Microsomal hemoproteins

In many mammals induction of monooxygenation by polycyclic aromatic hydrocarbons is accompanied by the formation of a hemoprotein not seen to any appreciable extent in non-induced animals. This leads to an alteration in the microsomal hemoprotein populations, a change in the metabolic activity of the microsomes and, hence, possible alterations in the toxicity of other chemicals (27, 8). 

In an attempt to resolve these questions, we have directed studies toward examining the interaction of chemicals with the hepatic microsomal monooxygenase system of fish and to determine whether multiple forms of hemoprotein(s) P-450 exist, and, if so, how the relative microsomal hemoprotein subpopulations are altered by xen ob i oti cs. 

Cytochrome b5 affects the kinetics of drug metabolism by certain CYP enzymes hence, coexpression of this microsomal hemoprotein (together with NADPH-CYP reductase) can affect the catalytic efficiency of certain recombinant CYP enzymes (76,109). For example, the presence of cytochrome b5 tends to increase Fmax for reactions catalyzed by CYP3 A4, whereas it tends to decrease Km for reactions catalyzed by CYP2E1. In both cases, cytochrome b5 increases Vmax/Km, which is a measure of in vitro intrinsic clearance. The fact that some commercially available recombinant CYP enzymes are expressed with cytochrome b5 while others are not complicates the interpretation of results of studies performed with recombinant human CYP enzymes. 

C. Ioannides and D. V. Parke, Drug Metab. Rev., 22,1 (1990). The Cytochrome P4501 Gene Family of Microsomal Hemoproteins and Their Role in the Metabolic Activation of Chemicals. 

Kuntzman R, Levin W, Schilling G, Alvares A. 1969. The effects of 3-methyl-cholanthrene and phenobarbital on liver microsomal hemoproteins and on the hy-droxylation of benzpyrene. In Microsomes and Drug Oxidations, ed. JR Gillette, AH Conney, GJ Cosmides, RW Estabrook, JR Fouts, GJ Mannering, pp. 349-69. New York Academic... 

Lu, A.Y. and Coon, M.J. (1968) Role of hemoprotein P-450 in fatty acid omega-hydroxylation in a soluble enzyme system from liver microsomes. Journal of Biological Chemistry, 243 (6), 1331-1332. 

Omura, T. and Sato, R. (1964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. Journal of Biological Chemistry, 239, 2370-2378. 

Sladek, N.E. and Mannering, C.J. Evidence for a new P-1+50 hemoprotein in hepatic microsomes from methylcholanthrene treated rats. Biochem. Biophys. Res. Commun. (1966) 2l+, 668-671+. 

Much work has demonstrated the presence of complex multienzyme monooxygenase systems within the endoplasmic reticulum of several mammalian species (for Reviews 1, 2, 3). These monooxygenase systems are responsible for the oxidative metabolism of many exogenous and endogenous substances, and the unusual non-specificity of these monooxygenase enzymes allows the metabolism of compounds with diverse chemical structures. Early work demonstrated that the terminal microsomal oxidase involved in xenobio-tic biotransformation was a hemoprotein, which has been subsequently named cytochrome P-450. 

Arylhydrocarbon (benzo[a]pyrene) hydroxylase, benzphetamine-N-demethylation, ethylmorphine-N-demethylation, ethoxycoumarin-0-deethylation and ethoxyresorufin-0-deethylation were performed by published procedures (31,32,33,34), but optimized for use with trout microsomes as described previously (30, 35). Hemoprotein P-450 was determined by the procedure of Estabrook et al. (36) to avoid spectral interference by hemoglobin. Microsomal protein content was estimated either by the method of Ross and Shatz (37) or Lowry et al. (38), using bovine serum albumin standards. 

Initial studies designed to obtain a valid subcellular fractionation scheme for rainbow trout liver illustrated the aryl-hydrocarbon (benzo[a]pyrene] hydroxylase activity separated with glucose-6-phosphatase (35). This observation indicated that the trout hemoprotein P-450-mediated monooxygenation system was located within the endoplasmic reticulum (microsomal fraction). 

Table II demonstrates the effect of two polychlorinated biphenyl mixtures (Aroclors 1254 and 1242), a polybrominated biphenyl mixture (Firemaster BP6), phenobarbital and -naphtho-flavone on various hemoprotein P-450-mediated monooxygenase activities of rainbow trout hepatic microsomes.

The alteration of hemoprotein(s) P-450 subpopulations in the rat may be observed spectrally, because after treatment of rats with polycyclic aromatic hydrocarbons, the Soret maximum of the carbonmonoxyferrocytochrome complex undergoes a hypsochromic shift from 450 to 448nm (50). This blue shift was not seen with rainbow trout hepatic microsomes (29,30). However, this does not preclude the induction of novel hemoproteins P-450 since (a) the induced hemoprotein(s) maty not differ spectrally from the constitutive enzymes and (b) the induced-hemoprotein may account for only a small proportion of total hemoprotein P-450, and hence its contribution to the position of the Soret maximum of carbon monoxide-treated reduced microsomes may be negligible. The latter suggestion is supported by the work of Bend et al. with the little skate. These workers have shown that hepatic microsomes from 1, 2,3,4-dibenzanthracene treated skates did not exhibit a hypsochromic shift when compared to control microsomes, however, partially purified hemoprotein exhibited an absorbance maxima at 448 nm (51). 

Schacter, B. A., H. S. Marver, and U. A. Meyer. Hemoprotein catabolism during stimulation of microsomal lipid peroxidation. Biochim. Biophys. Acta 279 221-227, 1972. 

Deferoxamine is isolated from Streptomycespilosus. It binds iron avidly but essential trace metals poorly. Furthermore, while competing for loosely bound iron in iron-carrying proteins (hemosiderin and ferritin), it fails to compete for biologically chelated iron, as in microsomal and mitochondrial cytochromes and hemoproteins. Consequently, it is the chelator of choice for iron poisoning (Chapters 33 and 59). Deferoxamine plus hemodialysis may also be useful in the treatment of aluminum toxicity in renal failure. Deferoxamine is poorly absorbed when administered orally and may increase iron absorption when given by this route. It should therefore be administered intramuscularly or, preferably, intravenously. It is believed to be metabolized, but the pathways are unknown. The iron-chelator complex is excreted in the urine, often turning the urine an orange-red color. 

In monooxygenation, the P-450 hemoprotein receives two electrons from cofactor NADPH or NADH, or both these electrons are received one at a time, usually via reductases (flavoproteins). In most organisms, the electron chain is deeply embedded principally in the endoplasmic reticulum (and to some degree in the inner mitochondrial membrane and nuclear envelope). After sheering of the membrane during homogenization, the endoplasmic reticulum is centrifuged at 100,000 for an hour, and the product is called the "microsomal pellet." The microsomal electron chain contains reductase and P-450. 

Cytochrome P-450 found in bacteria and lever microsomes is a kind of hemoprotein that activates molecular oxygen to catalyze the hydroxylation of organic compounds in drug metabolism. For example, camphor is incorporated into cytochrome P-450 (forming P-450 cam) and then oxygenated to form 5-exo-hydroxycamphor as shown in Reaction 8 (25). The substrate specificity of this enzyme is not strict. How-... 

Ravichandran, K. G., Boddupalli, S. S., Hasemann, C. A., Peterson, J. A., and Deisen-hofer, J. 1993. Crystal-Structure of Hemoprotein Domain of P450bm-3, a Prototype for Microsomal P450s. Science, 261,731-736. 

The hydroxylation reaction is directly effected by an enzyme-hemoprotein, monooxigenase, cytochrome P450 containing protocheme IX. The reduction of the enzyme involves flavin reductases and electron carriers, such as adrenodoxin, rubredoxin, and cytochrome b5. Dioxygen, being a weak one-electron oxidant, is activated after the reduction in the enzyme heme coordination sphere. The various forms of cytochrome P450 from liver microsomes and from Pseudomonas putida have a molecular mass of about 49000. One of the subunits of the enzyme from mitochondria of... 

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