Endoplasmic reticulum, cytochrome

The cytochromes are iron-containing hemoproteins in which the iron atom oscillates between Fe + and Fe + during oxidation and reduction. Except for cytochrome oxidase (previously described), they are classified as dehydrogenases. In the respiratory chain, they are involved as carriers of electrons from flavoproteins on the one hand to cytochrome oxidase on the other (Figure 12-4). Several identifiable cytochromes occur in the respiratory chain, ie, cytochromes b, Cp c, a, and (cytochrome oxidase). Cytochromes are also found in other locations, eg, the endoplasmic reticulum (cytochromes P450 and h, and in plant cells, bacteria, and yeasts. 

The cytochromes P-450 monooxygenase system is actually a collection of isoenzymes, all of which possess an iron protoporphyrin IX as the prosthetic group. The monomer of the enzyme has a molecular weight of 45,000 to 55,000. The enzyme is membrane bound within the endoplasmic reticulum. Cytochromes P-450 are closely associated with another vital component of the system, NADPH cytochrome P-450 reductase. This is a flavoprotein, which has 1 mol of FAD and 1 mol of FMN per mol of apoprotein. The monomeric molecular weight of the enzyme is 78,000. The enzyme transfers two electrons to cytochromes P-450, but one at a time. There only seems to be one reductase, which serves a group of isoenzymes of cytochromes P-450, and consequently, its concentration is 1/10 to 1/30 that of cytochromes P-450. 

Guillou, H., D Andrea, S., Rioux, V., Barnouin, R., Dalaine, S., Pedrono, F., Jan, S., and Legrand, P. Distinct roles of endoplasmic reticulum cytochrome b5 and fused cytochrome b5-like domain for rat Delta6-desaturase activity. J Lipid Res 45 (2004) 32-40. 

Dissociation of enzyme and membrane synthesis during regeneration has also been reported for the mitochondria of the rat adrenal cortex (Yago et al, 1972). After adrenal enucleation, cellular proliferation occurs, and, in about 20 days, the gland has regenerated to approximately its original size. Enucleation leads to rapid multiplication of mitochondria much smaller than normal. These mitochondria increase in size by accumulation of protein and phospholipid, the latter primarily in the inner mitochondrial membrane. In addition to the usual cytochromes, the inner membranes of adrenal cortical mitochondria of beef, swine, and rats possess a special cytochrome, cytochrome P-450, usually found in the endoplasmic reticulum. Cytochrome P-450 is carbon monoxide-sensitive... 

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... 

This impressive reaction is catalyzed by stearoyl-CoA desaturase, a 53-kD enzyme containing a nonheme iron center. NADH and oxygen (Og) are required, as are two other proteins cytochrome 65 reductase (a 43-kD flavo-protein) and cytochrome 65 (16.7 kD). All three proteins are associated with the endoplasmic reticulum membrane. Cytochrome reductase transfers a pair of electrons from NADH through FAD to cytochrome (Figure 25.14). Oxidation of reduced cytochrome be, is coupled to reduction of nonheme Fe to Fe in the desaturase. The Fe accepts a pair of electrons (one at a time in a cycle) from cytochrome b and creates a cis double bond at the 9,10-posi-tion of the stearoyl-CoA substrate. Og is the terminal electron acceptor in this fatty acyl desaturation cycle. Note that two water molecules are made, which means that four electrons are transferred overall. Two of these come through the reaction sequence from NADH, and two come from the fatty acyl substrate that is being dehydrogenated. 

Cytochrome P450 2C9 is a mixed-function oxidase localized in the endoplasmic reticulum which is responsible for the biotransformation of several nonsteroidal anti-inflammatory diugs, S-warfarin, several sulfonylurea antidiabetics and other diugs. 

Heme (C34H3204N4Fe) represents an iron-porphyrin complex that has a protoporphyrin nucleus. Many important proteins contain heme as a prosthetic group. Hemoglobin is the quantitatively most important hemoprotein. Others are cytochromes (present in the mitochondria and the endoplasmic reticulum), catalase and peroxidase (that react with hydrogen peroxide), soluble guanylyl cyclase (that converts guanosine triphosphate, GTP, to the signaling molecule 3, 5 -cyclic GMP) and NO synthases. 

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). 

Monooxygenases owe their catalytic properties to the hemeprotein cytochrome P450 (Fignre 2.3). Within the membrane of the endoplasmic reticulum (microsomal... 

The microsomal fraction consists mainly of vesicles (microsomes) derived from the endoplasmic reticulum (smooth and rough). It contains cytochrome P450 and NADPH/cytochrome P450 reductase (collectively the microsomal monooxygenase system), carboxylesterases, A-esterases, epoxide hydrolases, glucuronyl transferases, and other enzymes that metabolize xenobiotics. The 105,000 g supernatant contains soluble enzymes such as glutathione-5-trans-ferases, sulfotransferases, and certain esterases. The 11,000 g supernatant contains all of the types of enzyme listed earlier. 

Monooxygenases (MOs) Enzyme systems of the endoplasmic reticulum of many cell types, which can catalyze the oxidation of a great diversity of lipophilic xenobiotics, are particularly well developed in hepatocytes. Forms of cytochrome P450 constitute the catalytic centers of monooxygenases. 

The major mono oxygenases in the endoplasmic reticulum are cytochrome P450s—so named because the enzyme was discovered when it was noted that preparations of microsomes that had been chemically reduced and then exposed to carbon monoxide exhibited a distinct peak at 450 nm. Among reasons that this enzyme is important is the fact that approximately 50% of the drugs humans ingest are metabolized by isoforms of cytochrome P450 these enzymes also act on various carcinogens and pollutants. 

At least six isoforms of cytochrome P450 are present in the endoplasmic reticulum of human hver, each with wide and somewhat overlapping substrate specificities and acting on both xenobiotics and endogenous compounds. The genes for many isoforms of P450 (from both humans and animals such as the rat) have been isolated and smdied in detail in recent years. 

Lipids are also components of the cytochrome P450 system. The preferred lipid is phosphatidylcholine, which is the major lipid found in membranes of the endoplasmic reticulum. 

Cytochrome P450s are generally located in the endoplasmic reticulum of cells and are particularly enriched in liver. 

The subcellular location of PG was studied in cells disrupted by osmotic lysis through formation and disruption of sphaeroplasts from self-induced anaerobically-grown cells. A discontinuous sucrose-density gradient produced four bands labelled I, II, III and IV. Band I included many vesicles and a peak of alkaline phosphatase activity (a vacuolar marker in yeasts), NADPH cytochrome c oxidoreductase activity, an endoplasmic reticulum marker, and... 

SADP or sulfo-SADP also have been used to study the phenylalanine-methionine-arginine-phenylalanine-amide-activated sodium channel (Coscoy et al., 1998), various apolipoprotein E isoforms (Mann et al., 1995), the high-affinity phenylalkylamine Ca2+ antagonist binding protein from guinea pig (Moebius et al., 1994), the interaction of non-histone proteins with nucleosome core particles (Reeves and Nissen, 1993), and the interactions among cytochromes P-450 in the endoplasmic reticulum (Alston et al., 1991). See Chapter 28 for methods of using photoreactive heterobifunctional crosslinkers to study protein interactions. 

Alston, K., Robinson, R.C., Park, S.S., Gelboin, H.V., and Friedman, F.K. (1991) Interactions among cytochromes P-450 in the endoplasmic reticulum. Detection of chemically cross-linked complexes with monoclonal antibodies./. Biol. Chem. 266, 735-739. 

Many of the morphological and biochemical changes that occur in cells that die by necrosis are very different from those that occur in apoptosis. During necrosis cells swell, mitochondria and endoplasmic reticulum lose their structure and become dysfunctional and the nuclear membrane becomes disrupted (Fig. 35-1). Necrotic death is independent of premitochondrial apoptotic proteins such as Bax, cytochrome c release and caspase activation. Necrosis is further distinguished from apoptosis by the fact that necrosis usually occurs as the result of a traumatic physical injury or stroke and cells die en masse, whereas apoptosis typically occurs in individual cells within a population of surviving neighbors. 

Bourdi, M. et al., Anti-liver endoplasmic reticulum autoantibodies are directed against human cytochrome P-450IA2. A specific marker of dihydralazme-induced hepatitis, J. Clin. Invest., 85, 1967, 1990. 

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