Mitochondria potential

Besides multiple staining protocols, a mitochondria potential assay was applied to further assess the toxicity, which was able to rapidly measure the effect of compound on mitochondria potential. After cells were treated with compounds for 60 min, the mitochondria-active dye JC-10 was added for 15-30 min incubation with reagents. In addition, the assay was able to test the effects on inducing autophagy and phospholipidosis after treating the cell with compounds for 24 and 48 h, respectively. The method is compatible for screening of hundreds of compounds within 72 h. 

Mitochondria are distinct organelles with two membranes. The outer membrane limits the organelle and the inner membrane is thrown into folds or shelves that project inward and are called cristae mitochondriales. The uptake of most mitochondrion-selective dyes is dependent on the mitochondrial membrane potential. Conventional fluorescent stains for mitochondria, such as rhodamine and tetramethylrosamine, are readily sequestered by functioning mitochondria. They are, however, subsequently washed out of the cells once the mitochondrion s membrane potential is lost. This characteristic limits their use in experiments in which cells must be treated with aldehyde-based fixatives or other agents that affect the energetic state of the mitochondria. To overcome this limitation, the research... 

The sequence of the carriers in the chain is shown in Figure 9.6. Each of the components of the chain reduces the next, in sequence, according to the redox potential (Table 9.3). The enzymes and their prosthetic groups are organised into complexes, which can be isolated by gentle disruption of the whole mitochondrion or its inner membrane. Ubiqui-... 

Oxidizible substrates from glycolysis, fatty acid or protein catabolism enter the mitochondrion in the form of acetyl-CoA, or as other intermediaries of the Krebs cycle, which resides within the mitochondrial matrix. Reducing equivalents in the form of NADH and FADH pass electrons to complex I (NADH-ubiquinone oxidore-ductase) or complex II (succinate dehydrogenase) of the electron transport chain, respectively. Electrons pass from complex I and II to complex III (ubiquinol-cyto-chrome c oxidoreductase) and then to complex IV (cytochrome c oxidase) which accumulates four electrons and then tetravalently reduces O2 to water. Protons are pumped into the inner membrane space at complexes I, II and IV and then diffuse down their concentration gradient through complex V (FoFi-ATPase), where their potential energy is captured in the form of ATP. In this way, ATP formation is coupled to electron transport and the formation of water, a process termed oxidative phosphorylation (OXPHOS). 

The internal [Na+] and [Ca2+] are both low, while the internal [K+] is high. These differences are also linked to the membrane potential (Eq. 8-2), which is ordinarily expressed as a negative voltage of the interior of a cell, mitochondrion, plastid, etc. with respect to a reference electrode in the external medium. 

The mitochondrial membrane potential Em (or Ay) is the potential difference measured across a membrane relative to a reference electrode present in the surrounding solution.176 For both mitochondria and bacteria Em normally has a negative value. The Gibbs energy change AyH+ for transfer of one mole of H+ from the inside of the mitochondrion to the outside, against... 

Let s now consider how much free energy is released by moving protons into the mitochondrion. Is it really enough to drive the synthesis of ATP The free energy change depends both on the ratio of the proton concentrations on the two sides of the membrane and on the difference between the electric potentials on the two sides. 

Calcium levels are believed to be controlled in part at least by the uptake and release of Ca2+ from mitochondria.172"174 The capacity of mitochondria for Ca2+ seems to be more than sufficient to allow the buffering of Ca2+ at low cytosol levels. Mitochondria take up Ca2+ by an energy-dependent process either by respiration or ATP hydrolysis. It is now agreed that Ca2+ enters in response to the negative-inside membrane potential developed across the inner membrane of the mitochondrion during respiration. The uptake of Ca2+ is compensated for by extrusion of two H+ from the matrix, and is mediated by a transport protein. Previous suggestions for a Ca2+-phosphate symport are now discounted. The possible alkalization of the mitochondrial matrix is normally prevented by the influx of H+ coupled to the influx of phosphate on the H - PCV symporter (Figure 10). This explains why uptake of Ca2+ is stimulated by phosphate. Other cations can also be taken up by the same mechanism. 

In the previous section we mentioned the ADP/ATP transporter, and it is also shown in Figure 17.2. This system allows the export of ATP and import of ADP. Because ATP and ADP have net charges of -4 and -3, respectively, at pH 7, the transport is not electroneutral and must occur at the expense of the membrane potential. Atractyloside and bongkrekic acid are inhibitors of this system, the former binding to the ADP binding site of the porter and the latter to the ATP site. Associated with ADP/ATP transport is the transport of P which must enter the mitochondrion to participate in ATP formation. Several systems for transport-... 

Supplementing the diet with carnitine may stimulate the uptake of long-chain fatty acids into affected cells. Fatty acid oxidation within the mitochondrion will generate acetyl-CoA, which when oxidized in the TCA cycle will produce NADH and FADH2 to feed into the ETC. Carnitine also shuttles potentially toxic fatty acid catabolic by-products out of the mitochondrial matrix to the kidney for excretion in the urine. 

The chemiosmotic model requires that flow of electrons through the electron-transport chain leads to extrusion of protons from the mitochondrion, thus generating the proton electrochemical-potential gradient. Measurements of the number of H+ ions extruded per O atom reduced by complex IV of the electron-transport chain (the H+/0 ratio) are experimentally important because the ratio can be used to test the validity of mechanistic models of proton translocation (Sec. 14.6). 

The translocator moves the two nucleotides in either direction. However, ATT is transported as ATP4- and ADP as ADP3-. Thus the equilibrium position of the exchange is dependent on the electrochemical potential difference across the membrane. When the electrical potential difference is 160 mV, the ratio of ATP/ADP in the medium outside the mitochondrion is 125/1. 

Moore then explained how mitochondria are biological fuel cells. The oxygen reduction taking place in a mitochondrion is exactly the same as in a standard fuel cell. Using several enzymes and only earth-abundant elements, the mitochondrion converts electrochemical potential to biochemical work with efficiency greater than 90 percent. This is a steady-state process in which protons are pumped across the membrane to maintain its electrical potential. If... 

The mitochondrion is the key cellular organelle responsible for transducing free energy from primary substrates into the ATP potential that drives the majority of energy-consuming processes in a cell. Thus the mitochondrion plays a central... 

It is apparent from this equation that the mitochondrion, with a membrane potential of up to 180 mV could, if a suitable pathway existed, accumulate divalent cations until equilibrium is attained with a 10 gradient of the free cation across the membrane. Mitochondria possess a pathway for divalent cation uniport which allows the passage of Ca, Sr, Ba and, at a lesser rate, Mn [40-42]. 

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