Characterization of the respiratory activity of mitochondria isolated from an insect cell line CP-1268 Laspeyresia pomonella

Mitochondria have been isolated from the codling moth Laspeyresia pomonella, CP-1268 cell line. The mitochondrial fraction was isolated from pooled 4 d, exponential growth phase, cultures. The mitochondria were determined to be intact based on the demonstration of respiratory control, the effects of 2,4 dinitrophenol and oligomycin on respiration, the inability to oxidize NADH, and the inability of cytochrome c to enhance respiration. The isolated mitochondria were able to oxidize succinate, pyruvate, malate, alpha-ketoglutarate, and alpha-glycerophosphate efficiently. Of the substrates tested, the CP-1268 mitochondria oxidized succinate most efficiently. The respiratory control ratios ranged from a high of 4.6 for pyruvate to a low of 1.7 with alpha-glycerophosphate. These findings confirm that the mitochondria were tightly coupled. The data also confirm the presence of three sites of oxidative phosphorylation because NAD-linked substrates had ADP-to-O ratios approaching 3 and flavoprotein linked substrates had values approaching 2.     

Characterization of the respiratory activity of mitochondria isolated from an insect cell line CP-1268Laspeyresia pomonella

Summary Mitochondria have been isolated from the codling mothLaspeyresia pomonella, CP-1268 cell line. The mitochondrial fraction was isolated from pooled 4 d, exponential growth phase, cultures. The mitochondria were determined to be intact based on the demonstration of respiratory control, the effects of 2,4 dinitrophenol and oligomycin on respiration, the inability to oxidize NADH, and the inability of cytochromec to enhance respiration. The isolated mitochondria were able to oxidize succinate, pyruvate, malate, α-ketoglutarate, and α-glycerophosphate efficiently. Of the substrates tested, the CP-1268 mitochondria oxidized succinate most efficiently. The respiratory control ratios ranged from a high of 4.6 for pyruvate to a low of 1.7 with α-glycerophosphate. These findings confirm that the mitochondria were tightly coupled. The data also confirm the presence of three sites of oxidative phosphorylation because NAD-linked substrates had ADP-to-O ratios approaching 3 and flavoprotein linked substrates had values approaching 2.     

Oxidative phosphorylation and ATPase activities of human tumor mitochondria

Studies were carried out with intact mitochondria isolated from human astrocytoma, oat cell carcinoma and melanoma which were propagated in athymic mice. These human tumor mitochondria were capable of coupled oxidative phosphorylation. They also showed significant uncoupler-stimulated ATPase if defatted bovine serum albumin was included in the assay media. However, the uncoupler response curves were different and the magnitude of the ATPase activity was lower than could be obtained with mitochondria of a normal tissue, such as liver. Some of these characteristics were also exhibited by mitochondria from several animal hepatomas and Ehrlich ascites tumor. In the three tumors studied, mitochondria from oat cell carcinoma were more labile, whereas higher respiratory control ratios and greater stimulation of ATPase by uncouplers were obtained with melanoma mitochondria.The mitochondrial ATPase was not the major cellular ATPase in any of the three tumors. This was indicated by a low inhibition of the ATPase activity of tumor cell homogenates by oligomycin. A very large fraction of the cellular ATPase activities was recovered in the microsomal fractions.     

Comparative Studies with Three Antibiotics Binding to Deoxyribonucleic Acid

The effects of the three antibiotics U-12,241, nogalamycin, and U-20,661 on (i) deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis in KB cell cultures and cell-free systems of bacterial and mammalian origin and on (ii) oxidative phosphorylation in rat liver mitochondria were compared. Nogalamycin and U-12,241 inhibited RNA synthesis more strongly than DNA synthesis in all test systems. Antibiotic U-20,661 inhibited DNA and RNA synthesis equally in whole mammalian cells and their corresponding cell-free systems. The RNA polymerase from Escherichia coli, however, was at least 100 times more sensitive to U-20,661 than was the DNA polymerase. U-12,241 caused significant uncoupling of oxidative phosphorylation in mitochondria.     

OXIDATIVE PHOSPHORYLATION AND ULTRASTRUCTURAL TRANSFORMATION IN MITOCHONDRIA IN THE INTACT ASCITES TUMOR CELL

We have examined the ultrastructure of mitochondria as it relates to energy metabolism in the intact cell. Oxidative phosphorylation was induced in ultrastructurally intact Ehrlich ascites tumor cells by rapidly generating intracellular adenosine diphosphate from endogenous adenosine triphosphate by the addition of 2-deoxyglucose. The occurrence of oxidative phosphorylation was ascertained indirectly by continuous and synchronous monitoring of respiratory rate, fluorescence of pyridine nucleotide, and 90° light-scattering. Oxidative phosphorylation was confirmed by direct enzymatic analysis of intracellular adenine nucleotides and by determination of intracellular inorganic orthophosphate. Microsamples of cells rapidly fixed for electron microscopy revealed that, in addition to oxidative phosphorylation, an orthodox → condensed ultrastructural transformation occurred in the mitochondria of all cells in less than 6 sec after the generation of adenosine diphosphate by 2-deoxyglucose. A 90° light-scattering increase, which also occurs at this time, showed a t ½ of only 25 sec which agreed temporally with a slower orthodox → maximally condensed mitochondrial transformation. Neither oxidative phosphorylation nor ultrastructural transformation could be initiated in mitochondria in intact cells by the intracellular generation of adenosine diphosphate in the presence of uncouplers of oxidative phosphorylation. Partial and complete inhibition of oxidative phosphorylation by oligomycin resulted in a positive relationship to partial and complete inhibition of 2-deoxyglucose-induced ultrastructural transformation in the mitochondria in these cells. The data presented reveal that an orthodox → condensed ultrastructural transformation is linked to induced oxidative phosphorylation in mitochondria in the intact ascites tumor cell.     

Specific inhibition of phosphate transport in mitochondria by N-ethylmaleimide

N-ethylmaleimide (NEM), a reagent that alkylates free sulfhydryl groups, was shown to be a highly effective inhibitor of the following coupled mitochondrial processes: oxidative phosphorylation, ATP-³²Pi exchange, Pi-induced light scattering and configurational changes, State III respiration, valinomycin-induced translocation of potassium with Pi as the anion, and calcium accumulation in presence of Pi. However, NEM was less effective or ineffective in inhibiting some processes that do not require inorganic Pi, namely electron transfer and ATPase activity, ADP binding, energized light scattering changes induced by arsenate and nonenergized light scattering changes induced by acetate. The rate of oxidative phosphorylation and of ATP-³²Pi exchange was normal in ETP_H particles prepared from NEM-treated mitochondria. Also NEM, even et levels 2–3 times greater than those required to inhibit oxidative phosphorylation in intact mitochondria, did not inhibit coupled processes in submitochondrial particles. We are proposing that NEM alkylates sulfhydryl groups in the mitochondrion that modulate Pi translocation, and that the suppression of Pi translocation blocks oxidative phosphorylation, the Pi-dependent energized configurational change in mitochondria and Pi-dependent transport processes.On leave of absence from the Department of Biochemistry, Cancer Institute Okayama University Medical School, Okayama, Japan.On leave of absence from the Department of Pathology, Nagoya University Medical School, Nagoya, Japan.     

Rapid isolation of muscle and heart mitochondria, the lability of oxidative phosphorylation and attempts to stabilize the process in vitro by taurine, carnitine and other compounds

We modified the isolation procedure of muscle and heart mitochondria. In human muscle, this resulted in a 3.4 fold higher yield of better coupled mitochondria in half the isolation time. In a preparation from rat muscle we studied factors that affected the stability of oxidative phosphorylation (oxphos) and found that it decreased by shaking the preparation on a Vortex machine, by exposure to light and by an increase in storage temperature. The decay was found to be different for each substrate tested. The oxidation of ascorbate was most stable and less sensitive to the treatments.When mitochondria were stored in the dark and the cold, the decrease in oxidative phosphorylation followed first order kinetics. In individual preparations of muscle and heart mitochondria, protection of oxidative phosphorylation was found by adding candidate stabilizers, such as desferrioxamine, lazaroids, taurine, carnitine, phosphocreatine, N-acetylcysteine, Trolox-C and ruthenium red, implying a role for reactive oxygen species and calcium-ions in the in vitro damage at low temperature to oxidative phosphorylation.In heart mitochondria oxphos with pyruvate and palmitoylcarnitine was most labile followed by glutamate, succinate and ascorbate.We studied the effect of taurine, hypotaurine, carnitine, and desferrioxamine on the decay of oxphos with these substrates. 1 mM taurine (n = 6) caused a significant protection of oxphos with pyruvate, glutamate and palmitoylcarnitine, but not with the other substrates. 5 mM L-carnitine (n = 6), 1 mM hypotaurine (n = 3) and 0.1 mM desferrioxamine (n = 3) did not protect oxphos with any of the substrates at a significant level.These experiments were undertaken in the hope that the in vitro stabilizers can be used in future treatment of patients with defects in oxidative phosphorylation. (Mol Cell Biochem 174: 61–66, 1997)     

Iron uncouples oxidative phosphorylation in brain mitochondria isolated from vitamin E-deficient rats

Few, if any, studies have examined the effect of vitamin E deficiency on brain mitochondrial oxidative phosphorylation. The latter was studied using brain mitochondria isolated from control and vitamin E-deficient rats (13 months of deficiency) after exposure to iron, an inducer of oxidative stress. Mitochondria were treated with iron (2 to 50 microM) added as ferrous ammonium sulfate. Rates of state 3 and state 4 respiration, respiratory control ratios, and ADP/O ratios were not affected by vitamin E deficiency alone. However, iron uncoupled oxidative phosphorylation in vitamin E-deficient mitochondria, but not in controls. In vitamin E-deficient mitochondria, iron decreased ADP/O ratios and markedly stimulated state 4 respiration; iron had only a modest effect on these parameters in control mitochondria. Thus, vitamin E may have an important role in sustaining oxidative phosphorylation. Low concentrations of iron (2 to 5 microM) oxidized mitochondrial tocopherol that exists in two pools. The release of iron in brain may impair oxidative phosphorylation, which would be exacerbated by vitamin E deficiency. The results are important for understanding the pathogenesis of human brain disorders known to be associated with abnormalities in mitochondrial function as well as iron homeostasis (e.g., Parkinson's disease).     

A 31P-NMR study of bovine adrenocortical mitochondrial metabolic activities

High-field 31P-NMR spectroscopy has been used to study the metabolic activities of coupled bovine adrenocortical mitochondria in vitro. These differentiated organelles use oxygen as a substrate to support both oxidative phosphorylation and specific steroid hydroxylation reactions. The NMR technique allowed the resolution of two inorganic phosphate signals, attributed to the matrix and external medium phosphate pools, at low and high field, respectively. These signals were used to calculate the respective Pi concentrations and to obtain the pH of the two corresponding compartments. In addition, the NMR spectra displayed resonance signals corresponding to ADP added to the medium and to ATP synthesized during oxidative phosphorylation. NMR analysis of the mitochondrial perchloric acid extracts identified the major phosphate-containing metabolites, namely NADP+, NAD+, phosphocholine, phosphoethanolamine, sn-glycero-(3)phosphocholine, AMP, ADP, ATP and Pi. Upon addition of ADP and malate to the oxygenated suspension, the kinetics of mitochondrial external Pi consumption and of ATP synthesis, along with the intra- and extraorganelle pH variations could be monitored over time periods of approximately 30 min, in the absence and presence of different steroid hydroxylation substrates. A major observation was that oxidative phosphorylation, which takes place in the absence of steroid, was markedly inhibited as soon as steroid hydroxylation was operating. These observations show the potential of 31P-NMR spectroscopy in the study of metabolic activities of isolated intact mitochondrial organelles. Such an approach appears promising for further determination of the underlying mechanisms in the balance between vital oxidative phosphorylation and differentiated steroid hydroxylation which are under hormonal control in adrenocortical mitochondria as well as in other steroidogenic cell systems.     

Isolation and some properties of mitochondria from yeast Candida lipolytica 695]

Mitochondria is obtained from yeast Candida lipolytica 695 grown in the presence of glucose, lactate or citrate. Yeast mitochondria were shown to be practically indistinguishable from animal tissue mitochondria in [ADP]/[O] values and in their sensitivity to electron transport inhibitors, to inhibitors and uncoupling agents of oxidative phosphorylation. The only exception was more low value of the respiration control under succinate oxidation. Mitochondria from yeast, grown in the presence of lactate or citrate were capable of the reduction of endogenous pyridine nucleotides under succinate oxidation for the expense of the reverse electron transport. No reverse electron transport from succinate to NAD(P) was observed in mitochondria from yeast grown in the presence of glucose, but it was found under oxidation of alpha-glycerophosphate. All three types of yeast mitochondria were not capable of the reverse electron transport coupled with the pyridine nucleotides reduction under lactate oxidation.     

Bioenergetics of Yarrowia lipolytica cells grown at alkaline conditions

Energy status of the novel alkalitolerant Yarrowia lipolytica yeast strain grown at alkaline conditions (pH 9.7) was examined. Cells grown under such severe conditions were found to preserve high respiratory activity. The oxidative phosphorylation system dominated in the energy budget of the cell. A procedure was specially design to isolate tightly coupled mitochondria from yeast cells grown at alkaline conditions. The isolated mitochondrial preparations met known criteria of physiological intactness, as inferred from their ability to maintain distinctive state 4-3 respiration transition upon addition of ADP, high respiratory rates, good respiratory control values, and ADP/O ratios close to the theoretically expected maxima for the substrates used.     

Mitochondrial function in glucocorticoid triggered T-ALL cells with transgenic bcl-2 expression

Independent of apoptosis, dexamethasone induced and a decrease of respiration and citrate synthase activity per cell in cells with and without transgenic Bcl-2 expression. The reduction of respiration, however, was slightly, but statistically more pronounced in apoptotic cells compared to non-apoptotic Bcl-2 over-expressing cells. A slight cytochrome c release was detected in apoptotic cells only. Importantly, the stimulatory effect of FCCP was maintained, indicating that oxidative phosphorylation remained coupled in active mitochondria. Coupled and uncoupled respiration were reduced to almost identical degrees as the activities of the marker enzymes citrate synthase (matrix) and cytochrome c oxidase (respiratory chain). Therefore, the reduction of cellular respiration was mainly caused by a decrease in mitochondrial content per cell. The functional integrity of mitochondria was preserved, apart from the slight degree of cytochrome c release, either through a pore formed by the oligomerisation of BAK in coupled mitochondria or by permeability transition of a small fraction of injured mitochondria.     

Pyruvate modifies glycolytic and oxidative metabolism of rat embryonic spinal cord astrocyte cell lines and prevents their spontaneous transformation

This study aimed to provide detailed data on mitochondrial respiration of normal astrocyte cell lines derived from rat embryonic spinal cord. Astrocytes in early passages (EP), cultured without pyruvate for more than 35 passages, defined here as late passages (LP), undergo spontaneous transformation. To study initial steps in cell transformation, EP data were compared with those of LP cells. LP cells had reduced glycolysis, fewer mitochondria and extremely low oxidative rates, resulting from a dysfunction of complexes I and II + III of the respiratory chain. Treatment of EP cells with pyruvate until they were, by definition, LP cultures prevented transformation of these cells. Pyruvate-treated EP cells had more mitochondria than normal cells but slightly lower respiratory rates. The increase of mitochondrial content thus appears to act as a compensatory effect to maintain oxidative phosphorylation in these LP 'non-transformed' cells, in which mitochondrial function is reduced. However, pyruvate treatment of transformed LP cells during additional passages did not significantly restore their oxidative metabolism. These data highlight changes accompanying spontaneous astrocyte transformation and suggest potential targets for the control of astrocyte proliferation and reaction to various insults to the central nervous system.     

Effect of bovine serum albumin on membrane potential in plant mitochondria

The membrane potential in highly coupled potato ( Solanum tuberosum L.) mitochondria, as measured by changes in safranine absorbance, was significantly increased by addition of bovine serum albumin. Purification of potato mitochondria on Percoll, in removing 50% of free unsaturated fatty acids, decreased the BSA-de-pendent membrane potential. The effect of added linoleic acid and of the natural accumulation of fatty acids during aging was studied. The response of membrane potential to addition of bovine serum albumin appeared to be directly correlated to the amount of free unsaturated fatty acids. Aging in vitro, in releasing free fatty acids, decreased respiratory control and ADP:O ratios and collapsed the membrane potential. During 2–3 h of incubation, addition of BSA completely restored membrane potential and oxidative phosphorylation.
It is concluded that both in fresh and in aged potato mitochondria the effect of bovine serum albumin on oxidative phosphorylation can be ascribed to an effect on membrane permeability to ions. BSA, in binding free unsaturated fatty acids, restored maximal membrane potential. The bovine serum albumin-dependent membrane potential appears to be a sensitive criterion of the functional integrity of the inner mitochondrial membrane.     

Genetic determination of the mitochondrial adenine nucleotide translocation system and ITS role in the eukaryotic cell

On integrating experimental data published previously, the following picture of the mitochondrial adenine nucleotide (AdN) translocation system is being presented: 1. The AdN translocation system serves not only to transport ATP synthesized within mitochondria into the cytosol but also to transport cytosolic ATP into the mitochondria when oxidative phosphorylation is not functioning. 2. The AdN translocator is coded for by nuclear genes and the mitochondrial protein synthesis is not involved in its formation. 3. The AdN translocation system must be preserved and functioning even in cells which could dispense with oxidative phosphorylation. It assures appropriate concentrations of intramitochondrial ATP. 4. The intramitochondrial ATP is required for normal replication of mitochondrial DNA. Tis supports the view that the mitochondrion is a self-replicating semi-autonomous organelle. 5. The appropriate concentration of ATP must be present in mitochondria to make possible cell growth or multiplication. This points to a direct or indirect role of mitochondria in the control of cell proliferation.     

On the Mechanism of Respiratory Control

Control of oxidation is the key mechanism in the regulation of energy metabolism. In glycolysis the oxidation of glyceraldehyde-3-phosphate is controlled by DPNH, which inhibits glyceraldehyde-3-phosphate dehydrogenase. In oxidative phosphorylation the inhibition of electron flow from DPNH to oxygen, called "respiratory control," is the subject of this paper. After a discussion of the physiological significance of the "tight coupling" between phosphorylation and oxidation, studies on "loosely coupled" submitochondrial particles are reported. These particles are capable of oxidative phosphorylation in the presence of a suitable phosphate acceptor system, but in contrast to controlled, intact mitochondria they oxidize DPNH in the absence of phosphate and ADP. The addition of o-phenanthroline to submitochondrial particles gives rise to an inhibition of respiration, which is partly reversed by phosphate and ADP or by dinitrophenol. The properties of this model system of respiratory control will be described.     

Differential effects of N-acetylglutamate on citrulline synthesis by coupled and uncoupled mitochondria

When rats were placed on a low-protein (5%) diet for 24 h or less, liver mitochondrial acetylglutamate decreased rapidly, carbamyl phosphate synthetase (ammonia) and ornithine transcarbamylase decreased little, and carbamyl phosphate synthesis (measured as citrulline) by isolated mitochondria occurred at very low rates. The matrix acetylglutamate content of these mitochondria, whether coupled or uncoupled, was increased similarly by preincubating them with added acetylglutamate, but citrulline synthesis increased from less than 1 to 2.3 nmol min-1 mg-1 in the coupled state, and from less than 1 to 35 nmol min-1 mg-1 in the uncoupled state. However, when coupled mitochondria were incubated with the substrates required for the synthesis of acetylglutamate in the matrix, citrulline synthesis increased to 48 nmol min-1 mg-1; this rate was similar to that of mitochondria from control rats (fed a normal diet). When mitochondria from controls were incubated with up to 5mM acetylglutamate, citrulline synthesis by coupled mitochondria was increased by 10 to 40%, while synthesis by uncoupled mitochondria was 1.5 to 4 times higher than that observed with the coupled mitochondria; matrix acetylglutamate in both conditions rose to levels similar to those in the medium. The reason for the different behavior of carbamyl phosphate synthetase (ammonia) in coupled and uncoupled mitochondria was not apparent; neither oxidative phosphorylation nor ornithine transport were limiting in the coupled system. These observations are an example of the restrictions imposed upon enzymatic systems by the conditions existing in the mitochondrial matrix, and of the different behavior of carbamyl phosphate synthetase in situ and in solution. In addition, they show that conclusions about the characteristics of the enzyme in coupled mitochondria based on observations made in uncoupled mitochondria are not necessarily justified.     

An animal model to study human muscular diseases involving mitochondrial oxidative phosphorylation

Mitochondria are producing most of the energy needed for many cellular functions by a process named oxidative phosphorylation (OXPHOS). It is now well recognized that mitochondrial dysfunctions are involved in several pathologies or degenerative processes, including cardiovascular diseases, diabetes, and aging. Animal models are currently used to try to understand the role of mitochondria in human diseases but a major problem is that mitochondria from different species and tissues are variable in terms of regulation. Analysis of mitochondrial function in three species of planarian flatworms (Tricladia, Platyhelminthes) shows that they share a very rare characteristic with human mitochondria: a strong control of oxidative phosphorylation by the phosphorylation system. The ratio of coupled OXPHOS over maximal electron transport capacity after uncoupling (electron transport system; ETS) well below 1.0 indicates that the phosphorylation system is limiting the rate of OXPHOS. The OXPHOS/ETS ratios are 0.62?±?0.06 in Dugesia tigrina, 0.63?±?0.05 in D. dorotocephala and 0.62?±?0.05 in Procotyla fluviatilis, comparable to the value measured in human muscles. To our knowledge, no other animal model displays this peculiarity. This new model offers a venue in which to test the phosphorylation system as a potential therapeutic control point within humans.     

Effect of insulin on oxidative phosphorylation in the liver and heart mitochondria of adrenalectomized rats

The effect of insulin was studied as applied to the inhibited under conditions of adrenalectomy process of oxidative phosphorylation in the rat liver and heart mitochondria. It is established that adrenalectomy does not change oxidative activity of mitochondria but inhibits the process of phosphorylation, which results in the decreased values of the ADP/O coefficient and the respiratory control. Insulin administered to the adrenalectomized rats 3h before the experiments reduces the disturbed oxidative phosphorylation in mitochondria of the liver and heart by intensifying the degree of ADP phosphorylation.     

Bioenergetics of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> Cells Grown at Alkaline Conditions

Energy status of the novel alkalitolerant Yarrowia lipolytica yeast strain grown at alkaline conditions (pH 9.7) was examined. Cells grown under such severe conditions were found to preserve high respiratory activity. The oxidative phosphorylation system dominated in the energy budget of the cell. A procedure was specially design to isolate tightly coupled mitochondria from yeast cells grown at alkaline conditions. The isolated mitochondrial preparations met known criteria of physiological intactness, as inferred from their ability to maintain distinctive state 4–3 respiration transition upon addition of ADP, high respiratory rates, good respiratory control values, and ADP/O ratios close to the theoretically expected maxima for the substrates used.