ATP浓度和缺氧暴露对大鼠脑线粒体RNA和蛋白质体外合成的影响

本文探讨介质中ATP浓度和急,慢性缺氧暴露对大鼠脑线粒体内RNA和蛋白质合成的影响。用差速离心法分离正常和低压舱模拟4000m高原急性连续缺氧暴露3d和慢性连续缺氧暴露40d大鼠脑线粒体,用体外无细胞（cell-free in vitro)^3H-UTP和^3H-Leucine掺入法分别测定线粒体RNA和蛋白质合成活性,结果显示,大鼠急性缺氧暴露后大脑皮质线粒体RNA体外合成活性降低40%,蛋白质合成活性降低60%;慢性缺氧暴露后线粒体RNA和蛋白质合成活性分别为对照的72%和76%;ATP对正常大鼠脑线粒体RNA以及蛋白质的体外合成活性的影响均呈双相性,大于或小于1mmol/L均可产生不同程度的抑制效应,结果提示,缺氧可在转录和翻译两个水平上影响脑线粒体mtDNA的表达,而慢性缺氧暴露时,线粒体半自主性功能的改善可能是机体对缺氧适应的细胞机制之一;ATP对脑线粒体内转录和释放活性的调节是一种经济有效的反馈调节方式。     

大鼠脑线粒体体外蛋白翻译体系的建立及蛋白产物的鉴定

目的 :建立大鼠脑组织线粒体的体外蛋白合成体系并对其合成产物进行电泳分离和分子量鉴定。方法 :分离大鼠脑组织线粒体 ,用3 H 亮氨酸掺入法探索线粒体体外翻译的最佳条件 ,3 5S 蛋氨酸掺入并对翻译后产物经SDS 聚丙烯酰胺凝胶电泳和放射自显影进行分子量鉴定。结果 :分离的线粒体氧化磷酸化偶联程度高 ,呼吸控制率(RCR)在 3.5～ 5 .5之间 ;体外3 H 亮氨酸的掺入活性在 6 0min内近似线性增长 ,而后维持在一相对稳定水平 ;3 H 亮氨酸的掺入活性随线粒体蛋白浓度而增加 ,而单位线粒体蛋白的掺入活性在 1mg/ml时最高 ;3 5S 蛋氨酸掺入SDS 聚丙烯酰胺凝胶电泳后可观察到清晰的 8条自显影带 ,分子量分别为 (单位Kda) 86、6 6、5 6、43、33、2 9、2 5、18。结论 :用此方法建立的脑线粒体离体翻译反应体系具有高活性和翻译忠实性等特点 ,是研究脑mtDNA在翻译水平的表达及调控的有效方法     

The dynamic equilibrium between ATP synthesis and ATP consumption is lower in isolated mitochondria from myotubes established from type 2 diabetic subjects compared to lean control

Although, most studies of human skeletal muscle in vivo have reported the co-existence of impaired insulin sensitivity and reduced expression of oxidative phosphorylation genes, there is so far no clear evidence for whether the intrinsic ATP synthesis is primarily decreased or not in the mitochondria of diabetic skeletal muscle from subjects with type 2 diabetes. ATP synthesis was measured on mitochondria isolated from cultured myotubes established from lean (11/9), obese (9/11) and subjects with type 2 diabetes (9/11) (female/male, n = 20 in each group), precultured under normophysiological conditions in order to verify intrinsic impairments. To resemble dynamic equilibrium present in whole cells between ATP synthesis and utilization, ATP was measured in the presence of an ATP consuming enzyme, hexokinase, under steady state. Mitochondria were isolated using an affinity based method which selects the mitochondria based on an antibody recognizing the mitochondrial outer membrane and not by size through gradient centrifugation. The dynamic equilibrium between ATP synthesis and ATP consumption is 35% lower in isolated mitochondria from myotubes established from type 2 diabetic subjects compared to lean control. The ATP synthesis rate without ATP consumption was not different between groups and there were no significant gender differences. The mitochondrial dysfunction in type 2 diabetes in vivo is partly based on a primarily impaired ATP synthesis.     

Some observations on mitochondrial-bound hexokinase and creatine kinase of the heart

A large part of the hexokinase activity of the rat brain 20,000g supernatant became mitochondrial bound when incubated with rat heart mitochondria which had been pretreated with glucose-6-phosphate. This binding was dependent on small-molecular compounds (as yet unidentified) of the brain supernatant. Divalent cations, spermine, and pentalysine strongly stimulated the binding of brain supernatant hexokinase to heart mitochondria. Inorganic phosphate, alpha-glycerophosphate, and fructose-1,6-diphosphate showed some stimulatory effect. No effect was observed with insulin or glucose. Mitochondria isolated from hearts of fasted rats had less specific hexokinase activity than mitochondria from fasted and then carbohydrate refed rats. This dietary treatment had no significant effect on the total heart hexokinase activity. Oligomycin did not inhibit the formation of creatine phosphate or glucose-6-phosphate by isolated rabbit heart mitochondria incubated in the presence of phosphoenolpyruvate and pyruvate kinase. However, the presence of creatine inhibited the formation of glucose-6-phosphate when the ATP/ADP ratio was low, indicating that creatine kinase has a greater access to ATP/ADP translocation than has hexokinase.     

Age-related effect of melatonin on permeability transition pore opening in rat brain mitochondria

Aging is accompanied by mitochondrial dysfunction related with lowering of the respiratory complex activity and decrease of ATP synthesis, as well as by an enhancement of oxidative stress and increased sensitivity to mitochondrial permeability transition pore (mPTP) opening in mitochondral triggering the programmed cell death. In the present work we studied the effect of natural antioxidant (melatonin) on parameters of mPTP detected in non-synaptic mitochondria isolated from the brain of young and old rats (3 and 18 months, resp.) with different melatonin treatments; namely, melatonin was either directly applied to the mitochondrial suspension or chronically administered to rats with drinking water. The data obtained have shown that mitochondria isolated from brain of old rats were more susceptive to induction of mPTP. Melatonin added directly to suspension of brain mitochondria isolated from young rats demonstrated a proapoptotic effect. A prolonged chronical treatment with melatonin of old rats produced an anti-apoptotic protective effect. Non-synaptic mitochondria isolated from the brain of old rats treated with melatonin were more resistant to the mPTP opening and demonstrated the activation of respiration of mitochondria as compared to the untreated rats.     

Biochemical and morphological observations on rat liver and kidneys six months after intravenous injection of a perfluorocompound emulsion.

The histological appearance of liver and kidneys and the energy metabolism of isolated liver and kidney mitochondria were evaluated in rats 6 months after intravenous administration of 1 ml of a perfluorocompound emulsion. Both liver and kidney specimens showed neither significant histological alteration nor the presence of intracytoplasmic perfluorocompound particles. A substantial depression of the rate of ATP synthesis was observed both in liver and kidney isolated mitochondria (with respect to control mitochondria) although the magnitude of the transmembrane electrical potential was unaltered. The depression of ATP synthesis in mitochondria isolated from perfluorocompound-treated rats appeared then unrelated to the presence of perfluorocompound micelles within the cells, and might result from the interaction of either the perfluorocompound or the emulsifying agent with the mitochondrial ATP synthetase.     

Failure of dietary erucic acid to impair oxidative capacity or APT production of rat heart mitochondria isolated under controlled conditions.

1. Male, 8-week old rats were fed Purina Rat Chow for semisynthetic diets containing 20% by weight of rapeseed oil or corn oil for 3 days. 2. The hearts from the animals fed the three diets were analyzed for total lipid, phospholipid, free fatty acids, cholesterol esters, tri-, di- and monoacylglyerols. There was a seven-fold increase in the levels of triacylglycerols in the hearts of rats fed rapeseed oil diet compared to the levels in the hearts of animals fed the other two diets. Smaller increases in the content of other neutral lipid fractions were also observed. 3. Heart mitochondria from the three groups of animals were isolated under controlled conditions in the presence or absence of heparin. The rats of oxidation of different substrates and of ATP synthesis by these mitochondria were compared. 4. Mitochondria isolated in the absence of heparin from rapeseed oil-fed rats had much lower rates of oxidation and ATP synthesis than mitochondria isolated similarly from rats fed the other two diets. 5. With mitochondria freshly isolated in the presence of heparin, no significant differences in rates of oxidation or ATP synthesis were found among the three groups of animals. 6. It is concluded that, when properly isolated, mitochondria from rapeseed oil-fed rats are functionally intact with respect to oxidation and energy-coupling capacity.     

Stimulation of mitochondrial functions by glucagon treatment, starvation and by treatment of isolated mitochondria with glycogen-bound enzymes

Liver mitochondria isolated from rats starved overnight, or fed rats injected with glucagon, exhibited a similar increase of the respiration rate with succinate (by 30-40%) and glutamate plus malate (by 20-30%), as compared to mitochondria from control fed animals. The content of mitochondrial adenine nucleotides was elevated by 30-45% by glucagon treatment or starvation. Mitochondrial respiration and citrulline synthesis were stimulated by 30-40% when mitochondria isolated from fed rats were briefly preincubated with the extract from liver glycogen granules, ATP and MgCl2. This effect was abolished by heating the extract at 100 degrees C.     

Failure of dietary erucic acid to impair oxidative capacity or ATP production of rat heart mitochondria isolated under controlled conditions

1. Male, 8-week old rats were fed Purina Rat Chow or semi-synthetic diets containing 20% by weight of rapeseed oil or corn oil for 3 days.2. The hearts from the animals fed the three diets were analyzed for total lipid, phospholipid, free fatty acids, cholesterol esters, tri-, di- and monoacylglycerols. There was a seven-fold increase in the levels of triacylglycerols in the hearts of rats fed rapeseed oil diet compared to the levels in the hearts of animals fed the other two diets. Smaller increases in the content of other neutral lipid fractions were also observed.3. Heart mitochondria from the three groups of animals were isolated under controlled conditions in the presence or absence of heparin. The rates of oxidation of different substrates and of ATP synthesis by these mitochondria were compared.4. Mitochondria isolated in the absence of heparin from rapeseed oil-fed rats had much lower rates of oxidation and ATP synthesis than mitochondria isolated similarly from rats fed the other two diets.5. With mitochondria freshly isolated in the presence of heparin, no significant differences in rates of oxidation or ATP synthesis were found among the three groups of animals.6. It is concluded that, when properly isolated, mitochondria from rapeseed oil-fed rats are functionally intact with respect to oxidation and energy-coupling capacity.     

Synthesis of adenosine triphosphate in isolated nuclei and intact cells

1. It has previously been demonstrated that nuclei isolated from normal and neoplastic lymphoid cells are capable of oxygen-dependent ATP synthesis. In this paper it is shown that also the corresponding intact cells can synthesize ATP under those conditions in which nuclei can synthesize ATP. 2. In nuclei isolated from liver, kidney, rhabdomyosarcoma and osteosarcoma, oxygen-dependent ATP synthesis could not be demonstrated. The cells isolated from these tissues or tumours could not synthesize ATP either. The alternatives that such nuclei lost their ability for oxidative phosphorylation during the isolation procedure or that the process does not occur in these nuclei were explored. 3. Janus Green B, a vital stain for mitochondria, was used as a differential inhibitor of mitochondrial and nuclear ATP synthesis in intact cells. 4. Oxidative phosphorylation in mitochondria isolated from cells that had been incubated with various concentrations of Janus Green B (1–10μm) was seriously uncoupled, whereas at these concentrations oxygen-dependent ATP synthesis in isolated nuclei and in isolated cells were only inhibited to a small extent. 5. The results suggest that oxygen-dependent ATP synthesis in isolated cells measured under `nuclear' conditions and in the presence of Janus Green B and Ca²⁺ is mainly due to nuclear oxygen-dependent ATP synthesis. The stimulation of cellular ATP synthesis by glucose was completely inhibited by Janus Green B. 6. It is tentatively concluded that the stimulation of ATP synthesis in isolated cells by glucose, which is not found in isolated nuclei, represents mitochondrial ATP synthesis, and nuclear and mitochondrial ATP synthesis can then be studied differentially in the intact cell. The possibility is considered that oxygen-dependent nuclear ATP synthesis is not a general property of cell nuclei.     

Open-Loop Control of Oxidative Phosphorylation in Skeletal and Cardiac Muscle Mitochondria by Ca2+

In cardiac muscle, mitochondrial ATP synthesis is driven by demand for ATP through feedback from the products of ATP hydrolysis. However, in skeletal muscle at higher workloads there is an apparent contribution of open-loop stimulation of ATP synthesis. Open-loop control is defined as modulation of flux through a biochemical pathway by a moiety, which is not a reactant or a product of the biochemical reactions in the pathway. The role of calcium, which is known to stimulate the activity of mitochondrial dehydrogenases, as an open-loop controller, was investigated in isolated cardiac and skeletal muscle mitochondria. The kinetics of NADH synthesis and respiration, feedback from ATP hydrolysis products, and stimulation by calcium were characterized in isolated mitochondria to test the hypothesis that calcium has a stimulatory role in skeletal muscle mitochondria not apparent in cardiac mitochondria. A range of respiratory states were obtained in cardiac and skeletal muscle mitochondria utilizing physiologically relevant concentrations of pyruvate and malate, and flux of respiration, NAD(P)H fluorescence, and rhodamine 123 fluorescence were measured over a range of extra mitochondrial calcium concentrations. We found that under these conditions calcium stimulates NADH synthesis in skeletal muscle mitochondria but not in cardiac mitochondria.     

The biosynthesis of the mitochondrial ADP, ATP translocator.

The biosynthesis of the ADP,ATP carrier was studied in mitochondria of Neurospora crassa. The carrier was isolated as the carboxyatractylate-protein complex and characterized in dodecylsulphate/polyacrylamide gel electrophoresis to have a Mr = 33 000. Applying the inhibitors chloramphenicol for the intramitochondrial translation and cycloheximide for extramitochondrial translation, the site of synthesis of this polypeptide was found to be extramitochondrially located.     

Protein synthesis in isolated castor bean mitochondria is stimulated by cyanide

Cyanide added to isolated castor bean (Ricinus communis L.) mitochondria supplemented with ATP and succinate (or NADH) significantly enhanced the rate and extent of organellar protein synthesis. Cyanide stimulated mitochondrial protein synthesis in a dose-dependent manner with an optimum stimulation of over twofold at 1 millimolar cyanide. At this concentration of cyanide, the mitochondrial respiratory activity, in the presence of succinate (or NADH) and ADP was inhibited by 90%. The stimulatory effect of cyanide on mitochondrial translation was reflected in the increased synthesis of all the proteins synthesized within the organelle. Preliminary evidence indicates a role for the alternative, salicylhydroxamic acid-sensitive, oxidase in the cyanide stimulation of protein synthesis.     

Localization of PTP-1B, SHP-2, and Src exclusively in rat brain mitochondria and functional consequences

An immunodetection study of protein tyrosine phosphatase 1B (PTP-1B), SHP-2, and Src in isolated mitochondria from different rat tissues (brain, muscle, heart, liver, and kidney) revealed their exclusive localization in the brain. Given this result, we sought whether mitochondria respond to ATP and to the general tyrosine phosphatase inhibitor orthovanadate and found little or no change in the tyrosine phosphorylation profile of mitochondria from muscle, heart, liver, and kidney. In contrast, ATP induced an enhancement in the tyrosine-phosphorylated protein profile of brain mitochondria, which was further greatly enhanced with orthovanadate and which disappeared when Src was inhibited with two inhibitors: PP2 and PP1. Importantly, we found that in brain mitochondria, ATP addition induced Src autophosphorylation at Tyr-416 in its catalytic site, leading to its activation, whereas the regulatory Tyr-527 site remained unphosphorylated. Functional implications were addressed by measurements of the enzymatic activity of each of the oxidative phosphorylation complexes in brain mitochondria in the presence of ATP. We found an increase in complex I, III, and IV activity and a decrease in complex V activity, partially reversed by Src inhibition, demonstrating that the complexes are Src substrates. These results complemented and reinforced our initial study showing that respiration of brain mitochondria was partially dependent on tyrosine phosphorylation. Therefore, the present data suggest a possible control point in the regulation of respiration by tyrosine phosphorylation of the complexes mediated by Src auto-activation.     

ATP hydrolysis by ischemic mitochondria

Cellular ATP levels are determined by the rates of ATP production and ATP hydrolysis. Both phenomena are affected by ischemia. Mitochondrial enzymes are damaged, inhibiting this organelle's ability to make ATP. Mitochondria are also uncoupled by ischemia and have the ability to hydrolyze ATP. We designed a series of experiments to determine whether decreased production or increased hydrolysis of ATP was the primary effect of mitochondrial damage. Rat hearts were subjected to 45 min of warm ischemia in order to induce irreversible cell damage. ATP or ADP was injected into cuvettes containing mitochondria isolated from normal myocardium or myocardium damaged by ischemia. Luciferin-luciferase, which fluoresces in the presence of ATP, was also added to the tubes as an indicator of ATP levels. Mixtures of uncoupled and coupled mitochondria were made and compared with the mitochondria damaged by ischemia. The results showed that mitochondria damaged by prolonged ischemia hydrolyze ATP more rapidly than normal mitochondria; however, normal mitochondria can easily compensate for increased ATP hydrolysis when in mixture with equal amounts of uncoupled mitochondria. These data suggests that the low cellular levels of ATP following irreversible ischemia are primarily due to decreased ATP synthesis and not to increased hydrolysis.     

Toxic action of 2'-chloro-2,4-dinitro-5',6-di(trifluoromethyl) diphenylamine in the rat.

2'-Chloro-2,4-dinitro-5',6-di(trifluoromethyl)diphenylamine (CDTD) is a potent uncoupler of oxidative phosphorylation in isolated rat liver or brain mitochondria. The concentration of CDTD causing 50% uncoupling in vitro is dependent on the mitochdonrial protein concentration and is 2 nM at 0.9 mg protein/ml for rat liver mitochondria. Oxidative phosphorylation can be restored to CDTD uncoupled liver mitochondria by the addition of a 10 000-fold molar excess of bovine serum albumin to DCTD. Rats given a lethal dose (7.0 mumol/kg) of CDTD intrapertioneally show signs of toxicity typical of uncoupling agents. Mitochondria isolated from the livers of these rats show almost complete inhibition of ATP synthesis and mitochondria obtained from the livers of rats at various times after a single oral dose show maximal inhibition of ATP synthesis 4 h after dosing with complete recovery by about 24 h. A single oral administration of 58 mumol/kg or above, but not intraperitoneal injection, of CDTD into rats produced an increase in the water content of the brain and spinal cord. The additional fluid has been shown to contain Na+ ions. The increase in cerebral fluid is dose related, no effect being seen at 23 mumol/kg. This extra fluid is thought to be responsible for the hind limb weakness observed in these rats. These observations suggest that there are two facets to CDTD toxicity: early deaths (within 2 h), which appear to be due to uncoupling of oxidative phosphorylation, and delayed deaths, 2--3 days after dosing which are probably related to an increase in fluid in the brain and spinal cord.     

Antibiotic inhibition of protein synthesis by mitochondria of higher plants and animals

The antibiotic sensitivity of protein synthesis by mitochondria isolated from chick brain, trout liver, potato tuber and corn root has been investigated. Like mitochondria isolated from rat liver, organelles from these sources show a much reduced antibiotic sensitivity spectrum when compared to mitochondria derived from yeast, being sensitive to chloramphenicol, carbomycin, spiramycin and mikamycin but resistant to erythromycin, lincomycin, paromomycin, neomycin C and viridiogrisein. In addition spectinomycin inhibited protein synthesis by both types of plant mitochondria but not those from chick brain, rat liver or yeast.     

ATPase complex and oxidative phosphorylation in chloramphenicol-induced megamitochondria from mouse liver.

1. Functional properties of the ATPase complex are investigated in megamitochondria isolated from livers of weanling mice fed a diet containing 2% chloramphenicol, as an inhibitor of mitochondrial protein synthesis. 2. Whereas the specific activity of ATPase remains unchanged in chloramphenicol-induced megamitochondria, about 40% of the enyzme activity is resistant to inhibition by oligomycin, triethyltin or venturicidin. It is concluded that the ATPase complex lacks one or more components whose synthesis or accumulation is dependent on mitochondrial translation. The inhibitor-resistant ATPase portion appears tightly bound to the mitochondrial membrane. 3. Respiratory chain phosphorylation is tightly coupled in isolated megamitochondria. ATP synthesis and ATP-Pi exchange are diminished by 40%, as compared to control mitochondria, but both processes are sensitive to oligomycin, triethyltin or venturicidin. 4. The decrease in ATP synthesis and ATP-Pi exchange in megamitochondria correlates quite well with the emergence of inhibitor-resistant ATPase. 5. The following electron transport activities in the megmitochondria are reduced: NADH-cytochrome c reductase, by 60%, cytochrome oxidase, by 80%; the amount of antimycin required to gain complete inhibition of the bc1-segment is diminished by more than 50%. On the other hand succinate dehydrogenase activity is increased by 50%. 6. Chloramphenicol-induced megamitochondria appear to be a useful system for studying the role of mitochondrial translation in the assembly of mammalian mitochondria.     

Ischemia-induced alterations of mitochondrial structure and function in brain, liver, and heart muscle of young and senescent rats

Young and senescent rats (3 and 28-30 months old) were subjected to complete ischemia at 37 degrees C in order to study function and structure of mitochondria isolated from liver, heart muscle, and brain. The rates of energy-coupled respiration and ATP synthesis were found to decrease progressively in relation to time of ischemia. The respiratory rates in the absence of ADP (state 4 respiration) did not increase after exposure to ischemia, suggesting that ischemia primarily affects electron transport rather than the energy coupling system. Mitochondria of heart muscle were more affected by ischemia than mitochondria of brain and liver. Liver and heart muscle mitochondria obtained from young rats were found to be slightly more sensitive to short periods of ischemia than those isolated from senescent animals.