Cells cytochromes

The most important enzyme involved in bio transformation is cytochrome P-450, which catalyzes many phase 1 reactions. This enzyme is located primarily in the SER (microsomal fraction) of the cell and is especially abundant in liver cells. Cytochrome P-450 primarily catalyzes oxidation reactions and consists of many isoforms (isozymes). These isoenzymes have overlapping substrate specificities. The most important subfamily in humans is CYP3A4, although there is considerable variation in CYP3A4 expression between individuals. 

For practical purposes in immunohistochemistry, endogenous peroxidase activity can be defined as any activity that results in the decomposition of H202. Such activity is a common property of all hemoproteins such as hemoglobin (red cells), myoglobin (muscle cells), cytochrome (granulocytes, monocytes) and catalases (liver and kidney). Peroxidase activity may also be encountered in tissue areas adjacent to vascularized areas due to the diffusion of blood prior to fixation. 

Illustrative examples for such a possibility are found with the cytochromes. The name of these proteins comes from the Greek words meaning colored substances in the cell. Cytochromes are intensely red-colored redox enzjunes containing a heme prosthetic group as their dominant chromophore. Hemes are iron complexes of protoporph5uin IX derivatives (10,26). One of the most frequently studied metalloproteins of this family is cytochrome c (27). The ribbon structure of a cytochrome c enzyme together with the protein-bound heme c cofactor 6 is shown in Fig. 4. 

Case study optimised anomalous dispersion large unit cell cytochrome C4 (Sawyer, Harding, Gould, Papiz and Helliwell as... 

Hales, D.B., L.L. Sha, and A.H. Payne (1987). Testosterone inhibits cAMP-induced de novo synthesis of Leydig cell cytochrome P-450(17a)... 

Major Applications Photoelectrochemical cells, electrochromic devices, display device, inks, redox indicator, oil product detection,io dye laser,n chemiluminescence detection, 12 cosmetics,i microbial fuel cells, " cytochrome P 450 assay, multiplex assays, analyzing cells, semen quality, testing prenatal abnormalities, detecting nucleic acids, " antitumor agent ... 

Fig. 16. Cyclic voltamogram (20mV/s) of cytochrome c dissolved in polyacrylamide gel-cell. Cytochrome c was sorbed into a dry polyacrylamide gel-cell which was redried by N2 and then equilibrated in humidified N2. The figure was adapted according to Oliver et al. [221].

Staining Applications Bacteria cells cytochrome c oxidase-reconstituted proteoliposomes " fungus sperms epithelial cells erythrocytes lipid bilayer lipid membranes lipid particles liposome lymphocytes lysosomes microorganisms " mitochondrial neuroblastoma cells neutrophils red blood cells tumor cells yeast ... 

Hydroxy vitamin D pools ia the blood and is transported on DBF to the kidney, where further hydroxylation takes place at C-1 or C-24 ia response to calcium levels. l-Hydroxylation occurs primarily ia the kidney mitochondria and is cataly2ed by a mixed-function monooxygenase with a specific cytochrome P-450 (52,179,180). 1 a- and 24-Hydroxylation of 25-hydroxycholecalciferol has also been shown to take place ia the placenta of pregnant mammals and ia bone cells, as well as ia the epidermis. Low phosphate levels also stimulate 1,25-dihydtoxycholecalciferol production, which ia turn stimulates intestinal calcium as well as phosphoms absorption. It also mobilizes these minerals from bone and decreases their kidney excretion. Together with PTH, calcitriol also stimulates renal reabsorption of the calcium and phosphoms by the proximal tubules (51,141,181—183). 

Clotrimazole and other azole derivatives have a different mode of action than the polyenes, eg, amphotericin B. The latter biad to the ergosterol present ia the membranes of yeasts and fungi, but azole derivatives inhibit the cytochrome P-450 dependent biosynthesis of ergosterol (8—11). This inhibition not only results in a reduction of ergosterol, but also in an accumulation of C-14 methyl sterols. They disturb membrane permeabiUty, inhibit cell rephcation, and are basically responsible, in combination with the reduction of ergosterol levels, for the antifungal action. 

Like the a2ole derivatives, it inhibits the biosynthesis of ergosterol. However, naftifine [65472-88-0] does not inhibit the cytochrome P-450 dependent C-14-demethylase, but the epoxidation of squalene. Squalene epoxidase cataly2es the first step in the conversion of squalene via lanosterol to ergosterol in yeasts and fungi or to cholesterol in mammalian cells. The squalene epoxidase in C. albicans is 150 times more sensitive to naftifine, C2 H2 N, than the en2yme in rat fiver (15). Naftifine is available as a 1% cream. 

D. Keilin, The ETstory of Cell Kespiration and Cytochrome, University Press, Cambridge, 1966. 

In the endoplasmic reticulum of eukaryotic cells, the oxidation of the terminal carbon of a normal fatty acid—a process termed ch-oxidation—can lead to the synthesis of small amounts of dicarboxylic acids (Figure 24.27). Cytochrome P-450, a monooxygenase enzyme that requires NADPH as a coenzyme and uses O, as a substrate, places a hydroxyl group at the terminal carbon. Subsequent oxidation to a carboxyl group produces a dicarboxylic acid. Either end can form an ester linkage to CoA and be subjected to /3-oxidation, producing a... 

The biological activity of the polymers was evaluated in vitro by the ability to stimulate the release of superoxide from DMSO-differentiated HL-60 cells [18-20]. The released superoxide was monitored by the cytochrome C method [21,22]. As shown in Figure 7, when the differen-... 

Figure 7 Differentiation of HL-60 cells by the incubation with dimethylsulfoxide [DMSO] (a), and the assay of superoxide release in the DMSO-differentiated HL-60 cells by cytochrome C method (b).

Cytochrome c is an enzyme found in the cells of all aerobic organisms. Elemental analysis of cytochrome c shows that it contains 0.43% iron. What is the minimum molecular weight of this enzyme ... 

Squalene epoxidase, like most enzymes responsible for the later steps of sterol biosynthesis [43, 51], is membrane-bound which makes its purification in native form challenging. The purification is additionally complicated by the presence of a large number of cytochrome P450 and other enzymes that have similar hydro-phobicity and size as squalene epoxidase and are hence difficult to remove [52]. Most studies have been carried out with rat liver microsome squalene epoxidase either partially purified or as a homogenate of the cell membrane fraction. In vitro reconstitution of squalene epoxidase activity is absolutely dependent on molecular oxygen, NADPH, FAD, and NADPH-cytochrome c reductase [52, 53]. In this respect, squalene epoxidase resembles the cytochrome P450 enzymes described... 

Anthracyclins. Figure 2 Mechanisms of anthracycline-induced apoptosis of tumor cells. ROS, reactive oxygen species topo II, topoisomerase II cyt c, cytochrome c. 

Active caspases 8, 9 and 10 can convert caspase-3, the most abundant effector caspase from its pro-form to its active cleaved form. Cleavage of a number of different substrates by caspase-3 and also by caspase-6 and -7 which are two other executioner caspases besides caspase-3 then results in the typical morphology which is characteristic of apoptosis. Yet, the activation of caspase-3 and also of caspase-9 can be counteracted by IAPs, so called inhibitor of apoptosis proteins. However, concomitantly with cytochrome C also other proteins are released from mitochondria, including Smac/DIABLO. Smac/DIABLO and potentially other factors can interact with IAPs and thereby neutralize their caspase-inhibitory activity. This releases the breaks on the cell death program and allows apoptosis to ensue. 

The process whereby cellular and tissue levels of one or more cytochrome P450 enzymes are increased in response to treatment of cells, or a whole organism,... 

Imidazole antimycotics, ketoconazole, clotrimazole, and miconazole are potent inhibitors of various cytochrome P450-isoenzymes that also affect the metabolism of retinoids. They were fust shown to inhibit the metabolism of RA in F9 embryonal carcinoma cells. When tested in vitm liarazole, a potent CYP-inhibitor, suppressed neoplastic transformation and upregulated gap junctional communication in murine and human fibroblasts, which appeared to be due to the presence of retinoids in the serum component of the cell culture medium. Furthermore, liarazole magnified the cancer chemopreventive activity of RA and (3-carotene in these experiments by inhibiting RA-catabolism as demonstrated by absence of a decrease in RA-levels in the culture medium in the presence of liarazole over 48 h, whereas without liarazole 99% of RA was catabolized. In vivo, treatment with liarazole and ketoconazole reduced the accelerated catabolism of retinoids and increased the mean plasma all-irans-RA-concentration in patients with acute promyelocytic leukemia and other cancels. 

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