Partial independence of synthesis and membrane attachment of mitochondrial and cytoplasmic precursors of electron transfer complexes III and IV in adapting Bakers' yeast.

When anaerobically grown Saccharomyces cerevisiae are aerated under conditions which may deplete them of cytoplasmically translated, mitochondrial inner membrane enzyme precursors, they show no immediate decrease in in vivo mitochondrial translational activity compared with cells which have not been so depleted. Similarly, cells depleted of mitochondrially translanted precursors show no immediate decrease in their cytoplasmic translation of mitochondrial inner membrane proteins. These experiments suggest that the synthesis and nonspecific membrane attachment of mitochondrially and cytoplasmically translated inner membrane proteins are not stringently delimited by a prior depletion of inner membrane precursors elaborated by the “other” genetic system. It is thus possible to demonstrate a degree of uncoupling of the activities of the two genetic systems. The oxygen inductions of reduced CoQ cytochrome c reductase (complex III) and of cytochrome c oxidase (complex IV) activities in cells which have been sequentially exposed first to cycloheximide and then to chloramphenicol, or first to chloramphenicol and then to cycloheximide reflect the levels to which specifically integrated, mitochondrial and cytoplasmic precursors of these complexes can accumulate in the absence of concomitant translational activity by the “other” genetic system. These data again suggest the degree to which the translational activities of the two genetic systems can be uncoupled. A detailed study of the inductions of these two complexes in cells exposed first to chloramphenicol shows that the modes of induction of the two complexes are different. Complex III develops approximately 50% of its activity as an expression of a precursor (presumably mitochondrially translated) which is already present in the anaerobic cells, but which requires oxygen-induced cytoplasmic translation for its expression. The remainder of the induced complex III activity appears to require oxygen-induced mitochondrial translation for its expression. There was no analogous anaerobically present component evident during complex IV induction.     

Role of the mitochondrial protein synthesis is the catabolite repression of the petite-negative yeast K.lactis.

The effect of glucose in two different strains of the petite-negative yeast $\text{K. lactis}$ is studied. The results obtained show that one strain ($\text{K. lactis}\text{CBS 2359}$) is glucose repressible for Glutamate Dehydrogenase and β-Galactosidase, whereas the other one (CBS 2360) is almost completely insensitive. The effect of Erythromycin on expression of catabolite repression in CBS 2359 is also analyzed. The results show that the dependence of catabolite repression on mitochondrial protein synthesis reflect the degree of interaction between the nuclear and mitochondrial compartments.     

Glucose repression of the inducible catabolic pathway for N-acetylglucosamine in yeast.

In order to investigate the mechanism of glucose repression of the N-acetylglucosamine metabolic enzymes in $\text{Candida}\text{albicans}$, an obligatory aerobic yeast, the activities of the following inducible enzymes were assayed: the N-acetylglucosamine uptake, N-acetylglucosamine kinase and glucosamine-6-phosphate deaminase. In the presence of glucose or other sugars e.g. succinate and glycerol, synthesis of these enzymes took place at a normal rate, suggesting that the hexose produces no catabolite repression in this organism. On the contrary, strong inhibition by glucose was observed on the activities of N-acetylglucosamine uptake and deaminase in N-acetylglucosamine-grown cells of $\text{Saccharomyces}\text{cerevisiae}$, a facultative aerobe. From the results, it is concluded that “glucose effect” or catabolite repression is absent in $\text{Candida}\text{albicans}$, a pathogenic strain of yeast.     

Oxygen dependence of promitochondrial and cytoplasmic protein synthesis in the formation of electron transfer complexes III and IV in adapting Bakers' yeast.

When anaerobically grown Saccharomyces cerevisiae cells are aerated in the presence of cycloheximide, they accumulate precursor components of electron transfer complexes III and IV. The formation of these precursors is dependent upon promitochondrial protein synthesis and can occur in the absence of concomitant cytoplasmic protein synthesis. The levels to which these precursor components can accumulate during the cycloheximide incubation (phase I) are three to fourfold greater when the cells are grown anaerobically in galactose rather than in glucose. When such galactose-grown cells are sequentially aerated first in cycloheximide and then in chloramphenicol, adaptation responses are elicited with respect to cyanide-sensitive oxygen consumption (QO₂), coenzyme QH₂-cytochrome c reductase (complex III) and cytochrome oxidase (complex IV), all of which are exhibited during the chloramphenicol incubation (phase II). These phase II adaptation responses for QO₂ and for both enzyme activities were observed to be dependent upon the continued presence of oxygen during both phase I (period of mitochondrial translation) and phase II (period of cytoplasmic translation). If one makes the assumption that mRNA's are neither imported into nor exported from promitochondria during adaptation, then one may conclude that oxygen independently and coordinately derepresses synthetic activity in both the mitochondrial and nucleo-cytoplasmic genetic systems. Other regulatory schemes are discussed.     

Tetrahydropterin: Reduction of cytochrome c and coupled phosphorylation at mitochondrial site 3

Incubation of rat liver mitochondria with tetrahydropterin results in ATP production with a P:O ratio of 0.85, consistent with the entry of reducing equivalents into the mitochondrial electron transport chain at cytochrome $\text{c}$. No evidence for an enzymatic reduction of cytochrome $\text{c}$ was found. The reduction of either soluble or mitochondrial cytochrome $\text{c}$ was not diminished by superoxide dismutase or anaerobic conditions, indicating that the reaction is not dependent on the autoxidation of the reduced pterin and the formation of an active species of oxygen. The experiments indicate a potential pathway for the production of ATP coupled to the oxidation of NADPH through the activity of NADPH-dependent pteridine reductases.     

Comparison of plasma membranes and endoplasmic reticulum fractions obtained from whole white adipose tissue and isolated adipocytes

The influence of the mode of preparation upon some of the characteristics of white adipose tissue plasma membranes and microsomes has been reported. Plasma membrane fractions prepared from mitochondrial pellet were shown to have higher specific activities of $\text{(Mg}{}^{\mathrm{2+}}\text{+ Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ than plasma membranes originating in crude microsomes. Isolation of fat cells by collagenase treatment was found to result in a decrease in specific activity of the plasma membrane enzymes; in plasma membranes prepared from isolated fat cells, the specific activity values obtained for $\text{(Mg}{}^{\mathrm{2+}}\text{+ Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ and 5′-nucleotidase were only 42% and 6.3% respectively of those obtained in plasma membranes prepared from whole adipose tissue. Purification of whole adipose tissue crude microsomes by hypotonic treatment caused extensive solubilization of the endoplasmic reticulum marker enzymes, NADH oxidase and NADPH cytochrome $\text{c}$ reductase. The lability of endoplasmic reticulum marker enzymes, however, was found to be greatly diminished in the preparations from isolated fat cells. The possibility that NADH oxidase and NADHPH cytochrome $\text{c}$ reductase activities found in the plasma membranes are microsomal enzymes adsorbed by the plasma membranes is discussed. The peptide patterns as well as the NADH oxidase and NADPH cytochrome $\text{c}$ reductase activity patterns of plasma membranes and purified microsomes were compared by means of sodium dodecyl sulfate or Triton X-100 polyacrylamide gel electrophoresis.     

Cobalt cytochrome c: enzymic assays.

An improved synthesis for cobalt-cytochrome $\text{c}$ has been developed; its half reduction potential is ?140 ± 20mV. Reduced ^Cocyt-$\text{c}$³ is oxidized by bovine heart cytochrome $\text{c}$ oxidase at a rate ～45% that of the native cytochrome $\text{c}$. It is not reduced by mitochondrial NADH or succinate cytochrome $\text{c}$ reductase nor by microsomal NADH or NADPH cytochrome $\text{c}$ reductase.     

Binding of cytochrome c to the cytochrome bc1 complex (complex III) and its subunits cytochrome c1 and b1.

Cytochrome $\text{c}$₁, the electron donor for cytochrome $\text{c}$, is a subunit of the mitochondrial cytochrome $\text{bc}$₁ complex (complex III, cytochrome $\text{c}$ reductase). To test if cytochrome $\text{c}$₁ is the cytochrome $\text{c}$-binding subunit of the $\text{bc}$₁ complex, binding of cytochrome $\text{c}$ to the complex and to isolated cytochrome $\text{c}$₁ was compared by a gel-filtration method under non-equilibrium conditions (a $\text{bc}$₁ complex lacking the Rieske ironsulfur protein was used; von Jagow et al. (1977) Biochim. Biophys. Acta $\text{462}$, 549–558). The approximate stoichiometries and binding affinities were found to be very similar. Binding of cytochrome $\text{c}$ to isolated cytochrome $\text{b}$ which is another subunit of the reductase was not detectable by the gel-filtration method. Further, the same lysine residues of cytochrome $\text{c}$ were shielded towards chemical acetylation in the complexes $\text{c}\text{:}\text{c}$₁ and $\text{c}\text{:}\text{bc}$₁. From this we conclude that the same surface area of cytochrome $\text{c}$ is in direct contact with cytochrome $\text{bc}$₁ and with cytochrome $\text{c}$₁ in the respective complexes and that therefore cytochrome $\text{c}$ is most probably the structural ligand for cytochrome $\text{c}$ in mitochondrial cytochrome $\text{c}$ reductase.     

Bound cytochrome C as proton donor and acceptor during enzymic oxidoreduction

When ferrocytochrome $\text{c}$ reacts with delipidated cytochrome oxidase under conditions which prevent oxidation, one proton is taken up per molecule of ferrocytochrome $\text{c}$ bound to cytochrome oxidase. When ferricytochrome $\text{c}$ reacts with delipidated Complex III, one proton is released per molecule of ferricytochrome $\text{c}$ bound to Complex III. From these data it can be concluded that the oxidation of ferrocytochrome $\text{c}$ by cytochrome oxidase leads to the release of a proton and an electron, whereas the reduction of ferricytochrome $\text{c}$ by Complex III leads to the uptake of a proton and an electron. Thus ferrocytochrome $\text{c}$ like QH₂ and NADH is both an electron and proton donor, and ferricytochrome $\text{c}$ like Q and O₂ is both an electron and proton acceptor. The pattern for the three mitochondrial electron transfer sequences NADH → Q, QH₂ → ferricytochrome $\text{c}$ and ferrocytochrome $\text{c}$ → O₂ involves separation of an electron and proton on the side of the membrane where electron transfer is initiated and recombination of an electron and a proton in the terminal acceptor on the side of the membrane where electron transfer terminates.     

Microcalorimetric studies of the interactions between cytochromes c and c1 and of their interactions with phospholipids

Thermotropic properties of purified cytochrome $\text{c}{}_1$ and cytochrome $\text{c}$ have been studied by differential scanning calorimetry under various conditions. Both cytochromes exhibit a single endothermodenaturation peak in the differential scanning calorimetric thermogram. Thermodenaturation temperatures are ionic strength, pH, and redox state dependent. The ferrocytochromes are more stable toward thermodenaturation than the ferricytochromes. The enthalpy changes of thermodenaturation of ferro- and ferricytochrome $\text{c}{}_1$ are markedly dependent on the ionic strength of the solution. The effect of the ionic strength of solution on the enthalpy change of thermodenaturation of cytochrome $\text{c}$ is rather insignificant. The formation of a complex between cytochromes $\text{c}$ and $\text{c}{}_1$ at lower ionic strength causes a significant destabilization of the former and a slight stabilization of the latter. The destabilization of cytochrome $\text{c}$ upon mixing with cytochrome $\text{c}{}_1$ was also observed at high ionic strength, under which conditions no stable complex was detected by physical separation. This suggests formation of a transient complex between these two cytochromes. When cytochrome $\text{c}$ was complexed with phospholipids, no change in the thermodenaturation temperature was observed, but a great increase in the enthalpy change of thermodenaturation resulted.     

Effect of thyroid hormone administration on synthesis and degradation of cytochrome c in rat liver.

Thyrotoxicosis can induce increases in the concentrations of the cytochromes of the inner mitochondrial membrane in rat liver. The purpose of this study was to determine whether the increase in hepatic cytochrome c concentration in thyrotoxic rats is maintained by an increase in the rate of synthesis, a decrease in the rate of degradation, or a combination of the two. The turnover of cytochrome c labeled with δ-amino [¹⁴C]levulinate was measured in the livers of thyrotoxic rats that were in steady state with respect to liver cytochrome c concentration, liver weight, and body weight. Cytochrome c concentration was increased 3.4-fold in the livers of the thyrotoxic animals. The $\text{t}\text{1}\text{2}$ of liver cytochrome c was 3.7 days in the thyrotoxic and 5.7 days in euthyroid animals. It was calculated that the 3.4-fold increase in cytochrome c concentration was maintained, in the face of a 63% increase in k_d, by a 5.5-fold increase in synthesis rate.     

Charge distribution in electron transport components: cytochrome c and cytochrome c oxidase mixtures

Mixtures of cytochrome $\text{c}$ oxidase and cytochrome $\text{c}$ have been titrated by coulometrically generated reductant, methyl viologen radical cation, and physiological oxidant, O₂. Charge distribution among the heme components in mixtures of these two redox enzymes has been evaluated by monitoring the absorbance changes at 605 and 550 nm. Differences in the pathway of the electron transfer process during a reduction cycle as compared to an oxidation cycle are indicated by variations found in the absorbance behavior of the heme components during successive reductive and oxidative titrations. It is apparent that the potential of the cytochrome $\text{a}$ heme is dependent upon whether oxidation or reduction is occurring.     

Cytochrome c interaction with membranes. I. Use of a fluorescent chromophore in the study of cytochrome c interaction with artificial and mitochondrial membranes

Quenching of 12-(9-anthroyl) stearic acid (AS) fluorescence by cytochrome c occurs through an energy-transfer mechanism and can be used to measure the binding of the cytochrome to artificial and mitochondrial membranes. The quenching of AS³ fluorescence is biphasic ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ below 25 msec and above 500 msec) and its extent diminishes at high salt concentration or at high pH and increases in the presence of negatively charged lipids.Addition of cytochrome c to cytochrome c-depleted mitochondria results in binding of the cytochrome to the membrane and quenching of AS fluorescence. The affinity of oxidized cytochrome c for cytochrome c-depleted mitochondria is 1.8 × 10⁶m, while the affinity constant for reduced cytochrome c is 0.5 × 10⁶m. The lower affinity of the reduced cytochrome c for mitochondrial membranes is in accordance with midpoint potential differences between the bound and free forms.     

Photoreduction of membrane bound cytochrome C by excited-state phenothiazine.

Ferricytochrome $\text{c}$ can be reduced in a photochemical reaction by excited state phenothiazine. This reaction is observed between phenothiazine which is solubilized by phospholipid artificial membranes and cytochrome $\text{c}$ which is adsorbed to the membrane surface. Under conditions when cytochrome $\text{c}$ is not bound to the phospholipid, the rate of reduction by phenothiazine is greatly reduced. The phosphorescence of phenothiazine is quenched in the presence of cytochrome $\text{c}$, implying that the excited triplet state interacts with cytochrome $\text{c}$. Oxygen inhibits the reaction since possibly, as a paramagnetic species, it increases intersystem crossing of the excited states of phenothiazine. On the basis of molecular models the proximity between the iron of ferricytochrome $\text{c}$ and phenothiazine is estimated to be over 20 Å.     

Biogenesis of mitochondria: The effects of altered steady-state membrane lipid composition on mitochondrial-energy metabolism in Saccharomyces cerevisiae

A chemostat culture technique has been developed for the growth of an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae. Any chosen steady-state cellular unsaturated fatty acid level between 75 and 15% of the total fatty acids could be established and maintained. In all cultures the steady-state glucose concentrations were maintained at levels below that which induces catabolite repression.The efficiency of oxidative phosphorylation as determined from the molar growth yield decreased as the cellular unsaturated fatty acid composition was lowered. The number of moles of ATP produced by oxidative phosphorylation per mole of glucose utilized was 7.2, 4.8, 0.7, and 0.4 for cells in which 75, 50, 44, and 34%, respectively, of the total fatty acids were unsaturated.The lesion in oxidative phosphorylation was a direct result of lowering the membrane unsaturated fatty acid composition as the respiratory activities and cytochrome content of cells and mitochondria were unaffected by a decrease in the cellular unsaturated fatty acid level from the wild-type value of about 75% down to about 34%.In cells which contained lipids with 22–28% unsaturated fatty acids, cyanide-sensitive respiration was absent, and the levels of all mitochondrial cytochromes were less than 10% of normal. The reduction in the levels of cytochromes aa₃ and b appeared to be a consequence of a loss of mitochondrial protein synthetic activity in such cells. The level of cytochrome c was also greatly decreased, indicating that the cellular unsaturated fatty acid composition was affecting either the synthesis in the cytoplasm of mitochondrial proteins or the assembly of these proteins in the mitochondria.     

Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane: role of cytochrome P-450.

In order to define the site of bioactivation of CCl₄, CHCl₃ and CBrCl₃ in the NADPH cytochrome $\text{c}$ reductase-cytochrome P-450 coupled systems of liver microsomes, the ¹⁴C-labeled hepatotoxins were incubated $\text{in}$$\text{vitro}$ with isolated rat liver microsomes and a NADPH-generating system. The covalent binding of radiolabel to microsomal protein was used as a measure of the conversion of the hepatotoxins to reactive intermediates. Omission of NADPH, incubation under CO:O₂ (8:2) and addition of a cytochrome $\text{c}$ reductase specific antisera mardedly reduced the covalent binding of all three compounds. When cytochrome P-450 was reduced to less than 25% of normal by pretreatment of rats with allylisopropylacetamide (AIA), but cytochrome $\text{c}$ reductase activity was unchanged, the covalent binding of CCl₄, CHCl₃, and CBrCl₃ was decreased by 63, 83, 70%, respectively. Incubation under an atmosphere of N₂ enhanced the binding of CCl₄, inhibited the binding of CHCl₃ and did not influence the binding of CBrCl₃. It is concluded that cytochrome P-450 is the site of bioactivation of these three compounds rather than NADPH cytochrome $\text{c}$ reductase and that CCl₄ bioactivation proceeds by cytochrome P-450 dependent reductive pathways, while CHCl₃ activation proceeds by cytochrome P-450 dependent oxidative pathways.     

Electron paramagnetic resonance studies of membrane proteins in hepatic microsomes

Hepatic microsomal membranes, prepared under various conditions that yield either ‘intact’ or ‘disrupted’ microsomal vesicles, have been labeled via the sulfhydryl groups of intrinsic membrane proteins using nitroxide analogs of $\text{N-}\text{ethylmaleimide}$. Electron paramagnetic resonance spectra revealed the presence of two dominant classes of bound label corresponding to differing degrees of immobilization, the ratio of which were quantitated using a parameter designated the ‘$\text{W/S}$’ ratio. For latent microsomes, the value of this parameter was determined to be $\text{0.65 ± 0.02}$ and was influenced by factors such as label/protein ratio, incubation period, nitroxide structure, temperature and pH. The $\text{W/S}$ ratio was also sensitive to the degree of membrane integrity as revealed by the latency of mannose 6-phosphate activity of glucose-6-phosphohydrolase. In addition, membrane disruption resulted in a corresponding decrease in the order parameter for nitroxide-labeled fatty acids intercalated within the lipid bilayer. The $\text{W/S}$ ratio was observed to be dependent upon the method of microsome preparation yielding values of $\text{1.02 ± 0.02}$ for ‘hypertonically disrupted’ vesicles and $\text{1.28 ± 0.02}$ for ‘mechanically disrupted’ vesicles. Microsomal marker enzymes such as cytochrome $\text{P-450}$ and FAD-containing monooxygenase retained significant levels of functionally following nitroxide incorporation.     

Changes in predominant energy substrate after hepatectomy

The changes in the energy substrate utilized by the remnant liver were studied in relation to the changes in the cellular energy status of 25 and 70% hepatectomized rabbits. In 25% hepatectomized rabbits, the energy charge $\text{(}\text{(}\text{ATP}\text{+0.5}\text{ADP}\text{)}\text{(}\text{ATP}\text{+}\text{ADP}\text{+}\text{AMP}\text{)}\text{)}$ level of the remnant liver remained unchanged, the energy substrate of which was predominantly glucose, rather than fatty acid. In contrast, in 70% hepatectomized rabbits, the energy production by the mitochondria was mainly dependent upon fatty acid oxidation at the early period after hepatectomy when the energy charge level decreased remarkably, and then upon glucose oxidation, concomitant with the restoration of the energy charge. It is suggested that the changes in the energy substrate utilized are closely related to those in the energy charge level and the mitochondrial phosphorylative activity of the remnant liver following hepatectomy.     

The cardiolipin-cytochrome c interaction and the mitochondrial regulation of apoptosis

While many studies have focused on cytochrome c release from mitochondria, little attention has been given to the specific interaction between cardiolipin (CL) and cytochrome c, the breaching of which likely represents a critical event in the initiation of mitochondrially mediated apoptosis. Mounting evidence suggests that a decrease in the level of CL affects cytochrome c binding to the inner membrane, thus leading to higher levels of soluble cytochrome c in the mitochondrial intermembrane space. Among the factors known to affect CL levels are thyroid status, plasma concentrations of free fatty acids, Ca²⁺ dysregulation, and reactive oxygen species (ROS). These factors, especially Ca²⁺ and ROS, have long been recognized as triggers of cell death and, more recently, as modulators of mitochondrially mediated apoptosis. In this review, we discuss the significance of the disruption of the CL-cytochrome c interaction for cytochrome c release and apoptosis.     

Cytochrome oxidase from an extreme thermophile. Thermus thermophilus HB8

The cytochrome oxidase (EC 1.9.3.1) of $\text{Thermus}\text{thermophilus}$ HB8 was isolated from the membrane fraction, and was highly purified. The oxidase contained heme $\text{a}$ and heme $\text{c}$ as the prosthetic groups. The purified preparation showed a single band in polyacrylamide gel electrophoresis, and three major polypeptides with apparent molecular weights of 52,000, 37,000 and 29,000 were observed in the presence of sodium dodecyl sulfate. The enzyme reacted rapidly with $\text{T}\text{.}\text{thermophilus}$ cytochrome c-552. The oxidation of $\text{T}\text{.}\text{thermophilus}$ cytochrome $\text{c}$-555,549 by the enzyme was very slow, and was stimulated by the addition of cytochrome $\text{c}$-552. The enzyme was highly stable to heat.