Endoplasmic NADPH oxidase

In an endoplasmic reticulum fraction from pig H-ver enriched in transitional endoplasmic reticulum vesicles capable of forming 50-60 nm buds in the presence of ATP and retinol, the NADPH oxidase activity was inhibited by micromolar and submicromolar concentrations of retinol (Sun et aL 2000). Retinol at 1 mM stimulated the activity. The inhibition was confined to two activity maxima separated... 

Enzymes responsible for metabolism are located at various subcellular sites, for example the cytosol, mitochondria and smooth endoplasmic reticulum. However, it is enzymes derived from endoplasmic reticulum, called mixed function oxidases or monooxygenases , which have been most intensely studied in the past two or three decades. These enzyme systems, which utilize a family of haemoprotein cytochromes, or P-450 as terminal oxidases, require molecular oxygen and reduced nicotinamide adenine dinucleotide phosphate (NADPH) for activity. The overall stoichiometry of the reactions catalyzed by these enzymes is normally represented by equation (1). 

The hepatic endoplasmic reticulum possesses oxidative enzymes called mixed-function oxidases or monooxygenase with a specific requirement for both molecular oxygen and a reduced concentration of nicotinamide adenine dinucleotide phosphate (NADPH). Essential in the mixed-function oxidase system is P-450 (Figure 1.12). The primary electron donor is NADPH, whereas the electron transfer involved P-450, a flavoprotein. The presence of a heat-stable fraction is necessary for the operation of the system. 

Oxidation is by far the most important Phase I metabolic reaction. One of the main enzyme systems involved in the oxidation of xenobiotics appears to be the so called mixed function oxidases or monooxygenases, which are found mainly in the smooth endoplasmic reticulum of the liver but also occur, to a lesser extent, in other tissues. These enzymes tend to be nonspecific, catalysing the metabolism of a wide variety of compounds (Table 9.2). Two common mixed function oxidase systems are the cytochrome P-450 (CYP-450) and the flavin monoxygenase (FMO) systems (Appendix 12). The overall oxidations of these systems take place in a series of oxidative and reductive steps, each step being catalysed by a specific enzyme. Many of these steps require the presence of molecular oxygen and either NADH or NADPH as co-enzymes. 

For most drugs, oxidative biotransformation is performed primarily by the mixed-function oxidase enzyme system, which is present predominantly in the smooth endoplasmic reticulum of the liver. This system comprises (1) the enzyme NADPH cytochrome P450 reductase (2) cytochrome P450, a family of heme-containing proteins that catalyze a variety of oxidative and reductive reactions and (3) a phospholipid bilayer that facilitates interaction between the two proteins. Important exceptions to this rule are ethyl alcohol and caffeine, which are oxidatively metabolized by enzymes primarily present in the soluble, cytosolic fraction of the liver. 

Endoplasmic reticulum systems also introduce double bonds into long-chain acyl CoAs. For example, in the conversion of stearoyl CoA into oleoyl CoA, a cis-A double bond is inserted by an oxidase that employs molecular oxygen and NADH or NADPH). 

Ethanol is also oxidized by the mixed-function oxidase of smooth endoplasmic reticulum, which requires NADPH, oxygen, and a cytochrome P-450 electron transport system (Chapter 14) ... 

There are two hepatic vitamin D 25-hydroxylases, the major one in the mitochondria and the other in the smooth endoplasmic reticulum (Figure 37-2). Both require NADPH and molecular oxygen. The microsomal enzyme appears to be a P-450 mixed-function oxidase. 25-Hydroxylase activity also occurs in intestine, kidney, and lung. 25-Hydroxylase is apparently regulated only by availability of its substrate, leading to a high plasma concentration of 25-(OH)D and a low concentration of vitamin D. 

The answer is d. (Murray, pp 123—148. Scriver, pp 2367—2424. Sack, pp 159—175. Wilson, pp 287-317.) Some monooxygenases found in liver endoplasmic reticulum require cytochrome P450. This cytochrome acts to transfer electrons between NADPH, O2, and the substrate. It can be an electron acceptor from a flavoprotein. In the mitochondrial electron transport chain, flavoproteins donate electrons to coenzyme Q, which then transfers them to other cytochromes. Flavoproteins that are oxidases often react directly with molecular oxygen to form hydrogen peroxide. Flavoproteins can be NADH dehydrogenases that oxidize NADH and transfer the electrons to coenzyme Q. The electron transfer centers of flavoproteins in the electron transport chain contain nonheme iron and sulfur. 

Phase I metabolism Phase I reactions (mainly oxidation, reduction, and hydrolysis) act as a preparation of the drug for the phase II reactions, i.e., a chemically reactive group is produced or uncovered on which the phase II reactions can occur, e.g., -OH, -NH2, -SH, -COOH. Most toxic metabolites are produced by phase I reactions. The P-450 isoenzymes (CYP enzymes), known collectively as the mixed function oxidase system, are found in the endoplasmic reticulum of many cells (notably those of liver, kidney, lung, and intestine) and perform many of these different functionalization reactions. The system requires the presence of molecular oxygen and co-factor nicotinamide adenine dinucleotide phosphate (NADPH) as well as cytochrome P450, NADPH-cytochrome P450 reductase, and lipid. 

One of the most commonly found mid-chain hydroxylated components is dihydroxyhexadecanoic acid, which has hydroxyl moieties at C-10, C-9, C-8 or C-7, and on C-16 (232, 243, 244). A crude cell-free preparation from excised epidermis of V. faba catalyzed C-10 hydroxylation of 16-hydroxyhexadecanoic acid (473). This hydroxylation reaction was also catalyzed by the endoplasmic reticulum fraction from the embryonic shoots of K faba. This preparation required O2 and NADPH to catalyze mid-chain hydroxylation and the activity was inhibited by the typical mixed-function oxidase inhibitors and also by CO (427). The inhibition by CO was photoreversible, as expected of a cytochrome P450 hydroxylase. 

Cytochrome P450 oxidases, cytochrome b5, and NADPH cytochrome P450 reductase are connected with the electron transport chain of the endoplasmic reticulum. This transport chain also represents an important ROS generator. 

In plants, the (w-hydroxylase system is responsible for synthesis of the o-hydroxy fatty acid components of cutin and suberin. Kolattukudy has studied the reactions in preparations from Vida faba. NADPH and O2 were cofactors and the enzyme showed typical properties of a mixed-function oxidase. However, the involvement of P450 in the plant system is unproven since, although the hydroxylation is inhibited by CO, the inhibition is not reversed by 420-460 nm light (a property typical for cytochrome P450 systems). Where dihydroxy fatty acids are being synthesized for cutin or suberin, the co-hydroxy fatty acid is the substrate for the second hydroxylation. Like tw-oxidation, mid-chain hydroxylation also requires NADPH and O2 and is located in the endoplasmic reticulum. 

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