Abbreviations: TMPD, N,N,N′,N′-tetramethylphenylenediamine; FCCP, p-trifluoromethoxyphenylhydrazone 相似文献
1. 1. In an assay medium containing Cl− at an alkaline pH, above 7.1, triethyltin inhibited both the ADP stimulated rate of oxygen uptake and the dinitrophenol-induced ATPase (EC 3.6.1.3) but had no effect on the dinitrophenol-stimulated rate of oxygen uptake. If the pH was reduced to below 6.9 the pattern of inhibition changed and both the ADP and dinitrophenol-stimulated rates of oxygen uptake were inhibited by triethyltin.
2. 2. In the absence of Cl− in the medium triethyltin inhibited both the ADP-stimulated rate of oxygen uptake and dinitrophenol-induced ATPase and had no effect on the dinitrophenol-stimulated rate of oxygen uptake at either pH 7.4 or 6.6.
3. 3. In either the presence or absence of Cl− the ability of triethyltin to inhibit ATP synthesis appears to markedly decrease as the pH is lowered from 7.4 to 6.6.
4. 4. The significance of these observations is discussed in relation to the operation of a Cl−/OH− antiport in the coupling membrane.
1. 1. The effect of Mg2+ on ATP-dependent processes catalysed by intact rat-liver mitochondria can be explained quantitatively by the formation of Mg-ATP complexes that cannot act as a substrate for the adenine nucleotide translocator.
2. 2. The dinitrophenol-induced ATPase is characterized by two affinities of ATP: Km(1) = 6.7 μM and Km(2) = 63 μM, which contribute to the extent of 70% and 30%, respectively, to the total ATPase activity under the standard conditions employed.
3. 3. Km(1) of ATP is competitively increased by atractyloside, and is insensitive to changes in cation concentration or to oligomycin or aurovertin.
4. 4. Km(2) is as sensitive to atractyloside as the Km(1) and is also insensitive to oligomycin. However, it is increased by decreasing the cation concentration, and disappears in the presence of aurovertin.
5. 5. It is proposed that two conformations of the adenine nucleotide translocator exist, characterized by their different affinities for ATP. The distribution of the enzyme over these two conformations appears to be a function of the energy state of the mitochondria (coupled or uncoupled).
Abbreviations: PEP, phosphoenol pyruvate 相似文献
1. 1. Tightly bound ATP and ADP, found on the isolated mitochondrial ATPase, exchange only slowly at pH 8, but the exchange is increased as the pH is reduced. At pH 5.5, more than 60% of the bound nucleotide exchanges within 2.5 min.
2. 2. Preincubation of the isolated ATPase with ADP leads to about 50% inhibition of ATP hydrolysis when the enzyme is subsequently assayed in the absence of free ADP. This effect, which is reversed by preincubation with ATP, is absent on the membrane-bound ATPase. This inhibition seems to involve the replacement of tightly bound ATP by ADP.
3. 3. Using these two findings, the binding specificity of the tight nucleotide binding sites was determined. iso-Guanosine, 2′-deoxyadenosine and formycin nucleotides displaced ATP from the tight binding sites, while all other nucleotides tested did not. The specificities of the tight sites of the isolated and membrane-bound ATPase were similar, and higher than that of the hydrolytic site.
4. 4. The nucleotide specificities of ‘coupled processes’ nucleoside triphosphate-driven reversal of electron transfer, nucleoside triphosphate-32Pi exchange and phosphorylation were higher than that of the hydrolytic site of the ATPase and similar to that of the tight nucleotide binding sites.
5. 5. The different nucleotide specificities of uncoupled ATP hydrolysis and coupled processes can be explained even if both processes involve a single common site on the ATPase molecule. This model requires that energy can be ‘coupled’ only when it is released/utilised in the nucleotide binding steps of the mechanism.
6. 6. Adenosine β,γ-imidotriphosphate (AMP-PNP) is not a simple reversible inhibitor of the ATPase, since incubation requires preincubation and is not reversed when the compound is diluted out, or by addition of ATP. This compound inhibits the isolated and membrane-bound ATPase equally well. Its guanosine analogue does not act in this way.
7. 7. In submitochondrial particles, ADP inhibited uncoupled hydrolysis of ATP much more effectively than coupled hydrolysis, the latter being measured both directly (from ATP hydrolysis in the absence of uncoupler) or indirectly, by monitoring ATP-driven reduction of NAD+ by succinate.
8. 8. The effects of ADP and AMP-PNP were interpreted as providing evidence for two of the intermediates in the proposed scheme for coupled triphosphate hydrolysis.
Abbreviations: ε-ATP, N1,N6-ethenoadenosine triphosphate; 8-BrATP, 8-bromoadenosine triphosphate; AMP-PNP, adenosine β,γ-imidotriphosphate; GMP-PNP, guanosine β,γ-imidotriphosphate; N1,O-ATP, adenosine-N1-oxide triphosphate; rro-ATP 2,2′[1-(9-adenyl)-1′-(triphosphoryl-oxymethyl)-dihydroxydiethyl ether; and similarly for the respective diphosphates; NTP, NDP, nucleoside tri-, diphosphate; ANS, 1-anilino-8-naphthalene sulphonate; FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone; HEPES, N-2-hydroxyethylpiperazine-N′-2-ethane sulphonic acid; MES, 2-(N-morpholino)-ethane sulphonic acid; TES, tris(hydroxymethyl)methylamino ethane sulphonic acid 相似文献
1. 1. Glutamate can be transported across the mitochondrial membrane in exchange for OH− (or together with H+).
2. 2. Intramitochondrial glutamate is not extruded from the mitochondria by addition of aspartate when the mitochondria are preloaded with glutamate.
3. 3. N-Ethylmaleimide is a specific inhibitor of the movement of glutamate across the mitochondrial membrane.
Abbreviations: DMO, 5,5′-dimethyloxazolidine-2,4-dione; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone 相似文献
1. 1. The ATPase activity of myosin is affected by salicylalation if the treatment is carried out at higher pH than 6.5. The Mg2+-activated ATPase shows a maximal curve with 250–380% maximal activation when 25–70 moles of salicylaldehyde are bound per mole of myosin. The EDTA-activated ATPase decreases with increasing salicylalation. Ca2+-activated ATPase shows a small increase with increasing salicylalation.
2. 2. Less salicylaldehyde is bound if the treatment is carried out in the presence of ATP, while that of PPi does not affect the degree of salicylalation. The enzymic properties of myosins salicylalated in the presence of ATP or PPi are not different from those of the samples treated in their absence.
3. 3. Salicylalation decreases ATP sensitivity of ATPase and superprecipitation of actomyosins reconstituted from salicylalated myosins only if more than 50 moles of salicylaldehyde are bound per mole myosin.
Abbreviations: TBS, 2,4,6-trinitrobenzene sulphonate 相似文献
1. 1.|The mitochondrial population in hypothalamic and hypophysial brain tissue from warm (30°C) and cold (5°C) acclimated goldfish (Carassius auralus L.) was analyzed using sterological techniques.
2. 2.|It was revealed that there is a significantly larger volume density (Vv) in the cold acclimated tissue, with no significant difference in either of the surface densities (Svext and Svint) from either of the brain areas.
3. 3.|The hypothalamic brain tissue has a significantly lower specific surface (S/V) in the cold acclimated tissue but there is not a significant difference in this parameter for the hypophysial brain tissue.
4. 4.|The values for these three parameters (Vv, Svext and SVint, and S/V) indicate that mitochondria from acclimated brain tissue undergo shape changes in response to thermal stress.
5. 5.|We suggest that the shape changes may be related to the change in the phospholipid composition of the inner mitochondrial membrane with acclimation temperature.
Author Keywords: Temperature acclimation; mitochondria; quantitative; stereology; goldfish; hypothalamus; hypophysis; brain 相似文献
1. 1.|The instantaneous addition of phospholipase C to goldfish brain inner mitochondrial vesicles resulted in a partial loss of ferricyanide reduction. The retained activity rose when the phospholipase concentration and incubation period were increased.
2. 2.|No impairment of ferricyanide reduction was observed when Antimycin A-treated inner membrane vesicles were incubated with the phospholipase at 0.05 and 0.2 U mg−1 to 20 min. However, higher concentrations of the hydrolytic enzyme caused a considerable elevation in reduction and is attributed to a deterioration in vesicular integrity.
3. 3.|The hydrolysis of phospholipids from the inner membrane revealed that the phospholipase, at concentrations above 0.2 U mg−1 (minimum of 10 min incubation), resulted in removal of over 50% lipid phosphate. It was suggested that the phospholipase, at such levels, is accessible to the interior of the vesicles.
4. 4.|With the exception of cardiolipin and phosphatidyl inositol, the phospholipase was observed to be non-specific to the phospholipids from mitochondrial inner membrane treated with Triton X-100.
5. 5.|The phospholipids are asymmetrically arranged across the inner membrane and their distribution is dependent on acclimation temperature.
6. 6.|It is suggested that the compositional redistribution of the phospholipids, when acclimation temperature was changed, is necessary to stabilize the bilayer conformation of the membrane in concurrence with maintaining fluidity and functional capacity.
Author Keywords: Phospholipase C; phospholipid; mitochondria; mitoplast; submitochondria; goldfish 相似文献
1. 1. Aerobic oxidation of ubiquinol and b cytochromes is accompanied in EDTA particles, obtained by sonication of beef-heart mitochondria, by synchronous proton uptake.
2. 2. The rapid proton uptake associated to oxidation of ubiquinol and b cytochromes is greatly stimulated by valinomycin plus K+, but is unaffected by carbonyl cyanide p-trifluoromethoxyphenylhydrazone.
3. 3. 4 gion H+ are taken up per mol ubiquinol oxidized by oxygen. This H+/2e− ratio, measured in the rapid anaerobic-aerobic transition of the particles is unaffected by carbonyl cyanide p-trifluoromethoxyphenylhydrazone.
4. 4. In intact mitochondria aerobic oxidation of oxygen-terminal electron carriers is accompanied by antimycin-insensitive synchronous proton release, oxidation of ubiquinol and reduction of b cytochromes. The amount of protons released is in excess with respect to the amount of ubiquinol oxidized.
5. 5. It is concluded that electron flow along complex III, from ubiquinol to cytochrome c, is directly coupled to vectorial proton translocation. The present data suggest that there exist(s) between ubiquinol and cytochrome c one (or two) respiratory carrier(s), whose oxido-reduction is directly linked to effective transmembrane proton translocation.
Abbreviations: FCCP; carbonyl cyanide p-trifluoromethoxyphenylhydrazone 相似文献
1. 1. Fuscin, a mould metabolite, is a colored quinonoid compound which reacts readily with −SH groups to give colorless addition derivatives.
2. 2. Binding of fuscin to mitochondria has been monitored spectrophotometrically. Fuscin binding is prevented by −SH reagents such as N-ehylmaleimide, N-Methylmaleimide, mersalyl or p-chloromercuribenzoate. Conversely, fuscin prevents the binding of −SH reagents as shown with N-[14C]ethylmaleimide. Once bound to mitochondria, fuscin is not removable by washing of mitochondria.
3. 3. High affinity-fuscin binding sites (Kd = 1 μM, N = 4–8 nmoles/mg protein) are present in whole mitochondria obtained from rat heart, rat liver, pigeon heart or yeast (Candida utilis). They are lost upon sonication but are still present in digitonin inner membrane + matrix vesicles. On the other hand, lysis of mitochondria by Triton X-100 does not increase the number of high affinity binding sites indicating that all these sites are accessible to fuscin in whole mitochondria. The number of fuscin high affinity sites appears to correlate with the glutathione content of mitochondrial preparations.
4. 4. Fuscin as well as N-ethylmaleimide and avenaciolide are penetrant SH-reagents;
5. 5. Fuscin interferes with the ADP-stimulated respiration of mitochondria on NAD-linked substrates, several functions of the mitochondrial respiratory apparatus being inhibited by fuscin in a non-competitive manner, but to various extents: (a) The electron transfer chain (Ki in the range of 0.1 mM); (b) the lipoamide dehydrogenase system (Ki = 5–10 μM); (c) the transport systems of phosphate (Ki ≈ 20 μM) and of glutamate (Ki = 3–5 μM); (d) the ADP transport, indirectly (Ki ≈ 10 μM).
6. 6. Like N-ethylmaleimide, fuscin inhibits the glutamate-OH− carrier, the inhibition of that carrier bringing about an apparent increase of aspartate entry in glutamate-loaded mitochondria by the glutamate-aspartate carrier.
7. 7. The inhibition of phosphate transport by fuscin probably accounts for the inhibition of the reduction of endogenous NAD by succinate in intact pigeon heart mitochondria.
8. 8. By binding the −SH groups of mitochondrial membrane specifically unmasked by addition of micromolar amounts of ADP, fuscin, like N-ethylmaleimide, prevents the functioning of ADP translocation.
9. 9. Because of their specific and analogous effects on some well defined mitochondrial functions such as glutamate transport and ADP transport, fuscin and N-ethylmaleimide can be distinguished from other −SH reagents. The lipophilic nature of fuscin and N-ethylmaleimide which accounts for the accessbility of these compounds to hydrophobic sites in the mitochondrial membrane or on the matrix side of this membrane may be partly responsible for their characteristic inhibitory effects on mitochondrial functions.
Abbreviations: DTNB, 5,5′-dithio-bis-(2-nitrobenzoic acid); PCMB, p-chloromercuribenzoate 相似文献
1. 1. It was found that respiring mitochondria catalyze the insertion of Fe(II) and Co(II) into deuteroporphyrin. When Fe(III) was used as the metal donor, the reaction revealed an absolute requirement for a supply of reducing equivalents supported by the respiratory chain.
2. 2. A close correlation was found between the disappearance of porphyrin and the formation of heme which allows an accurate estimate of the extinction coefficient for the porphyrin to heme conversion. The value Δ (mM−1 · cm−1) = 3.5 for the wavelength pair 498 509 nm, is considerably lower than previously reported.
3. 3. The maximal rate of deuteroheme synthesis was found to be approx. 1 nM · min−1 · mg−1 of protein at 37 °C, pH 7.4 and optimal substrate concentrations, i.e. 75 μM Fe(III) and 50 μM deuteroporphyrin.
4. 4. Provided the mitochondria are supplemented with an oxidizable substrate, the presence of oxygen has no effect on the rate of deuteroheme synthesis.
Abbreviations: EPPS, (4-(2-hydroxyethyl)-1-piperazine propane sulphonic acid); HEPES, N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid; PIPES, piperazine-N,N′-2-bis(2-ethanesulphonic acid) 相似文献
1. 1.|During the heating of a synthetic actomyosin suspension, the following sequence of events were observed. First, the rate of superprecipitation decreased; secondly the extent of superprecipitation decreased and finally the MgATPase activity was inhibited. At the same time the dissociating capability of actomyosin decreased in a solution of high ionic strength.
2. 2.|A similar lack of coincidence between the mechanical and the enzymatic activities of actomyosin was observed with an increasing proportion of inactivated myosin occurring in the reconstructed actomyosin complex.
3. 3.|The different heat resistance of contractility and MgATPase activity in muscle models may be caused by inactivated myosin bridges which form in the course of heat treatment so that the dissociating capacity of actomyosin in the presence of ATP is lost.
Author Keywords: heat resistance of mucle models; actomyosin ATPase; actomyosin superprecipitation; myosin bridges 相似文献
1. 1. When isolated mutant mitochondria were preincubated with substrate and phosphorylation was started by the addition of external ADP, low P/O ratios were obtained under standard conditions. The P/O ratios could be raised to normal values approaching 2 with citrate and succinate in the presence of unusually high concentrations of ADP. Under these conditions the Michaelis constant for ADP of respiration and phosphorylation was found to be 2.9 mM. When isolated mitochondria were added to a medium containing substrate and adenine nucleotide, the Michaelis constant for ADP was found to be lower, about 0.5 mM and maximal P/O ratios of only 0.8 were achieved.
2. 2. Adenine nucleotide translocation across the membrane of the mutant mitochondria was found to be different from that in wild-type mitochondria and dependent on the energy level in mitochondria. When the intramitochondrial nucleotide pool consisted mostly of ADP and AMP, the rate of adenine nucleotide translocation was approx. 30 times lower than in wild-type mitochondria and the Michaelis constants for ADP of the translocation process were similar in the two types of mitochondria, being lower than 10 μM. When the nucleotide pool was enriched in ATP, the translocation rate in mutant mitochondria was as high as in wild-type mitochondria but the Michaelis constant for external adenine nucleotide was more than 100 times higher in the former than in the latter.
3. 3. An examination of the effects of the uncoupler, oligomycin, valinomycin and nigericin on the translocation process in the mutant mitochondria provided additional evidence that varying energization of mutant mitochondria was responsible for the variations of the translocation rates and the Michaelis constants under different experimental conditions.
4. 4. The properties of the adenine nucleotide carrier of mutant mitochondria were studied and found to be different from those of wild-type mitochondria.
5. 5. It has been concluded that the modification of adenine nucleotide translocation across the mitochondrial membrane is responsible for oxidative phosphorylation deficiency in the op1 mutant. The implications of these findings for the understanding of the adenine nucleotide translocation mechanism and the role of the translocation system in the control of cellular syntheses and growth are discussed.
Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; TMPD, tetramethyl-p-phenylenediamine 相似文献
1. 1. Trinitrophenylation of myosin was inhibited by the presence of ATP and its analogues during the treatment in the order ADP > ATP > pyrophosphate > AMP.
2. 2. The alteration of the enzymic properties due to trinitrophenylation of myosin was prevented by the presence of ATP or ADP and somewhat less by that of pyrophosphate and AMP during trinitrophenylation, but only if Mg2+ was also present.
3. 3. Neither the degree of trinitrophenylation nor the enzymic properties of the trinitrophenylated myosin were influenced by the presence of actin during the treatment of myosin.
Abbreviations: TBS; 2; 4; 6-trinitrobenzene sulphonate 相似文献
1. 1.|Goldfish acclimated to a range of temperatures between 5 and 35°C were found to only compensate the specific activity of their myofibrillar ATPase enzyme between 10 and 30°C.
2. 2.|The preferred temperatures of goldfish acclimated to 5°C and to 30°C were determined to be about 10 and 26°C respectively.
3. 3.|It is conlcuded that goldfish are only able to acclimate their myofibrillar ATPase system to temperatures between 10 and 30°C, but acclimation to these temperatures enables them to tolerate extremes.
Author Keywords: Acclimation; myofibrillar ATPase activity; temperature preference; adaption and tolerance limits; goldfish; Carassus auratus 相似文献
1. 1.|The mitochondrial outer membrane from goldfish contains on average approximately six times more phosholipid and eleven times more cholesterol than the inner membrane.
2. 2.|When acclimation temperature was lowered the amount of cholesterol to protein significantly decreased in the outer membrane, but remained unchanged in the inner membrane.
3. 3.|Although the amount of total phosphorus to cholesterol was not temperature dependent for inner membrane, it was found to be negatively correlated (P < 0.001) with acclimation temperature for outer membrane.
4. 4.|It is concluded that fluidity in the mitochondrial outer membrane is regulated by cholesterol whereas in the inner membrane, which contain very low quantities of this lipid, cholesterol has little or no influence on the fluidity properties.
Author Keywords: Cholesterol; phospholipid; mitochondia; outer and inner membrane; goldfish; temperature 相似文献
1. 1.|Progresterone concentrations in peripheral plasma, corpus luteum, uterus and placenta were determined by radioimmunoassay, in pregnant heat-acclimated (HA) and control (C) rats.
2. 2.|Concentration of progesterone in plasma and uterus of HA rats were lower than C from day 5 to 18 and day 5 to mid-gestation respectively.
3. 3.|Number and weight of pups born to HA rats were lower than C by 11.1% and 12.3% respectively.
4. 4.|It is concluded that aberration in pregnancy development observed in HA rats is associated with lower progesterone concentration in plasma and uterus from day of implantation to mid-pregnancy.
Author Keywords: Environmental heat-load and pregnancy development; progesterone levels in plasma, uterus, placenta and corpus lecteum in pregnant heat-acclimated rat 相似文献
1. 1.|The thermal resistance of the activity of frontal cilia in the median gills of the fresh water mussel Anodonta anatina was studied.
2. 2.|The resistance acclimation appeared in 2 days in the gills of intact animals, but not in the isolated gills kept at 4, 14 and 24°C, for between 1 to 3 days.
3. 3.|Warm-acclimation increased the ACh sensitivity of the gills of intact mussels.
4. 4.|Isolation of the gills enhanced the thermal resistance.
5. 5.|ACh, choline and tetramethylammonium enhanced the thermal resistance in the isolated gills. whereas atropine and physostigmine diminished it.
6. 6.|It is concluded that in A. anatina the control if the thermal resistance is probably neural.
Author Keywords: Temperature acclimation; thermal resistance; ciliary activity; acetylcholine; choline; tetramethylammonium; atropine; physostigmine; denervation supersensitivity; gill tissue; Anodonta anatina anatina; Anodonta cygnea cellensis 相似文献