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

The kinetics of CO binding to human cytochrome P450 1A1 was used to probe the interaction of polycyclic aromatic hydrocarbons (PAHs) with the membrane-bound P450 expressed in baculovirus-infected SF9 insect cells. Biexponential kinetics was observed, indicating that P450 1A1 is composed of at least two kinetically distinguishable species. To define the substrate specificity of the individual species, we evaluated the effect of a series of PAHs of varying sizes and shapes on the CO binding kinetics of P450 1A1. The overall rate of CO binding was increased in the presence of the tricyclic PAHs phenanthrene and anthracene and the tetracyclic PAHs pyrene and 1,2-benzanthracene, but was decreased by the pentacyclic PAHs benzo[a]pyrene and 1,2:3,4-dibenzanthracene. A kinetic difference method was applied to kinetically define the individual P450 1A1 species. Two species differing in their PAH specificities were identified: a slowly reacting species sensitive to the smaller PAHs, and a rapidly reacting species responsive to larger PAHs. Upon PAH binding, CO binding to these species was accelerated and decelerated, respectively. The results furthermore suggest that the two species are interconvertable. In addition to PAHs, the interactions of P450 1A1 with 7-ethoxy- and 7-pentoxyresorufin were likewise examined for their effect on the CO binding kinetics. These compounds interacted with and decreased the rate of the rapidly and slowly reacting P450 1A1 species, respectively. The markedly variable effects of these PAHs and resorufins on the CO binding kinetics indicate differential modes of interaction with the two target P450 1A1 species, resulting in differential modulation of their conformations. These results demonstrate that multiple P450 1A1 species with distinct conformations and substrate recognition profiles coexist in a biological membrane and are resolvable using a rapid kinetic technique.
 ...
PMID:Interaction of polycyclic aromatic hydrocarbons with human cytochrome P450 1A1: a CO flash photolysis study. 895 73

Over 400 P450s have been identified to date in prokaryotes and eukaryotes, plants and animals, mitochondria and endoplasmic reticulum. These enzymes function in areas such as metabolism and steroidogenesis. The eukaryotic members of this gene superfamily of proteins have proved difficult to study because of the hydrophobic nature of their substrates, their various redox partners, and membrane association. To better understand the structure/function relationship of P450s-what determines substrate specificity and selectivity, what determines redox-partner binding, and which regions are involved in membrane binding-we have compared the three crystallized, soluble bacterial P450s (two class I and one class II) and a model of a steroidogenic, eukaryotic P450 (P450arom), to define which structural elements form a conserved structural fold for P450s, what determines specificity of substrate binding and redox-partner binding, and which regions are potentially involved in membrane association. We believe that there is a conserved structural fold for all P450s that can be used to model those P450s that prove intransigent to structural determination. However, although there appears to be a conserved structural core among P450s, there is sufficient sequence variability that no two P450s are structurally identical. NADPH-P450 reductase transfers electrons from NADPH to P450 during the P450 catalytic cycle. This enzyme has usually been thought of as a simple globular protein; however, sequence analysis has shown that NADPH-P450 reductase is related to two separate flavoprotein families, ferredoxin nucleotide reductase (FNR) and flavodoxin. Recent studies by Wolff and his colleagues have shown that the FAD-binding FNR domain and FMN-binding flavodoxin domain of human NADPH-P450 reductase can be independently expressed in Escherichia coli. The subdomains can be used to reconstitute, however poorly, the monooxygenase activity of the P450 system. We have been utilizing the reductase domain of P450BM-3 to study the mechanism of electron transfer from NADPH to P450 in this complex multidomain protein. We have overexpressed both the FNR subdomain and the flavodoxin subdomain in E. coli and fully reconstituted the cytochrome c reductase activity of this enzyme. Our studies have shown that electron transfer from NADPH through the reductase domain to the P450 requires shuttling of the FMN subdomain between the reductase subdomain and the P450. Studies of the factors that control the molecular recognition and interaction among these three proteins are complicated by the weakness of the association and changes in the strength of the interaction depending on the redox state of each of the components. How these structural and mechanistic studies of a soluble bacterial P450 can be extended to gain a better understanding of the control of membrane-bound eukaryotic P450-dependent redox systems is discussed.
 ...
PMID:P450BM-3; a tale of two domains--or is it three? 902 25

The sensitivity of pig cytochrome P450c17 (CYP17), an endoplasmic reticulum membrane-bound enzyme, towards heat denaturation (48 degrees C) was measured by the P450-to-P420 spectral transition indicating conformational labilization of the protein. Both sucrose and glucose have comparable and increasingly protective effects at concentrations ranging from 100 to 800 mM, while ectoine, a novel zwitterionic compatible solute which regulates bacterial osmoadaptation and stabilizes cytoplasmic enzymes, has a strong labilizing effect on CYP17 and favours its proteolytic inactivation possibly by electrostatic derangements. Sucrose or glucose, but not ectoine, can therefore eventually be proposed as compatible stabilizers of cytochrome P450 structures.
 ...
PMID:Effects of compatible solutes on mammalian cytochrome P450 stability. 909 73

Bovine adrenocortical cytochrome P450scc (CYP11A1) was selectively modified with diiodofluorescein iodoacetamide (DIFIA). Only Cys264 is labeled in the P450 polypeptide chain. The modification significantly affected the cholesterol-hydroxylating activity in the reconstituted system containing NADPH, adrenodoxin reductase, adrenodoxin, and soluble or membrane-bound P450scc. The inhibitory effect correlates with decreased affinity of cytochrome P450scc to intermediate electron carrier, adrenodoxin. Cytochrome P450scc is modified in liposomes and the modified membrane-bound protein is cleaved by trypsin forming two large fragments F1 and F2 corresponding to the N- and C-terminal regions of the molecule. The data indicate that the Cys264-containing region of the cytochrome P450scc molecule is exposed to the surface of protein globule, located outside of the membrane, and can participate in protein-protein interactions.
 ...
PMID:[Selective chemical modification of cytochrome P450scc (CYP11A1) with diiodofluorescein iodoacetamide in the study of the role and topology of interdomain hinge of the hemoprotein molecule]. 915 54

Microsomal NADPH-cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase. CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450. The structure of rat liver CPR, expressed in Escherichia coli and solubilized by limited trypsinolysis, has been determined by x-ray crystallography at 2.6 A resolution. The molecule is composed of four structural domains: (from the N- to C- termini) the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains. The FMN-binding domain is similar to the structure of flavodoxin, whereas the two C-terminal dinucleotide-binding domains are similar to those of ferredoxin-NADP+ reductase (FNR). The connecting domain, situated between the FMN-binding and FNR-like domains, is responsible for the relative orientation of the other domains, ensuring the proper alignment of the two flavins necessary for efficient electron transfer. The two flavin isoalloxazine rings are juxtaposed, with the closest distance between them being about 4 A. The bowl-shaped surface near the FMN-binding site is likely the docking site of cytochrome c and the physiological redox partners, including cytochromes P450 and b5 and heme oxygenase.
 ...
PMID:Three-dimensional structure of NADPH-cytochrome P450 reductase: prototype for FMN- and FAD-containing enzymes. 923 90

To evaluate the antioxidant effects of beta-carotene and astaxanthin, rat liver microsomes were exposed to a mixture of chelated iron (Fe3+/ADP) and NADPH. The carotenoids (190 pmol/mg protein) were incorporated into some of these microsomal membranes, and phospholipid hydroperoxides (PLOOH), thiobarbituric acid reactive substances (TBARS) and endogenous alpha-tocopherol content were measured over time after the initiation of oxidant stress. In control microsomes, oxidant stress led to accumulation of 1,865 (+/- 371) pmol PLOOH/mg protein during the initial 10-min peroxidation reaction, followed by a more gradual decrease during the subsequent 20-min of reaction. PLOOH accumulation during the initial 10-min reaction period was reduced to 588 (+/- 169) pmol/mg protein with beta-carotene present and 800 (+/- 288) pmol/mg protein with astaxanthin present. During the following 20-min of incubation, PLOOH levels declined in the carotenoid-supplemented microsomes but continued to increase at a slower rate in control preparations. TBARS did not show such large accumulation as observed in PLOOH during the initial 10-min incubation in any microsomal sample. The presence of carotenoids in the microsomal membrane partially inhibited the loss of alpha-tocopherol, especially during the later phase of oxidant stress. When lipid peroxidation is generated by membrane-bound cyt-P450, the specific measurement of PLOOH clearly demonstrates that the presence of carotenoids provides antioxidant protection.
 ...
PMID:Inhibition of beta-carotene and astaxanthin of NADPH-dependent microsomal phospholipid peroxidation. 926 22

Catalysis by microsomal cytochromes P450 requires the membrane-bound enzyme NADPH-cytochrome P450 reductase (P450 reductase), which transfers electrons to the P450 heme via a flavodoxin-like domain. Previously, we reported that Escherichia coli flavodoxin (Fld), a soluble electron transfer protein, directly interacts with bovine cytochrome P450 17alpha-hydroxylase/17,20-lyase (P450c17) and donates electrons to this enzyme when reconstituted with NADPH-ferredoxin (flavodoxin) reductase (FNR) (Jenkins, C. M., and Waterman, M. R. (1994) J. Biol. Chem. 269, 27401-27408). To investigate whether flavodoxins can serve as useful models of the analogous domain in P450 reductase, we have examined the FNR-Fld system from the cyanobacterium Anabaena. Mutagenesis of two acidic Anabaena Fld residues (D144A and E145A) significantly decreased flavodoxin-supported P450c17 progesterone 17alpha-hydroxylase activity. Specifically, D144A exhibited only 15% of the activity of wild-type Fld, whereas the adjacent mutation, E145A, caused a 40% loss in activity. P450-dependent hydrogen peroxide/superoxide production by wild-type FNR-Fld was measurably higher than that generated by FNR-D144A or FNR-E145A, indicating that the mutations do not lead to P450 heme-mediated electron uncoupling. Interestingly, the D144A and E145A mutants bind with equal or even greater affinity to P450c17 than wild-type Fld. Furthermore, these mutations (D144A and E145A) actually increased cytochrome c reductase activity (35 and 100% higher than wild type). Anabaena Fld residues Asp144 and Glu145 align closely with rat P450 reductase residue Asp208, which has been shown by mutagenesis to be important in electron transfer to P4502B1 but not to cytochrome c (Shen, A. L., and Kasper, C. B. (1995) J. Biol. Chem. 270, 27475-27480). Thus, these residues in flavodoxins and P450 reductase appear to have similar functions in P450 recognition and/or electron transfer, supporting the hypothesis that flavodoxins represent valid models for the FMN-binding domain of P450 reductase.
 ...
PMID:Negatively charged anabaena flavodoxin residues (Asp144 and Glu145) are important for reconstitution of cytochrome P450 17alpha-hydroxylase activity. 927 3

Cytochrome P4501A1 is a hepatic, microsomal membrane-bound enzyme that is highly induced by various xenobiotic agents. Two NH2-terminal truncated forms of this P450, termed P450MT2a and MT2b, are also found localized in mitochondria from beta-naphthoflavone-induced livers. In this paper, we demonstrate that P4501A1 has a chimeric NH2-terminal signal that facilitates the targeting of the protein to both the ER and mitochondria. The NH2-terminal 30-amino acid stretch of P4501A1 is thought to provide signals for ER membrane insertion and also stop transfer. The present study provides evidence that a sequence motif immediately COOH-terminal (residues 33-44) to the transmembrane domain functions as a mitochondrial targeting signal under both in vivo and in vitro conditions, and that the positively charged residues at positions 34 and 39 are critical for mitochondrial targeting. Results suggest that 25% of P4501A1 nascent chains, which escape ER membrane insertion, are processed by a liver cytosolic endoprotease. We postulate that the NH2-terminal proteolytic cleavage activates a cryptic mitochondrial targeting signal. Immunofluorescence microscopy showed that a portion of transiently expressed P4501A1 is colocalized with the mitochondrial-specific marker protein cytochrome oxidase subunit I. The mitochondrial-associated MT2a and MT2b are localized within the inner membrane compartment, as tested by resistance to limited proteolysis in both intact mitochondria and mitoplasts. Our results therefore describe a novel mechanism whereby proteins with chimeric signal sequence are targeted to the ER as well as to the mitochondria.
 ...
PMID:Targeting of NH2-terminal-processed microsomal protein to mitochondria: a novel pathway for the biogenesis of hepatic mitochondrial P450MT2. 934 77

The diverse function of human placental aromatase including estradiol 6alpha-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6alpha-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli. Aromatase cytochrome P450 was reconstituted and incubated with [6alpha,7alpha-(3)H2,4-(14)C]estradiol, 7-ethoxycoumarin, and [N-methyl-(3)H3]cocaine. 6Alpha-hydroxy[7alpha-(3)H,4-(14)C]estradiol was isolated as the metabolite of estradiol and the 3H-water release method based on the 6alpha-3H label was established. The initial rate kinetics of the 6alpha-hydroxylation gave Km of 4.3 microM, Vmax of 4.02 nmol min(-1) mg(-1), and turnover rate of 0.27 min(-1). Testosterone competed dose-dependently with the 6alpha-hydroxylation and showed the Ki of 0.15 microM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed Km of 200 microM, Vmax of 12.5 nmol min(-1) mg(-1) and turnover rate of 1.06 min(-1). The N-demethylation of cocaine was analysed by the 3H-release method, giving Km of 670 microM, Vmax of 4.76 nmol min(-1) mg(-1), and turnover rate of 0.49 min(-1). All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 +/- 83 pmol min(-1) mg(-1) (n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min(-1).
 ...
PMID:Diverse function of aromatase and the N-terminal sequence deleted form. 936 80

Prostaglandin I2 synthase (PGIS) produces prostaglandin I2 (PGI2) which has opposite actions on platelet aggregatory and vasoconstrictive properties compared to thromboxane A2 (TXA2) produced from the same substrate by another P450 enzyme, thromboxane A2 synthase (TXAS). PGIS and TXAS have only 16% amino acid sequence identity. Hydropathy analysis suggests that the putative NH2-terminal membrane anchor domain of PGIS is similar to many other membrane-bound microsomal P450s, which are believed to be anchored by a single transmembrane segment, and thus different from the TXAS anchor, which appears to have two transmembrane segments. To characterize the membrane anchor function of the PGIS NH2-terminal region, we have used the peptidoliposome reconstitution assay to identify the membrane anchor segment in the PGIS NH2-terminal domain and compared it with the anchor segment of P450 2C1. Four peptides, mimicking putative NH2-terminal membrane anchor segments of PGIS and P450 2C1, containing residues 1-28 (PGIS-LP1 and P450 2C1-LP1) or residues 25-54 (PGIS-LP2 and P450 2C1-LP2), were synthesized and their ability to insert in a lipid bilayer was evaluated. The results indicated that both LP1 peptides of PGIS and P450 2C1 became bound to the lipid bilayer, whereas both LP2 peptides did not bind the lipid. The two LP1 peptides were further characterized as to their conformation using CD spectroscopy. Helical structure induced in these peptides by addition of trifluoroethanol, dodecylphosphocholine, or incorporation into liposomes indicated that these segments tend to adopt a helical structure in a hydrophobic environment and thus could function as membrane anchor segments. These results support the hypothesis that PGIS and TXAS interact with the endoplasmic reticulum membrane in different ways, in which the NH2-terminal anchor domain of PGIS, as with P450 2C1, appears to have a single transmembrane segment.
 ...
PMID:Characterization of the secondary structure and membrane interaction of the putative membrane anchor domains of prostaglandin I2 synthase and cytochrome P450 2C1. 952 18


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>