Mitochondrial Cytochrome Oxidase Activity Research Articles

Differentiation of promyelocytic (HL-60) cells into mature granulocytes: mitochondrial-specific rhodamine 123 fluorescence.

Rhodamine 123, a fluorescent dye which binds as a result of the transmembrane potential, was used to stain the mitochondria of HL-60 cells, a cell line established from human promelocytic leukemia cells. The DMSO-induced differentiation of promyelocytic cells into mature granulocytes caused a fourfold decrease in fluorescence intensity that paralleled the disappearance of S-phase and G2M cells. This suggests that upon myeloid differentiation whereby the cells enter an irreversible quiescent state, the mitochondrial mass of the cells has decreased. This suggestion is corroborated by electron microscopy, which shows a decrease in the number of mitochondria, and by decreases in total mitochondrial protein and cytochrome oxidase activity. The respiratory rate of isolated mitochondria did not change, suggesting that the transmembrane potential remained the same. Undifferentiated cells in exponential phase of growth exhibit an intracellular heterogeneity of fluorescence intensity. This heterogeneity appears to have a cell age basis, as late S/G2M cells, obtained by centrifugal elutriation, yielded twice the fluorescence intensity of early G1 cells.

Effect of dietary ethanol and cholesterol on metabolic functions of hepatic mitochondria and microsomes from the monkey, Macaca nemestrina.

Monkeys (Macaca nemestrina) were divided into four groups, and each group was fed a particular diet. The variables in the diets were as follows: diet A, 0.3 mg cholesterol/kcal nutrient; diet B, 1.0 mg cholesterol/kcal nutrient; diet C, 0.3 mg cholesterol/kcal nutrient, ethanol (36% of calories); diet D, 1.0 mg cholesterol/kcal nutrient, ethanol (36% of calories). Monkeys on the diets containing ethanol developed fatty liver. Mitochondria from ethanol-fed animals demonstrated significant decreases in uncoupler-stimulated, state 3, and state 4 succinate oxidation activity; respiratory control ratio; and ATP content. Liver microsomes isolated from the ethanol-fed groups demonstrated increased ethanol oxidizing activity with either NADPH or H2O2 as cosubstrate. Aniline hydroxylase and aminopyrine-N-demethylase activities were also elevated in ethanol-fed animals. The alterations in these functional properties were related primarily to ethanol in the diets. Cholesterol, while being less of a perturbant than ethanol, did elicit a significant decrease in cytochrome oxidase activity of mitochondria and a small but statistically significant increase in microsomal-associated ethanol oxidation activity. It appeared to potentiate the effect of ethanol in lowering mitochondrial respiratory control and ATP concentrations.

The effect of triton X-100 on cytochrome oxidase activity in carp, frog and rat liver mitochondria

1. 1. Maximal activity of cytochrome oxidase was observed in frog and rat liver mitochondria in the presence of 0.016% (w/v) Triton X-100 and in carp liver mitochondria in the presence of 0.035% (w/v) of this detergent. 2. 2. Osmotic disruption of mitochondria from those species and centrifugation through a discontinuous sucrose density gradient indicated that carp mitochondria are less susceptible to Triton X-100 than frog and rat mitochondria. 3. 3. Triton X-100 inhibited cytochrome oxidase activity in mitochondria and in mitochondrial inner membrane fraction under investigation.

Some aspects of the inhibition of cytochrome [formula omitted] oxidase by phosphine in susceptible and resistant strains of Rhyzopertha dominicia

(1) Phosphine (0.1 mM) when added to preparations of mitochondria from susceptible or resistant R. dominica inhibits the cytochrome c oxidase activity by up to 90%. (2) Cytochrome oxidase activity in mitochondria or crude homogenates of R. dominica of either strain which have been treated in vivo with phosphine shows little effect. (3) Possible mechanisms of resistance are described in the light of this and other evidence.

Changes in alternative oxidase activity and other mitochondrial parameters with callus formation by potato tuber tissue discs

Incubation of potato tuber tissus discs on agar with hormones (kinetin, KN and naphthyleneacetic acid, NAA) leads to callus induction which doubles the fresh weight in approx. 3 weeks. Changes during this incubation in some characteristics of mitochondrial isolated from this tissue have been compared with those isolated from tissue placed on agar without hormones. A considerable increase in mitochondrial state III respiration is found with both treatments. Antimycin-A resistant respiration (alternative oxidase activity) which is not detectable in freshly-sliced tissue, increases during the first days of incubation on the agar. It stabilises then at a level which is 3–4 times higher in callus-forming tissue than in tissue placed on agar without hormones. Cytochrome oxidase activity of mitochondria from callus forming tissue increases steadily during the first 3 weeks of callus induction. In mitochondria from tissue, not treated with hormones, cytochrome oxidase activity increases only during the first days and then stabilises. The increase in alternative oxidase activity (expressed as a percentage of state III respiration) in mitochondria from tissue placed on agar without hormones is greater when older tubers are used as a source for the potato tuber tissue. Finally, after approx. 10 months of storage of the tubers, this relative increase equals that of callus-forming tissue. Experiments with tubers during the first months after harvest indicate that the higher level of alternative oxidase in mitochondria from callus-forming tissue in this period, might be caused by the presence of the auxin in the agar.

Increases in Mitochondrial Steroidogenesis after Short Term Incubation of Porcine Ovarian Follicles with Luteinizing Hormone*

[4-14C]Cholesterol side chain cleavage, progesterone synthesis, and cytochrome oxidase activities were measured in mitochondria from unincubated and short term incubated large (8-10 mm) follicles isolated from porcine ovaries. Compared to the activity of mitochondria from unincubated follicles, specific [4-14C]cholesterol side chain cleavage activity in mitochondria from follicles incubated with LH (0.05 microgrogram/ml) did not change significantly after 12 h, but increased almost 2-fold after 18 h and 5-fold after 24 h. Also, specific mitochondrial progesterone synthesis activity increased dramatically after incubation of follicles for 24 h with LH. In comparison, mitochondria prepared from follicles incubated without LH showed no significant change in specific [4-14C]cholesterol side chain cleavage or progesterone synthesis activities after 18 h of incubation. While both of these activities increased after incubating follicles 24 h without LH, the values were significantly lower than those observed for preparations from follicles incubated with LH. In contrast to these changes in mitochondrial steroidogenesis, specific cytochrome oxidase activity in mitochondria did not change after incubation of follicles without or with LH. It is concluded that incubation of follicles with LH stimulates the development of mitochondrial steroidogenesis but initially does not affect some components of the respiratory chain.

Cytochemical analysis of organelle degradation in phagosomes and apoptotic cells of the mucoid epithelium of mice

The activity of mitochondrial cytochrome oxidase and peroxisomal catalase in the phagolysosomes and apoptotic bodies of mucoid epithelial cells was analysed. Tissue from 2-6 day old mice was used. The activity of acid phosphatase in lysosomes was also estimated. Cytochrome oxidase was demonstrated in well-preserved mitochondria inside phagosomes. Mitochondria in cells exhibiting apoptotic death also show activity of cytochrome oxidase. The enzyme activity in swollen mitochondria ceases before the membranes of the cristae disappear completely. Apoptotic bodies are phagocytosed by sister mucoid cells and, later on, they are digested inside the cell. Phagosomes which contain already degraded mitochondria show still active catalase in sequestered peroxisomes. The acid phosphatase involved in degradation of phagocytosed material originates from endocytosed lysosomes and primary and secondary lysosomes which fuse with the membranes of phagosomes.

Mitochondrial cytochrome content and cytochrome oxidase activity of some Amazonian fish

1. The cytochrome content of Keilin-Hartree particles isolated from water-breathing and facultative air-breathing fishes is reported and compared with data for mammalian mitochondria.2. Steady-state kinetic measurements were performed on the oxidase system. The results may be interpreted in terms of two, kinetically significant cytochromec binding sites associated with cytochrome oxidase withKm values of ∼ 1 × 10−6 and ∼5 × 10−8 M. These values are close to those reported for beef mitochondria (Ferguson-Milleret al., 1976).3. The turnover numbers for the cytochrome oxidases from a variety of fishes are compared, and are found to be similar and close to the value found for the mammalian enzyme.4. The constancy of the cytochrome content and the kinetic parameters from species to species suggests that the cellular respiratory processes constitute a conservative system from the standpoint of evolution.

INHIBITION OF MITOCHONDRIAL RESPIRATION BY SODIUM NITROPRUSSIDE AND THE MECHANISM OF CYANIDE LIBERATION

The influence of sodium nitroprusside (SNP) on mitochondrial respiration was examined in rat liver mitochondria. The addition of SNP 1 mmol litre-1 during state 3 respiration inhibited the oxygen uptake by 63.4%. A mixture of SNP 1 mmol litre-1 and glutathione (GSH) 1 mmol litre-1 inhibited the oxygen uptake more markedly (by 75.9%). The cyanide concentrations were 0.01 mmol litre-1 with SNP alone and 0.15 mmol litre-1 with the mixture of SNP and GSH. Cyanide production from SNP in the presence of various reducing agents was studied in potassium phosphate 0.1 mol litre-1 buffer solution (pH 7.4) incubated at 37 degrees C. Cyanide was liberated markedly from SNP in the presence of GSH or ascorbate. Less cyanide was produced in the presence of NADH or NADPH. The rate of production of cyanide was dependent entirely upon the concentration of each reducing agent added. No cyanide was liberated when sodium dithionite or the oxidized forms of GSH, NAD or NADP were used. It was concluded that SNP is degradated to cyanide by a hydrogen donor and that the cyanide liberated in this manner inhibits the cytochrome oxidase activity of mitochondria in vivo.

Cytochrome oxidase deficiency during development of amphibian nucleocytoplasmic hybrids

The activity of mitochondrial cytochrome oxidase was investigated during the embryonic development of nucleocytoplasmic hybrids containing a nuclear genome derived from R. pipiens and a mitochondrial genome derived from R. palustris. Using a quantitative cytochemical approach, we found that the activity of cytochrome oxidase failed to increase during the development of these embryos. Control embryos containing a haploid chromosomal complement, derived from the same species as that from which the maternally inherited mitochondria is derived and hybrid crosses between R. palustris and R. pipiens, showed a significant increase in cytochrome oxidase activity during development. Oxygen uptake data from diploid and haploid R. pipiens embryos were in agreement with the data obtained by the cytochemical method. These results indicate that a normal pattern of cytochrome oxidase activity during embryonic development requires a nuclear genome which contains a haploid chromosomal complement derived from the same species as that from which the mitochondrial genome is derived.

CYTOCHROMES IN BRAIN MITOCHONDRIA FROM LAMBS WITH ENZOOTIC ATAXIA

AbstractMitochondria from the cerebral cortex of untreated lambs from areas in which enzootic ataxia occurred showed a lower content of haem a (from cytochromes a and a 3) than mitochondria from control lambs with adequate copper but the haems of cytochromcs b2 c1 and c were not depleted. Cytochrome oxidase activity was closely correlated with the content of haem a (P<0.001) but there was no correlation with total mitochondrial copper which was always present in molar exccss of haem a. It is concluded that the lowered cytochrome oxidase activity in mitochondria from untreated lambs is immediately attributable to depletion of haem a. In clinically ataxic lambs that showed degradation of myelin in thc spinal cord, brain cytochrome oxidase was depressed by no more t han 60%. Arguments are advanced that this depletion was not sufficiently severe to have led to respiratory constraint

Functional change in hepatic mitochondria of swine fed ethylchlorophenoxyisobutyrate

Hepatic mitochondria of swine fed ethylchlorophenoxyisobutyrate (CPIB) at a level of 0.3% (w/w) for 8 weeks showed partial uncoupling of oxidative phosphorylation and increased state 4 QO 2 values. Both cytochrome oxidase and the calculated theoretical mitochondrial protein were increased per gram of total liver. Whether related or not these changes were accompanied by the appearance of tubular spiral structures within the mitochondria. Swine fed CPIB for 4 weeks showed similar but fewer functional and morphologic changes. Feeding the drug 1 week produced no abnormalities of oxidative phosphorylation or QO 2 values, and no morphologic changes were present. However, at this time calculated theoretical mitochondrial protein, cytochrome oxidase, and monoamine oxidase activities were already increased per unit of liver weight.

Mitochondrial cytochrome oxidase and monoamine oxidase activities in the developing chick cerebral hemispheres.

Mitochondrial cytochrome oxidase and monoamine oxidase activities in the developing chick cerebral hemispheres.

Mitochondrial proliferation during neonatal renal compensatory growth

Unilateral nephrectomy is associated with an increase in kidney mass in the contralateral kidney of young rabbits, progressively increasing from 8% at 1 day post-nephrectomy to 35% at 4 days post-nephrectomy. There is a preferential early increase in specific activity of mitochondrial cytochrome oxidase compared to monamine oxidase or malate dehydrogenase. Compensatory hypertrophy also stimulates quite early incorporation of radioactivity into mitochondrial inner membrane from 3H-leucine and 14C-glycerol.

Effects of transitory maternal pteroylglutamic acid (PGA) deficiency on respiratory control ratios, phosphorylative capacity, and cytochrome oxidase activities of mitochondria from rat embryos.

AbstractCytochrome oxidase activities and protein contents of homogenates and mitochondrial fractions of embryos from untreated and 9‐methyl PGA treated pregnant rats were determined on various days of gestation. Rates of respiration, ADP:O, and respiratory control ratios were determined with the mitochondrial fractions. The cytochrome oxidase specific activities of homogenates or mitochondrial fractions of treated embryos were similar to those of controls of the same age. However, total mitochondrial protein and cytochrome oxidase activities of the treated embryos were similar to those of controls even though the treated embryos were smaller than controls. The amount of oxygen consumed by the treated embryos was not significantly different from that consumed by controls and the ADP:O and respiratory control ratios calculated for mitochondrial fractions of treated and control embryos were not significantly different.

Inhibition of mitochondrial electron transport by nor-dihydroguaiaretic acid (NDGA)

The effects of various anti-oxidants on the activities of the mitochondrial NADH-oxidase and succinoxidase systems were determined. It was concluded that nor-dihydroguaiaretic acid (2,3-dimethyl-1, 4-di [3,4-dihydroxyphenyl] butane) (NDGA) was a potent inhibitor of both mitochondrial electron transport systems. NDGA did not inhibit mitochondrial cytochrome oxidase activity or the coenzyme Q-cytochrome c reductase system. The probable sites of inhibition of mitochondrial electron transport are complexes I and II (NADH-coenzyme Q reductase and succinate-coenzyme Q reductase systems).

Respiration of the Spadix From the Aroid Zantedeschia Aethiopica Spreng.

Spadix tissue of a number of aroid species has been shown to be an outstanding exception to the general pattern of plant respiration. In the work reported here the respiration of whole tissue and mitochondria from the spadix of another aroid species Zantedeschia aethiopica Spreng. was studied. The respiration of both whole tissue and mitochondria was found to be strongly inhibited by the metal-complexing agents cyanide and azide. Cytochrome oxidase and cytochromes band c were spectroscopically indentified in mitochondrial suspensions and cytochrome oxidase activity was demonstrated in extracts. The results indicate that the respiratory characteristics of the Zantedeschia spadix tissue are similar to those of most other plant tissues. Such characteristics are, however, in marked contrast to the respiratory pattern reported for other aroid spadices.

Cytochrome oxidase activity in mitochondria during amphibian development

A study has been made of the development of cytochrome oxidase in homogenates and isolated mitochondria of the embryos of Xenopus laevis. Cytochrome oxidase activity increases in the embryo throughout the prefeeding period of development. At all stages tested, the enzyme appears to be localized in the mitochondrial fraction of embryo homogenates. During development, the specific activity of the mitochondrial fraction (on a protein nitrogen base) shows a substantial increase.

Enzyme inhibition by derivatives of phenothiazine. IV. Inhibition of succinoxidases activity of rat-liver mitochondria.

The succinoxidase activity of rat-liver mitochondria was strongly inhibited by the following compounds (concentration for 50% reduction in rate of oxygen uptake is given in brackets): phenothiazine (1.4 × 10−5 M), phenothiazine sulphoxide (2.8 × 10−5 M), and phenothiazone (5.4 × 10−5 M). Thionol was only slightly inhibitory. The cytochrome oxidase activity of mitochondria was not inhibited by any of these compounds.