Integrity of mitochondrial membranes

See also Electron Transport, P/O Ratio, Chemiosmotic Coupling, Integrity of Mitochondrial Membranes, Uncoupling ETS and Oxidative Phosphorylation, The FIFO Complex, Oxidation as a Metabolic Energy Source (from Chapter 12)... 

See also Integrity of Mitochondrial Membranes, Uncoupling ETS and Oxidative Phosphorylation, Figure 15.15... 

See also The FOFl Complex, Integrity of Mitochondrial Membranes, Chemiosmotic Coupling... 

The well-known fact that in irreversibly damaged cells, respiratory control is lost and is accompanied by oxidation of cytochromes a and as, as well as NADH (Taegtmeyer et al., 1985), was originally thoug it to be due to substrate deficiency (Chance and Williams, 1955) but may be due to an enzymatic defect resulting in an inability to metabolize NADH-linked substrates (Pelican etal., 1987). It seems likely therefore that return of function is dependent on preservation of mitochondrial membrane integrity, and the structure and activities of respiratory chain (R.C) complexes I-IV (Chance and Williams, 1955). 

Much progress has been made in understanding the different mechanisms that can cause mitochondrial dysfunction, such as (i) uncoupling of electron transport from ATP synthesis by undermining integrity of inner membrane (ii) direct inhibition of electron transport system components (iii) opening of the mitochondrial permeability transition pore leading to irreversible collapse of the transmembrane potential and release of pro-apoptotic factors (iv) inhibition of the... 

Haferkamp I, Hackstein JHP, Voncken FGJ, Schmit G, Tjaden J (2002) Functional integration of mitochondrial and hydrogenosomal ADP/ATP carriers in the Escherichia coli membrane reveals different biochemical characteristics for plants, mammals and anaerobic chytrids. Eur J Biochem 269 3172-3181... 

An example of the use of a highly specialised cell type to study targeted toxic effects on the cellular metabolism is the recently developed boar spermatozoon motility inhibition test (Andersson et al., 1998). The motility of a spermatozoon depends on the integrity of mitochondrial functions, and thus the action of toxins affecting the energy metabolism is very rapidly detected as reduction of motility. Other end points that can be measured are plasma membrane integrity, astrodome function, and total cellular ATP and NAD reduction. This test has been particularly useful in the detection of certain types of bacterial toxins from various enviromnental and food sources. 

Peroxidation of lipid molecules invariably changes or damages lipid molecular structure. In addition to the self-destructive nature of membrane lipid peroxidation, the aldehydes that are formed can cross-link proteins. When the damaged lipids are the constituents of biologic membranes, the cohesive lipid bilayer arrangement and stable structural organization is disrupted (see Fig. 24.7). Disruption of mitochondrial membrane integrity may result in further free radical production. 

Monoamine oxidases are integral outer mitochondrial membrane proteins that catalyze the oxidative deamination of primary and secondary amines as well as some tertiary amines. MAO occurs as two enzymes, MAO-A and MAO-B, which differ in substrate selectivity and inhibitor sensitivity (Abell and Kwan, 2001 Edmondson et al., 2004 Shih et al., 1999). A number of MAO inhibitors have been developed for clinical use as antidepressants and as neuroprotective drugs. Clinically used drug substances include, among others, moclobemide, a relatively selective reversible MAO-A inhibitor, and L-deprenyl, an irreversible selective inhibitor of MAO-B. In vitro, clorgyline and L-deprenyl are used as selective irreversible inhibitors of MAO-A and B, respectively. (Note For in vitro studies using irreversible inhibitors, preincubation of the irreversible inhibitor with the enzyme prior to initiation of the substrate reaction is required for optimal inhibition.) Expressed MAO-A and MAO-B are not readily available via commercial resources however, MAO-A and MAO-B have been evaluated and are active in subcellular fractions. While monoamine oxidases are located in the mitochondria, many microsomal preparations are contaminated with monoamine oxidases during the preparation of the microsomal subcellular fraction and thus microsomes are sometimes used to evaluate monoamine oxidase activity in combination with selective inhibitors. 

In response to ischemia and reperfusion, mitochondrial membranes undergo depolarization and elevated levels of Ca in the intermembrane space. The control of mitochondrial membrane depolarization depends, in part, on Bcl-2 family member proteins. The proapoptotic group of Bcl-2 members consists of the Bax-subfamily (Bax, Bak, and Bok) and the BH3-only proteins (Bid, Bim, Bik, Bad, Bmf, Hrk, Noxa, Puma, Blk, BNIP3, and Spike) (Cory and Adams, 2002 Mund et al., 2003). It appears that the main function of the Bcl-2 family proteins is to guard mitochondrial integrity and to control the release of mitochondrial proteins into the cytoplasm (Cory and Adams, 2002). It is believed that the proapoptotic Bax and Bak provoke or contribute to the perme-abilization of the outer mitochondrial membrane, either by forming channels by themselves (Antonsson et al., 2000) or by interacting with components such as VDAC (Tsujimoto and Shimizu, 2000). 

Mitochondria play a central role in response to apoptotic stimuli. There is increasing evidence that altered mitochondrial function is linked to apoptosis and a decreasing mitochondrial transmembrane potential ( Pm) is associated with mitochondria dysfunction. The MPT (mitochondria permeability transition) is a permeability increase of the mitochondria membrane coupled with depolarization of the membrane and disruption of mitochondrial membrane integrity. We measured A Pm using a fluorescent probe DiOC6(3) which specially accumulated in polarized membranes and was monitored by flow... 

In yeast, it is possible to selectively synthesize either the cyto-plasmically or mitochondrially derived subunits of the ATPase by using site-specific inhibitors or protein synthesis. When cells are exposed to a drug that specifically inhibits the synthesis of Fi and OSCP, the hydro-phobic proteins of the membrane unit continue to be made and inserted into the membrane. The proper integration of the membrane unit of the ATPase in the drug-uncoupled system can be deduced from the observation... 

The studies show that if the cell unsaturated fatty acid content is low enough, the assembly of the mitochondrial membrane is profoundly affected. We have concentrated mainly on the mitochondrially synthesized components, but the mitochondrial level of cytochrome c, which is synthesized in the cytoplasm, was also reduced under lipid-depleted conditions. Since petite mutant cells, which lack a mitochondrial protein-synthesizing system, contain normal levels of cytochrome c when derepressed (Marzuki and Linnane, unpublished observation), this effect cannot be due to a simple feedback mechanism which coordinates the synthesis of cytoplasmically synthesized cytochrome components with mitochondrially synthesized components. More likely, it is a reflection of the altered physical structure of the membrane under these conditions, which may not allow the integration of cytochrome c into the membrane. This system offers great potential for resolving some of the problems of the mechanism of mitochondrial membrane assembly. 

Complex II is perhaps better known by its other name—succinate dehydrogenase, the only TCA cycle enzyme that is an integral membrane protein in the inner mitochondrial membrane. This enzyme has a mass of approximately 100 to 140 kD and is composed of four subunits two Fe-S proteins of masses 70 kD and 27 kD, and two other peptides of masses 15 kD and 13 kD. Also known as flavoprotein 2 (FP2), it contains an FAD covalently bound to a histidine residue (see Figure 20.15), and three Fe-S centers a 4Fe-4S cluster, a 3Fe-4S cluster, and a 2Fe-2S cluster. When succinate is converted to fumarate in the TCA cycle, concomitant reduction of bound FAD to FADHg occurs in succinate dehydrogenase. This FADHg transfers its electrons immediately to Fe-S centers, which pass them on to UQ. Electron flow from succinate to UQ,... 

The mechanism of ATP synthesis discussed here assumes that protons extruded during electron transport are in the bulk phase surrounding the inner mitochondrial membrane (intermembrane and extramitochondrial spaces). An alternative view is that there are local proton circuits within or close to the respiratory chain and complex V, and that these protons may not be in free equilibrium with the bulk phase (Williams, 1978), although this has not been supported experimentally (for references see Nicholls and Ferguson, 1992). The chemiosmotic mechanism is both elegant and simple and explains all the known facts about ATP synthesis and its dependence on the structural integrity of the mitochondria, although the details may appear complex. This mechanism will now be discussed in more detail. 

Henry, M.A., Nodes, E.E., Gao, D., Mazur, P., Critser. J.K. (1993). Cryopreservation of human spermatozoa. IV. The effects of cooling rate and warming rate on the maintenance of motility, plasma membrane integrity, and mitochondrial function. Fertil. and Steril. 60,911-918. 

Rats fed a purified nonlipid diet containing vitamins A and D exhibit a reduced growth rate and reproductive deficiency which may be cured by the addition of linoleic, a-linolenic, and arachidonic acids to the diet. These fatty acids are found in high concentrations in vegetable oils (Table 14-2) and in small amounts in animal carcasses. These essential fatty acids are required for prostaglandin, thromboxane, leukotriene, and lipoxin formation (see below), and they also have various other functions which are less well defined. Essential fatty acids are found in the stmctural lipids of the cell, often in the 2 position of phospholipids, and are concerned with the structural integrity of the mitochondrial membrane. 

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