Thermodynamic Driving Forces of Redox-Dependent CPR Insertion into Biomimetic Endoplasmic Reticulum Membranes.

Cytochrome P450 reductase (CPR) is a NADPH-dependent membrane-bound oxidoreductase found in the endoplasmic reticulum (ER) and is the main redox partner for most cytochrome P450 enzymes. Presented are the measured thermodynamic driving forces responsible for how strongly CPR partitions into a biomimetic ER with the same lipid composition of a natural ER. Using temperature-dependent fluorescence correlation spectroscopy and fluorescence single-protein tracking, the standard state free energies, enthalpies, and entropies of the CPR insertion process were all measured. The results of this study demonstrate that the thermodynamic driving forces are dependent on the redox states of CPR. In particular, the partitioning of CPRox into a biomimetic ER is an exothermic process with a small positive change in entropy, while CPRred partitioning is endothermic with a large positive change in entropy. Both resulted in negative free energies and strong association to the biomimetic ER, but the KP of CPRox insertion is measurably smaller than that of CPRred. Using this new information and known results from literature sources, we also present a phenomenological model that accounts for membrane-protein interactions, protein orientation relative to the membrane, and protein conformation as a function of the redox state.

An activatable probe for detecting alcoholic liver injury via multispectral optoacoustic tomography and fluorescence imaging.

A probe has been developed for imaging alcoholic liver injury through detecting the overexpressed cytochrome P450 reductase in hypoxia in the hepatic region. Upon response to the enzyme, the activated probe displays turn-on fluorescence and near-infrared absorption and generates prominent optoacoustic signals.

Screening for cytochrome P450 expression in Pichia pastoris whole cells by P450-carbon monoxide complex determination.

Cytochrome P450 (CYP) enzymes are useful biocatalysts for the pharmaceutical and biotechnological industries. A high-throughput method for quantification of CYP expression in yeast is needed in order to fully exploit the yeast expression system. Carbon monoxide (CO) difference spectra of whole cells have been successfully used for the quantification of heterologous CYP expressed in Escherichia coli in the 96-well format; however, very few researchers have shown whole-cell CO difference spectra with yeast cells using 1-cm path length. Spectral interference from the native hemoproteins often obscures the P450 peak, challenging functional CYP quantification in whole yeast cells. For the first time, we describe the high-throughput determination of CO difference spectra using whole cells in the 96-well format for the quantification of CYP genes expressed in Pichia pastoris. Very little interference from the hemoproteins of P. pastoris enabled CYP quantification even at relatively low expression levels. P. pastoris strains carrying a single copy or three copies of both hCPR and CYP2D6 integrated into the chromosomal DNA were used to establish the method in 96-well format, allowing to detect quantities of CYP2D6 as low as 6 nmol gCDW(-1 ) and 12 pmol per well. Finally, the established method was successfully demonstrated and used to screen P. pastoris clones expressing Candida CYP52A13.

Production of human cytochrome P450 2D6 drug metabolites with recombinant microbes--a comparative study.

The processes of drug development require efficient strategies to produce the respective drug metabolites, which are often difficult to obtain. Biotransformations employing recombinant microorganisms as whole-cell biocatalysts have become an attractive alternative to the chemical syntheses of such metabolites. For the first time, the potential of four different microbial systems expressing the human cytochrome P450 2D6 (CYP2D6), which is one of the most important drug-metabolizing enzymes, were compared and evaluated for such applications. The microbial host Pichia pastoris was the most efficient at expressing CYP2D6. Without additional over-expression of chaperons, the achieved yield of CYP2D6 was the highest of microbial hosts reported so far. Therefore, the system described in this study outperformed the previously reported expression of the N-terminally modified enzyme. It was also shown that the activities of the whole-cell conversions of bufuralol in recombinant P. pastoris were significantly higher than the Escherichia coli catalyst, which expressed the same unmodified gene.

Continuous spectrofluorometric and spectrophotometric assays for NADPH-cytochrome P450 reductase activity using 5-cyano-2,3-ditolyl tetrazolium chloride.

Mammalian NADPH-cytochrome P450 reductase (CPR) transfers electrons from NADPH to cytochrome P450 enzymes and other several microsomal enzymes. It also catalyzes the one-electron reduction of many chemicals and drugs. Reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) by CPR was assessed as a method for monitoring CPR activity. The electrons released from NADPH by CPR were transferred to CTC in the reaction medium, and CTC reduction activity could be assessed spectrophotometrically and spectrofluorometrically. The reduction kinetics of CTC follows classical Michaelis-Menten kinetics (K(m) = 50 microM, k(cat) = 2,520 min(-1)). This method offers a continuous assay of the enzymatic activity of CPR.

Localization of pyruvate:NADP+ oxidoreductase in sporozoites of Cryptosporidium parvum.

Cryptosporidium parvum contains a unique fusion protein pyruvate:NADP+ oxidoreductase (CpPNO) that is composed of two distinct, conserved domains, an N-terminal pyruvate:ferredoxin oxidoreductase (PFO) and a C-terminal cytochrome P450 reductase (CPR). Unlike a similar fusion protein that localizes to the mitochondrion of the photosynthetic protist Euglena gracilis, CpPNO lacks an N-terminal mitochondrial targeting sequence. Using two distinct polyclonal antibodies raised against CpPFO and one polyclonal antibody against CpCPR, Western blot analysis has shown that sporozoites of C. parvum express the entire CpPNO fusion protein. Furthermore, confocal immunofluorescence and transmission electron microscopy confirm that CpPNO is localized within the cytosol rather than the relict mitochondrion of C. parvum. The distribution of this protein is not, however, strictly confined to the cytosol. CpPNO also appears to localize posteriorly within the crystalloid body.

Defensive nature of Sargassum polycystum (Brown alga) against acetaminophen-induced toxic hepatitis in rats: role of drug metabolizing microsomal enzyme system, tumor necrosis factor-alpha and fate of liver cell structural integrity.

AIM: To assess the defensive nature of Sargassum polycystum (S. polycystum) (Brown alga) against acetaminophen (AAP)-induced changes in drug metabolizing microsomal enzyme system, tumor necrosis factor (TNF-alpha) and fine structural features of the liver during toxic hepatitis in rats. METHODS: Male albino Wistar strain rats used for the study were randomly categorized into 4 groups. Group I consisted of normal control rats fed with standard diet. Group II rats were administered with acetaminophen (800 mg/kg body weight, intraperitoneally). Group III rats were pre-treated with S. polycystum extract alone. Group IV rats were orally pre-treated with S. polycystum extract (200 mg/kg body weight for 21 d) prior to acetaminophen induction (800 mg/kg body weight, intraperitoneally). Serum separated and liver was excised and microsomal fraction was isolated for assaying cytochrome P450, NADPH Cyt P450 reductase and b(5). Serum TNF-alpha was detected using ELISA. Fine structural features of liver were examined by transmission electron microscopy. RESULTS: Rats intoxicated with acetaminophen showed considerable impairment in the activities of drug metabolizing microsomal enzymes, such as cytochrome P450, NADPH Cyt P450 reductase and b(5) when compared with the control rats. The rats intoxicated with acetaminophen also significantly triggered serum TNF-alpha when compared with the control rats. These severe alterations in the drug metabolizing enzymes were appreciably prevented in the rats pretreated with S. polycystum. The rats pretreated with S. polycystum showed considerable inhibition in the elevation of TNF-alpha compared to the rats intoxicated with acetaminophen. The electron microscopic observation showed considerable loss of structural integrity of the endoplasmic reticulum, lipid infiltration and ballooning of mitochondria in the acetaminophen-intoxicated rats, whereas the rats treated with S. polycystum showed considerable protection against acetaminophen-induced alterations in structural integrity. CONCLUSION: These observations suggest that the animals treated with S. polycystum extract may have the ability to protect the drug metabolizing enzyme system and mitochondrial functional status from free radical attack, thereby showing its defense mechanism in protecting hepatic cells from acetaminophen toxic metabolite N-acetyl-para-benzoquinone-imine (NAPQI).

Cytochrome P450 epoxygenases provide a novel mechanism for penile erection.

Erectile dysfunction (ED) is estimated to affect more than 30 million American men and 152 million men worldwide. Therapeutic agents targeting the nitric oxide/cyclic GMP signaling pathway have successfully treated patients with ED; however, the efficacies of these treatments are significantly lower in specific populations such as patients with diabetes. The goal of this study was to discover and identify new endothelium-derived relaxing factors involved in the regulation of erectile function, providing alternative therapeutic targets for treatment of ED. Immunoblotting results showed that protein expressions of epoxygenases from cytochrome P450 (CYP)2B, 2C and 2J subfamilies, as well as NADPH CYP reductase were present in rat corpora cavernosa, which was confirmed by immunohistochemical analysis. Furthermore, CYP2C was localized in cavernosal endothelial cells using double immunolabeling. CYP epoxygenase activity was analyzed by reverse-phase high-pressure liquid chromatography; and the results showed that 11,12- epoxyeicosatrienoic acid (EET) was the major product metabolized by CYP epoxygenases in rat corpora cavernosa. Inhibition of EETs function by injection of an EETs antagonist into rat penis significantly decreased intracavernosal pressure-induced by electrical stimulation of the major pelvic ganglion in vivo. In conclusion, our results suggest that EETs, produced by CYP epoxygenases, in penile endothelial cells serve as vasodilators. Inhibition of this pathway attenuated erectile function, suggesting that EETs are required for normal erection.

Cytochrome P4501A is induced in endothelial cell lines from the kidney and lung of the bottlenose dolphin, Tursiops truncatus.

Marine mammals respond to the presence of polycyclic and planar halogenated aromatic hydrocarbons (PAH or PHAH) with the induced expression in endothelium of cytochrome P4501A1, regulated through the aryl hydrocarbon receptor (AHR) transcription factor. Physiological responses in other animals, such as edema and inflammation indicate that the endothelium may be compromised by exposure to AHR agonists, which are ubiquitous in the marine environment. In other mammals and fish the cellular and molecular consequences of exposure to AHR agonists have been elucidated in cultured endothelial cells. We have cultured and characterized cetacean endothelial cells (EC) and used them in induction studies. Endothelial cells were cultured from the lung and kidney of the bottlenose dolphin, Tursiops truncates, and exposed to the AHR agonists beta-naphthoflavone (betaNF) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). betaNF (1-3 microM) induced significant increases in CYP1A1 (O-deethylation of 7-ethoxyresorufin to resorufin; EROD) activity to 3.6 and 0.92 pmol/mg/min in lung and kidney EC, respectively. TCDD was more potent than betaNF, and more efficacious, with maximum induction of CYP1A1 activity of 10.1 and 15.2 pmol/mg/min in lung and kidney EC at 3-10 nM TCDD. The differential response indicates that the lung and kidney endothelial cells in culture retain the ability to respond in a selective manner to specific stimuli. Both the molecular mechanisms of induction and the physiological consequences, especially in the vasculature, of toxicant exposure can be studied in this system.

Quantifying immunohistochemical staining of phospho-eIF2alpha, heme oxygenase-2 and NADPH cytochrome P450 reductase in oligodendrocytes during experimental autoimmune encephalomyelitis.

As a consequence of inflammation associated with multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), stress responses are induced in many cells within the CNS, however, those that occur within the primary pathological target, the oligodendrocyte, are not fully established. Recently, we found that phosphorylated eukaryotic initiation factor-2alpha (eIF2alpha), an inhibitor of protein translation associated with the stress response, is expressed in a greater number of oligodendrocytes in EAE animals compared to controls. However, since numerous oligodendrocytes in control animals also expressed phospho-eIF2alpha, a method was developed to detect expression levels within oligodendrocytes that did not rely on the number of oligodendrocytes that were stained. This method utilized a high dilution of the primary antibody so that the staining density was kept below a maximum plateau which could eliminate expression differences. Furthermore, the staining density within oligodendrocytes, as determined by image analysis, was corrected by the background density or that within neurons. In either case, the density of staining was greater in oligodendrocytes from EAE animals versus controls. The expression of heme oxygenase-2 and NADPH cytochrome P450 reductase also were examined, but unlike phospho-eIF2alpha, neither was increased in oligodendrocytes from EAE animals compared to controls. In summary, a protocol involving a high dilution of primary antibody and image analysis revealed that the expression of phospho-eIF2alpha within oligodendrocytes was increased in EAE animals compared to control animals.

The occurrence of NADPH-ferrihemoprotein reductase in Corbula caribea, from a natural oil seep at La Brea, Trinidad.

Corbula caribea is the most common non-polychaete macrofaunal organism identified at a large natural oil seep at La Brea in south Trinidad. It is hypothesized that these animals may possess (NADPH-ferrihemoprotein reductase) a component of the Mixed Function Oxygenase system (MFO), which may allow them to ameliorate the potentially deleterious effects resulting from exposure to the high levels of petroleum hydrocarbons within this environment. This study was designed to determine whether organisms from the seep site showed greater enzyme activity when compared to organisms from a non-seep reference site. NADPH-ferrihemoprotein reductase activity was determined by incubating 10 microm cryostat sections with nitro-blue tetrazolium. The reaction product was determined by visual assessment and quantified by measuring the relative mean stain intensity. The intense staining, indicative of enzyme activity was evident in the digestive epithelia of seep animals. Observations indicated that organisms from the seep showed more intense staining, indicating greater enzyme activity, when compared to animals from a non-seep reference site. The relative stain intensity of NADPH-ferrihemoprotein reductase determined for organisms from the seep was 61.30. This was significantly higher than the stain intensity determined for organisms from the non-seep reference site (7.11). This supported visual assessments, which suggested that the seep organisms showed higher enzyme activity than organisms from the non-seep site. The results suggest that NADPH-ferrihemoprotein reductase may be present in Corbula caribea from the seep site and not in those from the non-seep site. It is possible that this enzyme may contribute to these animals ability to tolerate chronic exposure to petroleum hydrocarbons and offer then a selective advantage for survival the seep environment.

R25 is an intracellular membrane receptor for a snake venom secretory phospholipase A(2).

Ammodytoxin is a presynaptically neurotoxic (beta-neurotoxic) snake venom secretory phospholipase A(2) (sPLA(2)). We detected a 25 kDa protein which binds the toxin with very high affinity (R25) in porcine cerebral cortex. Here we show that R25 is an integral membrane protein with intracellular localisation. It is the first sPLA(2) receptor known to date that localises to intracellular membranes. Centrifugation on sucrose gradients was used to fractionate porcine cerebral cortex. The subcellular composition of the fractions was determined by following the distribution of organelle-specific markers. The distribution of R25 in the fractions matched the distribution of the mitochondrial marker succinate dehydrogenase, but not the markers for plasma membrane, lysosomes, endoplasmic reticulum, synaptic and secretory vesicles. R25 most likely resides in mitochondria, which are known to be targets for sPLA(2) neurotoxins in the nerve ending and are potentially implicated in the process of beta-neurotoxicity.

Automatic identification of proteins with a MALDI-quadrupole ion trap mass spectrometer.

A matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometer of new design is described. The instrument is based on a commercial Finnegan LCQ ion trap mass spectrometer to which we have added a MALDI ion source that incorporates a sample stage constructed from a compact disk and a new ion transmission interface. The ion interface contains a quadrupole ion guide installed between the skimmer and the octapoles of the original instrument configuration, allowing for operation in both MALDI and electrospray ionization modes. The instrument has femtomole sensitivity for peptides and is capable of collecting a large number of MALDI MS and MALDI MS/MS spectra within a short period of time. The MALDI source produces reproducible signals for 10(4)-10(5) laser pulses, enabling us to collect MS/MS spectra from all the discernible singly charged ions detected in a MS peptide map. We describe the different modes of the instrument operation and algorithms for data processing as applied to challenging protein identification problems.

[Cytochrome P-450 content and activity of NADPH-cytochrome P-450 c-reductase in experimental subarachnoid hemorrhage]. / Soderzhanie tsitokhroma P-450 i aktivnost' NADFN-tsitokhrom c-reduktazy pri eksperimental'nom subarakhnoidal'nom krovoizliianii.

The condition was studied of the brain monooxigenase system (MOS) in experimental subarachnoidal hemorrhage in rats. The results show that the above hemorrhage leads to a significant depression of activity of brain MOS enzymes (cytochrome P-450, NADPN-cytochrome C-reductase). Depression of activity of the enzymic system is manifest from day 1 and has a tendency toward reduction but at day 14 and 21 the indices are still below the control values. Under the exposure to phenobarbital the reduction of activity of the enzyme system in subarachnoidal hemorrhage gets accelerated, with normalization of the indices occurring by day 7 after the hemorrhage.

Isolation of peroxisomes from tissues and cells by differential and density gradient centrifugation.

Peroxisome purification depends on a two-step procedure: differential centrifugation to prepare a light mitochondrial fraction and fractionation on a density-gradient medium preferably iodixanol or Nycodenz, to isolate the peroxisome enriched fraction. The iodixanol gradient may be a preformed continuous gradient or a self-generating gradient. Alternatively a continuous Nycodenz gradient or a simple Nycodenz barrier may be used for the second step. The unit contains protocols for peroxisome isolation from rat liver, tissue culture cells (HepG2 cells), and yeast spheroplasts. The extent of endoplasmic reticulum contamination of the prep can be assessed using an assay for the marker enzyme NADPH-cytochrome creductase.

Ca-ATPase of human myometrium plasma membranes.

We determined and characterized the Mg2+-dependent, Ca2+-stimulated ATPase (Ca-ATPase) activity in cell plasma membranes from the myometrium of pregnant women, and compared these characteristics to those of the active Ca2+-transport already demonstrated in this tissue. Similarly to the Ca2+-transport system, the Ca2+-ATPase is Mg2+-dependent, stimulated by calmodulin, and inhibited by vanadate. The Km for Ca2+ activation is 0.40 microM, very similar to that found for active calcium transport, i.e. 0.25 microM. Consequently, this Ca2+-ATPase can be responsible for the active calcium transport across the plasma membranes of smooth muscle cells.

Lanosterol 14alpha-demethylase (CYP51), NADPH-cytochrome P450 reductase and squalene synthase in spermatogenesis: late spermatids of the rat express proteins needed to synthesize follicular fluid meiosis activating sterol.

Lanosterol 14alpha-demethylase (CYP51) is a cytochrome P450 enzyme involved primarily in cholesterol biosynthesis. CYP51 in the presence of NADPH-cytochrome P450 reductase converts lanosterol to follicular fluid meiosis activating sterol (FF-MAS), an intermediate of cholesterol biosynthesis which accumulates in gonads and has an additional function as oocyte meiosis-activating substance. This work shows for the first time that cholesterogenic enzymes are highly expressed only in distinct stages of spermatogenesis. CYP51, NADPH-P450 reductase (the electron transferring enzyme needed for CYP51 activity) and squalene synthase (an enzyme preceding CYP51 in the pathway) proteins have been studied. CYP51 was detected in step 3-19 spermatids, with large amounts in the cytoplasm/residual bodies of step 19 spermatids, where P450 reductase was also observed. Squalene synthase was immunodetected in step 2-15 spermatids of the rat, indicating that squalene synthase and CYP51 proteins are not equally expressed in same stages of spermatogenesis. Discordant expression of cholesterogenic genes may be a more general mechanism leading to transient accumulation of pathway intermediates in spermatogenesis. This study provides the first evidence that step 19 spermatids and residual bodies of the rat testis have the capacity to produce MAS sterols in situ.

Developmental changes in steroidogenic enzymes in human postnatal adrenal cortex: immunohistochemical studies.

Adrenarche is considered to occur as a result of intra-adrenal changes in steroidogenic enzymes involved in C19 steroid production. The present study was conducted because developmental changes in steroidogenic enzymes have not been examined well in human postnatal adrenal. Twenty-four specimens of nonpathological human adrenals from 7 months to 62 years retrieved from autopsy files. Immunohistochemistry for P450 side-chain cleavage (P450scc), 17alpha hydroxylase (P450c17), dehydroepiandrosterone sulfotransferase (DHEA-ST), P450 oxidoreductase, cytochrome b5, and 3beta-hydroxysteroid dehydrogenase (3betaHSD) was per-formed in these specimens, and the immuno-intensity was evaluated using CAS 200 computed image analysis system. Immunoreactivity of P450scc was marked in the zona glomerulosa, fasciculata and reticularis in the adrenal glands of all the cases examined. P450c17 and DHEA-ST immunoreactivity was weak in the zona fasciculata and reticularis in the adrenals of age 7 months to 5 years, but thereafter became prominent in the zona reticularis. Immunoreactivity of P450 oxidoreductase and cytochrome b5, components of the electron transfer system hypothesized to regulate the 17-20 lyase activity of P450c17, was weak in all three zones of adrenal cortex from 7 months to 5 years, and became more marked in the zona reticularis after age 5 years. 3betaHSD immunoreactivity was marked in all three zones of the adrenal cortex from 7 months to 8 years but thereafter decreased in the zona reticularis. These data suggest that the human adrenal zona reticularis markedly begins to develop morphologically and functionally at around 5 years of age. The increased level of P450c17, DHEA-ST, P450 oxidoreductase, and cytochrome b5, and the decreased level of 3betaHSD in the reticularis is likely to contribute to increased C19 steroid production during adrenarche.

A comparative study of the isolation of type II epithelial cells from rat, hamster, pig and human lung tissue.

Data is reported on the reproducibility and purity of alveolar type II cell isolations from 4 species. Human and pig type II cells were isolated using a tissue slice method to remove blood and contaminating cells, whilst rat and hamster cells were isolated using the method of protease instillation. All cells were purified on Percoll gradients and by differential attachment. Cell type purity was assessed by phase contrast microscopy, electron microscopy (EM), percentage of cells alkaline phosphatase (AP) positive and percentage of cells staining strongly for NADPH dependent nitro blue tetrazolium reductase (NBT). These enzymes are considered as markers for type II and Clara cells respectively. The purity of all cell preparations was enhanced following 24 h culture on a biomatrix and whilst plating efficiency was similar for all species, the human tissue consistently yielded the highest purity of type II cells. All cells with lamellar bodies did not contain AP, and activity was variable between species. Further studies are needed to determine if NBT is equally nonspecific as a cell marker enzyme. In summary, sufficient type II cells of high purity can be isolated thus permitting interspecies comparative studies to investigate the effects of selective and non-specific pulmonary toxins, but more specific marker enzymes are required to identify Type II and Clara cells.

Dietary oxidized linoleic acid modifies lipid composition of rat liver microsomes and increases their fluidity.

The effect of dietary oxidized oil on the lipid composition, fluidity and function of rat liver microsomes was studied. Male growing rats were fed diets containing 10 g/100 g of a fresh (control) or oxidized (experimental) linoleic acid-rich preparation for 4 wk. High levels of fluorescent compounds and of thiobarbituric acid reactive substances indicated the occurrence of substantial lipid peroxidation in the microsomes of the experimental rats. The fluidity of the liver microsomes derived from rats fed the experimental diet was significantly higher than that of the membranes of the controls. This was due to profound differences in lipid composition of the liver microsomes, namely, a lower cholesterol to phospholipid molar ratio and a greater arachidonic acid content in the phospholipids of the rats fed the experimental diet. The fluidity differences were accompanied by greater activity of the microsomal enzymes, aldehyde dehydrogenase and NADPH cytochrome C reductase. The study demonstrated that ingestion of oxidized lipids caused profound alterations in membrane composition, fluidity and function. These alterations are likely to be associated with an enhanced cholesterol turnover, as indicated by the greater cholesterol excretion observed for the experimental rats.