The Mitochondrial Membranes and Compartments

It is contended that the renal slice technique measures primarily basolateral uptake of substrates or nephrotoxins, based on histological evidence of collapsed tubular lumens. This results in the inaccessibility of brush-border surfaces for reabsorptive transport (Burg and Orloff, 1969 Cohen and Kamm, 1976). This observation limits the ability of this model to accurately reflect reactions to nephrotoxins that occur as the result of brush-border accumulation of an injurious agent. Ultrastructurally, a number of alterations, particularly in the plasma membrane and mitochondrial compartments, have been shown to occur over a 2-h incubation period (Martel-Pelletier et al., 1977). This deterioration in morphology is very likely a consequence of the insufficient diffusion of oxygen, metabolic substrates, and waste products in the innermost regions of the kidney slice (Cohen and Kamm, 1976). Such factors also limit the use of slices in studying renal metabolism and transport functions. 

In the absence of a death signal, most of the pro- and anti-apoptotic members are located in separate subcellular compartments. Anti-apoptotic proteins are inserted in intracellular membranes, mainly the mitochondrial membrane, while some proapoptotic members are located in the cytoplasm or cytoskeleton in an inactive form. They are activated and translocated by apoptotic stimuli to their place of action to perform their functions (Gross et al., 1999). 

An appreciation of Ca2+ messenger function requires an understanding of cellular Ca2+ metabolism [1-4]. Our present view of cellular Ca2+ metabolism is represented schematically in Fig. 1. There are three membranes and four cellular compartments to consider. The membranes are the plasma membrane, the inner mitochondrial membrane, and the membrane of the endoplasmic reticulum (or a component of it). The compartments are the extracellular, the cytosolic, the mitochondrial matrix and the endoplasmic reticulum. The Ca2+ concentrations in these... 

Electron microscopic studies by George Palade and Fritjof Sjostrand revealed that mitochondria have two membrane systems an outer membrane and an extensive, highly folded inner membrane. The inner membrane is folded into a series of internal ridges called cristae. Hence, there are two compartments in mitochondria (1) the intermembrane space between the outer and the inner membranes and (2) the matrix, which is hounded by the inner membrane (Figure 18.3). Oxidative phosphorylation takes place in the inner mitochondrial membrane, in contrast with most of the reactions of the citric acid cycle and fatty acid oxidation, which take place in the matrix. 

The electrons are passed from NADH or FADH2, through an electron transport system located in the inner mitochondrial membrane, and finally to the terminal electron acceptor, molecular oxygen (O2). The transfer of electrons through the electron transport system causes protons (H+) to be pumped from the mitochondrial matrix into the intermembrane compartment. The result is a high-energy reservoir. 

FAAH has been found mainly in microsomal and mitochondrial fractions of rat brain and liver (Deutsch and Chin, 1993 Desarnaud et al., 1995), and of porcine brain (Ueda et al., 1995). Recent studies performed with confocal microscopy, showed that FAAH is localized intracellularly as a vesicular-like staining, that has no association with the plasma membranes and is partially co-localized with the endoplasmic reticulum (Fig. 4.5). These morphological data were corroborated by biochemical assays of FAAH activity in subcellular fractions, showing that AEA hydrolysis was primarily confined to the endomembrane compartment (Oddi et al., 2005). Moreover, by means of reconstituted vesicles derived from purified membrane fractions, it was demonstrated that transport activity is retained by plasma membrane vesicles devoid of FAAH, thereby indicating that AEA hydrolase activity is not necessary for AEA membrane transport. Overall, by means of confocal microscopy, subcellular fractionation, and... 

Most organelles have a characteristic architecture that is important for function, but may serve as an additional impediment to diffusion within the cell compartment. Organelle membranes within the cell can serve as local barriers (Table 4.9), which affect the rate of diffusion of molecules locally. This effect has been estimated for diffusion in the mitochondrial matrix and the endoplasmic reticulum (Figure 4.28a and b). The mitochondrial matrix was modeled as a closed cylinder with multiple barriers occluding the lumen to simulate the mitochondrial cristae the presence of multiple barriers produced a modest decrease in the rate of diffusion down the axis of the cyUnder (Figure 4.28a) this calculation is consistent with recent measurements of green fluorescent protein (GFP) diffusion in the mitochondrial matrix, which was approximately three-fold slower than diffusion in saline. The endoplasmic reticulum was modeled as an array of interconnected cylinders with a continu-... 

Yes. Because CoA cannot freely cross membranes, not only do the cytoplasmic and mitochondrial compartments contain separate pools of CoA, but also the acetyl CoA-to-CoA ratio can be quite different in each compartment. Similarly, mitochondrial and cytosolic NAD and NADH pools are wholly separate and the NADH to NAD ratio on one side of the mitochondrial membrane can be different from that on the other side. 

Despite the problems involved in the interpretation of measured metabolite concentration gradients across the mitochondrial membrane, knowledge of the approximate magnitude of mitochondrial and cytosolic metabolite concentrations has already provided important information with regard to the regulation of key enzymes by certain effector metabolites in each of the two compartments. 

The respiratory chain and other enzymes involved in oxidative phosphorylation comprise an organized group, or respiratory assembly, associated with the inner mitochondrial membrane (Lehninger, 1951). These respiratory assemblies seem to be distributed uniformly over or within the surface of the inner membrane, with center-to-center distance between assemblies of the order of 20 nm. Thus, there is one complete respiratory assembly for each 400 to 500 nm of inner membrane surface (Klingenberg, 1967 Lehninger, 1970). The metabolic activity of mitochondria is, therefore, proportional to the amount of inner mitochondrial membranes and to the number of respiratory assemblies they contain (Dempsey, 1956 Palade, 1956 Munn, 1969). Obviously, there is a reciprocal relationship between the amount of membrane and matrix in mitochondria with large numbers of cristae, the matrix compartment is materially reduced. Table I summarizes some typical figures. 

To determine whether OPAl is an integral or peripheral membrane protein, we extracted the mitochondrial membrane with alkahne sodium carbonate. This method was developed by Lazarow and colleagues to release peripheral membrane proteins from within membrane-bound compartments by converting the limiting membranes into flat sheets (Fujiki et al, 1982). After centrifugation, integral membrane proteins are pelleted, while peripheral membrane proteins are released into the supernatant. 

Fatty acids are oxidized only in the form of fatty a< l-CoA derivatives, and mitochondria from mammalian tissues contain the full equipment of enzymes necessary for the synthesis and the degradation of fatty acyl-CoA. The enzymes involved in the oxidative process are located in the mitochondrial matrix, and the inner mitochondrial membrane sequesters the oxidative process from the rest of these organelles. On the contrary, the fatty acids activating enzymes (thiokinase) seem to be present in different compartments of the mitochondrion and widely distributed among the subcellular fractions. The significance of this may lie in the fact that the conditions required for fatty acyl-CoA oxidation differ from those required for other CoA—SH dependent pathways. 

CoASH and thus reforming the true immediate substrate for the oxidatioa According to this hypothesis, acyl carnitine, which is not a substrate for the enzymes of the oxidative process, is an admirable substrate for fatty acid degradation in intact mitochondrial systems. In addition, mitochondria depleted of their endogenous energy donors (ATP, GTP) are unable to oxidize add fatty acids unless ATP and carnitine are both present in the system. This observation clearly proves that acyl-CoA is formed outside of the inner mitochondrial membrane—i.e. outside of the oxidation compartment—and is transported to the inner compartment via the carnitine-linked transport mechanism. In agreement with these results an ATP-dependent thiokinase has been identified in the outer mitochondrial membrane, and an acyl-camitine transferase has been found in the inner mitochondrial membrane. 

ATP synthase is an energy coupling protein, composed of a H carrier (the Eg subunit) that is located in the inner mitochondrial membrane, and a motor protein (the Fj subunit). The F, subunit generates ATP from Pi and ADP, a process that is driven by the flow of H+ ions through the Fg subunit, down their concentration gradient from the intermembrane space to the matrix compartment of the mitochondria. 

Freshly isolated, intact mitochondria contain considerable amounts of adenine nucleotides which are resistant to removal by repeated washings with isotonic sucrose. This indicates that these compounds are in a compartment—presumably within the inner mitochondrial membrane—which is inaccessible to the sucrose solution. When exogenous adenine nucleotide is added to the mitochondria, there is a rapid exchange with endogenous adenine nucleotides with no net increase in the concentration of adenine nucleotides in the mitochondria. ADP exchanges most rapidly, followed by ATP and then by AMP, which is relatively impermeable. It is the inner mitochondrial membrane through which the adenine nucleotides do not permeate and which contains the specific adenine-nucleotide transporting system. The movement of ATP across the inner mitochondrial membrane (and hence out of the mitochondria) depends directly on the translocation of ADP in the presence of adenylate kinase in the outer compartment of the mitochondria. 

We have measured the fractional cell volumes of the various compartments in isolated wheat protoplasts and developed a mathematical model of the cell which relates the values of the potentials across cellular membranes to the distribution of permeant ions in these compartments. This information has been used to predict how, or if, the mitochondrial membrane potential changes with illumination. The results show that it 4-ncreases in value, implying a greater limitation on mitochondrial respiration in the light. Whether the increase in membrane potential is due to adenylate control remains to be clarified. These results have considerable implication on the mechanism of oxidation of NADH produced by glycine decarboxylase. 

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