P-450 Hemoprotein Research Articles

Purification and properties of S-protein (hemoprotein 559) from human erythrocytes

1. 1. A procedure employing mild conditions has been developed for purifying S-protein (hemoprotein 559, erythrocyte), a protein present in the stromal fraction of human red blood cells. S-protein, solubilized by extracting the stroma of red blood cells with pH 8.2 buffer, was purified by chromatography on DEAE-cellulose and Bio-Gel P-60. 2. 2. The oxidized and reduced forms of this protein show visible absorption spectra typical of b- type cytochromes. The oxidized form of the protein shows absorbance maxima at 412, 533, and 565 mμ, and the dithionite-reduced spectrum shows maxima at 426, 530, and 559 mμ. The prosthetic group has been isolated and identified as protoheme IX by spectral and chromatographic techniques. The spectral properties of this protein differ from those of cytochrome b 5 . 3. 3. The reduced form of the protein binds CO, and the resulting complex shows absorbance maxima at 421, 540, and 568 mμ. This spectrum, the CO-reduced minus reduced difference spectrum, and the spectra of the oxidized and reduced forms of the free protein are all indistinguishable from the corresponding spectra of hemoprotein P-420, the altered form of the microsomal hydroxylase, hemoprotein P-450. The spectral properties, together with other physical properties, suggest that S-protein is P-420 from erythrocytes.

Phosphatidylcholine Requirement in the Enzymatic Reduction of Hemoprotein P-450 and in Fatty Acid, Hydrocarbon, and Drug Hydroxylation

Abstract The heat-stable factor required for fatty acid, hydrocarbon, and drug hydroxylation in a reconstituted liver microsomal enzyme system containing hemoprotein P-450 has been identified as phosphatidylcholine. Synthetically prepared dioleoylglyceryl-3-phosphorylcholine was fully active when substituted for the microsomal factor, as judged by the rate of laurate, hexane, octane, ethylmorphine, or benzphetamine hydroxylation in the presence of TPNH, oxygen, hemoprotein P-450, and TPNH-hemoprotein P-450 reductase. Various acyl derivatives of glyceryl-3-phosphorylcholine showed, at their optimal concentrations, the following increasing order of activity with benzphetamine as the substrate: distearoyl; a mixture of 1-monopalmitoyl and 1-monostearoyl; dipalmitoyl; dioleoyl; and a mixture of dilauroyl and monolauroyl. Electron transfer from TPNH to hemoprotein P-450, as determined from the carbon monoxide reduced difference spectrum under anaerobic conditions, was completely dependent upon the presence of microsomal lipid. Stopped flow measurements showed that the rate of electron transfer was biphasic in the presence of the lipid, with a rapid phase largely completed in less than 1 sec and having a first order rate constant of about 100 min-1, followed by a slow phase having a first order rate constant of about 6 min-1, but only the slow rate could be detected in the absence of the lipid. Under similar conditions, but with air as the gas phase, the turnover number (moles of benzphetamine hydroxylated per mole of hemoprotein P-450) was 22 min-1. These results show that the lipid is essential for the enzymatic reduction of hemoprotein P-450 and that only the rapid phase of the reaction is adequate to support the observed rate of substrate hydroxylation.

Ligand interactions with hemoprotein P-450. Equilibria between high- and low-spin forms of P-450 in bovine adrenal mitochondria.

Ligand interactions with hemoprotein P-450. Equilibria between high- and low-spin forms of P-450 in bovine adrenal mitochondria.

Effect of caffeine on hepatic microsomal cytochrome P-450

Previously we reported that pretreatment of rats with caffeine did not result in any observable increase in the P-450 hemoprotein content of the liver microsomes. However, the measurement of ethyl isocyanide difference spectra of dithionite reduced microsomes showed a small but definite increase in the 455-nm peak in the caffeinetreated group. There was no significant increase in the 430-nm peak. In this respect, caffeine action resembled that of 3-methylcholanthrene. Caffeine treatment also increased the rate of NADPH-cytochrome P-450 reductase activity. Microsomal enyzme induction by caffeine was not obliterated by adrenalectomy. The preferential increase in the 455-nm peak was more pronounced in the microsomes of the adrenalectomized animal after caffeine pretreatment.

Zonation of hemoprotein P-450 and cytochrome b5 in the adrenocortex of mammals.

Zonation of hemoprotein P-450 and cytochrome b5 in the adrenocortex of mammals.

Microsomal epoxidation of aldrin in lepidopterous larvae

Epoxidation of aldrin occurred in microsomal preparations from a number of species of lepidopterous larvae. The epoxidation process required NADPH and oxygen and was inhibited by carbon monoxide and 2-(diethylamino)ethyl 2,2-diphenyl-valerate hydrochloride (SKF-525-A). The presence of the hemoprotein P-450 in larval microsomes was determined by difference spectra, and the characteristic peak at 450 run that was produced by the addition of carbon monoxide to the reduced microsomal preparation appears to arise from a substance that is present in the reduced microsomes having an absorbance at 407 nm.

Preparation and physicochemical properties of functional hemoprotein P450 from mammalian tissue microsomes

Functional hemoprotein P450 particles were isolated from rabbit liver and pig and bovine adrenocortical microsomes by tryptic digestion in 0.1 M potassium-phosphate buffer (pH 7.0) containing 2 mM EDTA and 20% (v/v) glycerol. The optical and magnetic properties of the particles were studied at three widely different temperatures. The absolute absorption spectra of functional hemoprotein P450 of liver microsomes in the oxidized and reduced forms in the visible region had absorption peaks at 360, 414, 532 and 568 mμ in the oxidized form, at 416 and 555 mμ in the reduced form and at 450 and 555 mμ in the CO complex form at room temperature. The absolute absorption spectra of hemoprotein P450 in the visible region at the temperature of liquid N 2 had absorption peaks at 350, 414, 538 and 570 mμ in the oxidized form, at 415 and 550 mμ in the reduced form and at 447 and 550 mμ in the CO complex form. The electron paramagnetic resonance (EPR) spectrum of oxidized hemoprotein P450 at the temperatures of liquid N 2 and liquid H 2 showed a low spin form, and its g galues were identical with those of hemoprotein P450 of whole microsomes, whereas the EPR spectrum of hemoprotein P420 which had been highly purified by treatment with snake venom showed a pure high spin form at these temperatures. The same results were obtained with preparations of hemoprotein P450 from pig and bovine adrenocortical microsomes. The light absorption difference spectra in the visible region of the hemoprotein P450 complex with nitrosobenzene showed peaks at 452–453 and 554–558 mμ in the oxidized form and at 422, 455, 520 and 577 mμ in the reduced form. The absorption spectra of both forms changed with the pH. the absorption spectra of complexes of hemoprotein P450 with imidazole or triazole derivatives were also investigated. The absorption spectra of the reduced hemoprotein P450-triazole complex, unlike that of the complex of reduced hemoprotein P450 with imidazole, showed splitting of the Soret bands at 425 and 445 mμ, and these were pH dependent. On the other hand the Soret band of the oxidized hemoprotein P450-triazole complex showed neither splitting nor pH dependence. The unique structure of hemoprotein P450 is not formed only on reduction, for even the oxidized form has a unique structure. The EPR spectra of oxidized hemoprotein P450 complexes with nitrogenous ligands were studied. The optical and magnetic properties of hemoprotein P450 and its complexes with nitrogenous ligands were compared withi those of hemoprotein P420 and other hemoproteins.

Ligand interactions with hemoprotein P-450. II. Influence of phenobarbital and methylcholanthrene induction processes on P-450 spectra.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTLigand interactions with hemoprotein P-450. II. Influence of phenobarbital and methylcholanthrene induction processes on P-450 spectraColin R. E. Jefcoate and James L. GaylorCite this: Biochemistry 1969, 8, 8, 3464–3472Publication Date (Print):August 1, 1969Publication History Published online1 May 2002Published inissue 1 August 1969https://doi.org/10.1021/bi00836a050RIGHTS & PERMISSIONSArticle Views60Altmetric-Citations130LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (896 KB) Get e-Alerts Get e-Alerts

Hypertrophic, Hypoactive Smooth Endoplasmic Reticulum: A Sensitive Indicator of Hepatotoxicity Exemplified by Dieldrin

Rats with hypertrophic smooth endoplasmic reticulum (ER) and increased activities of the drug-handling enzymes induced by dieldrin were stressed with larger doses of the pesticide. The activity of the drug-handling enzymes was thus reduced, but liver weight, smooth ER, and P-450 hemoprotein remained elevated. While no changes were apparent by light microscopy, the hypertrophic, hypoactive smooth ER was recognized as tight clusters of tubular membranes associated with abnormalities of the mitochondrial membrane. Similar but not identical morphologic changes were noted in human liver diseases associated with hepatic insufficiency. Hypertrophic, hypoactive smooth ER may indicate transition from adaptation of injury, and can be used as a sensitive parameter of toxicity.

Role of Hemoprotein P-450 in Fatty Acid ω-Hydroxylation in a Soluble Enzyme System from Liver Microsomes

An enzyme system which catalyzes the ω-hydroxylation of fatty acids in the presence of reduced triphosphopyridine nucleotide and molecular oxygen has been obtained in a soluble form from rabbit liver microsomes. Three components have been separated from the microsomal extract and shown to be required for the conversion of laurate to ω-hydroxylaurate. These have been identified as hemoprotein P-450 (the carbon monoxide-binding pigment of microsomes), TPNH-cytochrome c reductase, and a heat-stable factor.

Electron-transfer mechanism associated with fatty acid desaturation catalyzed by liver microsomes.

Abstract Suitable assay conditions are described for the NADPH-dependent oxidative desaturation of stearyl-CoA by rat-liver microsomes. NADH is an even more effective electron donor than NADPH. Ascorbate at high concentrations also acts as a donor, but with low efficiency. Unlike the NADPH-dependent drug hydroxylations, the desaturation reaction does not seem to involve the microsomal hemoprotein P-450. Instead, a hitherto unknown cyanide-sensitive factor appears to be involved in the desaturation mechanism, regardless of the electron donors employed. The microsomal NADPH-specific flavoprotein with a cytochrome c reductase activity seems to participate, not only in the NADPH-dependent drug hydroxylations, but also in the NADPH-supported desaturation. Microsomal oxidation of methanol, which requires NADPH and is sensitive to cyanide, appears to be catalyzed by a mechanism which differs from that involved in desaturation. On the basis of these findings the electron-transfer mechanisms associated with these microsomal reactions are discussed.

Evidence for a new P-450 hemoprotein in hepatic microsomes from methylcholanthrene treated rats

Human body encounters thousands of chemicals in the form of drugs, food items, toxins, and other environmental pollutants. These agents are treated with the help of biomolecules called enzymes or more specific drug-metabolizing enzymes (DMEs). These exogenous substances have to pass certain chemical steps in order to become active, inactive, less toxic, more toxic, or easy to excrete out of body compartments after their biological actions. DMEs decide when, where, and for how long a given substance will pose its effects endogenously. Metabolism of drugs (xenobiotics) is always a core issue of bioscience research for its importance in fate of a chemical substance inside the body. The marked problems with these enzymes are certain disorders and agents enhancing or alleviating their biological roles termed as enzymes inducers and enzymes inhibitors, respectively. Such disorders mostly affecting the role of DMEs are those of major organs such as liver, kidneys, and gastrointestinal tract where DMEs are predominantly housed. Information provided in this chapter will help healthcare professionals to carry out dose adjustment, therapeutic drug monitoring, and prescription modification better than before. This will improve the treatment outcomes of a number of therapies related to in vivo enzymatic actions. The major chronic and acute diseases interfering with the functions of enzymes involving drug metabolism and the challenges and strategies about the fate of these enzymes involving drug metabolism have been discussed in this chapter.

Evidence for two forms of P-450 hemoprotein in microsomal membranes

Evidence for two forms of P-450 hemoprotein in microsomal membranes