Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:O95477 (membrane-bound)
 29,236 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Peptitergented P450 2a-4 (Pepti-P450), a water-soluble form of the mouse microsomal P450 2a-4, was genetically engineered and expressed in Escherichia coli. The NH2-terminal hydrophobic sequence (positions 2 to 19) of Pepti-P450 was replaced by a peptitergent PD1, amphipathic peptide consisting of 24 residues (C. E. Schafmeister, L. J. Miercke, and R. M. Stroud (1993) Science 262, 734-738). The expression level of Pepti-P450 (90,000 molecules/cell) was at least four times greater than that of wild-type P450 2a-4. Since Pepti-P450 was quite stable and was expressed as a peripheral membrane protein, it can be easily purified from the membrane fraction treated with Na2CO3 without using any detergents during the chromatographic steps. The purified Pepti-P450 retained the spectral and catalytic properties of the unmodified enzyme with a similar Km value for steroid 15 alpha-hydroxylase activity (19.7 microM in comparison to 14.2 microM of the wild-type). Gel permeation chromatography showed that the purified Pepti-P450 in the detergent-free buffer was an oligomer with an approximate molecular mass of 450 kDa. The replacement of the hydrophobic anchor domain with an amphipathic helix such as peptitergent, therefore, may provide a general method for engineering membrane-bound P450s to soluble enzymes.
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PMID:Molecular engineering of microsomal P450 2a-4 to a stable, water-soluble enzyme. 757 85

NADPH-cytochrome-P450 reductase both purified from rat hepatic microsomes and involved in microsomal fraction was inactivated by treatment with alpha-lipoic acid. Since alpha-lipoic acid contains disulfide bond, it reacts with SH-groups of the reductase via the reaction of thiol-disulfide exchange resulting in the loss of the enzyme reducing activity. NADP+ completely protected reductase from the inactivation. The modification of reductase was reversible: the modified enzyme was partially reactivated with dithiothreitol and dihydrolipoic acid in the case when cytochrome c was used as a substrate of reductase. In the case when inorganic substrate, K3Fe(CN)6, was used for assay the activity of modified reductase no reactivation was observed. It was found that the order of the reaction of inactivation of membrane-bound microsomal reductase is equal to 1.2 +/- 0.2, which is in an agreement with pseudo-first order kinetics, and the second-order-rate constant of 26 M-1min-1. The results have shown that well known therapeutic agent alpha-lipoic acid is an efficient inhibitor of both purified and microsomal reductase.
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PMID:Reversible inhibition of NADPH-cytochrome P450 reductase by alpha-lipoic acid. 757 37

All major classes of biologically active steroid hormones (progestins, mineralocorticoids, glucocorticoids, and sex steroids) are synthesized from cholesterol through 11 different bioconversions. With the exception of 5 alpha-reductase, all the enzymes mediating these reactions fall into two classes, cytochromes P450 and short-chain dehydrogenases. Cytochromes P450 are heme-containing membrane-bound proteins with molecular weights of approximately 50,000 that utilize molecular oxygen and electrons from NADPH-dependent accessory proteins to hydroxylate substrates. Short-chain dehydrogenases have molecular weights of 30,000-40,000, have tyrosine and lysine residues at the active site, and remove a hydride from the substrate, transferring the electrons of the hydride to NAD+ or NADP+. In most cases, this reaction is reversible so that the dehydrogenase can also function as a reductase under appropriate conditions. Inherited disorders in enzymes required for steroid biosynthesis have varying effects. Defects that prevent cortisol from being synthesized are referred to collectively as congenital adrenal hyperplasia. Because the enzymes required for cortisol biosynthesis in the adrenal cortex are in many cases required for the synthesis of mineralocorticoids and/or sex steroids, these classes of steroids may also not be synthesized normally. Thus, cholesterol desmolase and 3 beta-hydroxysteroid dehydrogenase deficiencies affect synthesis of all classes of steroids in both the adrenals and gonads. Steroid 21-hydroxylase deficiency, the most common cause (> 90% of cases) of congenital adrenal hyperplasia, can affect both mineralocorticoid and glucocorticoid synthesis, but androgen secretion is usually abnormally high due to shunting of accumulated precursors into this pathway. Excessive secretion of androgens and mineralocorticoids occurs in 11 beta-hydroxylase deficiency (the second most frequent form of congenital adrenal hyperplasia). Mineralocorticoid excess is also seen in 17 alpha-hydroxylase deficiency, but in this disorder sex steroid synthesis is defective. All defects that affect estrogen synthesis (deficiencies of cholesterol desmolase, 3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, aromatase, and 17 beta-hydroxysteroid dehydrogenase) are very rare, suggesting that the inability to synthesize placental estrogens may adversely affect fetal survival. A number of enzymes are expressed at sites of steroid action and regulate the amount of active steroid available to steroid receptors. Steroid 5 alpha-reductase converts testosterone to the more active dihydrotestosterone. Deficiency of this activity leads to incomplete development of male genitalia; 17 beta-hydroxysteroid dehydrogenase deficiency has similar phenotypic effects.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Genetic diseases of steroid metabolism. 781 70

Microsomal fractions from porcine ocular tissues synthesized 12(S)-5,8,10,14-hydroxyeicosatetraenoic acid [12(S)-HETE] from arachidonic acid by a membrane-bound lipoxygenase and 12(R)-HETE by the cytochrome P450-dependent monooxygenase system. Both activities were the highest in corneal microsomes. The 12(R)-HETE synthesizing activity of corneal microsomes was dependent on NADPH and inhibited by 0.1 mM SKF-525A, an inhibitor of P450 enzymes. The activity to form 12(R)-enantiomer was significantly enhanced by treatment of corneal epithelium with 3-methylcholanthrene or clofibrate. The induced activity was suppressed by cycloheximide, indicating that the induction of enzyme activities involved a translational process. The effect of these inducers on 12(R)-HETE synthesizing activity appeared to be additive. The activity to form 12(S)-enantiomer was markedly stimulated by 3 mM CaCl2. The 12-lipoxygenase of corneal microsomes was capable of oxygenating linoleic acid in addition to arachidonic acid, a characteristic of 12-lipoxygenases of the leukocyte type. 12(R)-HETE at 10(-6) M inhibited almost completely the Na,K-ATPase of corneal epithelium but had little or no effect on ciliary epithelial enzymic activity. 12(S)-HETE at 10(-6) M also inhibited corneal enzymic activity but to a lesser extent, and had no significant effect on ciliary epithelial Na,K-ATPase activity.
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PMID:Synthesis of 12(R)- and 12(S)-hydroxyeicosatetraenoic acid by porcine ocular tissues. 783 61

The kinetics of CO binding to human cytochrome P450 3A4 was examined by the flash photolysis technique, employing the membrane-bound P450 expressed in baculovirus-infected SF9 insect cells. Triexponential kinetics was observed, indicating that P450 3A4 is composed of multiple, kinetically distinguishable conformers. To define the substrate specificity of individual P450 3A4 conformers we evaluated the effect of a series of substrates of varying sizes and structures on the CO binding kinetics. The rate of CO binding to the total mixture of P450 3A4 conformers was increased in the presence of nifedipine and erythromycin, decreased by quinidine, testosterone, and warfarin, and unaffected by cimetidine and 17 alpha-ethynylestradiol. A recently developed kinetic difference method (Koley, A. P., Robinson, R. C., Markowitz, A., and Friedman, F. K. (1994) Biochemistry 33, 2484-2489) was used to define the kinetic parameters of individual P450 3A4 conformers. The results showed that different conformers have distinct substrate specificities. The substrates had markedly variable effects on the CO binding kinetics of their target P450 3A4 conformers and thus differentially modulate their conformations. These results demonstrate that the interaction of a particular substrate with a specific P450 3A4 conformer can be assessed in the presence of multiple conformers.
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PMID:CO binding kinetics of human cytochrome P450 3A4. Specific interaction of substrates with kinetically distinguishable conformers. 789 Jun 8

Human cytochrome P450 (P450) 2E1 is of interest because of its role in the oxidation of numerous drugs and carcinogens. The purification of the protein from human liver is difficult, and we report the development of a system for relatively high-level expression in Escherichia coli. A cDNA was prepared from liver cDNA by polymerase chain reaction methods and several variants with modified 5'-termini were constructed. Analysis of seven of these indicated that the highest levels of expression were found when the first 21 codons of the native sequence were deleted and the Trp immediately following the resulting N-terminal Met was changed to Ala (GCT). Levels of 40-nmol membrane-bound P450 2E1 (liter culture)-1 were routinely recovered. The recombinant P450 2E1 was purified to electrophoretic homogeneity from the bacterial membranes in two ion-exchange steps in > 80% yield. Ferric P450 2E1 was isolated in a mixed spin state. The enzyme was active in chlorzoxazone 6-hydroxylation; the addition of human liver cytochrome b5 lowered the Km for the substrate and increased Vmax. N-Terminal amino acid sequence analysis yielded the expected first 21 residues. The expression system should facilitate the availability of human P450 2E1 and antibodies for studies of the enzyme.
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PMID:Expression of modified human cytochrome P450 2E1 in Escherichia coli, purification, and spectral and catalytic properties. 803 Nov 47

Human cytochrome P450 (P450) 1A1 is primarily an extrahepatic enzyme and is important because of its roles in the activation of polycyclic hydrocarbons and other xenobiotic chemicals. Purification of active enzyme from human tissues has not been successful. We report the expression and purification of the recombinant enzyme from Escherichia coli. A full-length cDNA of human cytochrome P450 1A1 and several modified constructs were engineered into a pCW vector and used to transform E. coli cells. Little expression was observed with the native sequence and several modified constructs, but successful expression (20-25 nmol membrane-bound P450 1A1 per liter of culture) was achieved with a construct in which the Ala codon GCT was placed in the second position and the 5'-terminal codons were maximized for AT content and minimized for the potential of secondary structure formation of the mRNA transcript. alpha-Naphthoflavone was found to protect against denaturation by detergents during solubilization and was added to buffers used for purification. The recombinant P450 1A1 was purified to electrophoretic homogeneity after two ion-exchange chromatography steps in approximately 50% yield. N-Terminal amino acid sequence analysis verified the expected first 21 residues, with the exception of the terminal Met. The isolated human ferric P450 1A1 was predominantly in the high spin state, in contrast to the orthologous rat and rabbit enzymes. Recombinant P450 1A1 catalyzed 7-ethoxyresorufin O-deethylation and benzo[a]pyrene 3-hydroxylation with Km values of 0.58 and 15 microM and Vmax values of 8.3 and 2.5 nmol min-1 (nmol P450 1A1)-1, respectively. The successful expression and purification of human P450 1A1 should increase the availability of this enzyme and the generation of antibodies for further biochemical and other biological studies.
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PMID:Expression of modified human cytochrome P450 1A1 in Escherichia coli: effects of 5' substitution, stabilization, purification, spectral characterization, and catalytic properties. 803 57

A T7 expression system is described for the high-level production in Escherichia coli of the membrane-bound form of human and rat cytochrome b5. The cDNAs of b5 have been engineered to contain a coding sequence for a four-member histidine domain at the amino-terminus of the recombinant protein permitting the use of a nickel-chelate affinity column for rapid purification of the detergent-solubilized hemoprotein. Results are presented demonstrating the ability of the purified recombinant b5 proteins to stimulate the rate of oxidation of 17 alpha-hydroxypregnenolone to dehydroepiandrosterone, catalyzed by bovine P450 17A, and to stimulate the 6 beta-hydroxylation of testosterone, catalyzed by human P450 3A4. These P450-catalyzed reactions have been used to compare the properties of different forms of b5. Purified b5 can serve as a "coupling protein" as illustrated by its inhibition of NADPH oxidation, catalyzed by a fusion protein containing the heme domain of P450 3A4 linked to rat NADPH-P450 reductase, and the associated inhibition of hydrogen peroxide formation. Kinetic studies show the formation of a complex of the flavoprotein, NADPH-P450 reductase, with b5 for the rapid transfer of electrons from NADPH.
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PMID:The high-level expression in Escherichia coli of the membrane-bound form of human and rat cytochrome b5 and studies on their mechanism of function. 803 71

A full-length human cytochrome P450 (P450) 1A2 cDNA clone and four derivatives in which the 5'-terminus was modified were inserted into the pCW vector and used to transform Escherichia coli cells. Low levels of expression were seen with most of the constructs but high expression levels (245 nmol membrane-bound P450 recovered per liter culture) were achieved when the N-terminus was MALLLAVFL, as reported earlier by Fisher et al. (C. W. Fisher, D. L. Caudle, C. Martin-Wixtrom, L. C. Quattrochi, R. H. Tukey, M. R. Waterman, and R. W. Estabrook, 1992, FASEB J. 6, 759-764). The expressed human P450 1A2 in bacterial membranes was rapidly denatured to cytochrome P420 in the presence of detergents. This denaturation was blocked by the inhibitory ligand alpha-naphthoflavone (alpha NF, 7,8-benzoflavone). Human P450 1A2 was solubilized using high concentrations of sodium cholate and Triton N-101 and could be purified to near homogeneity in high yield in two steps. alpha NF was included in the buffer in the first step and then removed in the second chromatography step along with the detergent. The purified human P450 1A2 was found to be almost completely in the high spin iron configuration, in contrast to P450 1A2 enzymes isolated from rats and rabbits. The enzyme was catalytically active toward the known substrates 7-ethoxyresorufin and phenacetin. The N-terminal appears to be blocked, as is the case for other P450s we have expressed that contain the sequence MALLLAVFL in E. coli. Previously this human P450 has only been available in limited amounts; the methods presented here should facilitate further biochemical and practical studies on this interesting enzyme.
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PMID:Expression of modified human cytochrome P450 1A2 in Escherichia coli: stabilization, purification, spectral characterization, and catalytic activities of the enzyme. 811 5

The cytosolic domain of microsomal P450 52A3 (P450Cm1) was isolated as a soluble and functionally active protein. The NH2-terminal region that anchors the P450 protein to the endoplasmic reticulum was removed by sequence-specific proteolysis at a designed cleavage site. For that purpose, P450Cm1 was genetically engineered to establish at position 63-66 the sequence Ile-Glu-Gly-Arg, which is recognized by the restriction protease factor Xa. The modified P450 was produced in high yields as an integral membrane protein in Saccharomyces cerevisiae. In the microsomal fraction, it was accessible to factor Xa digestion, releasing a readily soluble, shortened P450 protein. For large scale preparation of the cytosolic domain, the modified P450Cm1 was first purified and then subjected to sequence-specific proteolysis. The highly purified delta(1-66)P450Cm1 exhibited unchanged spectral characteristics and catalyzed the hydroxylation of n-hexadecane with 85% of the activity determined for full-length wild-type P450Cm1. The method developed for the preparation of the cytosolic domain of P450Cm1 may be more generally applicable to facilitate structure-function studies on membrane-bound P450 forms.
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PMID:Generation of the soluble and functional cytosolic domain of microsomal cytochrome P450 52A3. 817 90


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