Hydroxylation of prostaglandin E1 by kidney cortex microsomal monooxygenase in the guinea pig

The incubation of [5,6-³H]prostaglandin E₁ ([³H]PGE₁) with guinea pig kidney cortex microsomes in the presence of NADPH in an atmosphere of air, resulted in chromatographically polar metabolites. The incubation products were treated with base which converted PGE₁ derivatives into PGB₁ derivatives, with a λ_max = 278 nm and the products were analyzed by TLC and high pressure-liquid chromatography (HPLC). Based on UV absorption, mobility on TLC and retention time in HPLC, as compared with authentic compounds, it was concluded that the two polar UV-absorbing peaks in HPLC represented 19-hydroxy-PGB₁ (19-OH-PGB₁) and 20-hydroxy-PGB₁ (20-OH-PGB₁). Further identification of the metabolites was obtained by derivatizing the incubation products as methyl esters and t-butyldimethylsilyl ethers, followed by co-injection with similarly derivatized authentic compounds in HPLC and gas chromatography. Finally, the derivatized metabolites were identified by comparing their mass fragmentation with that of similarly derivatized authentic compounds. There was an absolute requirement for NADPH, and NADH did not significantly support the hydroxylation of PGE₁. Inhibitors of microsomal monooxygenase (SKF 525A, metyrapone, and cytochrome c) inhibited the hydroxylation of PGE₁ by kidney cortex microsomes. By contrast, carbon monoxide at a CO:O₂ ratio of 5:1 did not inhibit the hydroxylation of PGE₁, pointing to a low or lack of CO sensitivity of the hydroxylation of PGE₁. The addition of PGE₁ or laurate to guinea pig kidney cortex microsomes elicited Type I spectral changes. The spectral dissociation constant (K_s) for PGE₁ was 2.4 × 10^?4m. The kinetic constants for 19- and 20-hydroxylations of PGE₁ were determined. The K_M values for the 19- and 20-hydroxylation pathways were found to be identical, being 3.3 × 10^?4m, suggesting that the same enzyme is involved in both hydroxylations; however, the V_max values for 19-hydroxylation and 20-hydroxylation of PGE₁ were 50 nmol/hr and 20.8 nmol/hr respectively. These results demonstrate that PGE₁ is a substrate for the kidney cortex microsomal monooxygenase. The similarities and differences of the kidney monooxygenase in the guinea pig with that in the rat are discussed.     

Interactions of prostaglandins with hepatic microsomal cytochrome P-450

The spectral changes associated with the addition of prostaglandins (PGs) to hepatic microsomes from guinea pigs and rats were examined. PGA₁, PGA₂, PGE₁, PGE₂, PGF_1α and PGF_2α when added to guinea pig liver microsomes exhibited type I spectra. The binding affinities as determined from spectral dissociation constants (K_s) were highest with PGA₁ and PGA₂. With liver microsomes from control or 3-methyl-cholanthrene (MC)-treated rats, PGs did not yield type I spectra; however, in this case a $\text{weak}$ spectrum, designated here as type “II” was at times observed, With microsomes from phenobarbital (Pb)-treated rats only PGA₁ and PGA₂ yielded type I spectra; again in absence of type I spectrum, a weak type “II” was occasionally observed. The addition of PGA₁ and PGA₂ to liver microsomes from Pb-treated rats inhibited the microcomal mediated hydroxylation of hexobarbital. The inhibition by PGA₁ was competitive; the K_i = 8.2 × 10^?4 M was found to be similar in magnitude to the K_s = 7.3 × 10^?4 M of PGA₁ observed with rat liver microsomes. These observations suggested that PGs particularly of the A series interact with the hepatic microsomal cytochrome P-450 monooxygenase system.     

Interaction of prostaglandins with adrenal microsomal cytochrome P-450 in the guinea pig

Studies were carried out to investigate the effects of prostaglandins (PG) $\text{in vitro}$ on adrenal microsomal steroid and drug metabolism in the guinea pig. The addition of PGE₁, PGE₂, PGA₁, PGF_1α or PGF_2α to isolated adrenal microsomes produced typical type I difference spectra. The sizes of the spectra (ΔA_385–420) produced by prostaglandins were smaller than those produced by various steroids including progesterone, 17-hydroxyprogesterone and 11β-hydroxyprogesterone. However, the affinities of prostaglandins and steroids for adrenal microsomal cytochrome P-450, as estimated by the spectral dissociation constants, were similar. Prior addition of prostaglandins to isolated adrenal microsomes did not affect steroid binding to cytochrome P-450 or the rate of steroid 21-hydroxylation. In contrast, prostaglandins inhibited adrenal metabolism of ethylmorphine and diminished the magnitude of the ethylmorphine-induced spectral change in adrenal microsomes. The results indicate that prostaglandins inhibit adrenal drug metabolism by interfering with substrate binding to cytochrome P-450. Since 21-hydroxylation was unaffected by PG, different cytochrome P-450 moieties are probably involved in adrenal drug and steroid metabolism.     

Effect of inducers and inhibitors of monooxygenase on the hydroxylation of prostaglandins in the guinea pig. Evidence for several monooxygenases catalyzing omega- and omega-1-hydroxylation.

The incubation of prostaglandins (PG's) with liver microsomes from guinea pigs treated with inducers of monooxygenase (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), benzo[alpha]pyrene (benzpyrene), or a mixture of chlorinated biphenyls (Aroclor 1254)) exhibited marked elevation of 19-hydroxylation of PGE1, PGE2, PGA1, and PGA2 without affecting significantly 20-hydroxylation. However, with respect to effects on hydroxylation of a variety of xenobiotics, benzpyrene and Aroclor treatments differed markedly; whereas Aroclor treatment elevated the demethylation of ethylmorphine, benzphetamine, and p-chloro-N-methylaniline (PCMA), benzpyrene treatment had no effect on demethylation of ethylmorphine and only a marginal effect on that of PCMA. Both inducers elevated benzpyrene hydroxylation. By contrast, treatment with phenobarbital did not affect the hepatic microsomal PG's hydroxylation, although the hydroxylation of benzpyrene and the demethylation of ethylmorphine, benzphetamine, and PCMA were enhanced. Also, the hydroxylation of PG's by kidney cortex microsomes was not affected by either benzpyrene or Aroclor treatment. Inhibitors of monooxygenase were used to help delineate the type of monooxygenases induced. At low levels of alpha-naphthoflavone (ANF), benzpyrene hydroxylation in control- and Aroclor-treated guinea pigs was only little affected; by contrast, the same concentration of ANF markedly inhibited benzpyrene hydroxylation in benzpyrene-treated guinea pigs. On the other hand, metyrapone was most inhibitory in control guinea pigs. Support for the conclusion that benzpyrene induces in the guinea pig a hepatic monooxygenase with different characteristics than that found in control animals was provided by the observation that ANF (10 MICROM) inhibited PGE1 hydroxylation more pronouncedly in liver microsomes from benzpyrene-treated than from Aroclor-treated guinea pigs or controls. In addition, in benzpyrene and Aroclor-treated guinea pigs, ANF inhibited the (omega-1)-hydroxylation more pronouncedly than that of omega-hydroxylation. By contrast, metyrapone appeared to inhibit omega-hydroxylation more effectively than (omega-1)-hydroxylation. These results indicate that in the guinea pig, hydroxylation of PG's at the omega (20-) and omega-1 (19-) positions is catalyzed by different monooxygenases and that the inducers tested affect several hepatic monooxygenases with different specificities toward xenobiotics; however, with respect to PG's only the enzyme(s) involved in the 19-hydroxylation is affected.     

Hydrogen peroxide formation and stoichiometry of hydroxylation reactions catalyzed by highly purified liver microsomal cytochrome P-450.

The stoichiometry of hydroxylation reactions catalyzed by cytochrome P-450 was studied in a reconstituted enzyme system containing the highly purified cytochrome from phenobarbital-induced rabbit liver microsomes. Hydrogen peroxide was shown to be formed in the reconstituted system in the presence of NADPH and oxygen; the amount of peroxide produced varied with the substrated added. NADPH oxidation, oxygen consumption, and total product formation (sum of hydroxylated compound and hydrogen peroxide) were shown to be equimolar when cyclohexane, benzphetamine, or dimethylaniline served as the substrate. The stoichiometry observed represents the sum of two activities associated with cytochrome P-450. These are (1) hydroxylase activity: NADPH + H⁺ + O₂ + RH → NADP⁺ + H₂O + ROH; and (2) oxidase activity: NADPH + H⁺ + O₂ → NADP⁺ + H₂O₂. Benzylamphetamine (desmethylbenzphetamine) acts as a pseudosubstrate in that it stimulates peroxide formation to the same extent as the parent compound (benzphetamine), but does not undergo hydroxylation. Accordingly, when benzylamphetamine alone is added in control experiments to correct for the NADPH and O₂ consumption not associated with benzphetamine hydroxylation, the expected 1:1:1 stoichiometry for NADPH oxidation, O₂ consumption, and formaldehyde formation in the hydroxylation reaction is observed.     

Hydroxylation of the carcinostatic 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) by rat liver microsomes

Ring hydroxylation of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea was shown to occur in the presence of liver microsomes prepared from both normal and phenobarbital induced rats. The metabolite was identified by mass spectrometry after selective extraction and purification by liquid chromatography. The microsomal catalyzed reaction was oxygen and NADPH dependent, inhibited by carbon monoxide and induced 4–5 fold by $\text{in vivo}$ phenobarbital pre-treatment. Phenobarbital induced microsomes hydroxylated the substrate at a rate of 17.6 nmoles/min/mg protein at 37°. A Type I difference spectrum was observed with phenobarbital induced microsomes that also displayed a substrate binding constant (K_s of 4 × 10^?5 M.     

Microbial Oxidation of Hydrocarbons: Properties of a Soluble Methane Monooxygenase from a Facultative Methane-Utilizing Organism, Methylobacterium sp. Strain CRL-26

Methylobacterium sp. strain CRL-26 grown in a fermentor contained methane monooxygenase activity in soluble fractions. Soluble methane monooxygenase catalyzed the epoxidation/hydroxylation of a variety of hydrocarbons, including terminal alkenes, internal alkenes, substituted alkenes, branched-chain alkenes, alkanes (C₁ to C₈), substituted alkanes, branched-chain alkanes, carbon monoxide, ethers, and cyclic and aromatic compounds. The optimum pH and temperature for the epoxidation of propylene by soluble methane monooxygenase were found to be 7.0 and 40°C, respectively. Among various compounds tested, only NADH₂ or NADPH₂ could act as an electron donor. Formate and NAD⁺ (in the presence of formate dehydrogenase contained in the soluble fraction) or 2-butanol in the presence of NAD⁺ and secondary alcohol dehydrogenase generated the NADH₂ required for the methane monooxygenase. Epoxidation of propylene catalyzed by methane monooxygenase was not inhibited by a range of potential inhibitors, including metal-chelating compounds and potassium cyanide. Sulfhydryl agents and acriflavin inhibited monooxygenase activity. Soluble methane monooxygenase was resolved into three components by ion-exchange chromatography. All three compounds are required for the epoxidation and hydroxylation reactions.     

Purifications and properties of L-mandelate- 4-hydroxylase from Pseudomonas convexa.

An inducible l-mandelate-4-hydroxylase has been partially purified from crude extracts of Pseudomonas convexa. This enzyme catalyzed the hydroxylation of l-mandelic acid to 4-hydroxymandelic acid. It required tetrahydropteridine, NADPH, Fe²⁺, and O₂ for its activity. The approximate molecular weight of the enzyme was assessed as 91,000 by gel filtration on Sephadex G-150. The enzyme was optimally active at pH 5.4 and 38 °C. A classical Michaelis-Menten kinetic pattern was observed with l-mandelate, NADPH, and ferrous sulfate and K_m values for these substrates were found to be 1 × 10^?4, 1.9 × 10^?4, and 4.7 × 10^?5m, respectively. The enzyme is very specific for l-mandelate as substrate. Thiol inhibitors inhibited the enzyme reaction, indicating that the sulfhydryl groups may be essential for the enzyme action. Treatment of the partially purified enzyme with denaturing agents inactivated the enzyme.     

NADPH-cytochrome P450 reductase.

The rate of reduction of cytochrome P450 in hepatic microsomes in the presence of NADPH has been measured with a dual wavelength stopped-flow spectrophotometer. The results obtained, with microsomes prepared from phenobarbital-pretreated rats, indicate that the reduction process is biphasic and most probably composed of two concurrent first-order reactions. The rate constant for the reduction of cytochrome P450 in the fast phase in the presence of ethylmorphine is 1.74 s^?1. Since approximately 50% or more of the cytochrome P450 is reduced in the fast phase under these conditions, the rate of reduction of cytochrome P450 is approximately 150 nmol min^?1 (mg of protein)^?1. Under similar conditions the rate of ethylmorphine N-demethylation is 8.6 nmol min^?1 (mg of protein)^?1. Thus the rate-limiting step in ethylmorphine N-demethylation cannot be the introduction of the first electron into cytochrome P450 by NADPH-cytochrome P450 reductase.     

Monooxygenase activity in Cunninghamella bainieri: evidence for a fungal system similar to liver microsomes

The fungus Cunninghamella bainieri effects the oxidative N-demethylation of aminopyrine, O-demethylation of 4-nitroanisole and anisole, the aryl hydroxylation of anisole, aniline, and naphthalene, and the reduction of nitro and azo groups. The hydroxylation of 4-[²H]-anisole and 2-[²H]-anisole proceeds with migration and retention of isotopic hydrogen (NIH shift). The above reactions and the formation of the trans-dihydrodiol of naphthalene and the incorporation of oxygen-18 from ¹⁸O₂ into the trans-dihydrodiol and hydroxylated anisole are characteristic of reactions catalyzed by the cytochrome P₄₅₀ monooxygenases of hepatic microsomes. The product ratios in these hydroxylations are very similar to those obtained using liver microsomes providing further evidence that the C. bainieri monooxygenase enzymes are similar to the liver monooxygenases. Furthermore, an epoxide hydrase enzyme similar to that present in hepatic microsomes must also be present in C. bainieri.     

Stoichiometric cooperation of NADPH and hexobarbital in hepatic microsomes during the catalysis of hydrogen peroxide formation

The interaction of NADPH and hexobarbital during catalysis of microsomal mixed function oxidase-dependent hydrogen peroxide formation has been investigated in hepatic microsomes from phenobarbital-treated rabbits. The application of Job's method (25) of continuous variation revealed optimal conditions for the rate and extent of hydrogen peroxide formation when hexobarbital and NADPH were in equimolar amounts. The formation of a complex of 1 mol NADPH with cytochrome c-reductase and 1 mol hexobarbital with cytochrome P-450 seems to be responsible for limitation of hydrogen peroxide formation. Rate and extent of hydrogen peroxide formation are directly proportional to the amount of hexobarbital and NADPH present and are governed by the mass action equation in a manner similar to that reported for interaction of purified enzymes (G. T. Miwa, S. B. West, M. T. Huang, and A. H. Lu, 1979,J. Biol. Chem.254, 5695–5700). Depending on either the NADPH concentration maintained by a generating system or the hexobarbital concentration, the extent of hydrogen peroxide formation could be shown to be a function of either compound alone, as long as the other one is in excess. The question whether the formation of hydrogen peroxide depends on the availability of two independent one-electron transfer reactions forming O₂^? or of one simultaneous two-electron transfer forming O₂^2? might thus become rather a matter of association of substrate and cosubstrate to a catalytically active complex in which the substrate augments the availability of reducing equivalents.     

The drug and carcinogen metabolism system of rat colon microsomes

The hydroxylation of N- and O-methyl drugs and polycyclic hydrocarbons has been demonstrated in microsomes prepared from colon mucosal cells. The hydroxylation of the drugs benzphetamine, ethylmorphine, p-nitroanisole, and p-nitrophenetole by colon microsomes is inducible two- to fourfold by pretreatment with phenobarbital/hydrocortisone. Colon microsomal benzo[α]pyrene hydroxylation is inducible 35-fold by pretreatment with β-naphthoflavone. Phenobarbital/hydrocortisone pretreatment also induces a fourfold increase in the specific content of colon microsomal cytochrome P-450, while β-naphthoflavone pretreatment causes a shift in the reduced CO difference spectrum peak to 448 nm and an eightfold increase in the specific content of this cytochrome. SKF 525-A inhibits the hydroxylation of the drug benzphetamine by colon microsomes or liver microsomes by 77% at a concentration of 2.0 mm. 7,8-Benzoflavone, on the other hand, inhibits the hydroxylation of the polycyclic hydrocarbon benzo[α]pyrene by colon microsomes by 76% and by liver microsomes by 44% at a concentration of 10 μm. Carbon monoxide, an inhibitor of oxygen interaction with cytochromes P-450 and P-448, inhibits benzphetamine hydroxylation and benzpyrene hydroxylation by colon microsomes 30 and 51%, respectively, at an oxygen to carbon monoxide ratio of 1:10. The K_m values of colon microsomal cytochrome P-450 reductase for the artificial electron acceptors cytochrome c, dichloroindophenol, and ferricyanide (10–77 μm) are in agreement with those for purified rat liver cytochrome P-450 reductase. These data support the conclusions that hydroxylation of drugs and polycyclic hydrocarbons is catalyzed by colon mucosal microsomes and that the hydroxylation activity is attributable to a cytochrome P-450-dependent drug metabolism system similar to that found in liver microsomes.     

Cytochrome p-450-dependent hydroxylation of lauric Acid at the subterminal position and oxidation of unsaturated analogs in wheat microsomes

Microsomes from etiolated wheat (Triticum aestivum L. cv Etoile de Choisy) shoots catalyzed the reduced nicotinamide adenine dinucleotide phosphate-dependent hydroxylation of lauric acid predominantly at the subterminal or (ω-1) position (65%). Minor amounts of 10-hydroxy- (31%) and 9-hydroxylaurate (4%) were also formed. The reaction was catalyzed by cytochrome P-450, since enzyme activity was strongly inhibited by tetcyclacis, carbon monoxide, and antibodies against NADPH-cytochrome c (P-450)-reductase. The apparent K_m for lauric acid was estimated to be 8.5 ± 2.0 μm. Seed treatment with the safener naphthalic acid anhydride or treatment of seedlings with phenobarbital increased cytochrome P-450 content and lauric acid hydroxylase (LAH) activity of the microsomes. A combination of both treatments further stimulated LAH activity. A series of radiolabeled unsaturated lauric acid analogs (8-, 9-, 10-, and 11-dodecenoic acids) was used to explore the regioselectivity and catalytic capabilities of induced wheat microsomes. It has been found that wheat microsomes catalyzed the reduced nicotinamide adenine dinucleotide phosphate-dependent epoxidation of sp2 carbons concurrently with hydroxylation at saturated positions. The regioselectivity of oxidation of the unsaturated substrates and that of lauric acid were similar. Preincubation of wheat microsomes with reduced nicotinamide adenine dinucleotide phosphate and 11-dodecenoic acid resulted in a partial loss of LAH activity.     

Quantitative determination of prostaglandins E1 and F1α by multiple ion analysis

Quantitative assays for prostaglandins (PG) E₁ and PGF_1α are described using [3,3,4,4,5,6-²H₆]labeled prostaglandins as carriers and methyl ester-O-methyloxime-acetate (PGE₁) and methyl ester-acetate (PGF_1α) derivatives for gas - liquid chromatography/mass spectrometric analysis. Thin-layer argentation chromatography was used to separate PGE₁ from PGE₂ and 13, 14-dihydro-PGE₂. These latter compounds, which do not separate from PGE₁ using conventional thin-layer chromatography or under the gas - liquid chromatographic conditions used, can significantly interfere with the quantitative analysis of PGE₁. The method described prevents this interference and is therefore suitable for the accurate analysis of PGE₁ in biological samples containing a high concentration of PGE₂ and/or 13, 14-dihydro-PGE₂.     

Interaction of prostaglandins with adrenal microsomal cytochrome P-450 in the guinea pig.

Studies were carried out to investigate the effects of prostaglandins (PG) in vitro on adrenal microsomal steroid and drug metabolism in the guinea pig. The addition of PGE1, PGE2, PGA1, PGF1 alpha or PGF2 alpha to isolated adrenal microsomes produced typical type I difference spectra. The sizes of the spectra (delta A385-420) produced by prostaglandins were smaller than those produced by various steroids including progesterone, 17-hydroxyprogesterone and 11 beta-hydroxyprogesterone. However, the affinities of prostaglandins and steroids for adrenal microsomal cytochrome P-450, as estimated by the spectral dissociation constants, were similar. Prior addition of prostaglandins to isolated adrenal microsomes did not affect steroid binding to cytochrome P-450 or the rate of steroid 21-hydroxylation. In contrast, prostaglandins inhibited adrenal metabolism of ethylmorphine and diminished the magnitude of the ethylmorphine-induced spectral change in adrenal microsomes. The results indicate that prostaglandins inhibit adrenal drug metabolism by interfering with substrate binding to cytochrome P-450. Since 21-hydroxylation was unaffected by PG, different cytochrome P-450 moieties are probably involved in adrenal drug and steroid metabolism.     

IDentification of a microsomal retinoic acid synthase as a microsomal cytochrome P-450-linked monooxygenase system

• 1.1. To characterize an enzyme which metabolizes retinal in liver microsomes, several properties of the enzymatic reaction from retinal to retinoic acid were investigated using rabbit liver microsomes.
• 2.2. The maximum pH of the reaction in the liver microsomes was 7.6.
• 3.3. The K_m and V_max values for all-trans, 9-cis and 13-cis-retinals were determined.
• 4.4. The reaction proceeded in the presence of NADPH and molecular oxygen.
• 5.5. The incorporation of one atom of molecular oxygen into retinal was confirmed by using oxygen-18, showing that the reaction comprised monooxygenation, not dehydrogenation.
• 6.6. The monooxygenase activity was inhibited by carbon monoxide, phenylisocyanide and antiNADPH-cytochrome P-450 reductase IgG, but not by anti-cytochrome b₅ IgG.
• 7.7. The enzymatic activity inhibited by carbon monoxide was photoreversibly restored by light of a wavelength of around 450 nm.
• 8.8. The retinal-induced spectra of liver microsomes with three isomeric retinals were type I spectra.
• 9.9. The microsomal monooxygenase activity induced by phenobarbital or ethanol were more effective than that by 3-methylcholanthrene, clotrimazole or β-naphthoflavone.
• 10.10. These results showed that the monooxygenase reaction from retinal to retinoic acid in liver microsomes is catalyzed by a cytochrome P-450-linked monooxygenase system.

     

Nociceptive Response to Prostaglandins and Analgesic Actions of Aspirin and Morphine

VANE et al.^1–3 have proposed that aspirin and allied antiinflammatory drugs act by inhibiting the production of prostaglandins in the tissues. Because, however, prostaglandins E₁ and E₂ (PGE₁ and PGE₂) had been reported not to elicit pain in human skin at doses inducing inflammation^{4, 5}, Vane did not suggest that the inhibition of prostaglandin production fully explains the analgesic action of aspirin-like drugs. Nonetheless, PGE₁ PGE₂ or PGF_2α irritates pulmonary^{6, 7} or ocular⁸ mucous membrane and, when injected by the subcutaneous or intramuscular route, PGE₂ or PGF_2α causes pain⁹.     

Microsomal electron transport. I. Reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase and cytochrome P-450 as electron carriers in microsomal NADPH-peroxidase activity

An electron transport system that catalyzes the oxidation of NADPH by organic, hydroperoxides has been discovered in microsomal fractions. A tissue distribution study revealed that the microsomal fraction of rat liver was particularly effective in catalyzing the NADPH-peroxidase reaction whereas microsomes from adrenal cortex, lung, kidney, and testis were weakly active. The properties of the hepatic microsomal NADPH-peroxidase enzyme system were next examined in detail.The rate of NADPH oxidation by hydroperoxides was first-order with respect to microsomal protein concentration and a K_m value for NADPH of less than 3 μm was obtained. Examination of the hydroperoxide specificity revealed that cumene hydroperoxide and various steroid hydroperoxides were effective substrates for the enzyme system. Using cumene hydroperoxide as substrate, the reaction rate showed saturation kinetics with increasing concentrations of hydroperoxide and an apparent K_m of about 0.4 mm was obtained. The NADPH-peroxidase reaction was inhibited by potassium cyanide, half-maximal inhibition occurring at a cyanide concentration of 2.2 mm. NADH was able to support the NADPH-dependent peroxidase activity synergistically.Evidence compiled for the involvement of NADPH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase, EC 1.6.2.3) in the NADPH-peroxidase reaction included: (1) an identical pH optimum for both activities; (2) stimulation of NADPH-peroxidase activity by increasing ionic strength; (3) inhibition by 0.05 mm, p-hydroxymercuribenzoate with partial protection by NADPH; (4) inhibition by NADP⁺; and (5) inactivation by antiserum to NADPH-cytochrome c reductase. In contrast, antibody to cytochrome b₅ did not inhibit the NADPH-peroxidase activity. Evidence for the participation of cytochrome P-450 in the NADPH-peroxidase reaction included inhibition by compounds forming type I, type II, and modified type II difference spectra with cytochrome P-450; inhibition by reagents converting cytochrome P-450 to cytochrome P-420; and marked stimulation by in vivo phenobarbital administration. The NADPH-reduced form of cytochrome P-450 was oxidized very rapidly by cumene hydroperoxide under a CO atmosphere.It was concluded that the NADPH-peroxidase enzyme system of liver microsomes is composed of the same electron transport components which function in substrate hydroxylation reactions.     

Prostaglandin synthesis in human placenta and fetal membranes

The conversion of exogenous arachidonic acid into prostaglandins was studied in human placenta and fetal membrane microsomes. Only one prostaglandin was formed, prostaglandin E₂ (PGE₂), in fetal membrane microsomes. In placental microsomes PGE₂ was further transformed into 15 keto-PGE₂. Cofactor requirements and some characteristics of the system were studied. 1 to 3% conversion of arachidonic acid into prostaglandins was observed in placental microsomes and 5 to 8% conversion in fetal membrane microsomes.     

Temporal effect of prostaglandins E and F on human adrenal cAMP and cortisol output.

The $\text{in vitro}$ modulating effects of the E and F series prostaglandins upon the cAMP and cortisol output of normal human adrenal dice were evaluated with time and compared to the effects of ACTH. PGE₁ and PGE₂ significantly increased human adrenal cAMP levels; cortisol output increased in a dose related manner. Although the cortisol levels produced by E prostaglandins and ACTH were quantitatively similar, on a molar basis ACTH was greater than 10⁶ fold more effective. PGE_1α, PGF_2α, PGF_1β and PGF_2β depressed adrenal cAMP, except PGF_2α and PGF_2β at 100 μg/ml. PGF_1α and PGF_2α depressed cortisol levels at all doses. Similarly, PGF_1β and PGF_2β also depressed cortisol output, except PGF_2β at 100 μg/ml which was stimulatory. In both series of prostaglandins the temporal responses were dose related in regard to the cyclic nucleotide and steroid alterations. The findings demonstrate the E and F series prostaglandins act antagonistically in respect to cAMP and cortisol output. In addition, as the cAMP level and cortisol output are not always correlated, it appears that these prostaglandin mediated events are separable. The relationship between adrenal prostaglandins and cyclic nucleotides, therefore, invites a more sophisticated second messenger concept in terms of adrenocortical function, than currently proposed.