Mitochondria separation

Aconitase, an unstable enzyme,4 is concerned with the reversible conversion of cis-aconitate to either citric acid or isocitric acid. It may be noted that the entire system of tricarboxylic cycle enzymes are present in the mitochondria separated from cells, and, furthermore, it has been found that the mitochondrial enzymes differ from the isolated enzymes in that the former require no addition of D.P.N. (co-enzyme I) or T.P.N. (co-enzyme II) for activity. Peters suggests that the citrate accumulation is caused by the competitive reaction of the fluorocitrate with aconitase required for the conversion of citrate to isocitrate. This interference with the tricarboxylic acid... 

Somlyo There is another problem. We only have relatively low-resolution information about where these channels are. We can say that they are on the SR. But if you ask me about a 15 nm wide SR section, whether the InsP3 receptor is on the plasma-membrane-related side or not, I couldn t say. Or more to the point, where the SR is surrounding a mitochondrion, separated from it by about the same distance, I couldn t tell you whether the InsP3 release channels are adjacent and close to the mitochondrion or whether they are on the other surface, away from the mitochondrion. This will make a lot of difference whether the SR releases Ca2+ close to the mitochondrion or acts as a barrier protecting the mitochondrion from Ca2+. High-resolution information is not available. 

Fig. 2.6 The moqjhological events of sporulation in Saccharomyces cerevisiae. (a) starved cell V, vacuole LG, lipid granule ER, endoplasmic reticulum CW, cell wall M, mitochondrion S, spindle pole SM, spindle microtubules N, nucleus NO, nucleolus, (b) Synaptonemal complex (SX) and development of polycomplex body (PB) along with division of spindle pole body in (c). (d) First meiotic division which is completed in (e). (f) Prepararation for meiosis II. (g) Enlargement of prospore wall, culminating in enclosure of separate haploid nuclei (h). (i) Spore coat (SC) materials produced and deposited, giving rise to the distinct outer spore coat (OSC) seen in the completed spores of the mature ascus (j). Reproduced from the review by Dickinson (1988) with permission from Blackwell Science Ltd.

Fig. 7.5. A schematic indication of some of the different membrane separated compartments in an advanced cell. PEROX is a peroxisome MITOCHLORO is either a mitochondrion or a chloroplast CHROMO is a vesicle of, say, the chromaffin granule ENDO is a reticulum, e.g. the endoplasmic reticulum. Other compartments are lysosomes, vacuoles, calcisomes and so on. Localised metal concentrations are shown. The figure is of a transverse section. To appreciate a cell fully it is necessary to have serial plane sections in parallel along the. "-direction.

Figure 11.1 Ultrastructure of the human lung alveolar barrier. The tissue specimen is obtained via lung resection surgery. (A) Section through a septal wall of an alveolus. The wall is lined by a thin cellular layer formed by alveolar epithelial type I cells (ATI). Connective tissues (ct) separate ATI cells from the capillary endothelium (en) within which an erythrocyte (er) and granulocyte (gc) can be seen. The minimal distance between the alveolar airspace (ai) and erythrocyte is about 800-900 nm. The endothelial nucleus is denoted as n. (B) Details of the lung alveolar epithelial and endothelial barriers. Numerous caveolae (arrows) are seen in the apical and basal plasma membranes of an ATI cell as well as endothelial cell (en) membranes. Caveolae may partake transport of some solutes (e.g., albumin). (C) ATII cells (ATII) are often localised in the comers of alveoli where septal walls branch off. (D) ATII cells are characterised by numerous multilamellar bodies (mlb) which contain components of surfactant. A mitochondrion is denoted as mi.

This three-step process for transferring fatty acids into the mitochondrion—esterification to CoA, transesterification to carnitine followed by transport, and transesterification back to CoA—links two separate pools of coenzyme A and of fatty acyl-CoA, one in the cytosol, the other in mitochondria These pools have different functions. Coenzyme A in the mitochondrial matrix is largely used in oxidative degradation of pyruvate, fatty acids, and some amino acids, whereas cytosolic coenzyme A is used in the biosynthesis of fatty acids (see Fig. 21-10). Fatty acyl-CoA in the cytosolic pool can be used for membrane lipid synthesis or can be moved into the mitochondrial matrix for oxidation and ATP production. Conversion to the carnitine ester commits the fatty acyl moiety to the oxidative fate. 

FIGURE 19-1 Biochemical anatomy of a mitochondrion. The convolutions (cristae) of the inner membrane provide a very large surface area. The inner membrane of a single liver mitochondrion may have more than 10,000 sets of electron-transfer systems (respiratory chains) and ATP synthase molecules, distributed over the membrane surface. Heart mitochondria, which have more profuse cristae and thus a much larger area of inner membrane, contain more than three times as many sets of electron-transfer systems as liver mitochondria. The mitochondrial pool of coenzymes and intermediates is functionally separate from the cytosolic pool. The mitochondria of invertebrates, plants, and microbial eukaryotes are similar to those shown here, but with much variation in size, shape, and degree of convolution of the inner membrane. 

In eukaryotes, most of the reactions of aerobic energy metabolism occur in mitochondria. An inner membrane separates the mitochondrion into two spaces the internal matrix space and the intermembrane space. An electron-transport system in the inner membrane oxidizes NADH and succinate at the expense of 02, generating ATP in the process. The operation of the respiratory chain and its coupling to ATP synthesis can be summarized as follows ... 

Some proteins, especially those destined for the eukaryotic mitochondria and chloroplasts, are transported after their synthesis on free polysomes is complete. Such transport is known as posttranslational transport. In the case of posttranslational transport it is believed that the polypeptide to be transported must be unfolded from its native folded configuration by a system of polypeptide-chain-binding proteins (PCBs) before it can pass through the membrane. Posttranslational transport into the mitochondrion requires both ATP and a proton gradient. Presumably the energy from one or both of these sources is used to unfold the protein or separate it from the PCB system so that it can pass through the membrane. 

Most differences in elongation result from the fact that the eukaryotic cell has different compartments, which are separated by membranes. Both prokaryotic and eukaryotic cells, of course, have an inside and outside however, eukaryotic proteins can be targeted to, for example, the mitochondrion. 

The eukaryotic cell, as we have seen, is chock full of complex molecular machines tidily separated into many discrete compartments. The biggest compartment is the nucleus, which could be seen even with the crude microscopes of the seventeenth century. Smaller compartments were not discovered until improved microscopes became available in the later nineteenth and twentieth centuries. One of the smaller compartments is the mitochondrion. 

Answer The transport of fatty acid molecules into mitochondria requires a shuttle system involving a fatty acyl-carnitine intermediate. Fatty acids are first converted to fatty acyl-CoA molecules in the cytosol (by the action of acyl-CoA synthetases) then, at the outer mitochondrial membrane, the fatty acyl group is transferred to carnitine (by the action of carnitine acyl-transferase I). After transport of fatty acyl-carnitine through the inner membrane, the fatty acyl group is transferred to mitochondrial CoA. The cytosolic and mitochondrial pools of CoA are thus kept separate, and no labeled CoA from the cytosolic pool enters the mitochondrion. 

Answer Pyruvate dehydrogenase is located in the mitochondrion, and glyceraldehyde 3-phosphate dehydrogenase in the cytosol. Because the mitochondrial and cytosolic pools of NAD are separated by the inner mitochondrial membrane, the enzymes do not compete for the same NAD pool. However, reducing equivalents are transferred from one nicotinamide coenzyme pool to the other via shuttle mechanisms (see Problem 21). 

These organelles are the sites of energy production of aerobic cells and contain the enzymes of the tricarboxylic acid cycle, the respiratory chain, and the fatty acid oxidation system. The mitochondrion is bounded by a pair of specialized membranes that define the separate mitochondrial compartments, the internal matrix space and an intermembrane space. Molecules of 10,000 daltons or less can penetrate the outer membrane, but most of these molecules cannot pass the selectively permeable inner membrane. By a series of infoldings, the internal membrane forms cristae in the matrix space. The components of the respiratory chain and the enzyme complex that makes ATP are embedded in the inner membrane as well as a number of transport proteins that make it selectively permeable to small molecules that are metabolized by the enzymes in the matrix space. Matrix enzymes include those of the tricarboxylic acid cycle, the fatty acid oxidation system, and others. 

Mitochondria are about the size of bacteria. They have a diameter of 0.2 to 0.5 gm and are 0.5 to 7 p.m long. They are bounded by two lipid bilayers, the inner one being highly folded. These folds are called cristae. The innermost space of the mitochondrion is called the matrix. They have their own DNA in the form of at least one copy of a circular double helix (Chap. 7), about 5 p.m in overall diameter it differs from nuclear DNA in its density and denaturation temperature by virtue of being richer in guanosine and cytosine (Chap. 7). The different density from nuclear DNA allows its separation by isopycnic centrifugation. Mitochondria also have their own type of ribosomes that differ from those in the cytoplasm but are similar to those of bacteria. 

In addition to the foregoing more general concerns are questions concerning the localization of an enzyme activity. The location of an enzyme can determine the type of cell lysis, since it could be more advantageous to lyse the cell completely or in such a manner that the organelles are left intact. For example, some lysis methods such as sonication completely disrupt mitochondria, nuclei, and Golgi systems. If an activity is localized in an organelle such as a mitochondrion, it would seem sensible to adopt a method that leaves these structures intact, to facilitate their separation from the rest of the cellular debris. Thus, for the isolation of mitochondrial enzymes, sonication is not the method of choice for cell lysis. 

Carnitine is used mainly for facilitating the transport of long-chain fatty adds into the mitochondria. As shown in Figure4.53, this transport system requires the participation of two different carnitine acyl transferases. One is located on the outside of the mitochondrial membrane, the other on the inner side. Once fatty acyl-camitine is inside the organelle, its carnitine is released. A separate transport system is used to transport this carnitine from the interior of the mitochondrion back to the cytoplasm for reuse. 

The nucleus, mitochondrion, and chloroplast are bounded by two bIlayer membranes separated by an intermembrane space. All other organelles are surrounded by a single membrane. 

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