitochondria isolated from coenzyme Q deficient yeast cells had no detectable NADH:cytochrome c reductase or succinate:cytochrome c reductase activity but contained normal amounts of cytochromes b and c1 by spectral analysis. Addition of the exogenous coenzyme Q derivatives including Q2, Q6, and the decyl analogue (DB) restored the rate of antimycin- and myxothiazole-sensitive cytochrome c reductase with both substrates to that observed with reduced DBH2. Similarly, addition of these coenzyme Q analogues increased 2-3-fold the rate of cytochrome c reduction in mitochondria from wild-type cells, suggesting that the pool of coenzyme Q in the membrane is limiting for electron transport in the respiratory chain. Preincubation of mitochondria from the Q-deficient yeast cells with DBH2 at 25 degrees C restored electrogenic proton ejection, resulting in a H+/2e- ratio of 3.35 as compared to a ratio of 3.22 observed in mitochondria from the wild-type cell. Addition of succinate and either coenzyme Q6 or DB to mitochondria from the Q-deficient yeast cells resulted in the initial reduction of cytochrome b followed by a slow reduction of cytochrome c1 with a reoxidation of cytochrome b. The subsequent addition of antimycin resulted in the oxidant-induced extrareduction of cytochrome b and concomitant oxidation of cytochrome c1 without the "red" shift observed in the wild-type mitochondria. Similarly, addition of antimycin to dithionite-reduced mitochondria from the mutant cells did not result in a red shift in the absorption maximum of cytochrome b as was observed in the wild-type mitochondria in the presence or absence of exogenous coenzyme Q analogues.(ABSTRACT TRUNCATED AT 250 WORDS)

Further studies on the binding of DCCD to cytochrome B and subunit VIII of complex III isolated from beef heart mitochondria.

Complex III (the cytochrome b-c1 complex) from beef heart mitochondria was incubated with [14C]DCCD for various periods of time. The polypeptide profile of the complex was compared in both stained gels and their autoradiograms when three different methods were used to terminate the reaction. Precipitation with ammonium sulfate resulted in the formation of a new band with an apparent molecular weight of 39,000 in both incubated samples and the zero time controls. Reisolation of the complex by centrifugation through 10% sucrose or by precipitation with trichloroacetic acid did not result in any changes in the appearance of the subunit peptides of the complex. Subunit III (cytochrome b) and subunit VIII were the only bands labeled after termination of the reaction by centrifugation through sucrose, while both ammonium sulfate and trichloroacetic precipitation resulted in nonspecific labeling of several other subunits of the complex and increased labeling of subunit VIII relative to subunit III. Preincubation of the complex with antimycin prior to treatment with [14C]DCCD resulted in a 50% decrease in the binding of DCCD to both cytochrome b and subunit VIII. Furthermore, treatment of the complex III with DCCD resulted in a change in the red shift observed after antimycin or myxothiazol addition to the dithionite-reduced complex resulting in a broad peak with no sharp maximum. These results provide further confirmation that DCCD binds preferentially to cytochrome b and subunit VIII of complex III from beef heart mitochondria and suggest that cytochrome b may play a role in proton translocation.

Effect of temperature on protein synthesis and leucine transport by yeast mitochondria.

Protein synthesis in yeast mitochondria shows biphasic Arrhenius plots both in vivo and in vitro, with a twofold increase in the activation energy below the transition temperature suggesting a functional association between mitochondrial protein synthesis and the inner membrane. Analysis by gel electrophoresis of mitochondrial translation products labeled in vivo showed that the same proteins are synthesized and then inserted into the membrane above and below the transition temperature of the membrane. The rate of leucine uptake into mitochondria was decreased at least fivefold in the presence of chloramphenicol, suggesting that leucine is used mainly for protein synthesis. In the absence of chloramphenicol, the rate of leucine uptake was always slightly higher but comparable to the incorporation rate of leucine into protein at all temperatures studied, suggesting that the transport of leucine into mitochondria is not rate-limiting for protein synthesis. The ionophore valinomycin or the uncoupler carbonyl phenylhydrazone (CCCP) inhibited 75-80% of the leucine uptake in the presence of chloramphenicol. In addition, the omission of respiratory chain substrates and the ATP-regenerating system led to a 93% inhibition of uptake, suggesting that leucine uptake may occur by an active transport mechanism.

Inhibition by dicyclohexylcarbodiimide of proton ejection but not electron transfer in rat liver mitochondria.

The primary effect of dicyclohexylcarbodiimide (DCCD) at the cytochrome b-c1 region of the respiratory chain of rat liver mitochondria is an inhibition of proton translocation. No significant decrease was observed in the rate of electron flow from succinate to cytochrome c when measured as cytochrome c reductase, K3Fe(CN)6 reductase, or the rate of H+ release in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone after treatment with sufficient DCCD to abolish completely electrogenic proton ejection. The inhibitory effects of DCCD were time and concentration dependent and affected by the pH of the medium. Lowering the pH from 7.3 to 6.7 resulted in a progressively faster rate and extent of inhibition of proton ejection by DCCD. At pH 6.9, the H+/2e- decreased by 50% within 30 s after DCCD addition; however, at pH 7.3, a 50% decrease was not observed until 2 min after DCCD addition. DCCD did not act as an uncoupler as both the rate of proton ejection and back decay were decreased after incubation with DCCD. Treatment of rat liver mitochondria with DCCD under these same conditions also resulted in a broadening of the sharp spectral shift of cytochrome b observed after antimycin addition to mitochondria previously reduced with succinate suggesting that DCCD may modify cytochrome b in such a way that the binding of antimycin is altered.

Dicyclohexylcarbodiimide binds to cytochrome b and subunit VIII in soluble complex III from beef heart mitochondria.

Incubation of soluble complex III isolated from either yeast or beef heart mitochondria with 25-100 nmol of [14C]dicyclohexylcarbodiimide (DCCD)/nmol of cytochrome b followed by centrifugation through 10% sucrose or precipitation with trichloroacetic acid did not result in any changes in the appearance of the subunits of either complex. The [14C]DCCD was bound to cytochrome b and phospholipids in the yeast complex and with similar kinetics to both cytochrome b and subunit VIII (Mr = 4000-8000) plus phospholipids of the beef complex. Subunit VIII of the beef complex was partially extracted with chloroform:methanol; however, no subunit of this mobility was present in the yeast complex. Incubation of the beef complex in phosphate buffer for short times resulted in a doubling of the [14C]DCCD bound to cytochrome b relative to that to subunit VIII. Preincubation of both complexes with venturicidin prior to treatment with DCCD resulted in a 50% decrease in the binding of [14C]DCCD to cytochrome b. Reisolation of the beef complex III by precipitation with (NH4)2SO4 after incubation with [14C]DCCD resulted in the formation of a new band with an apparent molecular weight of 39,000 even in the zero time control. The [14C]DCCD was bound to subunit VIII and the core proteins but not to cytochrome b at all times, suggesting that precipitation with (NH)2SO4 in the presence of DCCD causes cross-linking of the subunits of complex III.

The preferential binding of dicyclohexylcarbodiimide to cytochrome b and phospholipids in soluble complex III from yeast mitochondria.

The binding of [14C]dicyclohexylcarbodiimide (DCCD) to soluble complex III from yeast mitochondria was examined under conditions which resulted in the inhibition of proton ejection but had a minimal effect on cytochrome c reductase activity. Incubation of the complex with 50-100 nmol of [14C]DCCD/nmol of cytochrome b at 12 degrees C did not result in any changes in the appearance of the high-molecular-weight subunits (I-V) after sodium dodecyl sulfate-gel electrophoresis, although a slight broadening of the three lowest molecular-weight subunits (VI-VIII) was observed. The [14C]DCCD was bound preferentially to subunit III (cytochrome b) and a wide band with an apparent low-molecular weight ranging from 8000 to 9000 to less than 2000 depending on the gel system used. Extraction of the [14C]DCCD-treated complex III with chloroform:methanol had no effect on subunit III but completely removed the low-molecular-weight radioactive band. Thin-layer chromatography of the chloroform:methanol extract revealed that the radioactive material extracted from the [14C]DCCD-treated complex III migrated with the same apparent RF as either free [14C]DCCD or cardiolipin. Amino acids were not detectable in an acid hydrolysate of the chloroform:methanol extract, suggesting the absence of protein. Digestion of the [14C]DCCD-treated complex III with either chymotrypsin or Staphylococcus aureus V8 protease resulted in the decrease of both staining intensity and labeling in subunit III but had no effect on the radioactivity in the low-molecular-weight material. These results confirm that DCCD binds preferentially to cytochrome b in complex III from yeast mitochondria and suggest that cytochrome b may play an important role in proton translocation at this site of the respiratory chain.

The presence of the iron-sulfur protein (subunit V) of complex III in mitochondria of heme-deficient yeast cells lacking iron-sulfur clusters detectable by electron paramagnetic resonance.

The presence of subunit V, the iron-sulfur protein, of complex III has been demonstrated in mitochondria from a mutant of Saccharomyces cerevisiae which lacks 5-aminolevulinic acid synthase and, hence, is devoid of heme. The mature form (24 K Da) of the iron-sulfur protein was observed in equal amounts in the heme-deficient and heme-sufficient cells with antiserum against subunit V and either the sensitive immuno-transfer technique or immunoprecipitation from dodecylsulfate-solubilized mitochondria. In addition, a slight shoulder with a molecular mass 1.5 kDa larger than the mature form was present in mitochondria from the heme-deficient cells. Electron paramagnetic resonance spectroscopy revealed the absence of iron-sulfur signals due to clusters S-1, S-2 and S-3 of succinate dehydrogenase or to Rieske's iron-sulfur cluster of complex III in mitochondria from the heme-deficient cells. The lack of iron-sulfur centers in these cells may be a consequence of the absence of sulfite reductase in the cells without heme.