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Effect of tonicity of the medium on the respiratory and phosphorylative activity of heart-muscle sarcosomes

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... ntration of sarcosine, an important osmolyte in skate tissues (Boyd, Cha, Forster & Goldstein, 1977) during osmotic stress. This study examines the mechanism which may be responsible for the osmotic sensitivity of sarcosine oxidation in skate liver mitochondria. Hyperosmotically induced inhibition of mitochondrial respiration is well estab- lished. Slater & Cleland (1953) and Witter, Watson & Cottone (1955) reported a reduction in oxygen consumption by rat mitochondria incubated in high concentrations of sucrose. Osmotic sensitivity of metabolite oxidation has been demonstrated in mitochondria from mammals (Atsmon & Davis, 1967; Joseph, McGivan & Meijer, 1981), elasmobranchs (Lewiston, Newman, Robin & Ho ...
... Hyperosmotically induced inhibition of mitochondrial respiration is well established. Slater & Cleland (1953) and Witter, Watson & Cottone (1955) reported a reduction in oxygen consumption by rat mitochondria incubated in high concentrations of sucrose. Osmotic sensitivity of metabolite oxidation has been demonstrated in mitochondria from mammals (Atsmon & Davis, 1967;Joseph, McGivan & Meijer, 1981), elasmobranchs (Lewiston, Newman, Robin & Holtzman, 1979;) and bivalve molluscs (Ballantyne & Storey, 1983, 1984Ballantyne & Moon, 1985;Ellis, Burcham, Paynter & Bishop, 1985). ...
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The mechanism responsible for the osmotic sensitivity of sarcosine oxidation by liver mitochondria from the little skate, Raja erinacea Mitchill, is examined. Assay medium tonicity, rather than a solute effect (urea or trimethylamine oxide), is probably responsible for the inverse relationship between osmolarity and the rate of oxidation of sarcosine by these mitochondria. Sarcosine oxidation proceeds through the flavin-linked sarcosine oxidase with the resultant glycine catabolized in the NAD-linked glycine cleavage system. The tonicity-sensitive component of the sarcosine oxidative pathway is not the glycine cleavage system. Sarcosine oxidation in the presence of rotenone is sensitive to medium tonicity. Oxidation of serine, which is also catabolized through the glycine cleavage system, is not as sensitive to tonicity as is sarcosine oxidation. Mitochondrial volume changes also appear to affect the transport of glycine. Although sarcosine does not appear to share the glycine transporter, it is possible that sarcosine transport is similarly sensitive to medium tonicity. The effects of osmolarity on the oxidation of dimethylglycine appear to support this hypothesis. Tonicity effects on sarcosine oxidase cannot yet be eliminated.
... Nevertheless, both the magnitude of the hyperosmotic thermal response and its critical dependence on oxygen (Fig. 4) are consistent with the possibility that hyperosmolality uncouples oxidative phosphorylation by short-circuiting the flow of protons through the F0-F1 ATP synthase. Studies of isolated mitochondria, however, convincingly demonstrate that hyperosmolality inhibits oxidative phosphorylation (Slater and Cleland, 1953;Nicholls et al., 1972), probably by inhibition of adenine nucleotide translocase (Chfivez et al., 1987). In fact, under isosmotic conditions, increased rates of oxidative phosphorylation are associated with an increase of mitochondrial volume (Halestrap, 1989) whereas mitochondria shrink in hypertonic media (Sperelakis and Rubio, 1971). ...
Article
We have measured the rate of heat production of isolated, quiescent, right ventricular trabeculae of the rat under isosmotic and hyperosmotic conditions, using a microcalorimetric technique. In parallel experiments, we measured force production and intracellular calcium concentration ([Ca2+]i). The rate of resting heat production under isosmotic conditions (mean +/- SEM, n = 32) was 100 +/- 7 mW (g dry wt)-1; it increased sigmoidally with osmolality, reaching a peak that was about four times the isosmotic value at about twice normal osmotic pressure. The hyperosmotic thermal response was: (a) abolished by anoxia, (b) attenuated by procaine, (c) insensitive to verapamil, ouabain, and external calcium concentration, and (d) absent in chemically skinned trabeculae bathed in low-Ca2+ "relaxing solution." Active force production was inhibited at all osmolalities above isosmotic. Passive (tonic) force increased to, at most, 15% of the peak active force developed under isosmotic conditions while [Ca2+]i increased, at most, 30% above its isosmotic value. We infer that hyperosmotic stimulation of resting cardiac heat production reflects, in large part, greatly increased activity of the sarcoplasmic reticular Ca2+ ATPase in the face of increased efflux via a procaine-inhibitable Ca(2+)-release channel.
... These, then were the circumstances that led me to remark at a symposium in 1953: ".. in complex biochemical systems, such as those carrying out oxidative phosphorylation (e.g. Slater & Cleland, 1953) the osmotic and enzymic specificities appear to be equally important and may be practically synonymous" (Mitchell, 1954). The general idea that I had in mind in the mid-nineteenfifties, illustrated by the hypothetical system for phosphoryl translocation reproduced in Fig. 7, was that of substrate-specific conformationally mobile enzyme and catalytic carrier systems catalysing the translocation, not only of solutes, but also of chemical groups. ...
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Abstract— The effects of altered osmolarity on respiration and fine structure were compared in isolated rat cerebral versus liver mitochondria. Polarographic study of cerebral mitochondria in hypo-osmolar media showed inhibition of State 3 (ADP-dependent) respiration which was not reversed by dinitrophenol. In hyperosmolar media, State 3 respiration was transiently inhibited and State 4 (ADP-independent) respiration increased with the NAD-linked substrate pair, glutamate and malate. With succinate as substrate, respiration was not affected by moderate hyperosmolarity. In the most hyperosmolar medium, State 3 respiration was inhibited with both substrates. In contrast to the results with cerebral mitochondria, State 4 respiration was increased in hypo-osmolar media and State 3 respiration was persistently inhibited in hyperosmolar media in liver mitochondria with both substrates. In both cerebral and liver mitochondria, cytochrome c oxidase (EC 1.9.3.1.) activity was mildly inhibited in hypo-osmolar media and increased in hyperosmolar media. Electron microscopy showed that liver mitochondria were swollen in hypo-osmolar media and condensed in hyperosmolar media. Cerebral mitochondria showed mild rarefaction in hypo-osmolar media and, in hyperosmolar media, more than half the mitochondria showed either no or minimal changes in fine structure. Our results suggest that there are differences in metabolic control and structure between mitochondria from different cell types, which may be important in the cellular metabolic response to pathologic changes in water or osmolarity.
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This chapter discusses a new technique of differential centrifugation in the study of cellular organization and the theoretical basis of the technique as well as the various factors of a practical nature. The limitations of differential centrifugation become particularly severe when the technique is applied to the study of tissue enzymes. The observed partitions provide only the roughest sort of information concerning the true intracellular distribution of enzymes. They can only be considered as clues, which have to be followed by many additional experiments to arrive at their real significance. A priori assumption that specific enzymes are entirely concentrated in a given cellular site has proved extremely profitable in guiding these additional experiments, even in those cases in which it is found not to hold true. The use of enzyme determinations to ascertain the composition of isolated fractions is also of great interest and deserves more frequent application. The main object of cellular physiology is to ascertain which of the many processes discovered by biochemists actually do take place within the cell.
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Respiratory chain metabolism was studied with isolated throacic muscle mitochondria (sarcosomes) of the adult Colorado potato beetle. The composition of the respiratory chain did not differ essentially from that found in housefly sarcosomes and rat liver mitochondria. Isolated sarcosomes readily oxidized various substrates with concomitant phosphorylation. Respiratory control through the phosphate acceptor level was demonstrated to operate in sarcosomes of the Colorado potato beetle, the housefly, the American cockroach, and the Moroccan locust. Observed differences between insect and mammalian mitochondrial activities are explained by differences in intactness of the mitochondria.
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Results are reported of experiments measuring the optical density, packed volume, swelling rates, and succinoxidase activity of lupine mitochondria under different osmotic conditions. The optically measured volume (1/O.D.) of mitochondria, with succinate and ATP added, varied inversely with osmolar concentration except for mitochondria isolated under very hypotonic conditions. However, the directly measured volume of sedimented mitochondria (packed volume) from the same preparations apparently still followed osmotic law. The data are consistent with two types of swelling, a gel swelling and a membrane-controlled swelling. The faster rates of swelling, measured by the time derivative of 1/O.D., were associated generally with the larger mitochondrial volumes. Isotonicity of the mitochondria, measured by swelling rates, corresponded to the concentration of the medium used for isolating the particles, except for concentrations appreciably lower than 0.4 M. Succinoxidase activity also decreased in media below 0.4 M tonicity under conditions used for measurements of packed volume and optical density. However a more complete characterization was required because either activation or inhibition was obtained depending upon the succinate concentration used. Low assay tonicity decreased the apparent first-order reaction constants for succinoxidase compared with those obtained in higher tonicities. Although the effects of cytochrome c and succinate prevented swelling and even promoted a contraction of the packed volume, the volume and rate of swelling measured by the indirect optical method was increased under similar conditions.
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Administration of a hypertonic NaCl (4%) solution or hypertonic solutions of MgCl2, Na2SO4 or sucrose (1 ml/16 g body weight) to overnight fasted female mice, caused a rapid (within 5–10 minutes) disaggregation of hepatic polyribosomes and a decrease in incorporation of (14C)leucine into proteins. Pretreatment with cycloheximide prevented the disaggregation of hepatic polyribosomes caused by 4% NaCl. Administration of a hypotonic solution, such as distilled water (1 ml/16 g body weight), shifted the hepatic polyribosomes from lighter to heavier aggregates and caused an increase in incorporation of (14C)leucine into proteins.
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1.Measurement of oxidative phosphorylation in rat-liver mitochondria was performed using a platinum oxygen electrode and ADP as acceptor substance in the absence of added alkali metal ions.2.Under these conditions both active respiration and high P/O ratios were observed with succinate and oxoglutarate as substrates (1.9 and 2.9 respectively).3.The addition of Na+ and K+ either singly or in combination up to a concentration of 75 mM, had little effect on mitochondrial respiration or phosphorylation.4.Intramitochondrial K+ was also measured during active respiration and phosphorylation, and showed a considerable fall to about 25% of the initial value during 30 min incubation. Respiration and phosphate esterification were estimated during this period, and were found to be linear. Intramitochondrial Na+ showed a similar but much less pronounced fall under these conditions.5.It is concluded from these experiments that Na+ or K+ do not stimulate respiration or phosphorylation in rat-liver mitochondria.
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1. Heavy bovine heart mitochondria were made deficient in cytochrome c by extraction with a solution of KCl. This preparation, containing about 15% of the normal complement of cytochrome c, was used in studies on the effect of exogenous cytochrome c on several energy-linked processes. 2. The P:O ratio of the preparation was increased as the oxidation rate was increased with small progressive additions of cytochrome c. The effect may have been caused by the participation of cytochrome c in the formation of an intermediate of oxidative phosphorylation, or to the fact that the P:O ratios were lowered as the rate at which electrons traversed the chain was lowered. 3. In the cytochrome c-deficient particles, the energy-linked systems implementing calcium translocation and pyridine nucleotide transhydrogenation were found to be intact and fully functional when adenosine triphosphate was the source of energy. However, the systems required cytochrome c when oxidation of substrate was the source of energy. 4. The study points out the unlikelihood that cytochrome c is a component part of an intermediate that functions in the steps between the breakdown of ATP and the formation of the high energy intermediate utilized by energy-requiring processes. It may, however, be a component part of an intermediate that functions at a point between the electron transfer chain and the formation of the high energy intermediate utilized by energy-requiring processes.
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Rat skeletal muscle mitochondria were isolated in three different isolation media: isotonic sucrose, isotonic KCl, and hypotonic sucrose medium. The use of isotonic KCl medium leads to mitochondrial suspensions which are more contaminated with protein of nonmitochondrial origin than the use of sucrose media. This could be concluded from electron microscopy and the specific activity of cytochrome oxidase. Furthermore, respiratory control index values and cytochrome content were lower in these mitochondria. The ratio between cytochrome c + c1 and cytochrome aa3 was similar after isolation in both isotonic media but was markedly lower after isolation in hypotonic sucrose.The highest rate of pyruvate oxidation was obtained with isotonic sucrose-isolated mitochondria when this rate was expressed on a protein base. When expressed on base of spectrally measured cytochrome aa3, reduced with succinate plus KCN, or on base of cytochrome oxidase activity, the rate of pyruvate oxidation was the same in isotonic sucrose-and isotonic KCl-isolated mitochondria.Exogenous cytochrome c stimulated pyruvate oxidation about 20% in mitochondria isolated in isotonic sucrose or KCl and about 100% in mitochondria isolated in hypotonic sucrose. In the latter case, oxidation rates in the presence of exogenous cytochrome c approached the values found with mitochondria isolated in isotonic sucrose. A clear inverse relationship between the content of endogenous cytochrome c + c1 and the stimulatory effect of exogenous cytochrome c is established.Implications of these results for clinical studies of oxidative capacity of skeletal muscle mitochondria are discussed.
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Respiration experiments with succinate as substrate were made with red beet mitochondria isolated in soluitions containing 0.25 to 1.25 M sucrose. The respiration was measured in reaction media adjusted to be 0.25, 0.50, 0.75 or 1.0 osmolar. With mitochondria isolated in 0.25 or 0.50 M sucrose the rate of succinate oxidation was completely dependent on the osmotic pressure of the reaction medium (decreasing with increasing osmotic pressures). Isolation in 0.75 M sucrose caused a slight after-effect of the osmotic pressure of the isolation medium, and by isolation in 1.0 M or 1.25, M sucrose the after-effect was complete. The rate of oxidation was low and independent of the osmotic pressure of the reaction medium. An electron microscopic examination of the state of the mitochondria before and after the respiration period showed that with the conditions used in the present experiments the structure of the mitochondria remained well preserved regardless of the osmotic pressures used.
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The oxidation of l-Malate to carbon dioxide was employed as a measure of malate transport into mitochondria. Conditions of incubation were optimized with regard to osmolarity, pH, phosphate concentration, and substrate concentrations. Under these conditions the rate of malate oxidation to carbon dioxide was 17.1 ± 2.1 μmol/min/g mitochondrial protein at 30°C. Previous workers have found that maximum oxidation of glucose by the isolated perfused rat heart is from 2.4 to 3.0 μmol/min/g dry weight of tissue which would require the transport of 22 to 28 μmol of malate/min/g mitochondrial protein. Since the perfused heart experiments were carried out at 37°C, it is apparent that malate transport into mitochondria can account for most, if not all, of the reducing equivalents generated during glycolysis.
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— The effects of altered osmolality on respiration and fine structure were studied in isolated cerebral mitochondria from mature rats (60-100 days of age) and from rat pups in the first month of postnatal life (5, 10, 20 and 30 days). In the mature cerebral mitochondria, ADP-dependent respiration was inhibited in media of decreased osmolality. There was a transient inhibition of ADP-dependent respiration and a sustained increase in ADP-independent respiration in media of increased osmolality. In contrast, cerebral mitochondria from 5-day-old rats showed both inhibition of ADP-dependent respiration and increased ADP-independent respiration in hypo-osmolal media. In these mitochondria, inhibition of ADP-dependent respiration was stable and ADP-independent respiration was unchanged in media of increased osmolality. The transition to the mature respiratory response occurring with altered osmolality took place between 10 and 30 days of age. During this same age period, cerebral mitochondria showed an increasing resistance to matrix condensation in media of normal and increased osmolality.
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Shark mitochondrial respiration was studied in media with osmolalities between 160 and 1500 milliosmoles. The respiratory control ratio, a marker for functional integrity of the isolated mitochondria, was maximal at 1000 millismoles and decreased during hypotonic or hypertonic exposure. Shark mitochondria function best at their native tonicity, a value that produces abnormal function in mammalian mitochondria.
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1. The rate of ADP-stimulated respiration with various substrates and the matrix volume of rat heart mitochondria were measured over a range of osmolarities of the medium. 2. The rate of oxidation of palmitoylcarnitine (in the presence of malate) was stimulated 7-fold by increasing the matrix volume from 0.6 to 1.0 μl/mg of protein. Oxidation of octanoate showed a similar sensitivity to the matrix volume, whereas oxidation of other substrates showed little sensitivity until the volume fell below 0.7 μl/mg of protein. 3. The matrix volume of heart mitochondria incubated under physiological conditions was about 0.8 μl/mg of protein. 4. Low concentrations of valinomycin added to mitochondria incubated under such physiological conditions could activate the rate of ADP-stimulated palmitoylcarnitine oxidation by at least 100%. 5. Decreasing the matrix volume increased the reduction of the electron-transferring flavoprotein (ETF), suggesting an effect on electron flow between ETF and ubiquinone, as has been observed for liver mitochondria. 6. A rapid decrease in light-scattering by heart mitochondria incubated in State 4 was induced by addition of Ca2+, reaching 50% of the maximal effect after about 30s at 30°C and with K0.5 for Ca2+ of 0.3 μM. This was not associated with a change in matrix volume, and is discussed in terms of a conformational change whose identity remains to be determined. 7. However, incubation of heart mitochondria at 37°C in the presence of 0.65 μM-Ca2+ for 4 min did increase the matrix volume significantly, by 0.181±0.029 μl/mg of protein (n = 7, P < 0.001), similar to the Ca2+-induced changes observed with liver mitochondria. 8. The possible significance of these results in the co-ordinate regulation of fatty acid oxidation and the citric acid cycle in the heart responding to increased work load or hormonal stimulation is discussed.
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The effects of acute versus chronic hyperthyroidism on rat heart mitochondria were explored. Acute, severe hyperthyroidism with an 18% loss of body weight was induced by injecting subcutaneously 10 µg/100g/day of triiodothyronine for 6 to 14 days, and chronic, anabolic, moderate hyperthyroidism was induced by placing triiodothyronine in the drinking water at a dose of 25 or 50 µg/100 ml for 15 to 60 days. Mitochondrial function was assessed polarographically using α-ketoglutarate as the oxidizing substrate, and mitochondrial structure was assessed indirectly from changes in rates of swelling in decimolar alkaline salt solutions in vitro. The P-O ratios of heart mitochondria isolated with Nagarse incubation from both types of hyperthyroid rats decreased slightly (15%) but significantly (P = 0.05). Simple dilution of the hyperthyroid mitochondrial suspensions effected a 75% increase in the P-O ratio of the acutely treated rats but only a 19% increase in that of the chronically treated rats. Significantly increased susceptibility to swelling in vitro was exhibited only by the mitochondria of the chronically triiodothyronine-treated rats. On the other hand, only those of the acutely treated rats showed significant increases in the activity of Mg²⁺-stimulated mitochondrial ATPase. These data suggest that the mechanisms whereby excess thyroid hormone in vivo affects the function and structure of isolated rat heart mitochondria vary with the mode of induction and the duration of the hyperthyroid state.
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At the invitation of the Editors, this paper gives a summary sketch of my position regarding some metabolic aspects of energy transduction and describes some present and anticipated perspectives from my point of view. To maintain as broad a horizon as possible, however, I have used this opportunity to describe how my views, and the rationale that I have developed to express them, have been derived from accepted or acceptable physicochemical theory and biochemical knowledge stemming from the creative and painstaking observations of my progenitors and colleagues.
Chapter
This chapter discusses the life and scientific achievements of E.C. Slater, who was born in St. Kilda, a suburb of Melbourne, on 16 January 1917. In the project, heart-muscle preparation, the experiments of Hopkins and Morgan on the inhibition of succinate dehydrogenase by oxidised glutathione (GSSG) and its reversal by glutathione (GSH), he found that GSSG slowly inhibited the succinate oxidase activity of the heart-muscle preparation. However, attempts to reverse the inhibition by incubation with GSH were only partially successful. He found that incubation with GSH of heart-muscle preparation, untreated with GSSG itself, caused a strong inhibition of the succinate oxidase activity. E.C. Slater undertook a systematic study of the inhibition of succinate oxidation by different types of compounds, including an arsenical known to react with thiol groups in proteins and the reversal of the inhibition by GSH and the thiol, 2,3-dimercaptopropanol. He also studied the system responsible for the oxidation of nicotinaminde adenine dinucleotide (NADH).
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Oxidation of some substrates requiring DPN (glutamate, malate, pyruvate, ketoglutarate) is strongly decreased in fatty livers of rats, obtained either by injection of steatogenic poisons (CCl 4, P) or by feeding with a diet deficient of choline. The decrease of oxidative power is particularly strong in mitochondria. Addition to these mitochondria of the supernatant fluid produces stimulation of the oxidative activity. Almost complete restoration of activity is produced both in homogenates and in mitochondria by addition of DPN. The concentration of pyridine nucleotides in fatty livers is strongly decreased. The extent of this decrease is particularly remarkable in mitochondria. A redistribution of pyridine nucleotides, with displacement of these substances from mitochondria into the supernatant fluid, occurs in fatty liver homogenates. The PN/PNH ratio is decreased in fatty livers. Synthesis of DPN in vitro through the Kornberg reaction and destruction of DPN are not modified. Both decrease of oxidation of glutamate and decrease and displacement of DPN occur in the livers of treated animals before the beginning of the accumulation of fat within the cells. The possible causes for the described phenomena and their importance for the pathogenesis of liver steatosis are discussed.
Article
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Chapter
Im Protoplasma laufen chemische Umsetzungen unaufhörlich nebeneinander ab und bilden untereinander ein äußerst komplexes Netzwerk. In diesem ständigen Stoffumsatz, der durch Reaktionen unterhalten wird, deren Aufeinanderfolge einer gewissen Ordnung unterliegt und deren Umfang gegenseitig fein abgestimmt ist, offenbart sich das Leben. Das Material, aus dem die Zell- und Organstrukturen bestehen, ist das Produkt dieser chemischen Umsetzungen; die Energie, die zum Aufbau der Strukturen benötigt und den errichteten Strukturen zur Ausübung ihrer Funktionen zugeführt werden muß, wird durch einen Oxydations-Reduktionsmechanismus bereitgestellt. Alle diese Prozesse gehen direkt oder indirekt auf die Wirkung von Enzymen zurück. Es ist die Leistung der Enzyme, den chemischen Umsatz mit ausreichender Geschwindigkeit zu erhalten, ohne daß Milieufaktoren, wie Temperatur, Wasserstoffionenkonzentration und Druck, wesentlich von der an der Erdoberfläche natürlicherweise vorkommenden Größenordnung abzuweichen brauchen. Ein Charakteristikum der biologischen Katalyse ist ihre kontrollierte Wirkung. Die Aktivität der Enzyme in einem gegebenen Reaktionssystem hängt nicht nur von physiko-chemischen Faktoren, wie Temperatur, Säuregrad und Druck, sondern vor allem auch von der Substratkonzentration, der Konzentration der Reaktionsprodukte und verschiedenartiger Begleitstoffe ab, die eine hemmende oder aktivierende Wirkung auf die katalytische Reaktion ausüben.
Chapter
An unresolved question of the molecular dynamics of electron-flow within the terminal portion of the eukaryotic electron transport chain concerns how cytochrome c, a water-soluble heme protein located in the intermembrane space of mitochondria, interacts with cytochrome c reductase and cytochrome c oxidase, protein complexes which are integral to the inner membrane. Recently, it was demonstrated that the interaction domains on the protein surface of cytochrome c for the reductase and oxidase are essentially identical (Ferguson-Miller et al., 1978; Speck et al., 1979a, b; Osheroff et al., 1979; Koppenol et al., 1980). These results suggest that cytochrome c must either rotationally and/or laterally move on the surface of the inner membrane to complete its redox cycle.
Chapter
Despite the great progress made by biochemistry in recent years, there are still problems presented by phenomena the significance of which is unknown. One of these is the role played by transitions in the configurational state of mitochondria. We know that mitochondria may oscillate between two distinct ultrastructural forms, but why is far from being understood. At present, we may say that our rather good knowledge of the mechanisms working at the “molecular level” of mitochondria decreases when we encounter more complex systems and is rather poor at the level of “supramolecular organization.”
Chapter
It is fitting that a volume commemorating the scientific achievements of David Green should contain a chapter on mitochondrial structure, since from the earliest days he has been involved in its elucidation. This account is not meant to be a history of how notions of mitochondrial structure have progressed, nor is it comprehensive; it is rather a perspective on some recent advances in our understanding of mitochondrial configurations. First, the role of osmosis is evaluated, followed by a discussion of other physical factors and membrane structural properties which could be involved in the determination of the shape of cristae.
Article
Aufgabe und Anliegen dieses Beitrages ist es, eine Reihe morphologisch faßbarer Veränderungen am und im Zelleib darzustellen, die bereits außerhalb der normalen, physiologischen Gestaltwandlungen stehen, aber doch noch nicht mit Notwendigkeit in den Zelluntergang münden oder gar als sicheres Kennzeichen einer tödlichen Schädigung anzusprechen sind.
Chapter
Der vorliegende Beitrag beschränkt sich auf Angaben zur Präparation tierischer Gewebe; dies entspricht nicht nur dem eigenen Interessen- und Erfahrungsbereich der Verfasser, sondern liegt vor allem auch an der wesentlich größeren Widerstandsfähigkeit der Membranen von pflanzlichen Zellen und Mikroorganismen, deren Verarbeitung daher meist schwieriger ist. Die für die Aufarbeitung von Geweben und Zellen in Fragekommenden Verfahren entstammen meistens der neueren und neuesten Zeit; daher gibt es kaum ältere, zusammenfassende Darstellungen über dieses Gebiet. Zu erwähnen sind1-3.
Chapter
Die Anwendung der von Linderstrom-Lang und Holter16 entwickelten Mikromethoden zum cytochemisehen Studium der einzelnen Zellelemente bietet Vorteile und Nachteile : Das Untersuchungsgut ist histologisch einheitlich, besteht also z.B. bei Geweben mit mehreren Zellarten nur aus einer dieser Formen; daher können genaue Beziehungen zwischen histologischem Erscheinungsbild und mikrochemischem Befund aufgestellt werden (s. z. B. die klassischen Unter­suchungen über die Bildungsstätte der Salzsäure in den Belegzellen der Magen­schleimhaut, S. 36). Da aber stets nur sehr geringe Mengen Untersuchungsgut erhältlich sind, lassen sich im allgemeinen nur Stoffwechselprozesse messend er­fassen, die mit hoher Intensität ablaufen. Für die Verfolgung kleinerer Umsätze reichen die Gewebemengen meist nicht aus. Obwohl also der Wert dieser Methoden unbestritten ist, ist die Anwendbarkeit häufig nur begrenzt.
Article
One of the major recent advances in cellular architectonics, the spatial and structural organization of reactions and processes, is the elucidation of the role of mitochondria in cell metabolism. Plant cell is not simply a bag of haphazardly arranged enzymes. The cell particulates possess a high degree of structural and functional organization, which is, under certain conditions, very labile. The fundamental processes carried out by the mitochondria appear to be the terminal transfer of electrons, the coupling of energy-trapping mechanisms (phosphorylations) to oxidations, and the Krebs cycle reactions and the numerous ancillary processes that eventually funnel through the cycle. In spite of the remarkable autonomy of the mitochondria, the overall activities of the cell are the result of intimate interactions among the various cellular components. Glycolysis involves the plastids, the soluble fraction, and possibly the nucleus as well as the mitochondria1 enzymes. Certain enzymes involved in the Krebs cycle (e.g., malic dehydrogenase) and in hydrogen transfer (e.g., cytochrome reductase) are not confined entirely to the mitochondria. The mitochondria interact with the nucleus in phosphorylation, with the chloroplasts in photosynthesis, and with the microsomes in protein synthesis. In close relation to the outer cell boundary, the mitochondria may participate actively in the movement of substances into the cell or in the growth of the cell wall.
Article
Ca++ inhibits the oxidation of DPN-linked substrates in KCl-sucrose suspensions of rat liver mitochondria incubated in the presence of ATP, orthophosphate, hexokinase, glucose and Mg++. The inhibition appears as a progressive decrease of the respiratory rate, which is more marked the higher the concentration of Ca++.
Article
In thin slices of leaves or potato tubers dehydrated in hypertonic sorbitol solutions, photosynthesis or dark-fixation of CO2 was almost fully inhibited at an osmotic potential of 50 bar, while respiratory oxygen uptake was less than 50% reduced. At the same osmotic potential, coupled oxidation of succinate and exogenous NADH by isolated leaf or tuber mitochondria suspended in media containing sorbitol or sucrose was between 20% and 60% inhibited. As in situ, oxidation was more sensitive to osmotic stress in tuber than in leaf mitochondrial preparations. Coupled oxidation of glycine or malate by leaf mitochondria was only insignificantly reduced when sorbitol was used as osmoticant, while strong inhibition was observed with sucrose or raffinose. The difference appeared to be due to penetration of sorbitol which caused less osmotic shrinkage of mitochondria thus preventing internal solutes from reaching injurious levels.
Article
An plasmolysierten Protoplasten vonTaraxacum officinale, Oenothera franciscana undHelodea densa wurde die gesamte Wasserführung in einen osmotischen und einen nichtosmotischen Anteil aufgegliedert. Mit Hilfe der plasmometrischen und der grenzplasmolytischen Methode ließen sich zwei verschiedene Komponenten der nichtosmotischen Wasserführung plasmolysierter Protoplasten aufzeigen:a) Bei Plasmolyse über mehrere Stunden läuft eine Wasseraufnahme ab (ΔV K ), die durch Azid (10−3 Mol), starke Plasmolyse oder osmotische Wasseraufnahme (kurzzeitige Erhöhung und Erniedrigung der Außenkonzentration) zu drosseln oder sogar in eine langsame Wasserabgabe umzuwandeln ist. Osmotische Wasseraufnahme und Azid gleichzeitig geboten, setzen die Wasserbilanz ganz besonders stark herab. Dieser Teil der nichtosmotischen Wasserführung ist also mit Stoffwechselleistungen der Zelle gekoppelt (Phosphorylierungen, Cytochromsystem). b) Bei wechselnder Plasmolyse läßt sich nach osmotischem Wasserentzug eine zusätzliche, neu entstandene nichtosmotische Wasserführung aufzeigen (ΔV S ), die durch Azid nicht zu beeinflussen ist. Dieser Wasserbetrag wird mit der wechselnden Hydratation von Vacuolenkolloiden in Verbindung gebracht.
Article
1.1. Inhibitor studies indicate that turtle-heart mitochondria have an excess of both the antimycin-sensitive factor and cytochrome oxidase and a limited amount of the oligomycinsensitive enzyme when compared to mammalian mitochondria.2.2. The evidence suggests that the phosphorylating system may be the rate-limiting factor of oxidative phosphorylation in turtle-heart mitochondria.3.3. NaF inhibits oxidative phosphorylation in a substrate specific manner.
Article
The authors studied the early changes in the mitochondria and lysosomes in the livers of rats given CCl4 by the mouth, their object being to establish whether uncoupling of oxidative phosphorylation may be a consequence of increased activity of acid RNAase. Decrease of the P/O ratio, and also release of respiratory control were seen as early as 2 h after feeding of CCl4. Activation of lysosomal RNAase, as well as its partial transfer to the supernatant fluid, did not occur until 6 h after the first dose of CCl4. The liver fat increased as early as 1 h after feeding of CCl4. The concentration of CCl4 in the liver was already high 1–2 h after the administration of it. The authors conclude that the mitochondrial changes are independent of RNAase activation, at least in the early phase of poisoning with CCl4In adrenalectomized rats, both the mitochondrial and RNAase changes occurred at times not different from those seen in normal animals. It was found, however, that the liver fat did not increase until 6 h after the administration of CCl4 began. It seems that the damage done to the mitochondria is due mainly to the action of CCL4 and it seems to be independent of the influence of the adrenal glands. The adrenal glands may play a part in the very first onset of fat accumulation, but they are not necessarily involved in the later stages of the damage done to the liver.
Article
The optimal conditions for the incorporation, of leucine-1-C14 into the protein of rat sarcosomes have been investigated. The incorporation is dependent upon ATP generated within the sarcosome by oxidative phosphorylation. The antibiotics puromycin and chloramphenicol suppress this incorporating ability almost completely, while RNase, DNase, and the soluble fraction of the cell have little or no effect on the process. Evidence has been presented that the capacity to incorporate labeled leucine is a property of the mitochondria per se and is not the result of contamination by either microsomes or bacterial growth. Treatment of the radioactive protein with 1-fluoro-2,4-dinitrobenzene indicates that the leucine-1-C14 most probably is incorporated into the interior of the peptide chain.
Article
The principal intracytoplasmic form of muscle mitochondria is rod shaped and irregular. The mitochondria of muscle are easily stained by the variety of techniques peculiar to mitochondrial staining, such as the procedures of Regaud, Altman, and Benda, and by Baker's method. The absence of structural membranes would bring the matrix of the mitochondrion into contiguity with the sarcoplasm and the adjacent myofibrillae. In transverse sections of skeletal muscle an interpenetration of myofibrillary substance and mitochondrial matrix is demonstrated. In addition to the approximation by contact, the mitochondria are linked to both the myofibrillae and the sarcosomes by delicate reticular strands. The ability of the muscle cyclophorase from both cardiac and skeletal muscles to oxidize all the citric acid substrates catalytically is not precisely paralleled by the mitochondria in pure suspension. There is sluggish citrate oxidation in particular in the cardiac mitochondria and some lag in skeletal muscle. This has been associated with diminution in the apparent isocitric acid dehydrogenase activity in mitochondria, because most of the activity of this enzyme observed in homogenates is recovered in the supernatant fraction.
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