Enzymatic Activity of Cytochrome C-Oxidase Inserted into Magnetoliposomes Differing in Surface Charge Density

The role played by the surface charge density of the phospholipid coat of nanometer-sized Fe₃O₄ colloids (so-called “magnetoliposomes”) in the catalytic activity of beef heart cytochrome c oxidase was investigated. Screening of various binary mixtures of the anionic dimyristoylphosphatidylglycerol and the zwitterionic dimyristoylphosphatidylcholine demonstrated that the highest degree of reactivation was found in the lower negative charge range. Pre-incubation of the charged colloidal biocatalytic particles with cytochrome c induced aggregation and reduced overall enzymatic activity. The results are interpreted in terms of a different affinity of the substrate for the various membrane types and of a reorganisation of the enzyme within the membrane matrices.     

Plasmalemma from the roots of cucumber: Isolation by two-phase partitioning and characterization

Plasmalemma was isolated from the roots of 2-week-old cucumber plants ( Cucumis sativus L. cv. Rhensk druv) by utilizing an aqueous polymer two-phase system with 6.5%:6.5% (w/w) Dextran T500 and polyethylene glycol (PEG) 3350 at pH 7.8. The plasmalemma fraction comprised ca 6% of the membrane proteins contained in the microsomal fraction. The specific activity of the plasma membrane marker enzyme (K⁺, Mg²⁺-ATPase) was 14- to 17-times higher in the upper (PEG-rich) than in the lower (Dextran-rich) phase, and the reverse was true for marker enzymes (cytochrome c oxidase, EC 1.9.3.1, and antimycin A-resistant NADPH cytochrome c reductase) of intracellular membranes. The ATPase was highly stimulated by the addition of detergent (Triton X-100), so that the isolated plasmalemma vesicles appear tightly sealed and in a right-side-out orientation. Further characterization of the ATPase activities showed a pH optimum at 6.0 in the presence of Mg²⁺. This optimum was shifted to pH 5.8 after addition of K⁺. K⁺ stimulated the ATPase activity below pH 6 and inhibited above pH 6. The ATPase activity was specific for ATP and sensitive to N,N-dicyclohexylcarbodiimide and sodium vanadate, with K⁺ enhancing the vanadate inhibition. The enzyme was insensitive to sodium molybdate, NO⁻₃, azide and oligomycin. No Ca²⁺-ATPase was detected, and even as little as 0.05 m M Ca²⁺ inhibited the Mg²⁺-ATPase activity.     

Sensitivity to drying in vitro of enzymes in mitochondrial subfractions from Vicia faba seed

In order to investigate the persistence of membrane and matrix functions following desiccation, enzymic activities were studied in Vicia faba L. seed mitochondrial subfractions subjected to drying and rehydration in vitro. Mitochondria were prepared after 0, 12 and 24 h of seed imbibition. These were fractionated into inner membranes ("submitochondrial particles"), outer membranes (12 and 24 h only) and the soluble matrix. Enzyme activities associated with the inner membrane and matrix were found to increase several-fold during the first 12 h of imbibition. The two matrix enzymes examined, malate dehydrogenase and glutamate dehydrogenase, were insensitive to in vitro drying at all stages of imbibition. The membrane-bound activities from 12 h and 24 h imbibed material, antimycin A-sensitive NADH: cytochrome c oxidoreductase and (F_o-F₁)-ATPase of the inner membrane and antimycin A-insensitive NADH: cytochrome c oxidoreductase of the outer membrane, were moderately sensitive to dehydration. The F₁-ATPase solubilized from the inner membrane (F_o-F₁) complex was much less sensitive to drying, provided this was done at room temperature.
Mitochondria posessing their outer membranes could not be prepared from dry seed. The antimycin A-sensitive NADH: cytochrome c oxidoreductase from inner mitochondrial membranes of unimbibed seed was extremely sensitive to desiccation in vitro, about 75 to 80% of the activity being lost. This loss could be somewhat reduced by addition of glycerol or sucrose before drying.
It is concluded that uncontrolled desiccation results in major damage to some of the membrane-bound enzymic systems in mitochondria, whereas activities in the soluble fraction are remarkably tolerant of desiccation.     

Effect of filipin on Rat-liver and yeast mitochondria

1. Under the appropriate conditions intact yeast and mammalian mitochondria exhibit a heretofore unobserved sensitivity to the polyene antibiotic, filipin. The activity of the “filipin complex” (Filipins I, II, III and IV) is shown to be primarily due to the component designated Filipin II.

2. Yeast mitochondria treated with filipin complex, or purified Filipin II, exhibit “uncoupled” succinate oxidation and inhibited -ketoglutarate oxidation. Maximum filipin effect is observed at a concentration of 4 mM Filipin II. Rat-liver mitochondria are more sensitive to filipin than yeast mitochondria, and respiratory inhibition is observed regardless of substrate.

3. In liver mitochondria filipin-inhibited respiration is not relieved by Mg²⁺, K⁺, Ca²⁺ or 2,4-dinitrophenol, but is reversed by cytochrome c.

4. It is proposed that filipin treatment leads to altered membrane permeability and that respiratory inhibition is due to a loss of endogenous respiratory cofactors or an inactivation of primary dehydrogenases. The filipin-uncoupled yeast respiration may likewise be attributed to an altered phosphate permeability of the yeast mitochondrial membranes.     

Comparative analysis of proapoptotic activity of cytochrome <Emphasis Type="Italic">c</Emphasis> mutants in living cells

A non-traumatic electroporation procedure was developed to load exogenous cytochrome c into the cytoplasm and to study the apoptotic effect of cytochrome c, its K72-substitued mutants and “yeast → horse” hybrid cytochrome c in living WEHI-3 cells. The minimum apoptosis-activating intracellular concentration of horse heart cytochrome c was estimated to be 2.7 ± 0.5 μM (47 ± 9 fg/cell). The equieffective concentrations of the K72A-, K72E- and K72L-substituted mutants of cytochrome c were five-, 15- and 70-fold higher. The “yeast → horse” hybrid created by introducing S2D, K4E, A7K, T8K, and K11V substitutions (horse protein numbering) and deleting five N-terminal residues in yeast cytochrome c did not evoke apoptotic activity in mammalian cells. The apoptotic function of cytochrome c was abolished by the K72W substitution. The K72W-substituted cytochrome c possesses reduced affinity to the apoptotic protease activating factor-1 (Apaf-1) and forms an inactive complex. This mutant is competent as a respiratory-chain electron carrier and well suited for knock-in studies of cytochrome c-mediated apoptosis.     

Influence du pH sur la synthèse de l''oxidase (cytochrome a3) chez Bacillus coagulans, microorganisme thermophile, capable d'' “adaptation respiratoire”

In a new series of experiments on Bacillus coagulans (ATCC 11.369), it was demonstrated that this organism possesses a respiratory system with cytochromes b, c₁, c, (a+a₃) and also cytochrome o. A small decrease in the pH of the growth medium from 6.5 to 5.5 increases the respiratory activity by a factor of 4 and induces a variation of the absorption ratio [₆₀₃ (a+a₃)]/[₅₆₀ (b+c)] resulting in a preponderant increase in the ₆₀₃ absorption. The kinetic studies of the respiratory system synthesis during the phenomenon of “respiratory adaptation” have shown that lowering the pH of the adaption medium has the same effect. Spectral studies of membrane fractions (red dithionite) with or without carbon monoxide showed a preferential synthesis of oxidase a₃.     

Human colon tumor cell line LS174T drug metabolizing system

The effects of culture variables on the specific content and activity of various enzymes of the drug mmetabolizing system were assessed in colon tumor cell line LS174T. The NADH reduced cytochrome b₅ (cyt b₅)⁴ spectrum of these cells was similar to rat liver cyt b₅. When released from the membrane by trypsin and concentrated, the cyt b₅ was found to cross react with rabbit antibody to rat liver cyt b₅ and human liver cyt b₅. The enzyme activities were found stable over limited cell passages with control values of 0.03 and 0.13 µol/min/mg protein for NADPH and NADH cytochrome c (cyt c) reducing activity, 0.05 nmol cyt b₅ and 0.013 nmol cytochrome P450 per milligram of microsomal protein. Phenobarbital/hydrocortisone showed a consistent, but not always significant increase in the NADPH and NADH cyt c reduction and benzanthracene an increase in the NADH cyt c reducing activity and cyt b₅ content. Griseofulvin lowered the NADH cyt c reducing activity. Delta-aminolevulinic acid (0.5 mM) caused a significant decrease in the specific activity of all enzymes, as judged by a student's t test, with a p<0.001.Abbreviations cyt b₅ cytochrome b₅ - cyt c cytochrome c - cyt P450 cytochrome P450 - PB Phenobarbital - HC Hydrocortisone - ALA -Aminolevulinic acid - GRIS Griseofulvin - PENT Pentagastrin - PASS Cell Passage - DMH Dimethylhydrazine - BA Benzanth Acene     

Cdk2 acts upstream of mitochondrial permeability transition during paclitaxel-induced apoptosis

Sequential activation of cyclin-dependent kinases (Cdks) controls mammalian cell cycle. Here we demonstrate that the upregulation of cyclin-dependent kinase 2 (Cdk2) activity coincides with the loss of mitochondrial membrane potential (MMP) in paclitaxel-induced apoptosis. Ectopic expression of the dominant negative Cdk2 (Cdk2-dn) and a specific Cdk2 inhibitor, p21^WAF1/CIP1, effectively suppresses the loss of MMP, the release of cytochrome c, and subsequent activation of caspase-3 in paclitaxel-treated cells. Whereas forced activation of Cdk2 by overexpression of cyclin A dramatically promotes these events. We further show that Cdk2 activation status does not interfere with a procedure that lies downstream of cytochrome c release induced by Bax protein. These findings suggest that Cdk2 kinase can regulate apoptosis at earlier stages than mitochondrial permeability transition and cytochrome c release.     

Degradation and restoration of mitochondria upon deaeration and subsequent aeration of aerobically grown Saccharomyces Cerevisiae cells

Changes in the mitochondria of aerobically grown Saccharomyces cerevisiae cells upon deaeration and subsequent aeration of the medium were studied.

1. It is shown that removal of oxygen at the end of the exponential phase of growth (after completion of mitochondria formation) causes a decrease in activity of the respiratory enzymes. The activity of the complete respiratory system decreases much more rapidly than the activities of its fragments (NADH: ferricyanide reductase, succinate:ferricyanide reductase, NADH:cytochrome c reductase, succinate:cytochrome c reductase and cytochrome oxidase). The activities are restored to their initial level upon aeration of the cell suspension. The addition of Tween-80 and ergosterol to the medium prior to deaeration does not prevent inactivation of the respiratory system.

All the changes in mitochondria described occurred under conditions where cell division was insignificant.

2. Deaeration of the medium decreases the content of cytochromes b and aa₃ in the mitochondrial fraction, cytochrome aa₃ “disappearing” more quickly. The concentration of cytochromes in this fraction increases upon subsequent aeration of the cells. The total cytochromal content of the cells remains practically unchanged under the same conditions.

3. According to electron microscopic data, anaerobiosis causes a certain disorganization of mitochondrial cristal membranes. The mitochondrial structures are recovered upon aeration of the yeast cell suspension. It may be reasoned that inactivation and reactivation of the respiratory system are associated with reversible changes in mitochondrial membrane structure.

4. The effect of protein synthesis inhibitors on the restoration of mitochondria was investigated. It is shown that chloramphenicol does not suppress this process. In the presence of cycloheximide, oxygen induces reactivation of the respiratory system and simultaneously the appearance of particles resembling mitochondria. However, these particles gradually undergo morphological changes and the respiratory activity of the mitochondrial fraction decreases. Cycloheximide added to yeast cells that had not been deaerated, did not affect their mitochondria.

5. The results described suggest that the functions of oxygen in the formation of mitochondria are not restricted to the induction of mitochondrial protein synthesis and to the participation in the synthesis of certain non protein membrane components. Evidently, oxygen has a direct effect on the assembly of the respiratory system and mitochondrial membranes as a whole.     

Isolation of an Escherichia coli mutant defective in cytochrome biosynthesis

Abstract A pleiotropic mutant of Escherichia coli affected in cytochrome biosynthesis was detected by anaerobic screening on a solid medium containing triphenyltetrazolium. When grown anaerobically on glycerol, nitrate and Casamino acids, this mutant exhibited a level of soluble cytochrome c ₅₅₂ which was ten times higher than that found in wild-type cells. The level of membrane-bound cytochrome b and the activity of nitrate reductase were about half the normal level. The mutant grew aerobically on succinate or d,l -lactate at a greatly reduced rate. The mutation impairing the growth ability at the locus sox (succinate oxidation) is also responsible for the deficiency of cytochrome b , nitrate reductase and formate dehydrogenase. Mapping by transduction placed sox at 86.7 min on the chromosome, very close to the glnA locus. Genetic analysis also indicated that the elevated level of cytochrome c ₅₅₂ was the result of a separate mutation, the location of which is yet to be determined.     

Role of Bradyrhizobium japonicum cytochrome c550 in nitrite and nitrate respiration

Bradyrhizobium japonicum cytochrome c ₅₅₀, encoded by cycA , has been previously suggested to play a role in denitrification, the respiratory reduction of nitrate to dinitrogen. However, the exact role of this cytochrome in the denitrification process is unknown. This study shows that cytochrome c ₅₅₀ is involved in electron transfer to the copper-containing nitrite reductase of B. japonicum , as revealed by the inability of a cycA mutant strain to consume nitrite and, consequently, to grow under denitrifying conditions with nitrite as the electron acceptor. Mutation of cycA had no apparent effect on methylviologen-dependent nitrite reductase activity. However, succinate-dependent nitrite reduction was largely inhibited, suggesting that c ₅₅₀ is the in vivo electron donor to copper-containing nitrite reductase. In addition, this study demonstrates that a cytochrome c ₅₅₀ mutation has a negative effect on expression of the periplasmic nitrate reductase. This phenotype can be rescued by extending the growth period of the cells. A model is proposed whereby a mutation in cycA reduces expression of the cbb ₃-type oxidase, affecting oxygen consumption rate by the cells and consequently preventing maximal expression of the periplasmic nitrate reductase during the first days of the growth period.     

Laser-induced reactions of P700 and cytochrome f in a blue-green alga, Plectonema boryanum

Kinetics of the absorption change of P700 (blue band) and cytochrome f in whole cells of a blue-green alga, Plectonema boryanum, have been studied by Q-switched ruby-laser flash excitation (694 nm; approx. 20 nsec) to elucidate the sequential relationship of these two components in photosynthetic electron transport. “P700” was photooxidized within 2 μsec and recovered in two phases t_1/2 10 μsec and 200 μsec). Under the same conditions cytochrome f was oxidized with a half time of 15 μsec. The magnitude of the fast phase of “P700” recovery, however, diminished at lower laser intensity while the cytochrome f change remained unaffected. The result suggests that cytochrome f and P700 may not be on the same electron-transport chain.     

Cytochrome c dissolved in 1-allyl-3-methylimidazolium chloride type ionic liquid undergoes a quasi-reversible redox reaction up to 140°C

Solubility of cytochrome c, a typical heme protein, in a various ionic liquids has been analyzed. The solubility has been discussed with polarity parameters of the ionic liquids. Both hydrogen bond basicity and dipolarity/polarizability of the ionic liquids were confirmed to be influential factors to control the solubilization of cytochrome c. Polar ionic liquids such as 1-butyl-3-methylimidazolium chloride and 1-allyl-3-methylimidazolium chloride solubilized cytochrome c at 80°C, and the dissolved cytochrome c was found to keep its redox activity in these ionic liquids. The redox response of the dissolved cytochrome c was detected in 1-allyl-3-methylimidazolium chloride up to 140°C.     

The nitrite oxidizing system of Nitrobacter winogradskyi

Abstract Cytochrome components which participate in the oxidation of nitrite in Nitrobacter winogradskyi have been highly purified and their properties studied in detail. Cytochrome a ₁ c ₁ is an iron-sulphur molybdoenzyme which has haems a and c and acts as a nitrite-cytochrome c oxidoreductase. Cytochrome c -550 is homologous to eukaryotic cytochrome c and acts as the electron mediator between cytochrome a ₁ c ₁ and aa ₃-type cytochrome c oxidase. The oxidase is composed of two kinds of subunits, has two molecules of haem a and two atoms of copper in the molecule, and oxidizes actively eukaryotic ferrocytochrome c as well as its own ferrocytochrome c -550. Further, a flavoenzyme has been obtained which has transhydrogenase activity and catalyses reduction of NADP⁺ with benzylviologen radical. This enzyme may be responsible for production of NADPH in N. winogradskyi . The electron transfer against redox potential from NO₂⁻ to cythochrome c could be pushed through prompt removal by cytochrome aa ₃ of H⁺ formed by the dehydrogenation of NO₂⁻+ H₂O. As cytochrome c in anaerobically kept cell-free extracts is rapidly reduced on addition of NO₂⁻, a membrane potential does not seem necessary for the reduction of cytochrome c by cytochrome a ₁ c ₁ with NO₂⁻ in vivo.     

N‐terminal cleavage of Bax by calpain generates a potent proapoptotic 18‐kDa fragment that promotes Bcl‐2‐independent cytochrome C release and apoptotic cell death

Upon apoptosis induction, the proapoptotic protein Bax is translocated from the cytosol to mitochondria, where it promotes release of cytochrome c, a caspase‐activating protein. However, the molecular mechanisms by which Bax triggers cytochrome c release are unknown. Here we report that before the initiation of apoptotic execution by etoposide or staurosporin, an active calpain activity cleaves Bax at its N‐terminus, generating a potent proapoptotic 18‐kDa fragment (Bax/p18). Both the calpain‐mediated Bax cleavage activity and the Bax/p18 fragment were found in the mitochondrial membrane‐enriched fraction. Cleavage of Bax was followed by release of mitochondrial cytochrome c, activation of caspase‐3, cleavage of poly(ADP‐ribose) polymerase, and fragmentation of DNA. Unlike the full‐length Bax, Bax/p18 did not interact with the antiapoptotic Bcl‐2 protein in the mitochondrial fraction of drug‐treated cells. Pretreatment with a specific calpain inhibitor calpeptin inhibited etoposide‐induced calpain activation, Bax cleavage, cytochrome c release, and caspase‐3 activation. In contrast, transfection of a cloned Bax/p18 cDNA into multiple human cancer cell lines targeted Bax/p18 to mitochondria, which was accompanied by release of cytochrome c and induction of caspase‐3‐mediated apoptosis that was not blocked by overexpression of Bcl‐2 protein. Therefore, Bax/p18 has a cytochrome c–releasing activity that promotes cell death independent of Bcl‐2. Finally, Bcl‐2 overexpression inhibited etoposide‐induced calpain activation, Bax cleavage, cytochrome c release, and apoptosis. Our results suggest that the mitochondrial calpain plays an essential role in apoptotic commitment by cleaving Bax and generating the Bax/p18 fragment, which in turn mediates cytochrome c release and initiates the apoptotic execution. J. Cell. Biochem. 80:53–72, 2000. © 2000 Wiley‐Liss, Inc.     

Proton translocation coupled to carbon monoxide-insensitive and -sensitive electron transport in Pseudomonas carboxydovorans

Abstract The respiration-driven proton translocation has been measured with the oxygen pulse method and whole cells of the carbon monoxide (CO)-insensitive bacterium Pseudomonas carboxydovorans . → H⁺/O ratios of 5 to 6 were determined with endogenous substrates, pyruvate or hydrogen. In the presence of 100% CO → H⁺/O ratios were lowered to about 4. The results indicate that the Co-sensitive electron transport via the cytochromes c and a conserves more energy than the CO-insensitive electron transport via the alternative pathway with cytochrome o as terminal oxidase. The CO-sensitive a -type cytochrome was found to translocate 2 protons per oxygen atom reduced, thus accounting for the difference of the energy yield of the CO-sensitive and the CO-insensitive electron transport in P. carboxydovorans .     

Shifts of bacteriochlorophyll and carotenoid absorption bands linked to cytochrome c-555 photooxidation in Chromatium

Shifts in the absorption bands of bacteriochlorophyll and carotenoids in Chromatium vinosum chromatophores were measured after short actinic flashes, under various conditions. The amplitude of the bacteriochlorophyll band shift correlated well with the amount of cytochrome c-555 that was oxidized by P₈₇₀⁺ after a flash. No bacteriochlorophyll band shift appeared to accompany the photooxidation of P₈₇₀ itself, nor the oxidation of cytochrome c-552 by P₈₇₀⁺. The carotenoid band shift also correlated with cytochrome c-555 photooxidation, although a comparatively small carotenoid shift did occur at high redox potentials that permitted only P₈₇₀ oxidation.

The results explain earlier observations on infrared absorbance changes that had suggested the existence of two different photochemical systems in Chromatium. A single photochemical system accounts for all of the absorbance changes.

Previous work has shown that the photooxidations of P₈₇₀ and cytochrome c-555 cause similar changes in the electrical charge on the chromatophore membrane. The specific association of the band shifts with cytochrome c-555 photooxidation therefore argues against interpretations of the band shifts based on a light-induced membrane potential.     

Effet de l'accumulation de Zn-protoporphyrine par la cellule de levure sur la synthese et le fonctionnement de son systeme respiratoireEffect of Zn-protoporphyrin accumulation by yeast cells on synthesis and activity of their respiratory system

Yeast cells harvested from aerobic or anaerobic culture are able to synthesize considerable amounts of Zn-protoporphyrin, by aeration of resting cells in phosphate buffer (pH 8).

In yeast cells harvested from aerobic growth, Zn-protoporphyrin accumulation inhibits respiratory activity and produces some letality. In yeast cells harvested from anaerobic growth this accumulation produces both a strong inhibition of cytochrome biosynthesis and of respiratory adaptation, accompanied by an important letality.

Zn-protoporphyrin is accumulated into the mitochondrial fraction and causes a total inhibition of O₂ consumption by isolated mitochondria. The “in vitro” addition of purified Zn-protoporphyrin to intact mitochondria induces a lost of respiratory control.     

The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.     

Cytochrome c release from rat brain mitochondria is proportional to the mitochondrial functional deficit: implications for apoptosis and neurodegenerative disease

Apoptosis may be initiated in neurons via mitochondrial release of the respiratory protein, cytochrome c. The mechanism of cytochrome c release has been studied extensively, but little is known about its dynamics. It has been claimed that release is all-or-none, however, this is not consistent with accumulating evidence of cytosolic mechanisms for 'buffering' cytochrome c. This study has attempted to model an underlying disease pathology, rather than inducing apoptosis directly. The model adopted was diminished activity of the mitochondrial respiratory chain complex I, a recognized feature of Parkinson's disease. Titration of rat brain mitochondrial respiratory function, with the specific complex I inhibitor rotenone, caused proportional release of cytochrome c from isolated synaptic and non-synaptic mitochondria. The mechanism of release was mediated, at least in part, by the mitochondrial outer membrane component Bak and voltage-dependent anion channel rather than non-specific membrane rupture. Furthermore, preliminary data were obtained demonstrating that in primary cortical neurons, titration with rotenone induced cytochrome c release that was subthreshold for the induction of apoptosis. Implications for the therapy of neurodegenerative diseases are discussed.