Cytosolic cytochrome

NO may react with superoxide to yield the highly reactive peroxynitrite, ONOO-. Superoxide may also be converted into H202 and the reactive hydroxyl radical, OH. In this way excessive activation of glutamate receptors leads to oxidative damage. The calcium influx has a major effect on mitochondria and causes them to depolarize and swell. This leads to a pore being formed in the outer mitochondrial membrane, which allows the escape of cytochrome c and procaspases from the mitochondria into the cytosol. Cytochrome c activates the caspase cascade, which leads to apoptotic cell death (Ch. 35). 

The immunocytochemical staining of cytochrome C offers another alternative since, upon exposure to apoptotic stimuli, cytochrome C is rapidly released into the cytosol, an event that may be required for the completion of apoptosis in some systems (L2). The effect of cytosolic cytochrome C is thought to be the activation of caspases. The immunocytochemical staining of cytochrome C localized in mitochondria in healthy cells or diffused in the cell cytoplasm with monoclonal antibody (Promega) after induction of apoptosis, as detected by fluorescence microscopy, can be used for monitoring apoptosis (L2, M7, S8, T6). It is also a simple, rapid, specific mefhod for quanfifafive assessment of apoptotic cells. 

In this model, cellular stress mediates the release of cytochrome C from the mitochondrion. The proapoptotic proteins Bax and BH3 proteins support the release of cytochrome C, while the antiapoptotic Bcl2 protein has an inhibitory effect. Cytosolic cytochrome C binds to the cofactor Apaf 1, which then associates via its CARD motif with procaspase 9 to a complex termed apopto-some. In this complex, procaspase 9 is processed to the mature caspase which subsequently activates downstream effector caspases and commits the cell to death. 

Measurement of cytochrome c revealed that TNF-a significantly increased the cytosolic cytochrome c levels in PC-12 cells at 6 h (Fig. (10)). The increase in cytochrome c was suppressed to near the control level by the presence of 1 or 10 pM crocin. Therefore, our present results show the possibility that crocin has a pharmacological effect that prevents apoptosis of neuronal cells [17]. 

Flg-(IO). ELISA of cytosolic cytochrome c levels in PC-12 cells, treated for 6 h with TNF-ct and/or crocin. 

Intrinsic (mitochondrial) pathway of caspase activation is initiated by the permeabilization of the mitochondrial outer membrane by proapoptotic members of the Bcl-2 family, resulting in a release of cytochrome c and other proteins from the intermembrane space of mitochondria into the cytosol. Cytochrome c translocation to the cytosol may follow a number of possible mechanisms. However, once in the cytosol, cytochrome c binds to apoptosis protease activating factor (Apaf-1) and in the presence of dATP or ATP facilitates Apaf-1 oligomerization and the recruitment of procaspase-9. The formation of this caspase-activating complex, termed the apoptosome, results in the activation of procaspase-9, and this in turn cleaves and activates the effector caspase-3 and -7. Activated effector caspases cleave key substrates in the cell and produce the cellular and biochemical events characteristic for apoptosis [33-35]. 

In cytosol, cytochrome c combines with very high affinity with a cytosolic protein called Apoptotic Protease-Activating Factor 1 (Apaf-1) and dATP. The complex, in turn, combines with an inactive protease precursor, procaspase 9, to form the apoptosome . As a result, several procaspase 9 molecules are placed near each other, and they cleave each other to form active caspases 9. When formed, caspase 9 attacks procaspase 3 and cleaves it to form active caspase 3, a protease that hydrolyses certain enzymes occupying key positions on the metabolic map. This causes cell death. 

In the cytosol, cytochrome c binds Apaf (pro-apoptotic protease activating factor). The Apaf/cytochrome c complex binds caspase 9, an initiator caspase, to form an active complex called the apoptosome. The apoptosome in turn activates execution caspases by zymogen cleavage. 

Figure 1. EPR spectra of cytochrome c549. A) partially purified cytosolic cytochrome, B) partially purified NaCl-solubilized cytochrome, C) detergent extract of thylakoid membranes, and D) whole membranes.

Solubilization of the membrane-bound cytochrome has little effect on its spectrum. The cytosolic cytochrome, however, has a somewhat different set of -values. This difference may reflect heterogeneity in the cytochrome vivo or heterogeneity induced by the different purification protocols. 

While this electron flow takes place, the cytochrome on the periplasmic side donates an electron to the special pair and thereby neutralizes it. Then the entire process occurs again another photon strikes the special pair, and another electron travels the same route from the special pair on the periplasmic side of the membrane to the quinone, Qb, on the cytosolic side, which now carries two extra electrons. This quinone is then released from the reaction center to participate in later stages of photosynthesis. The special pair is again neutralized by an electron from the cytochrome. 

Oxidation of this UQHg occurs in two steps. First, an electron from UQHg is transferred to the Rieske protein and then to cytochrome C. This releases two to the cytosol and leaves UQ , a semiquinone anion form of UQ, at... 

Cytochrome c, like UQ is a mobile electron carrier. It associates loosely with the inner mitochondrial membrane (in the intermembrane space on the cytosolic side of the inner membrane) to acquire electrons from the Fe-S-cyt C aggregate of Complex 111, and then it migrates along the membrane surface in the reduced state, carrying electrons to cytochrome c oxidase, the fourth complex of the electron transport chain. 

FIGURE 21.17 The electron transfer pathway for cytochrome oxidase. Cytochrome c binds on the cytosolic side, transferring electrons through the copper and heme centers to reduce O9 on the matrix side of the membrane. 

Mitochondrial permeability transition involves the opening of a larger channel in the inner mitochondrial membrane leading to free radical generation, release of calcium into the cytosol and caspase activation. These alterations in mitochondrial permeability lead eventually to disruption of the respiratory chain and dqDletion of ATP. This in turn leads to release of soluble intramito-chondrial membrane proteins such as cytochrome C and apoptosis-inducing factor, which results in apoptosis. 

Figure 7. Mechanism of the proton-translocating ubiquinol cytochrome c reductase (complex III) Q cycle. There is a potential difference of up to 150 mV across the hydrophobic core of this complex (potential barrier represented by the vertical broken line). Cytochromes hour and b N are heme groups on the same peptide subunits of complex III which can transfer electrons across the hydrophobic core. The movement of two electrons provides the driving force to transfer two protons from the matrix to the cytosol. Diffusion of UQ and UQHj, which are uncharged, in the hydrophobic core, and lipid bilayer of the inner membrane is not influenced by the membrane potential (see Nicholls and Ferguson, 1992).

Another pathway is the L-glycerol 3-phosphate shuttle (Figure 11). Cytosolic dihydroxyacetone phosphate is reduced by NADFl to s.n-glycerol 3-phosphate, catalyzed by s,n-glycerol 3-phosphate dehydrogenase, and this is then oxidized by s,n-glycerol 3-phosphate ubiquinone oxidoreductase to dihydroxyacetone phosphate, which is a flavoprotein on the outer surface of the inner membrane. By this route electrons enter the respiratory chain.from cytosolic NADH at the level of complex III. Less well defined is the possibility that cytosolic NADH is oxidized by cytochrome bs reductase in the outer mitochondrial membrane and that electrons are transferred via cytochrome b5 in the endoplasmic reticulum to the respiratory chain at the level of cytochrome c (Fischer et al., 1985). 

Although only two protons are pumped out of the matrix, two others from the matrix are consumed in the formation of H2O. There is therefore a net translocation of four positive charges out of the matrix which is equivalent to the extrusion of four protons. If four protons are required by the chemiosmotic mechanism to convert cytosolic ADP + Pj to ATP, then 0.5 mol ATP is made for the oxidation of one mol of ubiquinol and one mol ATP for the oxidation of 2 mols of reduced cytochrome c. These stoichiometries were obtained experimentally when ubiquinol was oxidized when complexes I, II, and IV were inhibited by rotenone, malonate, and cyanide, respectively, and when reduced cytochrome c was oxidized with complex III inhibited by antimycin (Hinkle et al., 1991). (In these experiments, of course, no protons were liberated in the matrix by substrate oxidation.) However, in the scheme illustrated in Figure 6, with the flow of two electrons through the complete electron transport chain from substrate to oxygen, it also appears valid to say that four protons are extmded by complex I, four by complex III, and two by complex 1. 

Figure26-2. Biosynthesis of squalene, ubiquinone, dolichol, and other polyisoprene derivatives. (HMG, 3-hydroxy-3-methylglutaryl x, cytokinin.) A farnesyl residue is present in heme a of cytochrome oxidase. The carbon marked with asterisk becomes C or C,2 in squalene. Squalene synthetase is a microsomal enzyme all other enzymes indicated are soluble cytosolic proteins, and some are found in peroxisomes.

Hereditary methemoglobinemia is classified into three types a red blood cell type (type I), a generalized type (type II), and a blood cell type (type HI). Enzyme deficiency of type I is limited to red blood cells, and these patients show only the diffuse, persistent, slate-gray cyanosis not associated with cardiac or pulmonary disease. In type II, the enzyme deficiency occurs in all cells, and patients of this type have a severe neurological disorder with mental retardation that predisposes them to early death. Patients with type III show symptoms similar to those of patients with type I. The precise nature of type III is not clear, but decreased enzyme activity is observed in all cells (M9). It is considered that uncomplicated hereditary methemoglobinemia without neurological involvement arises from a defect limited to the soluble cytochrome b5 reductase and that a combined deficiency of both the cytosolic and the microsomal cytochrome b5 reductase occurs in subjects with mental retardation. Up to now, three missense mutations in type I and three missense mutations, two nonsense mutations, two in-frame 3-bp deletions, and one splicing mutation in type n have been identified (M3, M8, M31). 

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