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1.
Calcium ions are accumulated by intact mitochondria isolated from Ehrlich ascites tumour cells in a buffered system supplemented with ATP or succinate. In the ATP-supplemented system, the tumour mitochondria, in contrast to rat liver mitochondria, retain the accumulated Ca2+, do not exhibit a marked “irreversible” ATPase and do not swell. In the succinate-supplemented system, added Ca2+ stimulates respiration in either the absence or presence of added inorganic phosphate. Whereas respiration by rat liver mitochondria, measured in the presence of added phosphate, remains continuously activated after the addition of only a small amount of Ca2+, that by the tumour mitochondria can be stimulated by several successive additions of 100 μM Ca2+ and at all times exhibit appreciable activation ratios.  相似文献   

2.
Mitochondria isolated from rat heart and kidney cortex by Polytron treatment of the tissues exhibit lower state 3 rates of respiration than mitochondria isolated by Nagarse method. Addition of cytochrome c to Polytron mitochondria isolated from heart, but not from kidney, increases oxygen uptake to values approaching those of Nagarse-treated preparations. Similar results were observed for Ca2+ uptake. Kidney Polytron mitochondria exhibited lower mitochondrial, but higher non-mitochondrial enzyme activities compared to kidney Nagarse mitochondria. Enzyme activities were the same in Polytron and Nagarse mitochondria from heart. The differences between Polytron and Nagarse mitochondria appear to be mainly due to lower cytochrome c content of Polytron mitochondria from heart and higher contamination of Polytron mitochondria from kidney.  相似文献   

3.
《BBA》1985,806(1):9-15
We have shown previously that heart mitochondria obtained by the Nagarse method from genetically diabetic mice (C57BL/KsJ db / db) exhibit a defect in oxidizing NAD+-linked substrates (Kuo, T.H., Moore, K.H., Giacomelli, F. and Wiener, J. (1983) Diabetes 32, 781–787). In this study, the oxidative phosphorylation characteristics of cardiac mitochondria isolated by the Polytron method were compared with that of Nagarse mitochondria. Evidence is presented here that in the diabetic heart both Nagarse and Polytron mitochondria have defective pyruvate oxidation, whereas only the former exhibit impaired fatty acid oxidation. Assay of two rate-limiting β-oxidation enzymes, namely β-hydroxyacyl-CoA dehydrogenase and β-ketothiolase, indicates no alteration in specific activities from diabetic mice vs. controls. The data suggest that two populations of mitochondria are present in myocardium and that the defective oxidative metabolism in the cardiac mitochondria of db / db mice may be linked to deficiencies in total NAD + NADH content.  相似文献   

4.
Modulation of cell calcium signals by mitochondria   总被引:4,自引:0,他引:4  
It is now clearer and clearer that mitochondria play a role, and perhaps an active role, in cell calcium signalling. The fact that mitochondria can exhibit a Ca2+>-induced Ca2+> release (mCICR, Ichas et al. [37]) reinforces this concept and makes the mitochondria an essential element in the relay of Ca2+> wave propagation. It must be emphasized that the modulation of cell Ca2+> signals by mitochondria depends upon their energetic status, thus making mitochondria an essential link between energy metabolism and calcium signalling inside the cell.  相似文献   

5.
Previous methods for isolating lung mitochondria, particularly from rabbits, have yielded preparations which exhibit low respiratory control ratios (RCRs). We now report a method for the isolation of lung mitochondria from rabbit, rat, and mouse with RCRs, ADP/O ratios, and rates of substrate oxidation comparable to those for liver mitochondria. These mitochondrial preparations fail to oxidize exogenously added NADH and exhibit RCRs, during succinate oxidation, which closely approximate those obtained with NADH-linked substrates. However, an otherwise latent Mg2+-stimulated ATPase activity can still be elicited when Mg2+ is added to the mitochondrial incubation medium. This ATPase activity is insensitive to oligomycin and atractyloside, indicating that the source is from contaminating endoplasmic reticulum. The pH and EDTA concentration for maximum substrate oxidation and RCR were found to be 7.2 and 0.1 mm, respectively. State 4 respiration was affected by pH and EDTA concentration while state 3 respiration appeared to be independent of these two factors over the ranges studied.  相似文献   

6.
Mitochondria isolated from stage 3 (about half-grown) oocytes of Xenopus laevis exhibit a DNA synthetic rate in vitro of 2.35 ± 0.28 pg/oocyte/h. Similarly, stage 6 (full-grown) oocyte mitochondria synthesize DNA (mtDNA) at 0.28 ± 0.02 pg/oocyte/h. By comparison, the rate of mtDNA synthesis by intact stage 6 oocytes following microinjection of [3H]-dTTP was calculated to be 0.43 ± 0.08 pg/oocyte/h, indicating that the observed in vitro rates may represent minimum values. Measurements of DNA polymerase activity associated with mitochondria isolated from stage 3 oocytes are almost three times those recorded with stage 6 oocyte mitochondria. It appears that active replication of complete mtDNA molecules, which accompanies accumulation of mitochondria by the egg, is terminated midway through oogenesis.  相似文献   

7.
The choleterol to phospholipid ratio in mitochodria from hepatomas AH-130, 3924A and 5123 is higher than in the particles isolated from adult or fetal rat livers. Nearly all the cholesterol of hepatoma mitochonda is located in membranes. As in liver mitochondria, in the particles isolated from hepatoma AH-130 there is more cholesterol in the outer than in the inner membrane.In mitochondria from cholesterol-enriched liver and hepatomas, there occur a decrease in extent of hypoosmotic and phosphate-induced sweeling and decrease of conformational linked energy states. The phenomenon is more marked in particles which exhibit higher cholesterol to phospholopid ratios. A statistically significant negative correlation exists between the cholesterol to phospholipid ratio and extent of volume or conformation changes. No significant modifications of these parameters were found in fetal liver mitochondria.Cholesterol content does not influence K+ uptake by cholesterol-enriched or hepatoma mitochondria. Nor does cholesterol content affect the respiratory increment related to this uptake. As a consequence of K+ uptake, total mitochodrial water exchangeable with tritiated water rises 20% while sucrose-impermeable water rises 42–48% in both adult rat liver and hepatoma AH-130 mitochondria. Absorbance changes linked to ion uptake do not correspond merely to variations in mitochobdrial water content. Water content. Water is apparently not influenced by the cholesterol to phospholipid ratio. However, the ratio is signifacantly correlated to both extent and initial rate of absorbance decrease of mitochondrial suspension during K+ uptake. The higher the ratio, the lower the extent and and initial rate of absorbance decrease.  相似文献   

8.
The kinetics of the processes accompanying the induction of Ca2+-dependent permeability (pore opening) of the inner membrane—swelling of organelles and Ca2+ release from the matrix—was studied in isolated liver mitochondria of mammals (mice, rats, and rabbits) and birds (pigeons and guinea fowls). It was found that the mitochondria of rats, pigeons, and guinea fowls of the gray-speckled population (GSP) are similar in terms of respiration and oxidative ATP synthesis, whereas mitochondria of rabbits exhibit a greater degree of coupling of respiration and ATP synthesis, and mitochondria of mice and Zagorskaya White breed (ZWB) guinea fowls, a lower degree of coupling. It was established that mammalian mitochondria energized by succinate oxidation and incubated with 1 mM of inorganic phosphate are able to swell upon the addition of 125 nmol of CaCl2 per 1 mg protein. Under these conditions, mitochondria of GSP and ZWB guinea fowls and pigeons are capable of swelling upon addition of at least 875, 875 and 1000 nmol of CaCl2 per 1 mg protein, respectively. Cyclosporin A (CsA, 1 μM) inhibits mitochondrial swelling. It was shown that mitochondria of mammalians and guinea fowls but not of pigeons are able to effectively absorb and retain Ca2+ in the matrix. Calcium retention capacity of mitochondria from rats, mice, rabbits, GSP, and ZWB guinea fowls were, respectively, 70, 57, 38, 844 and 793 nmol of CaCl2 per 1 mg of protein. In the presence of an oxidizing agent tert-butylhydroperoxide (TBH), the induction of the Ca2+-dependent pore in the mitochondria was observed upon addition of CaCl2 in substantially smaller quantities. TBH was most effective in the case of rabbit mitochondria and had the lowest efficiency in the case of guinea fowl and pigeon mitochondria.  相似文献   

9.
The fluorescent dyes DASPMI and rhodamine 6 GO specifically stain mitochondria in living cells. Dye concentrations from 10?8 to 5 × 10?6 mole l?1 can be used. Excitation and emission spectra, and quantum efficiency of DASPMI depend on solvent characteristics. Spectra of mitochondria in living cells correspond to those in phospholipids (excitation around 470 nm, emission 560–570 nm). Fluorescence intensity of DASPMI is a measure for the energization of mitochondria, as revealed by in vitro studies. In living cells uptake of the dye is strongly influenced by inhibitors of oxidative phosphorylation (i.e. by oligomycin, FCCP). Distribution of fluorescence intensity indicates an intracellular gradient in energy load of endothelial cells. Single mitochondria exhibit oscillations in fluorescence. Mitochondria loaded with DASPMI release the dye suddenly into the cytoplasm on treatment with FCCP. Cyanide and antimycin however, do not diminish fluorescence in vivo under optimal nutritional conditions, while they do so in mitochondrial suspension, indicating different mitochondrial behaviour in vivo and in suspension.  相似文献   

10.
31P-NMR studies of intact functional rat liver mitochondria at 37°C demonstrate that the large majority (?95%) of endogenous phospholipids exhibit motional properties consistent with bilayer structure. This property is unaffected by oxidative phosphorylation processes or the presence of Ca2+.  相似文献   

11.
12.
During the mid to late 4th instar period of larval development, the mitochondria of Rhynchosciara spermatocytes undergo highly characteristic morphological changes. In late meiosis the enlarged mitochondria fuse to form a single mitochondrial element which will ultimately extend the length of the spermatid tail. Our studies have shown that synthesis of a circular DNA occurs during this period of mitochondrial “differentiation.” This DNA has a density of 1.681 g/cm3; and its synthesis cannot be detected in somatic tissues such as salivary gland, fat body, or gastric cecum. From analysis of DNA extracted from mitochondrial pellets, we have shown that the circular DNA is associated with the mitochondria. The contour length of the mitochondrial DNA is 9 μm, equivalent to a molecular weight of 18 × 106. Although most metazoan mitochondrial DNAs exhibit contour lengths of approximately 5 μm (10 × 105 daltons), there is no extractable 5 μm circular DNA in these spermatocytes. Therefore, we conclude that either Rhynchosciara spermatocytes possess a distinct 9 μm mitochondrial DNA or that the spermatocyte mitochondrial DNA represents dimers of 5 μm monomers.  相似文献   

13.
P. Rustin  C. Queiroz-Claret 《Planta》1985,164(3):415-422
Kalanchoe blossfeldiana plants grown under long days (16 h light) exhibit a C3-type photosynthetic metabolism. Switching to short days (9 h light) leads to a gradual development of Crassulacean acid metabolism (CAM). Under the latter conditions, dark CO2 fixation produces large amounts of malate. During the first hours of the day, malate is rapidly decarboxylated into pyruvate through the action of a cytosolic NADP+-or a mitochondrial NAD+-dependent malic enzyme. Mitochondria were isolated from leaves of plants grown under long days or after treatment by an increasing number of short days. Tricarboxylic acid cycle intermediates as well as exogenous NADH and NADPH were readily oxidized by mitochondria isolated from the two types of plants. Glycine, known to be oxidized by C3-plant mitochondria, was still oxidized after CAM establishment. The experiments showed a marked parallelism in the increase of CAM level and the increase in substrate-oxidation capacity of the isolated mitochondria, particularly the capacity to oxidize malate in the presence of cyanide. These simultaneous variations in CAM level and in mitochondrial properties indicate that the mitochondrial NAD+-malic enzyme could account at least for a part of the oxidation of malate. The studies of whole-leaf respiration establish that mitochondria are implicated in malate degradation in vivo. Moreover, an increase in cyanide resistance of the leaf respiration has been observed during the first daylight hours, when malate was oxidized to pyruvate by cytosolic and mitochondrial malic enzymes.Abbreviations CAM Crassulacean acid metabolism - MDH malate dehydrogenase - ME malic enzyme  相似文献   

14.
Isolated brain mitochondria are a heterogeneous mixture from different cell types and these subsets may have differing sensitivities to Ca2+-induced membrane permeability transition (MPT) and to inhibition of the MPT by cyclosporin A (CsA). This study tested the hypothesis that mitochondria within primary cultures of astrocytes and neurons exhibit different energy-dependent Ca2+ uptake capacities and different degrees to which CsA increases their uptake capacity. Astrocytes and neurons were suspended in a cytosol-like medium containing respiratory substrates, ATP, and Mg2+ in the presence of digitonin to selectively permeabilize the plasma membrane. Uptake of added Ca2+ by mitochondria within the cells was measured by Calcium Green 5N fluorescent monitoring of the medium [Ca2+]. Permeabilized astrocytes had a fourfold higher Ca2+ uptake capacity, relative to neurons and a twofold higher content based on relative contents of mitochondria assessed by measurements of mitochondrial DNA and cytochrome oxidase subunit 1 protein. In astrocytes the Ca2+ uptake capacity was increased twofold by preincubation with 2–5 μM CsA, while in neurons CsA had no effect. Similar results were obtained using measurements of the effects of added Ca2+ on mitochondrial membrane potential. FK506, a drug similar to CsA but without MPT inhibitory activity, had no effect on either cell type. These results are consistent with the presence of a calcium-induced MPT in astrocytes, even in the presence of ATP, and indicate that the MPT in cerebellar granule neurons is resistant to CsA inhibition. Some of the protective effects of CsA in vivo may therefore be mediated by preservation of mitochondrial functional integrity within astrocytes.  相似文献   

15.
Ca 2+ transport activity in mitochondria from some plant tissues   总被引:8,自引:0,他引:8  
Mitochondria isolated from some 14 different higher plants and fungi were examined for their capacity to carry out respiration-dependent accumulation of Ca2+. Additions of Ca2+ give little or no stimulation of state 4 respiration of plant mitochondria, although the added Ca2+ was largely accumulated. Accumulation of Ca2+ required phosphate and, in most cases, was stimulated by Mg2+ and ADP or ATP. Ca2+ uptake was abolished by respiratory inhibitors and uncoupling agents. The ratio of Ca2+ ions taken up per pair of electrons per energy-conserving site was normal at about 2.0 for mitochondria from sweet potato and white potato; mitochondria from other plants showed somewhat lower ratios. Accumulated Ca2+ was only very slowly released from previously loaded plant mitochondria. Respiration-inhibited sweet potato mitochondria show both high-affinity and low-affinity Ca2+ binding sites sensitive to uncouplers, La3+, and ruthenium red and thus resemble animal mitochondria. Most other plant mitochondria lack high affinity sites. In general, mitochondria from sweet potato and white potato tubers resemble those from animal tissues, but mitochondria from carrots, beets, turnips, onions, cabbage, artichokes, cauliflower, avocados, mung bean and corn seedlings, and mushrooms show rather low affinity and activity in accumulation of Ca2+, probably due to lack of a specific Ca2+ carrier.  相似文献   

16.
Three DNA buoyant density species (nuclear, 1.692 g cm−3; mitochondria 1.705 g cm−3; and proplastid, 1.713 g cm−3) can be detected in extracts from castor bean endosperm. No other buoyant density species can be identified. DNA extracts from sucrose density gradient purified glyoxysomes exhibit varying amounts of each of the three identified DNAs but no other distinguishable DNA species. RNA synthesized in vitro by Escherichia coli RNA polymerase using purified castor bean nuclear DNA as a template, hybridizes equally well with its template and with the 1.692 g cm−3 species from glyoxysome fractions. These results are discussed in terms of their relevance to microbody biogenesis.  相似文献   

17.
1. 1. The 31P-NMR characteristics of intact rat liver mitochondria, mitoplasts and isolated inner mitochondrial membranes, as well as mitochondrial phosphatidylethanolamine and phosphatidylcholine, have been examined.
2. 2. Rat liver mitochondrial phosphatidylethanolamine hydrated in excess aqueous buffer undergoes a bilayer-to-hexagonal (HII) polymorphic phase transition as the temperature is increased through 10°C, and thus prefers the HII) arrangement at 37°C. Rat liver mitochondrial phosphatidylcholine, on the other hand, adopts the bilayer phase at 37°C.
3. 3. Total inner mitochondrial membrane lipids, dispersed in an excess of aqueous buffer, exhibit 31P-NMR spectra consistent with a bilayer arrangement for the majority of the endogeneous phospholipids; the remainder exhibit spectra consistent with structure allowing isotropic motional averaging. Addition of Ca2+ results in hexagonal (HII) phase formation for a portion of the phospholipids, as well as formation of ‘lipidic particles’ as detected by freeze-fracture techniques.
4. 4. Preparations of inner mitochondrial membrane at 4 and 37°C exhibit 31P-NMR spectra consistent with a bilayer arrangement of the large majority of the endogenous phospholipids which are detected. Approx. 10% of the signal intensity has characteristics indicating isotropic motional averaging processes. Addition of Ca2+ results in an increase in the size of this component, which can become the dominant spectral feature.
5. 5. Intact mitochondria, at 4°C, exhibit 31P-NMR spectra arising from both phospholipid and small water-soluble molecules (ADP, Pi, etc.). The phospholipid spectrum is characteristic of a bilayer arrangement. At 37°C the phospholipids again give spectra consistent with a bilayer; however, the labile nature of these systems is reflected by increased isotropic motion at longer (at least 30 min) incubation times.
6. 6. It is suggested that the uncoupling action of high Ca2+ concentrations on intact mitochondria may be related to a Ca2+-induced disruption of the integrity of the inner mitochondrial phospholipid bilayer. Further, the possibility that non-bilayer lipid structures such as inverted micelles occur in the inner mitochondrial membrane cannot be excluded.
Keywords: 31P-NMR; Inner mitochondrial membrane; Phosphatidylethanolamine; Ca2+; Hexagonal (HII) phase; Lipidic particle  相似文献   

18.
The morphology of testicular mitochondria changes markedly during spermatogenesis from a form normally seen in somatic cells to a “germ cell” form in which the matrix is diffuse and vacuolated and finally to a form with a condensed matrix seen in spermatozoa. Colloidal silica gel gradients and high-resolution, two-dimensional gel electrophoresis were used to define the changes in density and polypeptide composition that occur in testicular mitochondria during spermatogenesis. Similar densities were observed for mitochondria isolated from the same bovine or murine tissue, but mitochondria from different tissues usually had different densities. Mitochondria from testis of calf, bull, or sexually mature mouse had densities of 1.06 gm/cm3 while liver mitochondria were more dense, having a density of 1.09 gm/cm3. “Somatic-type” testicular mitochondria from calf and “germ cell-type” mitochondria from sexually mature mouse or bull had similar densities, 1.06 gm/cm3, while the density of mitochondria from ejaculated spermatozoa differed, ρ = 1.08 gm/cm3. Analysis of polypeptide composition of somatic and germ cell mitochondria from testes of prepuberal and sexually mature animals and from highly enriched populations of pachytene primary spermatocytes and round spermatids revealed a staining pattern of mitochondrial proteins that was markedly constant throughout development with most polypeptides being conserved and a few specific spots changing in abundance. Marked differences were detected, however, when mitochondria from ejaculated spermatozoa were compared with those from testis with many minor and major polypeptides missing and several new polypeptides present at high concentration.  相似文献   

19.
20.
The state of the Ca2+ signaling system has been assessed in proliferating and in differentiating C2C12 myoblasts. Proliferating myoblasts exhibit no features of a functional system: the intracellular ATP-controlled Ca2+ store is low (perhaps only mitochondrial) and no Ca2+ entry from the medium is registered upon its exhaustion, there is no cytosolic response to KCl-induced depolarization. The Ca2+ signaling system starts to form at the early stages of differentiation (within 10 h after transfer of cells to the differentiation medium). This is seen as appearance of capacitive and voltage-dependent Ca2+ entry and its accumulation in the endoplasmic reticulum. A small contribution to the ATP-evoked rise in cytosolic Ca2+ is also made by mitochondria.  相似文献   

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