Cytochrome c reactivity in its complexes with mammalian cytochrome c oxidase and yeast peroxidase

1.

1. The ascorbate reducibility of cytochrome c (beef or horse heart) in its complexes with cytochrome c oxidase (beef heart) and cytochrome c peroxidase (yeast) has been studied.     

Ligand binding to cytochrome c and other related haem proteins and peptides. Part I. Equilibrium studies

Investigation of ligand binding to native cytochrome c, carboxymethyl-Met 80-cytochrome c, myoglobin and haemhexapeptide revealed that the binding of exogenous ligands is modulated by the following factors:

• 1.Hydrophobicity of the haem environment.
• 2.Haem accessibility to exogenous ligands, termed the haem crevice ‘open-closed’ parameter.
• 3.Steric interactions between the protein and the bound ligand.

     

Charge interactions of cytochrome c with cytochrome c oxidase

• 1.1. The pyridoxal phosphate (PLP) modification of the lysine amino groups in cytochrome c causes decrease in the reaction rate with cytochrome c oxidase.
• 2.2. The rate constants for (PLP);-cyt. c, PLP(Lys 86)-cyt. c, PLP(Lys 79)-cyt. c and native cytochrome c (at pH 7.4, 1=0.02) are 3.6 × 10⁻³'sec-', 5.5 × 10⁻³, 5.2 × 10⁻³-'sec⁻¹ and 9.8 × 10⁻³sec⁻¹, respectively.
• 3.3. In spite of the same positive charge of singly PLP-cytochromes c the reaction between PLP(Lys 86)-cyt. c and cyt. c oxidase exhibits the ionic strength dependence that differs from those of the PLP(Lys 79)-cyt. c.
• 4.4. The rate constants at zero and infinite ionic strength for PLP(Lys 86)-cyt. c is 2-fold less than that for PLP(Lys 79)-cyt. c.
• 5.5. The positively charged cytochrome c lysines 86 and 79 form two from four or five predicted complementary charge interactions with carboxyl groups on cytochrome c oxidase.

     

Role of glutamate dehydrogenase reaction in the control of citrate pool in yeast

• 1.1. Role of NADP-glutamate dehydrogenase in the depletion of citrate was analyzed using permeabilized yeast cells.
• 2.2. Citrate was converted to 2-oxoglutarate, which was then metabolized to glutamate by NADP-glutamate dehydrogenase in the presence of ammonium ion.
• 3.3. Formation of 2-oxoglutarate plus glutamate was in good agreement with the concentration of citrate decreased. Glutamate formation can be a good indicator of the depletion of citrate, because 70% of the citrate decreased was converted to glutamate.
• 4.4. Glycolytic activity was closely correlated with the decrease in citrate under the in situ conditions.
• 5.5. NADP-glutamate dehydrogenase increased in anaerobically grown yeast cells.
• 6.6. An effective depletion of citrate by increased synthesis of NADP-glutamate dehydrogenase can explain the lowered mechanism of citrate causing glycolytic stimulation under the anaerobic growth conditions of yeast.

     

Properties of ferredoxin reductase and ferredoxin from the bovine corpus luteum

• 1.1. Ferredoxin reductase and ferredoxin were purified from the bovine corpus luteum and their properties compared to the corresponding adrenal proteins.
• 2.2. The luteal and adrenal proteins had similar absorbance spectra and molecular weights.
• 3.3. Evidence was obtained from spectrophotometric titrations for formation of 1:1 complexes between luteal ferredoxin reductase and ferredoxin and between ferredoxin and cytochrome P-450_scc.
• 4.4. Adrenal ferredoxin reductase and ferredoxin were equally as effective as luteal ferredoxin reductase and ferredoxin in supporting cholesterol side-chain cleavage by luteal cytochrome P-450_scc.

     

Responses of mixed-function oxygenase and antioxidase enzyme system of Mytilus sp. to organic pollution

• 1.1. Mixed-function oxidase (MFO) system components (cytochrome P-450, “418-peak”, cytochrome b₅ and NADPH-cytochrome c (P-450) reductase) and inducible antioxidant enzymes (catalase, Superoxide dismutase (SOD), glutathione peroxidase (GPX) and DT-diaphorase) has been determined in digestive glands of mussels (Mylilus galloprovincialis) collected from three Mediterranean coastal locations, exhibiting an organic pollution gradient.
• 2.2. Cytochrome P-450, the “418-peak”, catalase and SOD showed a good correlation with whole body tissue PAHs and, to a lower extent, with PCBs.
• 3.3. Microsomal NADPH-dependent DT-diaphorase, but not the NADH-dependent microsomal enzyme or the cytosolic DT-diaphorases, was indicated to increase with pollution exposure.
• 4.4. The application of such measurements to environmental monitoring is discussed. Given the magnitude of differences observed, and the state of knowledge on enzyme function and mechanisms of toxicity, a multiparameter approach is considered to offer current and future potential for detecting the impact of organic pollution on bivalve molluscs.

     

Structural analogies between different redox enzymes inserted into biological membranes

• 1.1. We have compared the primary structure and the predicted secondary structure of subunit I (COI) of cytochrome oxidase with those of other integral redox enzymes which contain membrane-buried iron centres.
• 2.2. Some striking analogies have been found between the deduced transmembrane folding for COI and the known three-dimensional structure of the photosynthetic reaction centre of Rhodobacter sphaeroides.
• 3.3. These structural analogies are paralleled by a fundamental functional analogy between these two redox systems, since they both oxidize reduced cytochrome c at the positive side of the membrane, transferring electrons to membrane-buried metal centres.
• 4.4. A statistical evaluation has been performed on the amino acid composition of the peptides containing known histidine ligands of the membrane-buried iron in cytochrome b of the bc 1 complex and in the bacterial reaction centre.
• 5.5. This evaluation was then applied to the peptides which contain conserved histidines in subunit I of cytochrome oxidase, subunit that is known to bind both haem a and a3, indicating which of these histidines are the most likely ligands of the membrane-buried iron of the a-haems.
• 6.6. A sequence homology has been found between the known oxygen binding site and the haem binding peptide in cyt P450 and two peptides which are conserved in all the sequences of COI, thus indicatingt the possible oxygen catalytic site of cytochrome oxidase.

     

Trehalose synthase and trehalase behaviour in yeast cells in anhydrobiosis and hydrobiosis

• 1.I. Trehalose synthase and trehalase behaviour has been analysed in cultured yeast cells isolated from baker's yeast to increase the understanding of the mechanisms involved in trehalose content modifications observed in anyhydrobiois and hydrobiosis.
• 2.2. After desiccating yeast cells to a constant weight, trehalose levels sharply increased, whereas the glycogen content decreased, trehalose synthase was stimulated and trehalase was significantly inhibited.
• 3.3. In desiccated cells after a rehydration for 15 min, trehalose levels dropped, the glycogen content further decreased, the activity of trehalose synthase declined while that of trehalase was dramatically stimulated.
• 4.4. After rehydration for 12hr, while the trehalose and glycogen content decreased even more, the behaviour of the two enzymes was completely reversed, trehalose synthase being activated and trehalase inhibited.
• 5.5. The reasons for such impressive enzyme activity alterations in desiccated and rehydrated cells for the moment remain unknown.

     

Effect of temperature on the kinetics of the yeast AMP deaminase

• 1.1. The temperature dependence of the kinetics of the yeast AM P deaminase was examined using the purified enzyme and the permeabilized yeast cells.
• 2.2. The increase in the enzyme affinity for the substrate AMP was accompanied by the decrease in the maximal velocity with the decreasing temperature in the absence and presence of ATP.
• 3.3. The apparent Km for AMP was lowest at 15–20°C, and the affinity was decreased below and above this temperature.
• 4.4. The rate of the AMP deaminase reaction remained constant over a wide range of temperature in the presence of physiological AMP concentrations.
• 5.5. The temperature dependent change in kinetic properties of AMP deaminase may contribute to the control of the yeast glycolytic flux under the condition of lower temperature environments.

     

Investigation of phycocyanin-membrane interaction: Promotion of membrane interactions

• 1.1. In a continuing investigation of phycocyanin-membrane surface interaction, fluorescence quenching experiments were performed with a mixture of two populations of fluorescence probe-encapsulated phospholipid bilayer vesicles in the presence and absence of phycocyanin.
• 2.2. These membrane vesicles were prepared with 1,2-dimyristoyl phosphatidylcholine (DMPC), cholesterol and a probe molecule.
• 3.3. A fluorophore was encapsulated in one population of membrane vesicles, while a quencher was encapsulated in another population of membrane vesicles.
• 4.4. The result was compared with those of experiments in the presence of other biomolecules, including albumin, cytochrome c, hemoglobin, myoglobin or RNA.
• 5.5. Interestingly, a one-third reduction of the fluorescence intensity was observed in the mixture of these two populations of membrane vesicles in phycocyanin's presence.
• 6.6. In contrast, the other biomolecules caused no significant reduction in the fluorescence intensity.
• 7.7. These findings were evidence of a phycocyanin-induced membrane perturbation.
• 8.8. This was further demonstrated by a phycocyanin-induced change in the thermotropic behavior of DMPC vesicles, as measured by differential scanning microcalorimetry.
• 9.9. Such a unique property of phycocyanin is believed to be associated with its known membrane surface-interacting character.
• 10.10. A possible phycocyanin-modulated membrane-membrane interaction was discussed.

     

Complement-like protein from the phylogenetically primitive vertebrate,Eptatretus Stouti,is a Humoral Opsonin

• 1.1. Serum from the Pacific hagfish,Eptatretus stouti,contains a complement-like protein (CLP).
• 2.2. CLP from unfractionated hagfish serum and from affinity-purified preparations binds to yeast cell surfaces.
• 3.3. Incubation with CLP enhances the phagocytosis of yeast by hagfish leukocytes.
• 4.4. CLP-mediated opsonization can be inhibited by anti-CLP antibody, EDTA, d(+)mannose and l(+)rhamnose.
• 5.5. Additional opsomic factors are also in hagfish serum.

     

A simple and reliable method for the purification of Pseudomonas aeruginosa phospholipase C produced in a high phosphate medium containing choline

• 1.1. Pseudomonas aeruginosa phospholipase C from culture supernatants of bacteria grown in high-P_i basal salt medium with choline, as the sole carbon and nitrogen source, was purified by precipitation with 70% saturation ammonium sulfate in the presence of celite.
• 2.2. The PLC activity was eluted of this mixture by the use of a reverse gradient of 70-0% ammonium sulfate.
• 3.3. The peak containing the PLC activity revealed a single protein after SDS-PAGE.
• 4.4. The method could also be applied to purify PLC produced in a low-P_i complex medium. The resultant preparation was not homogeneous.
• 5.5. The molecular weight for both PLC preparations was about 70 kDa.
• 6.6. Both PLC used phosphatydilcholine and sphingomyelin as substrates, displayed hemolytic activity an exhibited an apparent K_M of 25 mM for p-nitrophenylphosphorylcholine.
• 7.7. They were not inhibited by 1% sodium deoxycholate but were 30% inhibited by 1% Triton X-100.
• 8.8. 2% sodium dodecylsulfate and 1% tetradecyltrimethylammonium bromide inhibited the PLC from the HP_l-BSM plus choline but not the enzyme from the LP_l-CM.

     

The stimulation of yeast mitochondrial protein synthesis by low molecular weight cytoplasmic factors: Requirement for intact mitochondria and lack of effect by folate derivatives

• 1.1. Protein synthesis by GTP -supplemented yeast mitochondria is stimulated by a fraction of molecular weight less than 2,000 isolated from yeast high-speed supernatant (S-150).
• 2.2. The low molecular weight fraction works independently of the respiratory chain as the stimulation effect is not cyanide-sensitive.
• 3.3. Stimulation of mitochondrial protein synthesis by cytoplasmic factors is dependent upon the method of mitochondrial isolation.
• 4.4. The low molecular weight stimulatory factor(s) are not reduced folate derivatives which supply formyl groups required for initiation of mitochondrial protein synthesis.

     

Deleterious effects of disulfiram on the respiratory electron transport system of liver mitochondria

• 1.1. The mechanism of action of disulfiram on the respiratory electron transport system of the liver mitochondria was studied in vitro.
• 2.2. Disulfiram inhibited the respiration supported by malate-glutamate as well as succinate.
• 3.3. Mitochondrial respiration inhibition was dependent upon alteration of —SH groups.
• 4.4. The inhibitory action of disulfiram might be related to the crosslinking of several proteins of the inner mitochondrial membrane.
• 5.5. The effects described above could be attributed to disulfiram per se and not to the main metabolite diethyldithiocarbamate.

     

Comparative study of the endemic freshwater fauna of lake baikal—I. Phospholipid and fatty acid composition of two mollusc species,Baicalia oviformus and Benedictia baicalensis

• 1.1. Lipid and phospholipid compositions of endemic freshwater molluscs belonging to the class Gastropoda, Baicalia oviformus and Benedictia baicalensis, were studied.
• 2.2. The fatty acids composition of total lipids, neutral, glyco- and phospholipid fraction was investigated by capillary gas chromatography-mass spectrometry.
• 3.3. Ninety-five fatty acids were identified: 23 saturated (both iso- and anteiso-), 28 monoenoic, 14 dienoic and 30 polyenoic.
• 4.4. High percentage of the two main acids, 18:4 and 18:4(n-3) in phospholipid and glycolipid fractions were identified.
• 5.5. A number of unusual polyunsaturated fatty acids, such as 19:4, 18:5(n-3), 24:4(n-6), 24:5(n-6), 24:6(n-3), and furanoid acids, were found.

     

Genetic diversity in horses inferred from distribution of hemotypes—II. Genetic structure of mixed breed population

• 1.1. Fifteen populations of 10 horse breeds ranging from 110 to 2232 individuals yielded 61 phenotypes of electrophoretic variants at six blood loci.
• 2.2. The numbers of different hemotypes varied from 79 to 239, and the ratios of number of individuals to number of hemotypes varied from 1.38 to 9.34.
• 3.3. The genetic structure of the pooled mixed breed population comprising 11,393 individuals disclosed features similar to those of the least diversified component breeds.
• 4.4. The mean ratio of n individuals to n hemotypes was 11.33.
• 5.5. This figure represents a first direct attempt to estimate the individual genetic diversity in horses.

     

Temperature acclimation in respiratory and cytochrome c oxidase activity in common carp (Cyprinus carpio)

• 1.1. Common carp (Cyprinus carpio) exposed to experimental temperatures of 12, 18, 24, 30 or 36°C for a 4-week period were used to investigate the effect of temperature acclimation on the frequency of opercular movement (FOM), growth and cytochrome c oxidase (CCO) activity in heart, liver and muscle.
• 2.2. An exponential relationship between FOM and temperature after the first week (10₁₀ =1.76) disappeared after the second week.
• 3.3. The initially high FOM at temperatures of 30 or 36°C and the low FOM at 18 or 12°C changed over 4 weeks to approach the FOM of fish at 24°C.
• 4.4. This change in the relationship of FOM to temperature from highly dependent to independent appeared to be thermal compensation.
• 5.5. Heart and liver CCO activities were significantly affected by temperature, with the lowest activity at the approximate optimum temperature for growth, 24°C.
• 6.6. Highest CCO activities for heart and liver occurred at both the highest and lowest temperatures.
• 7.7. Among the three tissues, heart CCO activity was generally the highest and most affected by acclimation temperature.
• 8.8. Muscle tissue had the lowest CCO activity and was unaffected by temperature.
• 9.9. The high CCO activity at a cold acclimation of temperature 12°C was probably due to thermal compensation and the high activity at 36°C may have been a result of thermal stress.

     

Effects of inhibition of nadph: cytochrome p-450 reductase on benzo(a)pyrene metabolism in mouse liver microsomes

• 1.1. Effects of antioxidants (butylated hydroxytoluene and nor-dihydroguaiaretic acid), vitamin K-related quinones (vitamin K₁ and coenzyme Q₁₀) and inorganic copper (CuSO₄), in concentrations inhibiting NADPH: cytochrome P -450 reductase, were re-examined on benzo(a)pyrene metabolism in mouse liver uninduced microsomes.
• 2.2. It was found that all these compounds decrease production of the two-electron oxygenation products of benzo(a)pyrene (monophenoles, diols) and the amounts of glucuronides in a manner parallel to their inhibitory potency against NADPH: cytochrome P-450 reductase.
• 3.3. No correlation was found between amounts of one-electron oxidation products of benzo(a)pyrene and inhibition of NADPH: cytochrome P-450 reductase.
• 4.4. Without added UDPGA the compounds studied decreased protein associated benzo(a)pyrene metabolites in parallel to the decreased overall metabolism of this polyaromatic hydrocarbon.
• 5.5. The mode of action of the studied compounds is discussed.

     

Metabolic roles of carnitine expressed through the carnitine acetyltransferase system in Candida pintolopesii

• 1.1. The carnitine-responsive mutant yeast, Candida pintolopesii ATCC 26014 and the wild type strain (ATCC 22987) were used to investigate the role of carnitine and the carnitine acetyltransferase system.
• 2.2. [³H]l-Carnitine, supplied to the cells, was incorporated into acetylcamitine and [¹⁴C]pantothenate was incorporated into CoA and its derivatives.
• 3.3. Both bioautography and quantitative assays indicated that the relative amounts of CoA and acetylCoA were very different in the mutant and wild type cells.
• 4.4. The wild type yeast maintained an acetylCoA/CoA ratio of 0.33 ± 0.09 indicating that most of the CoA in the cell is in the free CoA form. Carnitine was not required to establish this ratio nor did its presence lower it further.
• 5.5. In contrast, the mutant cells contained a high acetylCoA/CoA ratio (12.8 ± 3.0).
• 6.6. In the mutant cells, carnitine lowered the ratio by decreasing the intracellular acetylCoA concentration and releasing free CoA.
• 7.7. These data indicated that wild type yeast possess an effective mechanism that is not related to the CAT system for regulating the acetylCoA/CoA ratio.
• 8.8. This mechanism appears to be lacking in the mutant. The CAT system decreased the acetylCoA/CoA ratio in the mutant cells but not to the value which is found in the wild type strain.
• 9.9. In both stains of Candida pintolopesii, in the presence of carnitine, an acetylcamitine pool can be created whose concentration exceeds that of acetylCoA.
• 10.10. The intracellular apparent equilibrium constant (K_app) for carnitine acetyltransferase for wild type Candida pintolopesii ATCC 22987 was 0.73 ± 0.12, close to the established value of 0.6, indicating that the CAT system ran close to equilibrium.
• 11.11. The K_app for the CAT system of the carnitine-responsive mutant yeast was 7.7 ± 1.7 indicating that this reaction was not at equilibrium.