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PROSTAGLANDINH SYNTHASE

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[29] Prostaglandin H Synthase

By JEFF A. BOYD Introduction Prostaglandin H synthase (PHS) (EC 1.14.99.1) is a m e m b r a n e - b o u n d glycoprotein complex, consisting o f two identical heme-containing subunits of Mr 70,000.1 T w o distinct activities, a c y c l o o x y g e n a s e and a peroxidase, are present in the single polypeptide chain, and h e m e is absolutely required for both activities. 2 The physiologic role of P H S is to initiate the conversion of arachidonic acid to the biologically active eicosanoids, including prostaglandins, t h r o m b o x a n e , and prostacyclin, s This reaction is a c c o m p l i s h e d through the c y c l o o x y g e n a s e - c a t a l y z e d bisdioxygenation of arachidonic acid to the unstable h y d r o p e r o x y endoperoxide PGG2, followed by the peroxidase-catalyzed reduction of PGG2 to the h y d r o x y e n d o p e r o x i d e PGH2 (Fig. l). 4-6 The c y c l o o x y g e n a s e reaction p r o c e e d s through a chain m e c h a n i s m of carbon- and o x y g e n - c e n t e r e d radicals, 7-9 whereas the h y d r o p e r o x i d a s e reaction generates radical species f r o m fatty acid hydroperoxide, reducing cofactors, and the e n z y m e itself. 10-12 The p e r o x i d a s e reaction of P H S is typical of heme-containing peroxidases, in that a cycle of native e n z y m e , c o m p o u n d I, and c o m p o u n d II m a y be observed. 13 During this reaction, both the cyclooxygenase and F. J. Van der Ouderaa, M. Buytenhek, F. J. Slikkerveer, and D. A. Van Dorp, Biochem. Biophys. Acta 572, 29 (1979).

T. Miyamoto, N. Ogino, S. Yamamoto, and O. Hayaishi, J. Biol. Chem. 251, 2629 (1976). s B. Samuelsson, M. Goldyne, E. Granstrom, M. Hamberg, S. Hammerstrom, and C. Malmsten, Annu. Rev. Biochem. 47, 997 (1978). 4 S. Ohki, N. Ogino, S. Yamamoto, and O. Hayaishi, J. Biol. Chem. 254, 829 (1979). M. Hamberg, J. Svensson, T. Wakabayashi, and B. Samuelsson, Proc. Natl. Acad. Sci. U.S.A. 71, 345 (1974). 6 D. H. Nugteren and E. Hazelhof, Biochem. Biophys. Res. Commun. 326, 448 (1973). 7 M. E. Hemler and W. E. M. Lands, J. Biol. Chem. 255, 6253 (1980). s N. A. Porter, in "Free Radicals in Biology" (W. A. Pryor, ed.), Vol. 4, p. 261. Academic Press, New York, 1980. 9 R. P. Mason, B. Kalyanaraman, B. E. Tainer, and T. E. Eling, J. Biol. Chem. 255, 5019 (1980). i0 B. Kalyanaraman, R. P. Mason, B. Tainer, and T. E. Eling, J. Biol. Chem. 257, 4764 (1982). N R. W. Egan, P. H. Gale, E. M. Baptism, K. L. Kennicot, W. J. A. Vanden-Heuvel, R. W. Walker, P, E. Fagerness, and F. A. Kuehl, Jr., J. Biol. Chem. 256 7352 (1981). 12T. A. Dix and L. J. Marnett, J. Am. Chem. Soc. 103, 6744 (1981). 13A.-M. Lambeir, C. M. Markey, H. B. Dunford, and L. J. Marnett, J. Biol. Chem. 260, 14894 (1985). METHODS 1N ENZYMOLOGY, VOL. 186

Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

284

ASSAY OF FORMATION OR REMOVAL OF OXYGEN RADICALS

COOH

COOH

Cyclooxygenos)e 02

Arochidonlc Acid

)x.

OOH PGG2 (or ROOH) ~

Prostoglondins Thromboxane = Prostacyclln

[29]

×.

~

COOH OH PGH2

(or ROH)

Cyclooxygenom7 .~Prostaglondin H Synthase Peroxidase FIG. I. Role of prostaglandin H synthase in the conversion of arachidonic acid to eicosanoids. The enzyme complex consists of two activities, both of which generate radical products.

the peroxidase undergo irreversible substrate-dependent self-deactivation, ~4,15as the result of an attack on the enzyme by a free radical generated during the peroxidase reaction.~6 This free radical derives from the enzyme itself and may be formed by the oxidation of an amino acid located near the iron of the heine group.=O Observations that certain organic compounds such as phenol, tryptophan, and serotonin stimulate both enzymatic activities ~5,17,18are now understood in the context of their protecting the enzyme from self-deactivation by donating an electron and undergoing oxidation, hence serving as reducing cofactors for the peroxidase reaction, l°,ll Numerous xenobiotics are among the several classes of compounds which may serve as reducing cofactors for the PHS peroxidase reaction.~9 A small number of other compounds are oxygenated during prostaglandin biosynthesis but do not serve as reducing cosubstrates; the oxidant in this 14 W. L. Smith and W. E. M. Lands, Biochemistry 11, 3276 (1972). 15 R. W. Egan, J. Paxton, and F. A. Kuehl, Jr., J. Biol. Chem. 251, 7329 (1976). 16 R. W. Egan, P. H. Gale, and F. A. Kuehl, Jr., J. Biol. Chem. 254, 3295 (1979). 17 T. Miyamoto, S. Kamamoto, and O. Hayaishi, Proc. Natl. Acad. Sci. U.S.A. 71, 3645 (1974). is C. J. Sih, C. Takeguchi, and P. Foss, J. Am. Chem. Soc. 92, 6670 (1970). ]9 L. J. Marnett and T. E. Eling, in "Reviews in Biochemical Toxicology" (E. Hodgson, J. R. Bend, and R. M. Philpot, eds.), Vol. 5, p. 135. Elsevier/North Holland, New York, 1983.

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system is a fatty acid peroxyl radical, 12and the source of oxygen is molecular O2.20 Thus, there are two major components of the PHS-catalyzed reaction, each involving radical formation, for which convenient assays exist: (1) the cyclooxygenase-dependent oxygenation of arachidonic acid and (2) the peroxidase-dependent oxidation of a reducing cosubstrate, in which the oxidant is a higher oxidation state of the enzyme. Assays for these reactions are presented below; an assay for the oxygenation of nonreducing cosubstrates was presented in an earlier volume of this series. 2~ Arachidonic Acid Oxygenation

Principle. Oxygenation of arachidonic acid (AA) to PGG2 by PHS cyclooxygenase is the rate-limiting step in prostaglandin biosynthesis. An initial hydrogen abstraction, resulting in a carbon-centered AA radical, leads to a series of sequential reactions in which two molecules of molecular oxygen are incorporated to give PGG2.9 The substrate requirement of the cyclooxygenase is quite specific, namely, a methylene-interrupted trienoic fatty acid in which the last double bond is located six carbon atoms from the methyl terminus. 22 Various PHS enzyme preparations may be tested using this assay, in which the uptake of molecular oxygen is monitored. This assay is also useful in preliminary assessment of cyclooxygenase inhibitors and peroxidase reducing cofactors: addition of the former results in decreased oxygen consumption, whereas addition of the latter results in increased oxygen consumption owing to protection of PHS from self-deactivation. Materials Potassium phosphate buffer, I M, pH 7.8 PHS enzyme preparation 23 (e.g., microsomes from ram seminal vesicle or rabbit kidney medulla, 10 mg/ml) Arachidonic acid, 20 mM in ethanol Protocol. Reaction mixtures consist of 0. I M phosphate buffer and 1 mg/ml of microsomal enzyme preparation at 37°. The reaction is initiated by addition of arachidonic acid to 100 /~M and can be monitored by following the utilization of molecular oxygen using a Clark-type oxygen electrode and strip chart recorder. Oxygenation is relatively rapid, becoming maximal during the first 20-30 sec and nearing completion in less 2o L. J. Marnett, M. J. Bienkowski, and W. R. Pagels, J. Biol. Chem. 254, 5077 (1979). 21 p. H. Siedlik and L. J. Marnett, this series, Vol. 105, p. 412. 22 S. Bergstrom, H. Danielson, and B. Samuelsson, Biochem. Biophys. Acta 90, 207 (1964). 23 j. A. Boyd and T. E. Eling, J. Pharmacol. Exp. Ther. 219, 659 (1981).

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than 2 min. The effect of cyclooxygenase inhibitors (e.g., indomethacin) or peroxidase reducing cofactors (e.g., phenol) may be assayed by adding the inhibitors to the reaction mixture 2-3 min prior to the addition of arachidonic acid. Comment. Purified PHS (>50,000 units/mg protein) is now commercially available, and may be assayed with a similar protocol. Generally, 12 x 103 units/ml of enzyme are used. A reducing cofactor such as phenol, 100/zM, may also be added prior to the addition of arachidonic acid. Reducing Cofactor Oxidation Principle. The peroxidase activity of PHS is much less substrate specific than that of the cyclooxygenase. In addition to PGG2, a wide variety of organic hydroperoxides may serve as substrates, there being a preference for alkyl hydroperoxides. 4 Following the reduction of peroxide to alcohol (PGG2 to PGH2), two molecules of suitable cofactor may donate one electron each in the sequential reduction of the peroxidase back to its native state) 3 The resultant free radical metabolites may then react with molecular oxygen, producing a measurable oxygen uptake response as described above for the cyclooxygenase reaction. The antiinflammatory drug phenylbutazone is one such substrate, 24 commonly employed in studies of PHS peroxidase. Materials Potassium phosphate buffer, 1 M, pH 7.0 PHS microsomal enzyme preparation, 10 mg/ml H202, 20 mM in water, or 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) 25 Phenylbutazone, 50 mM in ethanol (new stock solutions should be made before each experiment) Protocol. Reaction mixtures consist of 0.1 M phosphate buffer, 0.4 mg/ml of PHS microsomal enzyme preparation, and 500/zM phenylbutazone. The reaction is initiated by the addition of hydroperoxide to 100 /~M, and it may be monitored as above by following the consumption of molecular oxygen. A rapid time course is essentially complete after approximately 60 sec. Comment. Other compounds may serve as reducing cofactors for PHS peroxidase, but their one-electron oxidation products do not trap molecu54 L. J. Marnett, T. A. Dix, R. J. Sachs, and P. H. Siedlik, in "Advances in Prostaglandin, Thromboxane, and Leukotriene Research" (B. Samuelsson, R. Paoletti, and P. Ramwell, eds.), Vol. 11, p. 79. Raven, New York, 1983. M. O. Funk, R. Isaac, and N. A. Porter, Lipids 11, 113 (1976).

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lar oxygen to any appreciable degree; one example is 2-aminofluorene. 26 Such compounds may be identified as potential reducing cofactors for the PHS peroxidase by their inhibition of phenylbutazone oxygenation. The compound to be studied is placed in the reaction mixture with phenylbutazone prior to the addition of peroxide, and its effect on oxygen consumption is monitored. It should also be noted that for PHS peroxidase-dependent oxidations of reducing cofactors, classical Michaelis-Menton kinetics are inapplicable. A linear phase of reaction is not observed during oxygen uptake measurements; initial rates must therefore be extrapolated from points early in the reaction curves. Acknowledgments The author is indebted to Dr. Thomas E. Eling of the Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences. 26j. A. Boyd and T. E. Eling, J. Biol. Chem. 259, 13885 (1984).

[30] D T - D i a p h o r a s e : P u r i f i c a t i o n , P r o p e r t i e s , a n d F u n c t i o n B y CHRISTINA LIND, ENRIQUE CADENAS, PAUL HOCHSTEIN, and LARS ERNSTER

In 1958, Ernster and Navazio 1,2 reported the occurrence of a highly active diaphorase in the soluble fraction of rat liver homogenates, which catalyzed the oxidation of NADH and NADPH at equal rates. A partial purification and some properties of the enzyme were described in 1960 by Ernster et al. 3 They named the enzyme DT-diaphorase because of its reactivity with both NADH and NADPH (at that time DPNH and TPNH). The same authors subsequently published a detailed report on DTdiaphorase, which included its purification, assay conditions, data regarding kinetics, electron acceptors, activators, and inhibitors, as well as a comparison of the enzyme with various diaphorases and quinone reductases earlier described in the literature. 4 i L. Ernster and F. Navazio, Acta Chem. Scand. 12, 595 (1958). 2 L. Ernster, Fed. Proc., Fed. Am. Soc. Exp. Biol. 17, 216 (1958). 3 L. Ernster, M. Ljunggren, and L. Danieison, Biochem. Biophys. Res. Commun. 2, 88 (1960). 4 L. Ernster, L. Danielson, and M. Ljunggren, Biochim. Biophys. Acta 58, 171 0962).

METHODS IN ENZYMOLOGY, VOL. 186

Copyright © 1990 by Academic Press, Inc. All fights of reproduction in any form reserved.

Prostaglandin H synthase.

[29l PROSTAGLANDINH SYNTHASE 283 [29] Prostaglandin H Synthase By JEFF A. BOYD Introduction Prostaglandin H synthase (PHS) (EC 1.14.99.1) is a m e...
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