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1.
The deleterious action of Cd2+ on rat liver mitochondria was investigated in this work using spectroscopic and microscopic methods. The concentration dependence
of Cd2+ on mitochondrial swelling, membrane potential and membrane fluidity was studied. Our aim was to detect the active sites of
Cd2+ in the mitochondrial membrane treatments with cyclosporin A (CsA) and EGTA on the mitochondrial permeability transition (MPT)
induced by low and high concentrations of Cd2+. The protective effects of dithiothreitol, human serum albumin and monobromobimane+ on Cd2+-induced MPT were also monitored. All of these investigations indicated that Cd2+ can directly affect MPT at two separate localization sites at different concentrations: the classic Ca2+ triggering site and the thiol (–SH) groups of membrane proteins matched by MPT pore opening (defined as “S” site). At the
high concentration of Cd2+, other free –SH groups in the mitochondrial matrix may be involved in this process. These findings were supported by transmission
electron microscopy and shed light on the toxic mechanism of Cd2+ on mitochondria. 相似文献
2.
The adenine nucleotides ADP and ATP are probably the most important endogenous inhibitors of the mitochondrial permeability
transition (MPT). We studied the inhibitory effects of adenine nucleotides on brain MPT by measuring mitochondrial swelling
and Ca2+ and cytochrome c release. We observed that in the presence of either ADP or ATP, at 250 μM, brain mitochondria accumulated more than 1 μmol
Ca2+ × mg protein−1. ADP or ATP also prevented Ca2+-induced mitochondrial swelling and cytochrome c release. Interestingly, ATP lost most of its inhibitory effects on MPT when the experiments were carried out in the presence
of ATP-regenerating systems. These results indicate that MPT inhibition observed in the presence of added ATP could be mainly
due to hydrolysis of ATP to ADP. From mitochondrial swelling measurements, half-maximal inhibitory values (K
i) of 4.5 and 98 μM were obtained for ADP and ATP, respectively. In addition, a delayed mitochondrial swelling sensitive to
higher ADP concentrations was observed. Mitochondrial anoxia/reoxygenation did not interfere with the inhibitory effect of
ADP on Ca2+-induced MPT, but oxidative phosphorylation markedly decreased this effect. We conclude that ADP is a potent inhibitor of
brain MPT whereas ATP is a weaker inhibitor of this phenomenon. Our results suggest that ADP can have an important protective
role against MPT-mediated tissue damage under conditions of brain ischemia and hypoglycemia. 相似文献
3.
Resveratrol, a natural polyphenolic antioxidant, has been reported to possess the cancer chemopreventive potential in wide
range by means of triggering tumor cells apoptosis through various pathways. It induced apoptosis through the activation of
the mitochondrial pathway in some kinds of cells. In the present reports, we showed that resveratrol-induced HepG2 cell apoptosis
and mitochondrial dysfunction was dependent on the induction of the mitochondrial permeability transition (MPT), because resveratrol
caused the collapse of the mitochondrial membrane potential (ΔΨm) with the concomitant release of cytochrome c (Cyt.c). In addition, resveratrol induced a rapid and sustained elevation of
intracellular [Ca2+], which compromised the mitochondrial ΔΨm and triggered the process of HepG2 cell apoptosis. In permeabilized HepG2 cells, we further demonstrated that the effect
of the resveratrol was indeed synergistic with that of Ca2+ and Ca2+ is necessary for resveratrol-induced MPT opening. Calcium-induced calcium release from mitochondria (mCICR) played a key
role in mitochondrial dysfunction and cell apoptosis: (1) mCICR inhibitor, ruthenium red (RR), prevent MPT opening and Cyt.c
release; and (2) RR attenuated resveratrol-induced HepG2 cell apoptotic death. Furthermore, resveratrol promotes MPT opening
by lowering Ca2+-threshold. These data suggest modifying mCICR and Ca2+ threshold to modulate MPT opening may be a potential target to control cell apoptosis induced by resveratrol.
Xuemei Tian—Foundation item: Chinese National Natural Science Foundation (No.30300455). 相似文献
4.
Carlos Velez-Pardo Marlene Jimenez-Del-Rio Silvia Lores-Arnaiz Juanita Bustamante 《Neurochemical research》2010,35(9):1323-1332
The effects of cannabinoids in mitochondria after acute oxidative stress insult are not fully established. We investigated
the ability of CP55,940 and JWH-015 to scavenge reactive oxygen species and their effect on mitochondria permeability transition
(MPT) in either a mitochondria-free superoxide anion generation system, intact rat brain mitochondria or in sub-mitochondrial
particles (SMP) treated with paraquat (PQ). Oxygen consumption, mitochondrial membrane potential (Δψm) and MPT were determined as parameters of mitochondrial function. It is found that both cannabinoids effectively attenuate
mitochondrial damage against PQ-induced oxidative stress by scavenging anion superoxide radical (O2
∙−) and hydrogen peroxide (H2O2), maintaining Δψm and by avoiding Ca2+-induced mitochondrial swelling. Understanding the mechanistic action of cannabinoids on mitochondria might provide new insights
into more effective therapeutic approaches for oxidative stress related disorders. 相似文献
5.
McCranor BJ Bozym RA Vitolo MI Fierke CA Bambrick L Polster BM Fiskum G Thompson RB 《Journal of bioenergetics and biomembranes》2012,44(2):253-263
The role of zinc ion in cytotoxicity following ischemic stroke, prolonged status epilepticus, and traumatic brain injury remains
controversial, but likely is the result of mitochondrial dysfunction. We describe an excitation ratiometric fluorescence biosensor
based on human carbonic anhydrase II variants expressed in the mitochondrial matrix, permitting free zinc levels to be quantitatively
imaged therein. We observed an average mitochondrial matrix free zinc concentration of 0.2 pM in the PC12 rat pheochromacytoma cell culture line. Cytoplasmic and mitochondrial free zinc levels were imaged in a cellular
oxygen glucose deprivation (OGD) model of ischemia/reperfusion. We observed a significant increase in mitochondrial zinc 1 h
following 3 h OGD, at a time point when cytosolic zinc levels were depressed. Following the increase, mitochondrial zinc levels
returned to physiological levels, while cytosolic zinc increased gradually over a 24 h time period in viable cells. The increase
in intramitochondrial zinc observed during reoxygenation after OGD may contribute to bioenergetic dysfunction and cell death
that occurs with both in vitro and in vivo models of reperfusion. 相似文献
6.
Tsujimoto Y Shimizu S 《Apoptosis : an international journal on programmed cell death》2007,12(5):835-840
In recent years, the role of the mitochondria in both apoptotic and necrotic cell death has received considerable attention.
An increase of mitochondrial membrane permeability is one of the key events in apoptotic or necrotic death, although the details
of the mechanism involved remain to be elucidated. The mitochondrial membrane permeability transition (MPT) is a Ca2+-dependent increase of mitochondrial membrane permeability that leads to loss of Δψ, mitochondrial swelling, and rupture of
the outer mitochondrial membrane. The MPT is thought to occur after the opening of a channel that is known as the permeability
transition pore (PTP), which putatively consists of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocator
(ANT), cyclophilin D (Cyp D: a mitochondrial peptidyl prolyl-cis, trans-isomerase), and other molecule(s). Recently, significant progress has been made by studies performed with mice lacking Cyp
D at several laboratories, which have convincingly demonstrated that Cyp D is essential for the MPT to occur and that the
Cyp D-dependent MPT regulates some forms of necrotic, but not apoptotic, cell death. Cyp D-deficient mice have also been used
to show that the Cyp D-dependent MPT plays a crucial role in ischemia/reperfusion injury. The anti-apoptotic proteins Bcl-2
and Bcl-xL have the ability to block the MPT, and can therefore block MPT-dependent necrosis in addition to their well-established ability
to inhibit apoptosis. 相似文献
7.
Umberto De Marchi Lucia Biasutto Spiridione Garbisa Antonio Toninello Mario Zoratti 《BBA》2009,1787(12):1425-1432
The Ca2+- and oxidative stress-induced mitochondrial permeability transition (MPT) plays an important role in phenomena ranging from tissue damage upon infarction to muscle wasting in some forms of dystrophy. The process is due to the activation of a large pore in the inner mitochondrial membrane. Anti-oxidants are considered a preventive and remedial tool, and mitochondria-targeted redox-active compounds have been developed. Plant polyphenols are generally considered as anti-oxidants, and thus candidates to the role of mitochondria-protecting agents. In patch-clamp experiments, easily oxidizable polyphenols induced closure of the MPT channel. In swelling experiments with suspensions of mitochondria, high (20–50 μM) concentrations of quercetin, the most efficient inhibitor, promoted instead the onset of the MPT. Chelators of Fe2+/3+ and Cu+/2+ ions counteracted this effect. Fluorescent indicators of superoxide production confirmed that quercetin potentiates O2? generation by isolated mitochondria and cultured cells. Since this was not affected by chelating Fe and Cu ions, the MPT-inducing effect can be ascribed to a “secondary”, metal ion-catalyzed production of ROS. These results are a direct demonstration of the ambivalent redox character of polyphenols. Their mode of action in vivo cannot be taken for granted, but needs to be experimentally verified. 相似文献
8.
Mitochondrial Dysfunction in the Pathogenesis of Necrotic and Apoptotic Cell Death 总被引:18,自引:0,他引:18
Lemasters JJ Qian T Bradham CA Brenner DA Cascio WE Trost LC Nishimura Y Nieminen AL Herman B 《Journal of bioenergetics and biomembranes》1999,31(4):305-319
Mitochondria are frequently the target of injury after stresses leading to necrotic and apoptoticcell death. Inhibition of oxidative phosphorylation progresses to uncoupling when opening ofa high conductance permeability transition (PT) pore in the mitochondrial inner membraneabruptly increases the permeability of the mitochondrial inner membrane to solutes of molecularmass up to 1500 Da. Cyclosporin A (CsA) blocks this mitochondrial permeability transition(MPT) and prevents necrotic cell death from oxidative stress, Ca2+ ionophore toxicity,Reye-related drug toxicity, pH-dependent ischemia/reperfusion injury, and other models of cell injury.Confocal fluorescence microscopy directly visualizes onset of the MPT from the movementof green-fluorescing calcein into mitochondria and the simultaneous release from mitochondriaof red-fluorescing tetramethylrhodamine methylester, a membrane potential-indicatingfluorophore. In oxidative stress to hepatocytes induced by tert-butylhydroperoxide, NAD(P)Hoxidation, increased mitochondrial Ca2+, and mitochondrial generation of reactive oxygen speciesprecede and contribute to onset of the MPT. Confocal microscopy also shows directly thatthe MPT is a critical event in apoptosis of hepatocytes induced by tumor necrosis factor-.Progression to necrotic and apoptotic cell killing depends, at least in part, on the effect theMPT has on cellular ATP levels. If ATP levels fall profoundly, necrotic killing ensues. If ATPlevels are at least partially maintained, apoptosis follows the MPT. Cellular features of bothapoptosis and necrosis frequently occur together after death signals and toxic stresses. A newterm, necrapoptosis, describes such death processes that begin with a common stress or deathsignal, progress by shared pathways, but culminate in either cell lysis (necrosis) or programmedcellular resorption (apoptosis) depending on modifying factors such as ATP. 相似文献
9.
V. Battaglia S. Grancara J. Satriano S. Saccoccio E. Agostinelli A. Toninello 《Amino acids》2010,38(2):431-437
The arginine metabolite agmatine is able to protect brain mitochondria against the drop in energy capacity by the Ca2+-dependent induction of permeability transition (MPT) in rat brain mitochondria. At normal levels, the amine maintains the
respiratory control index and ADP/O ratio and prevents mitochondrial colloid-osmotic swelling and any electrical potential
(ΔΨ) drop. MPT is due to oxidative stress induced by the interaction of Ca2+ with the mitochondrial membrane, leading to the production of hydrogen peroxide and, subsequently, other reactive oxygen
species (ROS) such as hydroxyl radicals. This production of ROS induces oxidation of sulfhydryl groups, in particular those
of two critical cysteines, most probably located on adenine nucleotide translocase, and also oxidation of pyridine nucleotides,
resulting in transition pore opening. The protective effect of agmatine is attributable to a scavenging effect on the most
toxic ROS, i.e., the hydroxyl radical, thus preventing oxidative stress and consequent bioenergetic collapse. 相似文献
10.
Bambrick LL Chandrasekaran K Mehrabian Z Wright C Krueger BK Fiskum G 《Journal of bioenergetics and biomembranes》2006,38(1):43-47
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
11.
The mitochondrial permeability transition (MPT) initiated by reactive oxygen species (ROS) plays an essential role in ischemia–reperfusion (IR) injury. Iron is a critical catalyst for ROS formation, and intracellular chelatable iron promotes oxidative injury-induced and MPT-dependent cell death in hepatocytes. Accordingly, our aim was to investigate the role of chelatable iron in IR-induced ROS generation, MPT formation, and cell death in primary rat hepatocytes. To simulate IR, overnight-cultured hepatocytes were incubated anoxically at pH 6.2 for 4 h and reoxygenated at pH 7.4. Chelatable Fe2+, ROS, and mitochondrial membrane potential were monitored by confocal fluorescence microscopy of calcein, chloromethyldichlorofluorescein, and tetramethylrhodamine methyl ester, respectively. Cell killing was assessed by propidium iodide fluorimetry. Ischemia caused progressive quenching of cytosolic calcein by more than 90%, signifying increased chelatable Fe2+. Desferal and starch–desferal 1 h before ischemia suppressed calcein quenching. Ischemia also induced quenching and dequenching of calcein loaded into mitochondria and lysosomes, respectively. Desferal, starch–desferal, and the inhibitor of the mitochondrial Ca2+ uniporter (MCU), Ru360, suppressed mitochondrial calcein quenching during ischemia. Desferal, starch–desferal, and Ru360 before ischemia also decreased mitochondrial ROS formation, MPT opening, and cell killing after reperfusion. These results indicate that lysosomes release chelatable Fe2+ during ischemia, which is taken up into mitochondria by MCU. Increased mitochondrial iron then predisposes to ROS-dependent MPT opening and cell killing after reperfusion. 相似文献
12.
Oliveira KA Zecchin KG Alberici LC Castilho RF Vercesi AE 《Journal of bioenergetics and biomembranes》2008,40(4):307-314
In the present study we analyzed the mechanisms of simvastatin toxicity for the PC3 human prostate cancer cell line. At 10
μM, simvastatin induced principally apoptosis, which was prevented by mevalonic acid but not by cyclosporin A, the inhibitor
of calcineurin and mitochondrial permeability transition (MPT). At 60 μM, simvastatin induced the necrosis of PC3 cells insensitive
to mevalonic acid. Cell necrosis was preceded by a threefold increase in cytosolic free Ca2+ concentration and a significant decrease in both respiration rate and mitochondrial membrane potential. Both mitochondrial
dysfunction and necrosis were sensitive to the compounds cyclosporin A and bongkrekic acid, as well as the calcineurin inhibitor
FK506. We have concluded that simvastatin-induced PC3 cells apoptosis is dependent on 3-hydroxy-3-methylglutaryl coenzyme-A
reductase inhibition and independent of MPT, whereas necrosis is dependent on mitochondrial dysfunction caused, at least in
part, by calcineurin. 相似文献
13.
Xiaolei Shi Hikaru Osaki Yoshihiro Matsunomoto Chisako Fujita Daisuke Shinohe Naoko Ashida Hyunjin Choi Yoshihiro Ohta 《Biochemistry and Biophysics Reports》2016
Mitochondrial permeability transition (MPT) is thought to determine cell death under oxidative stress. However, MPT inhibitors only partially suppress oxidative stress-induced cell death. Here, we demonstrate that cells in which MPT is inhibited undergo cell death under oxidative stress. When C6 cells were exposed to 250 μM t-butyl hydroperoxide (t-BuOOH), the loss of a membrane potential-sensitive dye (tetramethylrhodamine ethyl ester, TMRE) from mitochondria was observed, indicating mitochondrial depolarization leading to cell death. The fluorescence of calcein entrapped in mitochondria prior to addition of t-BuOOH was significantly decreased to 70% after mitochondrial depolarization. Cyclosporin A suppressed the decrease in mitochondrial calcein fluorescence, but not mitochondrial depolarization. These results show that t-BuOOH induced cell death even when it did not induce MPT. Prior to MPT, lactate production and respiration were hampered. Taken together, these data indicate that the decreased turnover rate of glycolysis and mitochondrial respiration may be as vital as MPT for cell death induced under moderate oxidative stress. 相似文献
14.
A.P. Halestrap C.P. Connern E.J. Griffiths P.M. Kerr 《Molecular and cellular biochemistry》1997,174(1-2):167-172
When loaded with high (pathological) levels of Ca2+, mitochondria become swollen and uncoupled as the result of a large non-specific increase in membrane permeability. This process, known as the mitochondrial permeability transition (MPT), is exacerbated by oxidative stress and adenine nucleotide depletion. These conditions match those that a heart experiences during reperfusion following a period of ischaemia. The MPT is caused by the opening of a non-specific pore that can be prevented by sub-micromolar concentrations of cyclosporin A (CsA). A variety of conditions that increase the sensitivity of pore opening to [Ca2+], such as thiol modification, oxidative stress, increased matrix volume and chaotropic agents, all enhance the binding of matrix cyclophilin (CyP) to the inner mitochondrial membrane in a CsA-sensitive manner. In contrast, ADP, membrane potential and low pH decrease the sensitivity of pore opening to [Ca2+] without affecting CyP binding. We present a model of pore opening involving CyP binding to a membrane target protein followed by Ca2+-dependent triggering of a conformational change to induce channel opening. Using the ischaemic/reperfused rat heart we have shown that the mitochondrial pore does not open during ischaemia, but does do so during reperfusion. Recovery of heart during reperfusion is improved in the presence of 0.2 µM CsA, suggesting that the MPT may be critical in the transition from reversible to irreversible reperfusion injury. (Mol Cell Biochem 174: 167–172, 1997) 相似文献
15.
Dysregulation of Ca2+ has long been implicated to be important in cell injury. A Ca2+-linked process important in necrosis and apoptosis (or necrapoptosis) is the mitochondrial permeability transition (MPT). In the MPT, large conductance permeability transition (PT) pores open that make the mitochondrial inner membrane abruptly permeable to solutes up to 1500 Da. The importance of Ca2+ in MPT induction varies with circumstance. Ca2+ overload is sufficient to induce the MPT. By contrast after ischemia-reperfusion to cardiac myocytes, Ca2+ overload is the consequence of bioenergetic failure after the MPT rather than its cause. In other models, such as cytotoxicity from Reye-related agents and storage-reperfusion injury to liver grafts, Ca2+ appears to be permissive to MPT onset. Lastly in oxidative stress, increased mitochondrial Ca2+ and ROS generation act synergistically to produce the MPT and cell death. Thus, the exact role of Ca2+ for inducing the MPT and cell death depends on the particular biologic setting. 相似文献
16.
Stirpe A Rizzuti B Pantusa M Bartucci R Sportelli L Guzzi R 《European biophysics journal : EBJ》2008,37(8):1351-1360
There is growing evidence that metal ions can accelerate the aggregation process of several proteins. This process, associated
with several neuro-degenerative diseases, has been reported also for non-pathological proteins. In the present work, the effects
of copper and zinc ions on the denaturation and aggregation processes of β-lactoglobulin A (BLG-A) are investigated by differential
scanning calorimetry (DSC), fluorescence, electron paramagnetic resonance (EPR) and optical density. The DSC profiles reveal
that the thermal behaviour of BLG-A is a complex process, strongly dependent on the protein concentration. For concentrations
≤0.13 mM, the thermogram shows an endothermic peak at 84.3°C, corresponding to denaturation; for concentrations >0.13 mM an
exothermic peak also appears, above 90°C, related to the aggregation of the denaturated BLG-A molecules. The thioflavin T
fluorescence indicates that the thermally induced aggregates show fibrillar features. The presence of either equimolar Cu2+ or Zn2+ ions in the protein solution has different effects. In particular, copper binds to the protein in the native state, as evidenced
by EPR experiments, and destabilizes BLG-A by decreasing the denaturation temperature by about 10°C, whereas zinc ions probably
perturb the partially denaturated state of the protein. The kinetics of BLG-A aggregation shows that both metal ions abolish
the lag phase before the aggregation starts. Moreover, the rate of the process is 4.6-fold higher in the presence of copper,
whereas the effect of zinc is negligible. The increase of the aggregation rate, induced by copper, may be due to a site-specific
binding of the metal ion on the protein. 相似文献
17.
《Cryobiology》2018
When the mitochondria of somatic cells are exposed to pathological calcium overload, these trigger mitochondrial permeability transition (MPT) leading to mitochondrial dysfunction and cell death. Cryopreservation procedures expose mammalian spermatozoa to physical and chemical stressors, which affect plasma membrane integrity and induce a pathological calcium overload that gradually promotes loss of sperm quality and ultimately function. Although several studies highlight the role of calcium in many physiological and pathological processes, the MPT induced by an intracellular calcium increase and its effect on the cell quality of mammalian spermatozoa are unknown. The aim of this study was to evaluate the effects of cryopreservation on MPT and its relationship with the deterioration of sperm quality in a bovine model. To do this, frozen bovine spermatozoa were thawed and adjusted to 2 × 106 mL−1 and incubated for 4 h at 38 °C. Using flow cytometry, we evaluated MPT by the calcein-AM and cobalt chloride method, intracellular Ca2+ level using FLUO3-AM, plasma membrane integrity by exclusion of propidium iodide, mitochondrial membrane potential (ΔΨm) with tetramethylrhodamine methyl ester perchlorate and intracellular ROS production with dihydroethidium. ATP levels were assessed by a chemiluminiscent method. The results showed that thawed spermatozoa trigger MPT associated with an intracellular calcium increase and that this was accompanied by ΔΨm dissipation, decrease of ATP levels and ROS production, and deterioration of plasma membrane integrity. In conclusion, cryopreservation induces MPT and this is associated with a loss of sperm quality. 相似文献
18.
Jan B. Hoek Elisabeth Walajtys-Rode Xiaolan Wang 《Molecular and cellular biochemistry》1997,174(1-2):173-179
Ca2+ functions as an intracellular signal to transfer hormonal messages to different cellular compartments, including mitochondria, where it activates intramitochondrial Ca2+-dependent enzymes. However, excessive mitochondrial Ca2+ uptake can promote the mitochondrial permeability transition (MPT), a process known to be associated with cell injury. The factors controlling mitochondrial Ca2+ uptake and release in intact cells are poorly understood. In this paper, we investigate mitochondrial Ca2+ accumulation in intact hepatocytes in response to the elevation of cytosolic Ca2+ levels ([Ca2+]c) induced either by a hormonal stimulus (vasopressin), or by thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump. After stimulation, cells were rapidly permeabilized for the determination of the mitochondrial Ca2+ content (Ca2+_m) and to analyze the susceptibility of the mitochondria to undergo the MPT. Despite very similar levels of [Ca2+]c elevation, vasopressin and thapsigargin had markedly different effects on mitochondrial Ca2+ accumulation. Vasopressin caused a rapid (< 90 sec), but modest (< 2 fold) increase in Ca2+m that was not further increased during prolonged incubations, despite a sustained [Ca2+]c elevation. By contrast, thapsigargin induced a net Ca2+ accumulation in mitochondria that continued for up to 30 min and reached Ca2+_m levels 10–20 fold over basal. Accumulation of mitochondrial Ca2+ was accompanied by a markedly increased susceptibility to undergo the MPT. Both mitochondrial Ca2+ accumulation and MPT activation were modulated by treatment of the cells with inhibitors of protein kineses and phosphatases. The results indicate that net mitochondrial Ca2+ uptake in response to hormonal stimulation is regulated by processes that depend on protein kinase activation. These controls are inoperative when the cytosol is flooded by Ca2+ through artificial means, enabling mitochondria to function as a Ca2+ sink under these conditions. (Mol Cell Biochem 174: 173–179, 1997) 相似文献
19.
Chartchalerm Isarankura-Na-Ayudhya Tanawut Tantimongcolwat Hans-Joachim Galla Virapong Prachayasittikul 《Biological trace element research》2010,134(3):352-363
In the present study, spectroscopic determinations of copper ions using chimeric metal-binding green fluorescent protein (His6GFP)
as an active indicator have been explored. Supplementation of copper ions to the GFP solution led to a remarkable decrease
of fluorescent intensity corresponding to metal concentrations. For circumstances, rapid declining of fluorescence up to 60%
was detected in the presence of 500 μM copper. This is in contrast to those observed in the case of zinc and calcium ions,
in which approximately 10–20% of fluorescence was affected. Recovery of its original fluorescence up to 80% was mediated by
the addition of ethylenediamine tetraacetic acid. More importantly, in the presence of metal ions, the emission wavelength
maximum remains unchanged while reduction of the optical density of the absorption spectrum has been observed. This indicates
that the chromophore’s ground state was possibly affected by the static quenching process. Results from circular dichroism
measurements revealed that the overall patterns of circular dichroism spectra after exposure to copper ions were not significantly
different from that of the control, where the majority of sharp positive band around 195–196 nm in combination with a broad
negative deflection around 215–216 nm was obtained. Taken together, it can be presumed that copper ions exerted their static
quenching on the fluorescence rather than structural or conformational alteration. However, notification has to be made that
some peptide rearrangements may also occur in the presence of metal ions. Further studies were conducted to investigate the
feasibility of using the His6GFP as a sensing unit for copper ions. The His6GFP was encapsulated in Sol-gel and immobilized
onto the optical fiber connected with a fluorescence detecting device. The Sol-gel was doped into the metal solution where
the quenching of fluorescence could be monitored in real time. The sensing unit provided a high sensitivity of detection in
the range of 0.5 μM to 50 mM with high selectivity for copper ions. All these findings open up a high potential to apply the
fluorescent protein-based bioanalytical tool for copper determination in the future. 相似文献
20.
Posterior gills (No. 7 and 8) of shore crabsCarcinus maenas were homogenized and fractionated by means of differential and density gradient centrifugation. Employment of marker enzymes
Na-K-ATPase and carbonic anhydrase for plasma membranes and cytochrome oxidase for mitochondria showed that these structural
elements were separated. Ultramicroscopic investigations of combined fractions confirmed the presence of the respective mitochondrial
and vesicular plasma membrane structures. An ATPase which did not depend on the presence of sodium (20 mM) ions in the incubation
medium but on the presence of potassium (20 mM) ions only was found in the mitochondrial fractions. The mitochondrial ATPase
was tightly bound to cellular particulates and activated approximately threefold by bicarbonate (20 mM) ions. The activity
of this ATPase was nearly completely inhibited by oligomycin (1 μg ml−1) and greatly inhibited by low levels (5 mM) of thiocyanate and calcium ions, the Ki for Ca2+ being ca 4 mM. The results obtained confirm literature data on high mitochondrial densities in crab gills and allow the assumption
of significant rates of energy metabolism in these organs. Considering its properties the mitochondrial ATPase is clearly
distinct from crab gill Na-K-ATPase and can be measured specifically in samples containing Na-K-ATPase. Mitochondrial ATPase
is therefore considered a suitable and reliable marker enzyme for mitochondria. 相似文献