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.     

Spectral intermediates in the reaction of oxygen with purified liver microsomal cytochrome P-450

Stopped flow spectrophotometry has shown the occurrence of two distinct spectral intermediates in the reaction of oxygen with the reduced form of highly purified cytochrome P-450 from liver microsomes. As indicated by difference spectra, Complex I (with maxima at 430 and 450 nm) is rapidly formed and then decays to form Complex II (with a broad maximum at 440 nm), which resembles the intermediate seen in steady state experiments. In the reaction sequence, P-450_LM^red$\text{→}\text{O}{}_2$Complex I→Complex II→P-450_LM^ox the last step is rate-limiting. The rate of that step is inadequate to account for the known turnover number of the enzyme in benzphetamine hydroxylation unless NADPH-cytochrome P-450 reductase or cytochrome $\text{b}{}_5$ is added. The latter protein does not appear to function as an electron carrier in this process.     

Solubilization and partial purification of cytochrome P-450 from rat lung microsomes

Cytochrome P-450 from rat lung microsomes has been solubilized and purified 8-fold by using affinity chromatography on an ω-amino-$\text{n}$-octyl derivative of Sepharose 4B. The purified fraction was free of cytochrome $\text{b}$₅ and NADPH-cytochrome $\text{c}$ reductase and showed spectral characteristics similar to those of lung microsomal cytochrome P-450. When combined with NADPH-cytochrome $\text{c}$ reductase partially purified from liver microsomes, the cytochrome P-450 fraction supported the hydroxylation of benzo (α)pyrene and the activity was proportional to the content of the hemoprotein. No absolute requirement for phosphatidylcholine was found.     

Induction of hepatic microsomal propranolol N-desisopropylase activity by 3-methylcholanthrene and sudan III

Metabolism of propranolol in liver microsomes was markedly induced in rats and $\text{C57BL}\text{6J}$ mice treated with 3-methylcholanthrene (3-MC) or sudan III, inducers of cytochrome P-448. 7,8 Benzoflavone inhibited propranolol metabolism in microsomes from treated rats. 3-MC did not induce propranolol metabolism in genetically nonresponsive $\text{DBA}\text{2}$ mice. High-performance liquid chromatographical analysis of propranolol metabolites revealed a 6-fold increase in propranolol N-desisopropylase activities in liver microsomes from sudan III- or 3-methylcholanthrene-treated rats. It is concluded that propranolol N-desisopropylation is predominantly catalyzed by cytochrome P-448.     

Molecular cloning vectors derived from the CoLE1 type plasmid pMB1.

$\text{In}$$\text{vitro}$ recombination via restriction endonucleases and $\text{in}$$\text{vivo}$ genetic translocation of an impicillin resistance gene, have been utilized for the construction of a series of cloning vehicles. These vectors have been derived by combining the essential replication properties of plasmid pMBl, a CoLEl type plasmid, with one, two or three resistance markers (tetracycline, ampicillin, chloramphenicol and colicin El production). During the construction of these vectors, the position of restriction sites used for cloning DNA fragments, relative to the antibiotic resistance genes and the origin of DNA replication, have been determined. The use of the cloning vectors pMB9, pBR313, pBR322, pBR324, pBR325 permits the molecular cloning and easy selection of $\text{Eco}$ RI, $\text{Bam}$ HI, $\text{Bgl}$ II, $\text{Dpn}$ II, $\text{Hind}$ III, $\text{Sal}$ I, $\text{Xho}$ I, $\text{Xam}$ I, $\text{Taq}$ I, $\text{Pst}$ I, $\text{Hinc}$ II, $\text{Pvu}$ I, $\text{Sma}$ I, $\text{Xma}$ I, $\text{Ba}$l I, $\text{Hpa}$ I, $\text{Ava}$ I, $\text{Pvu}$ II, $\text{Hae}$ III, $\text{Alu}$ I, $\text{Hpa}$ II, $\text{Eco}$ RII and virtually any blunt-ended generated DNA fragment.     

Acyltransferases and the biosynthesis of pulmonary surfactant lipid in adenoma alveolar type II cells.

Acyltransferases are present in microsomes from alveolar type II cell adenomas (produced by urethan injections) that transfer palmitic acid in the presence of CoA, ATP, and Mg⁺⁺ to $\text{sn}$-glycerol-3-P to form phosphatidic acid, to dihydroxyacetone-P to form acyldihydroxyacetone-P, and to 1-acyl-$\text{sn}$-glycero-3-phosphocholine to form 3-$\text{sn}$-phosphatidylcholine. The data clearly demonstrate that the microsomal preparations can catalyze significant incorporation of palmitic acid into the 2-position of the disaturated species of 3-sn-phosphatidylcholine independently of phosphatidic acid formation as evidenced by the fact that $\text{sn}$-glycerol-3-P and calcium ions (which inhibit choline phosphotransferase) did not influence the incorporation of palmitic acid into the main surfactant lipid. Thus, a deacylation-acylation reaction involving 2-lysophosphatidylcholine appears to be an important pathway for the synthesis of surfactant lipid in alveolar type II cells; the control of acyl specificity at the 2-position is determined by the relative concentrations of the coparticipating substrates, l-palmitoyl-$\text{sn}$-glycero-3-phosphocholine and palmitoyl-CoA.     

The 520 nm absorbance changes in Scenedesmus obliquus and its relation to Photosystem I

The kinetics (region of seconds) of the light-induced 520 nm absorbance change and its dark reversal have been studied in detail in the wild type and in some pigment and photosynthetic mutants of Scenedesmus obliquus. The following 5 lines of evidence led us to conclude that the signal is entirely due to the photosystem I reaction modified by electron flow from Photosystem II.Gradual blocking of the electron transport with 3(3,4-dichlorophenyl)-1,1-dimethylurea resulted in diminution and ultimate elimination of the biphasic nature of the signal without reducing the extent of the absorbance change or of the dark kinetics. On the contrary, blocking electron flow at the oxidizing side of plastoquinone with 2, $\text{5-}\text{dibromo}\text{-3-}\text{methyl}\text{-6-}\text{isoprophyl}\text{-p-}\text{benzoquinone}$ or inactivating the plastocyanin with KCN, prolonged the dark reversal of the absorbance change apart from abolishing the biphasic nature of the signal.Action spectra clearly indicate that the main signal (I) is due to electron flow in Photosystem I and that its modification (Signal II) is due to the action of Photosystem II.Signal I is pH independent, whereas Signal II demonstrates a strong pH dependence, parallel to the O₂-evolving capacity of the cells.Chloroplast particles isolated from the wild type Scenedesmus cells demonstrated in the absence of any added artificial electron donor or acceptor and also under non-phosphorylation conditions the 520 nm absorbance change with approximately the same magnitude as whole cells. The dark kinetics of the particles were comparatively slower. Removal of plastocyanin and other electron carriers by washing with Triton X-100 slowed down the kinetics of the dark reversal reaction to a greater extent. A similar positive absorbance change at 520 nm and slow dark reversal was also observed in the Photosystem I particles prepared by the Triton method.Mutant C-6E, which contains neither carotenoids nor chlorophyll $\text{b}$ and lacks Photosystem II activity, demonstrates a normal signal I of the 520 nm absorbance change. This latter result contradicts the postulate that carotenoids are the possible cause of the 520 nm absorbance change.     

The involvement of cytochrome P-450 in the NADH-dependent O-demethylation of p-nitroanisole in phenobarbital-treated rabbit liver microsomes.

Addition of $\text{p}$-nitroanisole to a reaction mixture containing phenobarbital-pretreated rabbit liver microsomes brings about an increase the reoxidation rate of NADH-reduced cytochrome b₅. Addition of partially purified cytochrome b₅ to a solution containing microsomes results in a marked increase in both NADH- and NADPH-dependent O-demethylation of $\text{p}$-nitroanisole. $\text{p}$-Nitroanisole also increases the rate of NADH mediated cytochrome P-450 reduction. From these and other results described in the Discussion section, we confirm that electrons required for NADH-dependent O-demethylation of $\text{p}$-nitroanisole is transfered from NADH to cytochrome P-450 via cytochrome b₅ and that cytochrome P-450 is the enzyme which catalyzes $\text{p}$-nitroanisole O-demethylation.     

Cold lability and dissociation of the F1-ATPase from Bacillus stearothermophilus

Effects of inadequate vitamin E (E) and/or selenium (Se) nutrition on the activities of cytochrome P-450 mixed function oxidase system (heme hydroperoxidase, p-nitroanisole O-demethylase), and epoxide hydrolase have been investigated. Heme hydroperoxidase activity of liver and lung microsomes was significantly decreased in E deficiency. In the liver, Se deficiency resulted in a significant increase in hydroperoxidase activity. In contrast to the peroxidase activity, liver demethylase activity was only marginally affected in $\text{E}\text{Se}$ deficiency states. However, kidney demethylase activity was increased two fold in Se deficient states. Liver microsomal epoxide hydrolase activity was significantly increased in both E and Se deficiency states.     

Phospholipase A2 inactivation of microsomal 17β-hydroxysteroid oxidoreductase: Rates of phospholipid hydrolysis and enzyme inactivation,effects of hydrolysis products and properties of the phospholipase A2-treated enzyme

Exposure of guinea pig liver microsomes to phospholipase A₂ resulted in the nearly complete loss of 17β-hydroxy-steroid oxidoreductase (17β-HSD) activity, the time course of which correlated with phospholipid hydrolysis and lysolecithin formation. Lysolecithin and unsaturated fatty acids added to microsomes also inactivated 17β-HSD indicating that they may contribute to the inactivation by phospholipase A₂.If exposure to lysolecithin and fatty acids was minimized by including serum albumin in the reaction mixture, phospholipids were rapidly hydrolyzed; but in this case the extent of 17β-HSD inactivation was less and the rate of loss was significantly slower. The data suggest that phospholipid hydrolysis $\text{per se}$ results in a destabilization of 17β-HSD resulting in the subsequent activity loss.The inactivation of 17β-HSD by lysolecithin and fatty acids has not been reported previously and is suggestive of a possible control mechanism $\text{in vivo}$.     

A study of the modified type II spectral change produced by the interaction of agroclavine with cytochrome P-450

Previous work in this laboratory has demonstrated that agroclavine is metabolized by liver microsomes and that cytochrome P-₄₅₀ is involved as the terminal oxidase in the reaction. Addition of agroclavine to suspensions of liver microsomes in the same concentration as used in the metabolic studies produced a modified Type II spectral change with peak near 420 nm and trough near 390 nm. Using modifiers such as hexobarbital, this spectrum has now been shown to contain a Type 1 component. Furthermore, when sufficiently low concentrations of agroclavine were added to rat liver microsomes, a Type I spectral change appeared, which gradually turned into the modified Type II variety upon increase in agroclavine concentration.     

Membrane fluidity and drug metabolism in liver microsomes of lean, obob and dbdb mice

The thermally-induced changes of a cytochrome P-450 dependent activity (ethoxycoumarin-O-deethylase) and of the fluorescence polarization of diphenylhexatriene were compared in microsomes from lean, $\text{ob}\text{ob}$ and $\text{db}\text{db}$ mice. In lean mice, biphasic plots were obtained with break points in the same range of temperature by both methods, whereas, in $\text{ob}\text{ob}$ and $\text{db}\text{db}$ mice, no discontinuities were observed. These results may be related to a modified fatty acid composition of microsomal membranes in mutant mice. They exemplify the influence of the lipid environment on the monooxygenase system as also shown by the modified binding constants of cytochrome P-450 towards type II substrates in $\text{db}\text{db}$ mice.     

Membrane effects on drug monoxygenation activity in hepatic microsomes

The temperature dependence of drug monooxygenation in phenobarbital-induced rat liver microsomes has been investigated. With 7-ethoxycoumarin as a substrate the activity of the microsomes could be measured down to 0°C by the increase in fluorescence of the dealkylated reaction product 7-hydroxycoumarin (umbelliferone).Arrhenius plots of the activities at various temperatures between 0°C and 45°C showed a break in the activation energy around 20°C.Addition of deoxycholate or high concentrations of glycerol, known to solubilize membrane-bound enzymes, abolished the break of the activation energy. Cholesterol, incorporated into the microsomal membrane in amounts equimolar to the microsomal phospholipid content led to a decrease of the activation energy at low temperatures and to an increase at higher temperatures, resulting in a loss of the break.The activity of microsomal NADPH-cytochrome $\text{c}$ reductase with the water-soluble electron acceptor dichlorophenolindophenol showed no discontinuity in the Arrhenius plot. In addition the cumene hydroperoxide-mediated and cytochrome $\text{P-450-}\text{dependent}$ O-dealkylation of 7-ethoxycoumarin proceeded without a break in the activation energy.It is concluded that phospholipid phase transitions affect the electron transfer from the reductase to cytochrome $\text{P-450}$.     

The interaction of vinyl chloride with rat hepatic microsomal cytochrome P-450 in vitro.

In the presence of hepatic microsomes, vinyl chloride produces a ‘type I’ difference spectrum and stimulates carbon monoxide inhibitable NADPH consumption. A comparison of the binding and Michaelis parameters for the interaction of vinyl chloride with uninduced, phenobarbital and 3-methylcholanthrene induced microsomes indicates that the binding and metabolism of vinyl chloride is catalyzed by more than one type P-450 cytochrome, but predominantly by cytochrome P-450. Metabolites of vinyl chloride from this enzyme system decrease the levels of cytochrome P-450 and microsomal heme, but not cytochrome $\text{b}$₅ or NADPH-cytochrome $\text{c}$ reductase $\text{in vitro}$.     

The effect of extra bound cytochrome b-5 on cytochrome P-450-dependent enzyme activities in liver microsomes

Binding of increasing amounts of detergent-purified cytochrome $\text{b}{}_5$ to rabbit liver microsomes produces a progressive inhibition of NADPH-cytochrome P-450 reductase activity which is accompanied by a similar inhibition of NADPH-supported benzphetamine demethylation. In contrast, NADH-cytochrome P-450 reductase activity in the enriched microsomes is markedly enhanced and this stimulation is accompanied by a similar increase in NADH-peroxidase activity, suggesting that cytochrome $\text{b}{}_5$ in these two reactions functions as an intermediate electron carrier to cytochrome P-450.     

Direct fluorometric analysis of benzo(a)pyrene metabolite formation by mouse liver microsomes

We have examined the metabolites produced by in vitro incubation of benzo(a)pyrene with 3-methylcholanthrene-induced mice liver microsomes. Our objective was to observe directly a possible difference in microsomal enzyme systems of animal models having different susceptibility to chemical carcinogens. The metabolites produced by the two animal models,$\text{C57BL}\text{6J}$ and $\text{DBA}\text{2}$ mice, were analyzed by a highly sensitive, “three-dimensional” fluorescence plotting technique. The fluorescence spectra of the total ethyl acetate-soluble metabolites clearly indicate that the metabolites produced by $\text{DBA}\text{2}$ enzymes were predominantly monohydroxylated benzo(a)pyrene while those produced by the liver microsomes of $\text{C57BL}\text{6J}$ were highly enriched with the 7,8-dihydrodihydroxybenzo(a)pyrene type.     

Substrate-induced spectral changes in human normal and chronic myeloid leukemic granulocytes

Several drugs/chemicals were allowed to interact with the cytochrome P-450 dependent mixed function oxidase system in the postmitochrondrial supernatant fractions of Ficoll-Hypaque-separated granulocytes from human normal subjects and patients with chronic myeloid leukemia. The substrate-induced spectral changes were followed by recording the difference spectra. Compounds conventionally classified as type I and type II substrates, on addition to S1 fractions of both normal and leukemic granulocytes, caused spectral changes that were reverse to those reported for the rat liver microsomes. Aminopyrine, phenobarbital, and Tween 80 evoked a reverse type I spectral change with a peak at 420-430 nm and a trough at 380-400 nm, whereas aniline and pyridine induced a modified type I (a reverse type II) spectral change characterized by a peak at 408 nm and a trough at 421 nm. These changes were found to be quantitatively proportional to the amounts of substrate added. However, the magnitude of the peaks and troughs was considerably less in the S1 fraction of the leukemic granulocytes. Correspondingly, total heme content was significantly decreased in S1 fractions of CML granulocytes as compared to similar fractions of normal granulocytes.     

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.     

NADPH-dependent reductase solubilized from microsomes by peroxidation and its activity

Isolated rat liver microsomes were subjected to enzymatic or non-enzymatic lipid peroxidation $\text{in vitro}$. NADPH-dependent cytochrome $\text{c}$ reductase activity was released from the microsomes into the media during peroxidation. This activity could be recovered from the media by DEAE-cellulose chromatography. The recovered enzyme retained high activity for the reduction of cytochrome $\text{c}$ and a lower level of activity for the reduction of cytochrome P-450. The active fractions were capable of enzymatically supporting the peroxidation of isolated mitochondria in the presence of organically complexed Fe⁺³ and NADPH, and in this respect the specific activity was found to be about ten times higher than in microsomes.