Thin film voltammetry of metabolic enzymes in rat liver microsomes

We report herein thin film voltammetry and kinetics of electron transfer for redox proteins in rat liver microsomes for the first time. Films were made layer-by-layer from liver microsomes and polycations on pyrolytic graphite electrodes. Cyclic voltammograms were chemically reversible with a midpoint potential of -0.48 V vs SCE at 0.1 V s(-1) in pH 7.0 phosphate buffer. Reduction peak potentials shifted negative at higher scan rates, and oxidation-reduction peak current ratios were approximately 1 consistent with non-ideal quasireversible thin film voltammetry. Analysis of oxidation-reduction peak separations gave an average apparent surface electron transfer rate constant of 30 s(-1). Absence of significant electrocatalytic reduction of O(2) or H(2)O(2) and lack of shift in midpoint potential when CO is added that indicates lack of an iron heme cofactor suggest that peaks can be attributed to oxidoreductases present in the microsomes rather than cytochrome P450 enzymes.     

Oxygenation of alkyl sulfides with ferrous perchlorate/ascorbic acid/oxygen system

In the oxidation of alkyl sulfides bearing acidic α-hydrogens eiher with phenobarbital induced rabbit liver microsomal cytochrome P-450, or with the Udenfriend's model system (ferous perchlorate/ascorbic acid/oxygen system), both S-dealkylation and S-monoxygenation took place concurrently. Meanwhile, in the oxidation of simple alkyl sulfides, steeoselective S-monooxygenation was found of occur predominantly.     

DESTRUCTION OF MICROSOMAL CYTOCHROME P-450 BY REACTIVE OXYGEN SPECIES GENERATED DURING PHOTOSENSITIZATION OF HEMATOPORPHYRIN DERIVATIVE

Rat liver microsomal cytochrome P-450 undergoes rapid destruction in the presence of hematoporphyrin derivative (HpD) and solar radiation (∼ 400 nm). Destruction of cytochrome P-450 is associated with the formation of cytochrome P-420 and significant loss of microsomal haem. Quenchers of singlet oxygen including 2,5-dimethylfuran, histidine, ß-carotene, and ascorbic acid and inhibitors of the hydroxyl radical such as benzoate, mannitol, and ethanol prevent deterioration of the microsomal haem-protein, whereas superoxide dismutase and catalase are ineffective in this regard. These results indicate that generation of singlet oxygen during hematoporphyrin photosensitization is associated with destruction of microsomal cytochrome P-450 and haem.     

Enzymatic generation of alloxan radicals in rat liver microsomes: possible participation of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase.

Electron spin resonance studies showed that addition of rat liver microsomes to the reaction system of alloxan with reduced nicotinamide adenine dinucleotide phosphate (NADPH) resulted in a marked increase in the generation of alloxan radicals (AH.), whereas heat-denatured microsomes were without such effect. Oxidation of NADPH by alloxan was also stimulated by microsomes. The microsomes from rats treated with phenobarbital, an inducer of cytochrome P-450 reductase, greatly stimulated both the AH.generation and the NADPH oxidation. However, the microsomes from rats treated with 3-methylcholanthrene, an inducer of DT-diaphorase, did not have stimulative effect greater than the control microsomes. These results suggest the possibility that NADPH-linked AH.generations in microsomal membranes is catalyzed by NADPH-cytochrome P-450 reductase.     

A Solid Phase Radioimmunoassay for Yeast Cytochrwe P-450

Abstract

A radioimwunoassay for the quantitation of cytochrome P-450 (cyt P-450) from the yeast Candida maltosa in the microsomab fraction was established using a purified secondary antibody adsorbed to cellulose nitrate discs, a primary rabbit anti (cyt P-450) serum and [¹²⁵I] labeled cyt P-450. The present assay is highly sensitive, specific and simple, It allows the detection of as little as 5 f moles cyt P-450 in a volume of 40 μ1. Equal immunological reactivities were found for the highly purified, solubilized microsomal, denatured and the apo-form of the yeast cyt P-450.     

Use of A New HPLC Method in Rat Liver Microsomal Testosterone Monooxygenation and Its Application to Study the Sex Dependent Expression of Several Hydroxylases

Abstract

An HPLC method described by Mancilla and Gil [Analytical Letters 17, (B9), 1984, 873-886] has been applied to study the sex dependent expression of several rat liver testosterone hydroxylases. A sample clean up procedure has been developed using SEP-PACK C- 18 cartridges which retained testosterone and its microsomal oxidative products. Undesired components were not retained or selectivly eluted with organic solvents. The clean steroid sample was eluted with a mixture of n-hexane and 2-propanol. HPLC of testosterone microsomal oxidation products was performed by normal phase In a Lichrosorb diol column using an Isocratic mixture of n-hexane and 2-propanol. The main five testosterone metabolites produced by male and female rat liver microsomes were determined In only 24 min. The turnover rates for testosterone oxidation were similar in male and female microsomes, but significant differences were observed in the rate of production of different metabolites. Male microsomes catalized mainly oxidation at positions 2 α, and 7 α; whereas female microsomes produced mainly 7 α OHT and androstenedione. These results might be explained by the different contribution of some cytochrome P-450 isozymes in microsomes from the different sexes. This method provides a useful tool to study the P-450 isozymic contributions to microsomal activities in different tissues and might facilitate the comparison of P-450 isozymes purified in different laboratories.     

Analytical fractionation of microsomal cytochrome P-450 isoenzymes from rat liver by high-performance ion-exchange chromatography

Ion-exchange Fast Protein Liquid Chromatography (FPLC) on Mono Q and Mono S was optimized for the analytical separation of microsomal cytochrome P-450 species from rat liver. The effects of detergent, pH, gradient profile and column load on resolution are demonstrated. Successive application of anion- and cation-exchange chromatography leads to eleven separated P-450 fractions. The altered microsomal P450 pattern after treatment of rats with various inducers is reflected by distinct elution profiles. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and enzymatic analysis imply that several FPLC fractions contain more than one P-450 species. Preliminary results are presented showing the suitability of immobilized metal affinity chromatography (MAC) for general P-450 fractionation and thus for the further resolution of Mono Q and Mono S fractions. Scale-up for preparative P-450 fractionation is easily done by adapting the optimized analytical FPLC procedures to Q- and S-Sepharose Fast Flow.     

Measurement of enzyme activities of cytochrome P-450 isoenzymes by high-performance liquid chromatographic analysis of products.

A method is described for the qualitative and quantitative determination of the isoenzymes of cytochrome P-450 from rat liver microsomes. Microsomes are incubated with the endogenous steroid 17 beta-testosterone, which results in the formation of a number of stereo-specific hydroxylation products of testosterone. The hydroxylated products were identified using standards or by comparison with data from the literature. The products can be analysed by reversed-phase gradient high-performance liquid chromatography. The assay has been optimised for pH, linearity and time of incubation. An evaluation of the assay was performed for different kinds of microsomes, microsomal dilution and specificity for particular cytochrome P-450 isoenzymes.     

Determination of the cytochrome P-450 IV marker, omega-hydroxylauric acid, by high-performance liquid chromatography and fluorimetric detection

The formation of omega-hydroxylauric acid from lauric acid is an indicator of the activity of cytochrome P-450 IV family proteins. The two main metabolites of lauric acid, (omega-1)-and omega-hydroxylauric acid, have been completely separated by reversed-phase high-performance liquid chromatography. Measurement of lauric acid hydroxylase activity in microsomal liver samples, based on derivatization of the substrate and metabolites with the fluorescent agent 4-bromomethyl-6,7-dimethoxycoumarin, is a precise method (coefficient of variation = 7.6 and 10% for omega and (omega-1) metabolites, respectively) with good sensitivity (signal-to-noise ratio in microsomal samples of untreated rats greater than 20). In microsomal fractions from livers of rats treated with di-(2-ethylhexyl)phthalate the extent of omega-hydroxylation of lauric acid increased dose-dependently (ca. ten-fold). The (omega-1)-hydroxylase activity was not altered. A strong correlation between immunochemically determined cytochrome P-450 IVA1 and lauric acid omega-hydroxylase activity was found (r = 0.94, n = 30).     

Electrochemical Activation of the Natural Catalytic Cycle of Cytochrome P450s in Human Liver Microsomes

The natural catalytic cycle of cytochrome (cyt) P450 enzymes in human liver microsome (HLM) films was activated electrochemically via the electron transfer sequence electrode→cyt P450 reductase (CPR)→cyt P450. Cyclic voltammograms for HLM films had midpoint potentials of ?0.50 V vs. SCE at pH 7.4 characteristic of CPR, not cyt P450s. HLM and CPR microsomes without cyt P450s did not electrocatalytically reduce H₂O₂, and did not shift midpoint potential when CO was added, also indicating that the peaks do not correspond to iron heme cyt P450 enzymes. Electrochemical activation of the natural cyt P450 cycle for substrate conversion via CPR in HLM films was confirmed by catalytic electrolysis in an electrochemical microfluidic array designed to generate and detect reactive metabolites by measuring their reactivity with DNA.     

Immobilization of Cytochrome P-450 and its Application in Ehzikb Electrodes

Abstract

For application in enzyme electrodes liver microsomal cytochrome P-450 was immobilized in a membraneous form. The immobilization yielded 60% of activity and did not impair the functional stability of the enzyme. By coimmobilization of glucose oxidase with P-450 the cofactor NADPH could be replaced by H₂O₂ formed from the enzymatic glucose oxidation. Fixed to a graphite electrode the obtained preparations were employed for quantitative substrate analysis. The P-450 substrate aniline was measured by anodic oxidation of its hydroqlation product at +250mV. A linear dependence of: the current on aniline concentration up to 0.5mM was obtained.     

Application of chemical P-450 model systems to study drug metabolism. III. Metabolism of 3-isobutyryl-2-isopropylpyrazolo[1,5-alpha]pyridine

Oxidation of 3-isobutyryl-2-isopropylpyrazolo[1,5-alpha]pyridine (IBPP) was carried out with various chemical model systems for cytochrome P-450 in comparison with the liver microsomal system of rats or humans. Alpha-hydroxylation of side chains and ring hydroxylation at the 6 and 7 positions were the main reactions in both systems. A pattern analysis of products using two dimensional thin layer chromatography was employed to compare the functions of the chemical model systems with those of microsomal systems. The reaction profile of IBPP by the catalyst/Pt-colloid/H2, O2 system was most similar to that of human or rat microsomal system. The utility of these chemical models is discussed from the viewpoint of drug metabolism.     

Litcrosomal Nadph Oxidase and Hybrid Electrodes

Abstract

The NADPH oxidizing activity of rat liver microsomes was investigated and found to be mainly due to the cytochrome P-450 system. The XADPH oxidase was utilized for the development of several organelle electrodes. Gelatin membrane immobilized microsomes were combined with an O₂ membrane sensor for bioelectrochemical measurement of NADPH. The dependence of the current on substrate concentration was linear up to 1 mmol·1^?1 To assemble hybrid electrodes for determination of glucose-6-phoaphate, ATP and isocitrate pure enzymes were coimmobilized with the microsomal fraction.     

Inhibitory effect and interaction of stanozolol with pig testicular cytochrome P-450 (17 alpha-hydroxylase/C17,20-lyase)

The inhibitory effect of an anabolic steroid, stanozolol, on testicular microsomal cytochrome P-450 (17 alpha-hydroxylase/C17,20-lyase) (P-450(17 alpha/lyase] and the nature of the interaction were compared with those of other anabolic steroids, furazabol and mestanolone. Stanozolol markedly inhibited delta 16-C19-steroid synthesizing activity, 17 alpha-hydroxylase and C17,20-lyase activities, which were mediated by oxygenase activities of testicular microsomal cytochrome P-450(17 alpha/lyase). In addition, stanozolol was a competitive inhibitor of 17 alpha-hydroxylase (Ki = 6.31 microM) and C17,20-lyase (Ki = 1.30 microM) activities in the reconstituted enzyme system. The interaction of cytochrome P-450&17 alpha/lyase) with stanozolol induced a type I difference spectrum (peak at 387 nm and trough at 418 nm) with a dissociation constant (Ks) of 1.47 microM.     

Fractionation by High Performance Liquid Chromatography of Microsomal Cytochrome P-450 Induced by Hexachlorobiphenyl Isomers

Abstract

High performance liquid chromatography has been employed to fractionate rat liver microsomes under nondenaturing conditions. Selective detection at 405 nm allowed resolution of microsomal heme proteins into three peaks (A, B, and C). Cytochromes in the peaks retain their native property of binding CO after HPLC. Peak-A, first eluting, contains P-450 and is rich in cytochrome P-420. Peak-B is largely hemoglobin and peak-C is a major cytochrome P-450. The ratio of peak-C to A is increased by treatment of rats with phenobarbitone, β-naphthoflavone, 2,3,5,2′,3′,5′-hexachlorobiphenyl and 3,4,5,3′,4′,5′-hexachlorobiphenyl as compared to controls. The highest increment in the ratio is observed on feeding 3,4,5,3′,4′,5′-hexachlorobiphenyl. NADPH cytochrome c reductase elutes earlier than peak-C but cytochrome b₅ is not separated from the major cytochrome P-450 peak. The separations obtained are highly reproducible and considerably faster than conventional gel permeation chromatography. The data presented here are very promising in establishing the role of HPLC in the studies of insoluble proteins and enzymes in general and cytochrome P-450 in particular.     

Co-suppression by nicardipine, a calcium antagonist, of induction of microsomal lauric acid hydroxylation with peroxisome proliferation in clofibrate-treated rat liver

The in vivo effect of nicardipine, a well-known calcium antagonist, on microsomal omega-oxidation of laurate in clofibrate-treated rat liver was studied. The 15.3-fold induction of the activity by 2 weeks administration of 0.25% clofibrate in the diet was markedly suppressed to about 6-fold by co-administration of nicardipine at 100 mg/kg body weight. Similarly, the induction of peroxisomal beta-oxidation and carnitine acetyltransferase activities were also suppressed by this simultaneous administration by more than 50%. Although clofibrate also induced the activity of reduced nicotineamide adenine dinucleotide phosphate (NADPH)-cytochrome c reductase and increased the hepatic content of cytochrome P-450, no suppressive effect of nicardipine was observed. Contrarily, nicardipine induced the reductase activity and increased the hepatic content of cytochromes P-450 and b5. These results provide the first demonstration of a calcium antagonist, e.g. nicardipine acting as inhibitor of the induction of microsomal omega-oxidation, in association with the inhibition of peroxisome proliferation in animals. The suppression of drug-induced peroxisome proliferation and microsomal omega-oxidation by the calcium antagonist may help in elucidating the causal relationship of the induction mechanisms between peroxisomal and microsomal enzymes.     

Immobilization of pig liver microsomes

Microsomes from pig liver were covalently coupled to Sepharose activated by CNBr and to Sephadex activated by 1,1’-carbonyldiimidazole. Microsomes were also entrapped inside Ca-alginate andk-carrageenan gels. The concentration of immobilized cytochrome P-450 was determined by CO-difference spectra. The activity of the monooxygenase system was demonstrated by theN-demethylation of aminopyrine, theO-demethylation ofp-nitroanisole, and the hydroxylation of perhexiline maleate. Upon immobilization, a 30–40% and a 60–70% decrease in V _max ^app for theOandN-demethylations were respectively observed. The V _max ^app values for the hydroxylation of perhexiline maleate were essentially the same for the different immobilized forms and for the freely suspended microsomal cytochrome P-450. Under storage at 4°C, microsomes entrapped insidek-carrageenan and Ca-alginate were less stable than the free microsomes, whereas immobilization on CNBr-activated Sepharose improved the stability of the hepatic microsomal monooxygenase system at the same temperature. These types of immobilized microsomes have the advantage of being easily recovered and reused for other assays. Finally, microsomes entrapped insidek-carrageenan or Ca-alginate can be used to follow up the continuous metabolization ofp-nitroanisole for several hours in a stirred-batch reactor.     

The sequence homologies of cytochromes P-450 and active-site geometries

Summary The amino acid sequence alignment of 16 cytochrome P-450 proteins representative of the major families is reported. The sequence matching process has been carried out on the basis of maximum homology by residue type, retention of secondary structure and minimization of deletions/insertions except where additional loop regions exist. From the starting point of known reported sequence homology matching from the literature, a realignment on the basis of conserved residues involved in both structure and function gives rise to a self-consistent set of sequences which correlates with known mechanistic and structural data. Once fitted, these archetypal sequences form a straightforward template for the alignment of all P-450 subfamilies. Computer modelling of the active-site regions constructed from homology with the bacterial form of the enzyme (P-450_CAM) evinces the correct substrate specificity. Furthermore, the construction of the macromolecular assembly of components of the cytochrome P-450 system on the microsomal endoplasmic reticular membrane is presented from the evidence of site-directed mutagenesis, analysis by molecular probes, X-ray crystallography and molecular modelling.     

PHOTODYNAMIC EFFECTS OF NEW SILICON PHTHALOCYANINES: In vitro STUDIES UTILIZING RAT HEPATIC MICROSOMES AND HUMAN ERYTHROCYTE GHOSTS AS MODEL MEMBRANE SOURCES

Abstract— Photodynamic therapy (PDT) of cancer is a modality that relies upon the irradiation of tumors with visible light following selective uptake of a photosensitizer by the tumor tissue. There is considerable emphasis to define new photosensitizers suitable for PDT of cancer. In this study we evaluated six phthalocyanines (Pc) for their photodynamic effects utilizing rat hepatic microsomes and human erythrocyte ghosts as model membrane sources. Of the newly synthesized Pc, two showed significant destruction of cytochrome P-450 and monooxygenase activities, and enhancement of lipid peroxidation, when added to microsomal suspension followed by irradiation with ∼ 675 nm light. These two Pc named SiPc IV (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₂) and SiPc V (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₃¹ I) showed dose-dependent photodestruction of cytochrome P-450 and monooxygenase activities in liver microsomes, and photoenhancement of lipid peroxidation, lipid hydroperoxide formation and lipid fluorescence in rnicrosomes and erythrocyte ghosts. Compared to chloroaluminum phthalocyanine tetrasulfonate, SiPc IV and SiPc V produced far more pronounced photodynamic effects. Sodium azide, histidine, and 2,5-dimethylfuran, the quenchers of singlet oxygen, afforded highly significant protection against SiPc IV- and SiPc V-mediated photodynamic effects. However, to a lesser extent, the quenchers of superoxide anion, hydrogen peroxide and hydroxyl radical also showed some protective effects. These results suggest that SiPc IV and SiPc V may be promising photosensitizers for the PDT of cancer.     

Microsomal electrodes for reduced nicotinamide adenine dinucleotide and its phosphate,glucose-6-phosphate and ascorbate

Rat liver microsomes have been immobilized in a membrane by gelatin entrapment. The resulting membranes can be attached to an oxygen electrode to provide a sensor for several compounds. NADPH and NADH are determined by utilizing the liver microsomal NADPH oxidase activity, which is, at least in part, due to the cytochrome P-450 system. The calibration graph for NADPH is linear up to 1 mM. A multi-enzyme system localized in liver microsomes allows the determination of 20–800 μM glucose-6-phosphate. The non-enzymatic lipid peroxidation in liver microsomes, which is induced by ascorbate, allows 0.5–2.5 mM ascorbate to be determined.