Immunochemical evidence for the participation of cytochrome b5 in microsomal stearyl-CoA desaturation reaction

A rabbit antiserum was prepared against rat liver microsomal cytochrome b₅, and utilized in demonstrating the participation of this cytochrome in the microsomal stearyl-CoA desaturation reaction. The antiserum inhibited the NADH-cytochrome c reductase activity of rat liver microsorncs, but it did not inhibit either NADH-ferricyanide or NADPH-cytochrome c reductase activity of the microsomes. Thus, the inhibitory effect of the antiserum on the microsomal electron-transferring reactions seemed to be specific to those which require the participation of cytochrome b₅.The NADH-dependent and NADPH-dependent desaturations of stearyl CoA by rat liver microsomes were strongly inhibited by the antiserum. The reduction of cytochrome b₅ by NADH-cytochrome b₅ reductase as well as the reoxidation of the reduced cytochrome b₃ by the desaturase, the terminal cyanide-sensitive factor of the desaturation system, was also strongly inhibited by the antiserum. When about 90%, of cytochrome b₅ was removed from rat liver microsomes by protease treatment, the desaturation activity of the microsomes became much more sensitive to inhibition by the antiserum. These results confirmed our previous conclusion that the reducing equivalent for the desaturation reaction is transferred from NAD(P)H to the cyanidesensitive factor mainly via cytochrome b₅ in the microsomal membranes.     

Oxidation of ethanol by hepatic microsomes of acatalasemic mice

Hepatic microsomes of acatalasemic Cs^b mice subjected to heat inactivation displayed decreased catalatic activity but NADPH dependent microsomal ethanol oxidation (MEOS) remained active and unaffected. Even without heat inactivation, in the Cs^b strain, the NADPH dependent metabolism of ethanol was much more active than the H₂O₂ mediated one whereas microsomes of Cs^a control mice displayed equal rates of H₂O₂ and NADPH dependent ethanol oxidation. Addition of catalase to liver microsomes in vitro abolished this difference whereas the catalase inhibitor azide established in the Cs^a mice a pattern similar to that of the Cs^b, namely a much more active NADPH dependent than a H₂O₂ mediated ethanol oxidation. The selective persistence in the Cs^b mice of NADPH dependent ethanol oxidation contrasting with the reduction in the H₂O₂ mediated metabolism of ethanol supports the existence of a microsomal ethanol oxidizing system independent of catalase.     

Purification and properties of a highly active catalase from cabbage loopers,Trichoplusia ni

Using ethanol-chloroform fractionation in conjunction with standard column chromatography techniques catalase has been purified to electrophoretic homogeneity from mid-fifth instar larvae of the cabbage looper moth, Trichoplusia ni. The specific activity of purified catalase was 2.2 × 10⁵ units (IU = 1 μmol H₂O₂ decomposed mg protein⁻¹ min⁻¹). The purified enzyme's native molecular weight was in the 247,000–259,000 Da range and was tetrameric with an apparent molecular weight of 63,000 Da for each subunit. In addition, biochemical properties of the enzyme were studied with emphasis on substrate specificity, kinetics, and the mechanism of inactivation by the irreversible inhibitor 3-amino-1,2,4-triazole (AT). The apparent K_m of the purified catalase for H₂O₂ was 54.2 mM and 50% of the maximal rate occurred at 16 mM H₂O₂. Purified catalase was ineffective in metabolizing organic hydroperoxides and, unlike other catalases, lacked peroxidase activity. Lastly, AT in the presence and absence of H₂O₂ was an effective inhibitor of catalase activity (I₅₀ = 100 mM) suggesting that a portion of the purified catalase was complexed with hydrogen peroxide in a compound 1 configuration.     

Oxidation and reduction of membrane-bound cytochrome c in Hemophilus parainfluenzae. Reaction with oxygen,hydrogen peroxide and nitrate

Cytochromes of the a-, b-, c- and d-type become reduced when intact cells of Hemophilus parainfluenzae have become anaerobic following respiration with substrates such as formate or succinate, as shown previously (J. Biol. Chem. (1970) 254, 5096–5100). In the presence of formate after depletion of O₂, there is an unusual two-step time course of reduction of the membrane-bound cytochrome c. The proportion of the cytochrome c which is reduced during the second stage is oxidizable by either nitrate or H₂O₂ and is reduced again when the nitrate or H₂O₂ have been depleted. We conclude that the observed two-stage reduction of cytochrome c results from the presence of an oxidant, probably H₂O₂, produced by reaction of formate dehydrogenase with O₂. This was shown by the effects of cyanide, catalase and O₂. In addition, no evidence for the production of the oxidant is seen when succinate is the substrate oxidized. Although measurements of absorption spectra indicated only one species of cytochrome c, kinetic evidence is presented for some separation of the cytochrome c into more than one electron transport pathway.     

The dimeric structure of the cytochrome bc1 complex prevents center P inhibition by reverse reactions at center N

Energy transduction in the cytochrome bc₁ complex is achieved by catalyzing opposite oxido-reduction reactions at two different quinone binding sites. We have determined the pre-steady state kinetics of cytochrome b and c₁ reduction at varying quinol/quinone ratios in the isolated yeast bc₁ complex to investigate the mechanisms that minimize inhibition of quinol oxidation at center P by reduction of the b_H heme through center N. The faster rate of initial cytochrome b reduction as well as its lower sensitivity to quinone concentrations with respect to cytochrome c₁ reduction indicated that the b_H hemes equilibrated with the quinone pool through center N before significant catalysis at center P occurred. The extent of this initial cytochrome b reduction corresponded to a level of b_H heme reduction of 33%–55% depending on the quinol/quinone ratio. The extent of initial cytochrome c₁ reduction remained constant as long as the fast electron equilibration through center N reduced no more than 50% of the b_H hemes. Using kinetic modeling, the resilience of center P catalysis to inhibition caused by partial pre-reduction of the b_H hemes was explained using kinetics in terms of the dimeric structure of the bc₁ complex which allows electrons to equilibrate between monomers.     

Different contribution of rat liver microsomal pigments in the formation of superoxide anions and hydrogen peroxide during development.

Liver microsomes of adult rats produce, by an NADPH-dependent pathway, O₂^? radicals, as detected by the epinephrine cooxidation to adrenochrome (24.8 nmol/min/mg of protein). This production has also been measured during liver development (from 1 to 20 days after birth) and correlated to the enzyme content (NADPH-cytochrome c reductase, cytochrome b₅, and cytochrome P-450), with the aim of establishing the level at which Superoxide radicals are formed in the electron transport system. At 1 day the adrenochrome formation and the activity of NADPH-cytochrome c reductase are about 50 and 40% of those of the adult, respectively, whereas those of cytochromes b₅ and P-450 are approximately 10%. After 20 days of development cytochrome b₅ and the dehydrogenase reach the adult level, while cytochrome P-450 is about 80%. At this age O₂^? radicals have a 30% increment and reach only 60% of those of the adult; H₂O₂ production is also 60% and the N-demethylation of aminopyrine is only 30%. Thus, at birth the formation of O₂^? radicals is almost entirely dependent on the activity of the flavoprotein. The close correlation between the slight increase in the demethylase activity and adrenochrome formation from 1 to 20 days suggests that a portion of O₂^? radicals produced by the NADPH-dependent electron transfer is directly involved in the mixed function oxidation. Since about 50% of the radicals are formed at the flavoprotein level, these results indicate that in the adult liver the remaining amount may be generated at the level of cytochrome P-450.     

Changes in oxidation-reduction potential of cytochrome b observed in the presence of antimycin A

The cytochrome b of sonic particles of mitochondria or the isolated segment of the respiratory chain containing cytochromes b and c₁ (Complex III) was 80–95% reducible with Q₁H₂ (ubiquinol-5) in the presence of antimycin plus selected electron acceptors added externally (i.e., oxidants which reacted preferentially with respiratory components on the oxygen side of the point of inhibition by antimycin) such as oxygen or ferricyanide depending on whether sonic particles or isolated Complex III was used. In contrast, less than 40% of the cytochrome b was reduced by Q₁H₂ in the absence of either antimycin or the external electron acceptor. In the presence of antimycin ascorbate or mercaptoethanol, which behaved as mild reducing agents, completely inhibited the reduction of cytochrome b by Q₁H₂.     

Fatty acid desaturase system of yeast microsomes. Involvement of cytochrome b5-containing electron-transport chain.

The oxidative desaturation of palmitoyl CoA by microsomes from anaerobically grown Saccharomyces cerevisiae has been studied by using NADH as electron donor. The desaturation product was identified as palmitoleic acid by periodate oxidation. The desaturase activity was sensitive to relatively high concentrations of cyanide; the concentration of cyanide causing half-maximal inhibition was determined to be 7.1 mm. The rate of reoxidation of cytochrome b₅ in NADH-reduced microsomes was stimulated by the addition of palmitoyl CoA, and the amount of cytochrome b₅ reoxidized by the palmitoyl CoA added could be closely correlated to the amount of palmitoleate formed. No stimulation of the reoxidation of cytochrome b₅ was induced by palmitoyl CoA in microsomes prepared from the desaturase-repressed cells and from a desaturase-deficient mutant, strain KD-20. It is concluded that the fatty acyl CoA desaturase system of yeast microsomes involves cytochrome b₅ as an electron carrier and that the terminal desaturase is sensitive to relatively high concentrations of cyanide.     

The direct involvement of hydrogen peroxide in indoleacetic acid inactivation

Hydrogen peroxide appears to mask the chemical characteristics of indoleacetic acid. This was demonstrated by the Salkowski and Fluorescence tests. Stem elongation and root initiation were inhibited as a result of adding H₂O₂ to nutrient media containing IAA, however, upon the addition of purified catalase, most of the symptoms of IAA inactivation were reversed. It is suggested that in vivo IAA may be regulated partially by its conjugation with H₂O₂, and catalase may have a role in the IAA reactivation process. The accumulation of hydrogen peroxide in the cells as a result of catalase inhibition may lead to a temporary IAA inactivation, therefore effecting plant growth.     

THE DECOMPOSITION OF HYDROGEN PEROXIDE BY LIVER CATALASE

1. The velocity of decomposition of hydrogen peroxide by catalase as a function of (a) concentration of catalase, (b) concentration of hydrogen peroxide, (c) hydrogen ion concentration, (d) temperature has been studied in an attempt to correlate these variables as far as possible. It is concluded that the reaction involves primarily adsorption of hydrogen peroxide at the catalase surface. 2. The decomposition of hydrogen peroxide by catalase is regarded as involving two reactions, namely, the catalytic decomposition of hydrogen peroxide, which is a maximum at the optimum pH 6.8 to 7.0, and the "induced inactivation" of catalase by the "nascent" oxygen produced by the hydrogen peroxide and still adhering to the catalase surface. This differs from the more generally accepted view, namely that the induced inactivation is due to the H₂O₂ itself. On the basis of the above view, a new interpretation is given to the equation of Yamasaki and the connection between the equations of Yamasaki and of Northrop is pointed out. It is shown that the velocity of induced inactivation is a minimum at the pH which is optimal for the decomposition of hydrogen peroxide. 3. The critical increment of the catalytic decomposition of hydrogen peroxide by catalase is of the order 3000 calories. The critical increment of induced inactivation is low in dilute hydrogen peroxide solutions but increases to a value of 30,000 calories in concentrated solutions of peroxide.     

Efficiency of bovine liver catalase as a catalyst to cleave H2O2 added continually to buffer solutions

Empirical estimations of H₂O₂ concentration in a system containing bovine liver catalase and continually supplied with H₂O₂ were done to evaluate the efficiency of the enzyme to cleave H₂O₂. It was found that the continuous addition of H₂O₂ leads to the formation of steady-state concentrations of H₂O₂ in the medium. At a constant catalase concentration both the level and the duration of the steady state are dependent on the flow rate of H₂O₂. The increase of the catalase concentration in the medium does not change the steady-state level, it merely leads to the maintenance of the steady state for longer durations. At higher flow rates of H₂O₂, no steady state could be maintained, even when catalase was present in high excess. The incomplete cleavage of H₂O₂ by catalase under these conditions is due to the low affinity of catalase toward H₂O₂ (high K_m value, apparent K_m = 0.1M H₂O₂) and to the rapid inactivation of the enzyme during the continuous addition of H₂O₂.     

The Structure of Calf Liver Cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>5</Subscript> at 2.8 Å Resolution

CYTOCHROME b₅ is a haem-containing protein in the microsomes of liver tissue. It interacts specifically with a flavo-protein, cytochrome b₅ reductase, which catalyses the transfer of electrons from NADH to the haem iron of the cytochrome¹. The microsomal cytochrome b₅ system has been implicated in fatty acid desaturation reactions² and a similar system in erythrocytes may catalyse the reduction of methaemoglobin³. Calf liver cytochrome b₅, solubilized by pancreatic lipase, has a molecular weight of 11,000 and consists of ninety-three amino-acids in the sequence shown in Fig. 1 (refs. 4 and 5). The haem group is non-covalently bound to the protein and can be removed reversibly by acid acetone treatment⁶.     

Suicidal inactivation of microsomal cytochrome P-450 by halothane under hypoxic conditions

Under anaerobic conditions the addition of halothane to NADPH-reduced liver microsomes from phenobarbital-pretreated male rats resulted in a pronounced inactivation of microsomal cytochrome P-450, presumably produced by covalent binding of reactive halothane metabolites such as the CF₃CHCl-radical. Compared with microsomes from phenobarbital-pretreated rats, the loss of active cytochrome P-450 was markedly decreased in microsomes from both 3-methylcholanthrene-pretreated and untreated rats. Increasing the O₂-partial pressure decreased the amount of cytochrome P-450 inactivated by halothane metabolites. At an O₂-partial pressure of approximately 40 mm Hg the inactivation was virtually eliminated.     

Effects of inhibitors of catalase on photosynthesis and on catalase activity in unwashed preparations of intact chloroplasts

The catalase activity of unwashed preparations containing intact spinach (Spinacia oleracea L.) chloroplasts is inhibited both by cyanide and by azide at concentrations which also cause inhibition of photosynthetic CO₂- dependent O₂ evolution.

Aminotriazole can also be used to inhibit this contaminant catalase, and in this case inhibition of catalase can be achieved at aminotriazole concentrations which have little effect on the rate of photosynthetic CO₂ fixation. Aminotriazole may be used as a specific inhibitor of catalase in order to demonstrate inhibition of photosynthesis by added H₂O₂.

It is therefore concluded that inhibition of photosynthesis by cyanide and azide does not necessarily result from inhibition of catalase in the chloroplast preparation, and that intact chloroplasts do not produce inhibitory concentrations of H₂O₂ under the best experimental conditions for CO₂ fixation.

     

A Mechanism of Resistance to Hydrogen Peroxide in Vibrio rumoiensis S-1

A possible mechanism of resistance to hydrogen peroxide (H₂O₂) in Vibrio rumoiensis, isolated from the H₂O₂-rich drain pool of a fish processing plant, was examined. When V. rumoiensis cells were inoculated into medium containing either 5 mM or no H₂O₂, they grew in similar manners. A spontaneous mutant strain, S-4, derived from V. rumoiensis and lacking catalase activity did not grow at all in the presence of 5 mM H₂O₂. These results suggest that catalase is inevitably involved in the resistance and survival of V. rumoiensis in the presence of H₂O₂. Catalase activity was constitutively present in V. rumoiensis cells grown in the absence of H₂O₂, and its occurrence was dependent on the age of the cells, a characteristic which is observed for the HP II-type catalase of Escherichia coli. The presence of the HP II-type catalase in V. rumoiensis cells was evidenced by partial sequencing of the gene encoding the HP II-type catalase from this organism. A notable difference between V. rumoiensis and E. coli is that catalase is accumulated at very high levels (~2% of the total soluble proteins) in V. rumoiensis, in contrast to the case for E. coli. When V. rumoiensis cells which had been exposed to 5 mM H₂O₂ were centrifuged, most intracellular proteins, including catalase, were recovered in the medium. On the other hand, when V. rumoiensis cells were grown on plates containing various concentrations of H₂O₂, individual cells had a colony-forming ability inferior to those of E. coli, Bacillus subtilis, and Vibrio parahaemolyticus. Thus, it is suggested that when V. rumoiensis cells are exposed to high concentrations of H₂O₂, most cells will immediately be broken by H₂O₂. In addition, the cells which have had little or no damage will start to grow in a medium where almost all H₂O₂ has been decomposed by the catalase released from broken cells.     

Studies of cytochrome synthesis in rat liver

The incorporation of radioactive amino acids and of δ-amino[2,3-³H₂]laevulinate into rat liver cytochromes b₅ and c and cytochrome oxidase has been examined with and without protein-synthesis inhibitors. Cycloheximide promptly inhibits labelling of both haem and protein for cytochrome c in parallel fashion. Although incorporation of ¹⁴C-labelled amino acid into microsomal cytochrome b₅ is also rapidly inhibited, cycloheximide incompletely inhibits haem labelling of cytochrome b₅ and cytochrome a+a₃, and inhibition occurs only after repeated antibiotic injections. The possibility of apo-protein pools, or of haem exchange, with a rapidly renewed `free' haem pool, is considered. Consistent with this model is the observation of non-enzymic haem exchange in vitro between cytochrome b₅ and methaemoglobin. Chloramphenicol, injected intravenously over 5h, results in a 20–40% decrease in incorporation of δ-amino[2,3-³H₂]laevulinate into haem a+a₃ and haem of cytochromes b₅ and c. With the dosage schedule of chloramphenicol studied, amino acid labelling of total liver protein and of cytochrome c was not inhibited. Similarly, ferrochelatase activity was not decreased.     

Aniline is hydroxylated by the cytochrome P-450-dependent hydroxyl radical-mediated oxygenation mechanism

Hydroxylation of aniline, catalyzed by rabbit liver microsomal cytochromes P-450 in reconstituted systems, was inhibited by catalase, superoxide dismutase, catechol, mannitol, hydroquinone, dimethylsulfoxide and benzoate, whereas the cytochrome P-450-catalyzed O-demethylation of paranitroanisole, measured under the same conditions, was unaffected by these agents. A similar inhibition profile of the hydroxylation reaction was observed in reconstituted systems where cytochrome P-450 had been replaced by hemoglobin. The results indicate that aniline hydroxylation is mediated by hydroxyl radicals generated in an iron-catalyzed Haber-Weiss reaction between O₂^? and H₂O₂ and may explain some of the special properties of this reaction previously described.     

Studies on methemoglobin reductase. Immunochemical similarity of soluble methemoglobin reductase and cytochrome b5 of human erythrocytes with NADH-cytochrome b5 reductase and cytochrome b5 of rat liver microsomes.

An antibody preparation elicited against purified, lysosomal-solubilized NADH-cytochrome b₅ reductase from rat liver microsomes was shown to interact with methemoglobin reductase of human erythrocytes by inhibiting the rate of erythrocyte cytochrome b₅ reduction by NADH. The ferricyanide reductase activity of the enzyme was not inhibited by the antibody, suggesting that the inhibition of methemoglobin reductase activity may be due to interference with the binding of cytochrorme b₅ to the flavoprotein. Under conditions of limiting concentrations of flavoprotein, the antibody inhibited the rate of methemoglobin reduction in a reconstituted system consisting of homogeneous methemoglobin reductase and cytochrome b₅ from human erythrocytes. This inhibition was due to the decreased level of reduced cytochrome b₅ during the steady state of methemoglobin reduction while the rate of methemoglobin reduction per reduced cytochrome b₅ stayed constant, suggesting that the enzyme was not concerned with an electron transport between the reduced cytochrome b₅ and methemoglobin.An antibody to purified, trypsin-solubilized cytochrome b₅ from rat liver microsomes was shown to inhibit erythrocyte cytochrome b₅ reduction by methemoglobin reductase and NADH to a lesser extent than microsomal cytochrome b₅ preparations from rat liver (trypsin solubilized or detergent solubilized) and pig liver (trypsin solubilized). The results presented establish that soluble methemoglobin reductase and cytochrome b₅ of human erythrocytes are immunochemically similar to NADH-cytochrome b₅ reductase and cytochrome b₅ of liver microsomes, respectively.     

Nitric oxide increases toxicity of hydrogen peroxide against rat liver endothelial cells and hepatocytes by inhibition of hydrogen peroxide degradation

Nitric oxide (NO) and hydrogen peroxide (H₂O₂) show cooperativity in their cytotoxic action. The present study was performed to decipher the mechanisms underlying this phenomenon. In cultured liver endothelial cells and in cultured, glutathione-depleted hepatocytes, the combined exposure to NO (released by spermine NONOate, 1 mM) and H₂O₂ (released by glucose oxidase) induced cell injury that was far higher than the injury elicited by NO or H₂O₂ alone. In both cell types, the addition of the NO donor increased H₂O₂ steady-state levels, although with different kinetics: in hepatocytes, the increase in H₂O₂ levels was already evident at early time points while in liver endothelial cells it became evident after 2 h of incubation. NO exposure inhibited H₂O₂ degradation, assessed after addition of 50 µM, 200 µM, or 4 mM authentic H₂O₂, significantly in both cell types. However, again, early and delayed inhibition was observed. The late inhibition of H₂O₂ degradation in endothelial cells was paralleled by a decrease in glutathione peroxidase activity. Glutathione peroxidase inactivation was prevented by hypoxia or by ascorbate, suggesting inactivation by reactive nitrogen oxide species (NO_x). Early inhibition of H₂O₂ degradation by NO, in contrast, could be mimicked by the catalase inhibitor azide. Together, these results suggest that the cooperative effect of NO and H₂O₂ is due to inhibition of H₂O₂ degradation by NO, namely to inhibition of catalase by NO itself (predominant in hepatocytes) and/or to inhibition of glutathione peroxidase by NO_x (prevailing in endothelial cells). nitrogen monoxide; catalase; glutathione peroxidase