Peter J. O'Brien

Cytochrome P-450 as a microsomal peroxidase utilizing a lipid peroxide substrate☆

Abstract Linoleic acid hydroperoxide (LAHPO) when incubated with heme compounds or liver microsomes is rapidly decomposed, presumably by a free-radical mechanism, to yield a complex range of products. In this study, a spectrophotometric method has been developed for investigating the peroxidase reaction using N,N,N′,N′ -tetramethyl- p -phenylenediamine (TMPD) as hydrogen donor and LAHPO as substrate. An intracellular distribution study showed that the mitochondrial and microsomal fractions from rat liver exhibited the highest peroxidase activity per milligram protein. The microsomal peroxidase activity had a pH optimum of 4.7, was inhibited 50% by 1 m m cyanide, and was heat labile. Compounds that can form type I and type II spectra with cytochrome P-450 inhibited the peroxidase activity. Microsomes from phenobarbital-injected rats exhibited a 2.5-fold higher specific P-450 content and showed a similarly enhanced peroxidase activity. The peroxidase activity of microsomes was enhanced 2–8-fold by reagents that converted cytochrome P-450 to P-420 (e.g., lysolecithin, p -hydroxymercuribenzoate, N -bromosuccinimide, iodine, trypsin, deoxycholate). Isolation of the microsomal cytochromes showed that cytochrome b 5 had low peroxidase activity whereas microsomal “P-450 particles” containing P-450 as the sole protoheme constituent were very active. In the absence of a hydrogen donor, LAHPO destroyed cytochrome P-450 rapidly and inhibited demethylation activity in liver microsomes but did not affect cytochrome b 5 . It was concluded that cytochrome P-450 was responsible for most of the peroxidase activity of liver microsomes. A mechanism for the microsomal peroxidase activity is proposed in which LAHPO oxidizes the P-450 thiol ligand to form high spin P-420. The latter then acts as a peroxidase and decomposes LAHPO.

Cytochrome P-450 as a microsomal peroxidase in steroid hydroperoxide reduction.

The decomposition of steroid and other organic hydroperoxides by the microsomal fractions of rat liver and bovine adrenal cortex has been investigated. The peroxidase activity of microsomal fractions was measured using N,N,N′,N′-tetramethyl-pphenylene diamine (TMPD) as the hydrogen donor and following its rate of oxidation spectrophotometrically at 610 nm. A comparison of the hydroperoxide specificity of microsomal peroxidase revealed that the 17α-hydroperoxide derivatives of progesterone, pregnenolone, and allopregnanolone were very effective substrates. n nPhenobarbital or 3-methylcholanthrene pretreatment of rats enhanced the peroxidase activity of hepatic microsomes and cytochrome P-450 showed a parallel increase in specific content. Microsomal “P-450 particles” containing cytochrome P-450 as the sole protoheme constituent showed the same peroxidase activity per mole of P-450 as did original microsomes. Further evidence for cytochrome P-450 being a microsomal peroxidase was the inhibition of peroxidase activity by ligands forming type I and type II spectra with P-450 and inhibition by reagents that converted cytochrome P-450 to cytochrome P-420. A mechanism for microsomal peroxidase activity involving higher oxidation states of iron of the P-450 heme is suggested. n nIncubation of the 17α-hydroperoxide derivatives of progesterone and pregnenolone with microsomal fractions resulted in the formation of the corresponding 17α-hydroxy derivatives as the major products. The conversion of progesterone 17α-hydroperoxide by adrenocortical microsomes to 17α-hydroxyprogesterone was followed spectrally by means of induced difference spectra. Progesterone 17α-hydroperoxide did not give a type I-induced difference spectrum but the product, 17α-hydroxyprogesterone, gave a type I difference spectrum thus permitting direct observation of the conversion of the hydroperoxide to 17α-hydroxyprogesterone. n nThese results are discussed in light of previous suggestions that steroid hydroperoxides may be intermediates in steroid hydroxylations.

Microsomal electron transport

An electron transport system that catalyzes the oxidation of NADH by organic hydroperoxides has been discovered in rat liver microsomes. The rate of NADH oxidation by hydroperoxides was first-order with respect to microsomal protein concentration and a Km value for NADH of less than 3 μ m was obtained. Examination of the hydroperoxide specificity revealed that cumene hydroperoxide and various steroid hydroperoxides were effective substrates for the enzyme system. A Km value for cumene hydroperoxide of about 0.6 m m was obtained. The activity was inhibited 50% by 0.45 m m potassium cyanide but was insensitive to inhibition by carbon monoxide. NADH was a more efficient electron donor for peroxidase activity than NADPH. Evidence suggesting the involvement of NADH-cytochrome b5 reductase (EC 1.6.2.2) in the NADH-peroxidase reaction included: (1) a marked inhibition by 0.05 m m p-hydroxymercuribenzoate with partial protection by NADH; (2) inhibition by antibody to the flavoenzyme; (3) a similar Km for NADH in both activities; (4) a similar distribution of both activities in the supernatant and pellet fractions after digestion of liver microsomes with trypsin; and (5) a requirement of the flavoenzyme in the reconstituted enzyme system. Cytochrome b5 apparently was not involved in the NADH-peroxidase enzyme system since removal of the hemoprotein from liver microsomes by trypsin produced no inhibitory effect; addition of purified cytochrome b5 to either microsomes or a microsomal preparation devoid of the hemoprotein had no stimulatory effect; and antibody preparated against the hemoprotein did not inhibit the reaction rate. Evidence against the involvement of NADPH-cytochrome c reductase in the reaction was the lack of inhibition by NADP+ and by antibody prepared against the flavoprotein. Evidence for the participation of a cytochrome P-450 species in the NADH-peroxidase enzyme system included inhibition by type I, type II, and modified type II compounds; inhibition by reagents converting cytochrome P-450 to cytochrome P-420; and marked stimulation by in vivo phenobarbital treatment. The NADH-reduced form of cytochrome P-450 was oxidized very rapidly by cumene hydroperoxide under a CO atmosphere. The participation of two distinct species of cytochrome P-450 in the NADH- and NADPH-dependent peroxidase enzyme systems of liver microsomes is suggested.

[47] Superoxide production

Publisher Summary The methodologies currently employed for measuring superoxide generation in biological systems involve oxidation, reduction, or binding of superoxide to an indicator to form a stable product. Superoxide radicals have been shown to play an important role in host defenses against microorganisms and contribute to phagocytic bactericidal activity. Polymorphonuclear leukocytes (PMN), and other phagocytic cells ingest opsonized particles by encompassing them within phagocytic vesicles formed from the plasma membrane. A rapid cyanide-insensitive respiratory burst ensues forming superoxide, H 2 O 2 , and hydroxyl radicals. This system can also be activated by a wide variety of soluble agents without involving phagocytosis. Two principal methods have been used for measuring superoxide formation, one involving the superoxide dismutase inhibition of cytochrome c reduction and the other involving the trapping of the superoxide by 5,5- dimethylpyrroline N-oxide (DMPO).

Peroxidase-mediated irreversible binding of arylamine carcinogens to DNA in intact polymorphonuclear leukocytes activated by a tumor promoter

Addition of the tumor promoter phorbol myristate acetate to polymorphonuclear leukocytes results in the oxidation of the arylamine carcinogens; [14C]benzidine, N-[14C]methylaminoazobenzene and [14C]aminofluorene to reactive intermediate(s) that bind irreversibly to the leukocyte DNA. The binding was dependent on oxygen and was decreased by sulfhydryl inhibitors and phenolic antioxidants that inhibit the respiratory burst triggered by the phorbol myristate. Both the binding and the respiratory burst were increased by azide, presumably as a result of intracellular catalase inhibition. However higher concentrations of azide and cyanide prevented binding without affecting the respiratory burst indicating that myeloperoxidase is a catalyst for the binding. Granules isolated from the activated leukocytes and H2O2 catalyzed a cyanide sensitive benzidine binding to calf thymus DNA. Myeloperoxidase and H2O2 also catalysed extensive binding of these arylamines to calf thymus DNA. The leukocytes appear to be a useful model cell for studying one electron oxidation-catalyzed carcinogen activation.

The possible involvement of a peroxidase in prostaglandin biosynthesis

Abstract Sheep vesicular gland microsomes have been found to have an unusual peroxidase activity with a wide peroxide specificity and capable of oxidizing cofactors of prostaglandin synthetase. The peroxidase was also similar to the synthetase in its cellular location, its activation by hemin, inhibition by heme ligands and its inactivation by different peroxides. The inhibition by 2,7-naphthalenediol (K i = 2 μM) also suggests that the peroxidase is an integral part of the synthetase complex.

The possible involvement of singlet oxygen in prostaglandin biosynthesis.

Abstract It has been found that both the peroxidase and synthetase activity of sheep vesicular gland microsomes catalyze the oxygenation of singlet oxygen trapping or quenching agents. Furthermore the synthetase was also readily inactivated by these agents, particularly bilirubin, and suggests that singlet oxygen formed by the peroxidase activity may initiate prostaglandin biosynthesis. The singlet oxygen agents also protected the synthetase from self-catalyzed destruction or inactivation by peroxides and suggest that singlet oxygen may also be responsible for the inactivation.

Mechanisms of H2O2 formation by leukocytes: Evidence for a plasma membrane location☆

Abstract It is postulated that the increase in H 2 O 2 formation following phagocytosis in guinea pig polymorphonuclear leukocytes is due to the activation of a plasma-membrane-located NAD(P)H oxidase. The cyanide-resistant oxidase activity of intact leukocytes was markedly stimulated when the leukocytes were suspended in a hypotonic medium. Hydrogen peroxide was the principal product of the oxidase reaction. Evidence that the oxidase activity was located on the outside surface of the plasma membrane was the finding that added NAD(P)H was rapidly oxidized and the plasma membrane was impermeable to NADH or NADPH. Further evidence was the marked inhibition of the oxidase by p-CMB which also did not penetrate the plasma membrane. The oxidase was also inhibited on disruption of the plasma membrane. In addition, the enhanced oxidase activity under hypotonic conditions decreased to normal values when the medium was made isotonic and suggested that a reversible conformational change in the plasma membrane was responsible for the activation of oxidase activities.

Singlet oxygen formation during hemoprotein catalyzed lipid peroxide decomposition

Abstract The singlet oxygen trap diphenylfuran was rapidly oxidized to cis dibenzoylethylene during the decomposition of linoleic acid hydroperoxide catalyzed by ceric ions, methemoglobin or hematin. This conversion was enhanced in a deuterated medium and inhibited by other singlet oxygen quenchers or traps. The chemiluminescence accompanying the decomposition of the linoleic acid hydroperoxide was also markedly enhanced in a deuterated medium and inhibited by other singlet oxygen quenchers or traps. Antioxidants markedly inhibited these reactions. It is concluded that singlet oxygen is formed in substantial quantities during the metal catalyzed decomposition of linoleic acid hydroperoxide.

The cellular localisation of glutathione peroxidase and its release from mitochondria during swelling

Abstract 1. The intracellular and intramitochondrial localisation of rat liver GSH peroxidase (GSH: H2O2 oxidoreductase, EC 1.11.1.9) and its release from mitochondria have been investigated. 2. The peroxidase was localised in the mitochondria and the cytosol. 3. The matrix was established as the intramitochondrial site of GSH peroxidase. 4. GSH peroxidase was released from the mitochondria during the swelling initiated by GSH, GSH + GSSG, ascorbate and oleate. The extent of the release was proportional to the degree of swelling. The peroxidase was not released during the swelling induced by phosphate, Ca2+ or a mixture of phosphate + GSH. 5. The release of peroxidase during mitochondrial swelling was not specific since the specific activity of the enzyme released was similar to that released by sonication. 6. The significance of GSH peroxidase in the swelling-contraction cycle is discussed.