Biomedical subjects

H Michel

Publications and source records attributed to H Michel.

At least 55 records · Page 3Linked to original sources

PubMed1999

Refined crystal structures of reaction centres from Rhodopseudomonas viridis in complexes with the herbicide atrazine and two chiral atrazine derivatives also lead to a new model of the bound carotenoid.

In a reaction of central importance to the energetics of photosynthetic bacteria, light-induced electron transfer in the reaction centre (RC) is coupled with the uptake of protons from the cytoplasm at the binding site of the secondary quinone (QB). It has been established by X-ray crystallography that the triazine herbicide terbutryn binds to the QB site. However, the exact description of protein-triazine interactions has had to await the refinement of higher-resolution structures. In addition, there is also interest in the role of chirality in the activity of herbicides. Here, we report the structural characterisation of triazine binding by crystallographic refinement of complexes of the RC either with the triazine inhibitor atrazine (Protein Data Bank (PDB) entry 5PRC) or with the chiral atrazine derivatives, DG-420314 (S(-) enantiomer, PDB entry 6PRC) or DG-420315 (R(+) enantiomer, PDB entry 7PRC). Due to the high quality of the data collected, it has been possible to describe the exact nature of triazine binding and its effect on the structure of the protein at high-resolution limits of 2.35 A (5PRC), 2.30 A (6PRC), and 2.65 A (7PRC), respectively. In addition to two previously implied hydrogen bonds, a third hydrogen bond, binding the distal side of the inhibitors to the protein, and four additional hydrogen bonds mediated by two tightly bound water molecules on the proximal side of the inhibitors, are apparent. Based on the high quality data collected on the RC complexes of the two chiral atrazine derivatives, unequivocal assignment of the structure at the chiral centres was possible, even though the differences in structures of the substituents are small. The structures provide explanations for the relative binding affinities of the two chiral compounds. Although it was not an explicit goal of this work, the new data were of sufficient quality to improve the original model also regarding the structure of the bound carotenoid 1,2-dihydroneurosporene. A carotenoid model with a cis double bond at the 15,15' position fits the electron density better than the original model with a 13,14-cis double bond.

Atrazine↗

PubMed1999

Purification, cloning, and characterization of the 16S RNA m5C967 methyltransferase from Escherichia coli.

The methyltransferase that forms m5C967 in Escherichia coli small subunit ribosomal RNA has been purified, cloned, and characterized. The gene was identified from the N-terminal sequence of the purified enzyme. The gene is a fusion of two open reading frames, fmu and fmv, previously believed to be distinct due to a DNA sequencing error. The gene, here named rsmB, encodes a 429-amino acid protein that has a number of homologues in prokaryotes, Archaea, and eukaryotes. C-Terminal sequencing of the overexpressed and affinity-purified protein by mass spectrometry methods verified the sequence expected for the gene product. The recombinant protein exhibited the same specificity as the previously described native enzyme; that is, it formed only m5C and only at position 967. C1407, which is also m5C in natural 16S RNA, was not methylated. In vitro, the enzyme only recognized free 16S RNA. 30S ribosomal subunits were not a substrate. There was no requirement for added magnesium, suggesting that extensive secondary or tertiary structure in the RNA substrate may not be a requirement for recognition.

5-Methylcytosine↗

PubMed1999

Electrochemical and ultraviolet/visible/infrared spectroscopic analysis of heme a and a3 redox reactions in the cytochrome c oxidase from Paracoccus denitrificans: separation of heme a and a3 contributions and assignment of vibrational modes.

Cytochrome c oxidase from Paracoccus denitrificans was studied with a combined electrochemical and ultraviolet/visible/infrared (UV/vis/IR) spectroscopic approach. Global fit analysis of oxidative electrochemical redox titrations was used to separate the spectral contributions coupled to heme a and a3 redox transitions, respectively. Simultaneous adjustment of the midpoint potentials and of the amplitudes for a user-defined number of redox components (here heme a and a3) at all wavelengths in the UV/vis (900-400 nm) and at all wavenumbers in the infrared (1800-1250 cm-1) yielded difference spectra for the number of redox potentials selected. With an assumption of two redox components, two spectra for the redox potential at -0.03 +/- 0.01 V and 0.22 +/- 0.04 V (quoted vs Ag/AgCl) were obtained. The method used here allows the separation of the heme signals from the electrochemically induced visible difference spectra of native cytochrome c oxidase without the addition of any inhibitors. The separated heme a and a3 UV/vis difference spectra essentially correspond to spectra obtained for high/low-spin and 5/6-coordinated heme a/a3 model compounds presented by Babcock [(1988) in Biological Applications of Resonance Raman Spectroscopy (Spiro, T., Ed.) Wiley and Sons, New York]. Single-component Fourier transform infrared (FTIR) difference spectra were calculated for both hemes on the basis of these fits, thus revealing contributions from the reorganization of the polypeptide backbone, from the hemes, and from single amino acids upon electron transfer of the cofactors (heme a/a3, CuA, and CuB), as well from coupled processes such as proton transfer. A tentative assignment of heme vibrational modes is presented and the assignment of the signals to the reorganization of the polypeptide backbone and to perturbations of single amino acids, in particular Asp, Glu, Arg, or Tyr, is discussed.

Arginine↗

PubMed1998

Production and characterization of monoclonal antibodies directed against native epitopes of NhaA, the Na+/H+ antiporter of Escherichia coli.

Monoclonal antibodies (mAbs) recognizing native epitopes are an important tool for functional and structural studies of proteins, yet they have rarely been used with transport proteins. In an attempt to raise monoclonal antibodies against the NhaA Na+/H+ antiporter of Escherichia coli we encountered difficulties in the screening procedure, which is based on the standard enzyme-linked immunosorbent assay (ELISA). Here we report a rapid and efficient method of screening for anti-NhaA mAbs which recognize native epitopes of the antiporter. The method is based on the use of His-tagged protein, Ni2+-nitrilotriacetic acid coated plates and non-denaturing conditions in the assay. With this procedure four mAbs were obtained, three of which recognize the NhaA in its native conformation and one preferentially recognizes the denatured form. The latter mAb is Western blot positive, the others are Western blot negative and bind the detergent solubilized NhaA as assayed by gel filtration chromatography. Competition experiments show that the native epitopes are common to both the His-tagged and the wild-type protein. We suggest that in the standard ELISA the NhaA protein is not presented to the antibody in the native conformation whereas the His tag based protocol favors this presentation. Indeed, we could remarkably improve the low reactivity of the standard ELISA by coating the plates with anti-NhaA mAb and use it to present NhaA ('sandwich' ELISA or two antibodies assay). Remarkably, two of the mAbs (5H4, 2C5) which bind native NhaA inhibit drastically the deltapH driven 22Na uptake mediated by His-tagged NhaA reconstituted in proteoliposomes. Hence, these mAbs afford a new tool to study the structure/function relationship of the antiporter.

Animals↗

PubMed1998

Electrochemically induced FT-IR difference spectra of the two- and four-subunit cytochrome c oxidase from P. denitrificans reveal identical conformational changes upon redox transitions.

In order to study the role of subunits III and IV of the cytochrome c oxidase from P. denitrificans for electron and proton transfer, electrochemically induced FT-IR difference spectra of the two- and of the four-subunit enzyme have been compared. These spectra reflect the alterations in the protein upon electron and proton transfer. Since the spectra are essentially identical, they clearly indicate that the additional subunits III and IV do not contribute to the FT-IR difference spectra of the four-subunit oxidase. Subunits III and IV are thus not involved in the reorganization of the polypeptide backbone and of single amino acids upon electron transfer and coupled proton transfer observed in the difference spectra in addition to heme contributions. The subtle differences between the FT-IR difference spectra that are attributed to the influence of protein-protein interactions between the subunits are discussed.

Binding Sites↗

PubMed1998

The mechanism of proton pumping by cytochrome c oxidasex127e [comments].

Cytochrome c oxidase catalyzes the reduction of oxygen to water that is accompanied by pumping of four protons across the mitochondrial or bacterial membrane. Triggered by the results of recent x-ray crystallographic analyses, published data concerning the coupling of individual electron transfer steps to proton pumping are reanalyzed: Conversion of the conventional oxoferryl intermediate F to the fully oxidized form O is connected to pumping of only one proton. Most likely one proton is already pumped during the double reduction of O, and only three protons during conversion of the "peroxy" forms P to O via the oxoferryl form F. Based on the available structural, spectroscopic, and mutagenesis data, a detailed mechanistic model, carefully considering electrostatic interactions, is presented. In this model, each of the four reductions of heme a during the catalytic cycle is coupled to the uptake of one proton via the D-pathway. These protons, but never more than two, are temporarily stored in the regions of the heme a and a3 propionates and are driven to the outside ("pumped") by electrostatic repulsion from protons entering the active site during turnover. The first proton is pumped by uptake of one proton via the K-pathway during reduction, the second and third proton during the P --> F transition when the D-pathway and the active site become directly connected, and the fourth one upon conversion of F to O. Atomic structures are assigned to each intermediate including F' with an alternative route to O.

Amino Acid Sequence↗

PubMed1998

Electrical current generation and proton pumping catalyzed by the ba3-type cytochrome c oxidase from Thermus thermophilus.

Several amino acid residues that have been shown to be essential for proton transfer in most cytochrome c oxidases are not conserved in the ba3-type cytochrome c oxidase from the thermophilic eubacterium Thermus thermophilus. So far, it has been unclear whether the Th. thermophilus ba3-type cytochrome c oxidase can nevertheless function as an electrogenic proton pump. In this study, we have combined charge translocation measurements on a lipid bilayer with two independent methods of proton pumping measurements to show that enzymatic turnover of the Th. thermophilus cytochrome c oxidase is indeed coupled to the generation of an electrocurrent and proton pumping across the membrane. In addition to a 'vectorial' consumption of 1.0 H+/e- for water formation, proton pumping with a stoichiometry of 0.4-0.5 H+/e- was observed. The implications of these findings for the mechanism of redox-coupled proton transfer in this unusual cytochrome c oxidase are discussed.

Amino Acid Sequence↗

PubMed1998

The 18 kDa cytochrome c553 from Heliobacterium gestii: gene sequence and characterization of the mature protein.

The 18 kDa cytochrome c553 is the dominant c-type cytochrome in cell membranes of Heliobacterium gestii. After solubilization, this cytochrome was purified in three steps as a complex with two other proteins of 32 and 42 kDa. The redox midpoint potential of the cytochrome c553 was determined to be +215 mV. The EPR spectra clearly show the presence of an ascorbate-reducible low-spin heme with gz = 3.048 and gy = 2.238. The gx = trough could not be detected. In addition, a Cu(II) signal with g = 2.058 was observed, indicating that one component of the cytochrome c553 complex contains a bound copper ion. The gene for the 18 kDa cytochrome c553, cyhA, consists of 429 bp coding for a protein of 142 amino acids. The association of the cytochrome with the cytoplasmic membrane is mediated by two fatty acid molecules, one palmitate and one stearate, that could be identified by mass spectrometry. Both fatty acids are most likely bound to the cysteine residue of the N-terminally processed protein via a glycerol moiety. The amino acid sequence deduced from the DNA sequence exhibits partial identity to the membrane-bound cytochrome c551 from Bacillus PS3 [Fujiwara, Y., Oka, M., Hamamoto, T., and Sone, N. (1993) Biochem. Biophys. Res. Commun. 1144, 213-219] and to the cytochrome c subunit (NorC) of the nitrous reductase from Pseudomonas stutzeri [Zumft, W. G., Braun, C., and Cuypers, H. (1994) Eur. J. Biochem. 219, 481-490].

Amino Acid Sequence↗

PubMed1998

Involvement of glutamic acid 278 in the redox reaction of the cytochrome c oxidase from Paracoccus denitrificans investigated by FTIR spectroscopy.

The molecular processes concomitant with the redox reactions of wild-type and mutant cytochrome c oxidase from Paracoccus denitrificans were analyzed by a combination of protein electrochemistry and Fourier transform infrared (FTIR) difference spectroscopy. Oxidized-minus-reduced FTIR difference spectra in the mid-infrared (4000-1000 cm-1) reflecting full or stepwise oxidation and reduction of the respective cofactor(s) were obtained. In the 1800-1000 cm-1 range, these FTIR difference spectra reflect changes of the polypeptide backbone geometry in the amide I (ca. 1620-1680 cm-1) and amide II (ca. 1560-1540 cm-1) region in response to the redox transition of the cofactor(s). In addition, several modes in the 1600-1200 cm-1 range can be tentatively attributed to heme modes. A peak at 1746 cm-1 associated with the oxidized form and a peak at 1734 cm-1 associated with the reduced form were previously discussed by us as proton transfer between Asp or Glu side chain modes in the course of the redox reaction of the enzyme [Hellwig, P., Rost, B., Kaiser, U., Ostermeier, C., Michel, H., and Mäntele, W. (1996) FEBS Lett. 385, 53-57]. These signals were resolved into several components associated with the oxidation of different cofactors. For a stepwise potential titration from the fully reduced state (-0.5 V) to the fully oxidized state (+0.5 V), a small component at 1738 cm-1 develops in the potential range of approximately +0.15 V and disappears at more positive potentials while the main component at 1746 cm-1 appears in the range of approximately +0.20 V (all potentials quoted vs Ag/AgCl/3 M KCl). This observation clearly indicates two different ionizable residues involved in redox-induced proton transfer. The major component at 1746 cm-1 is completely lost in the FTIR difference spectra of the Glu 278 Gln mutant enzyme. In the spectrum of the subunit I Glu 278 Asp mutant enzyme, the major component of the discussed difference band is lost. In contrast, the complete difference signal of the wild-type enzyme is preserved in the Asp 124 Asn, Asp 124 Ser, and Asp 399 Asn variants, which are critical residues in the discussed proton pump channel as suggested from structure and mutagenesis experiments. On the basis of these difference spectra of mutants, we present further evidence that glutamic acid 278 in subunit I is a crucial residue for the redox reaction. Potential titrations performed simultaneously for the IR and for the UV/VIS indicate that the signal related to Glu 278 is coupled to the electron transfer to/from heme a; however, additional involvement of CuB electron transfer cannot be excluded.

Asparagine↗

PubMed1998

Redox dependent changes at the heme propionates in cytochrome c oxidase from Paracoccus denitrificans: direct evidence from FTIR difference spectroscopy in combination with heme propionate 13C labeling.

Specific isotope labeling at the carboxyl groups of the four heme propionates of cytochrome c oxidase from Paracoccus denitrificans was used in order to assign signals observed in electrochemically induced redox Fourier transform infrared (FTIR) difference spectra of this enzyme. For this purpose, the hemA gene of the P. denitrificans strain PD1222, coding for 5-aminolevulinate synthase, was deleted by partial replacement with a kanamycin resistance cartridge, resulting in a stable 5-aminolevulinic acid (ALA) auxotrophy. Normal growth of this deficient strain and cytochrome c oxidase yield comparable to that of P. dentrificans wild-type strain PD1222 could be obtained by supplementation with 0.1 mM ALA in the growth medium. Visible spectra and reduced-minus-oxidized FTIR spectra showed that the purified cytochrome c oxidase had spectral characteristics identical to those of the wild-type enzyme. The decrease of a negative signal at 1676 cm-1 in the reduced-minus-oxidized FTIR difference spectra of the 13C-labeled cytochrome c oxidase in comparison to those of the unlabeled protein allowed the assignment of this signal to a COOH vibration mode of at least one of the four heme propionates. Moreover, a negative band at approximately 1570 cm-1 shifted to smaller wavenumbers in the spectra of the 13C-labeled enzyme in comparison to the spectra of the unlabeled enzyme and was thus assigned to contributions from an antisymmetric COO- mode of one or more of the four heme propionates. Additionally, a positive signal at 1538 cm-1 shifted to approximately 1500 cm-1 in the spectra of the isotopically labeled protein and was therefore assigned to at least one antisymmetric COO- mode of the heme propionates. A negative signal at 1390 cm-1, which has been shifted to 1360 cm-1 in the spectra of the 13C-labeled enzyme, is due to a symmetric COO- mode from at least one heme propionate. These results suggest that at least two of the four heme propionates in cytochrome c oxidase undergo significant vibrational changes upon reduction of the enzyme, either by protonation/deprotonation or by environmental changes.

5-Aminolevulinate Synthetase↗

PubMed1998

Comparative biochemical and pharmacological characterization of the mouse 5HT5A 5-hydroxytryptamine receptor and the human beta2-adrenergic receptor produced in the methylotrophic yeast Pichia pastoris.

Over the last few years, Pichia pastoris has been developed into a powerful expression system for a multitude of foreign genes. Here, we demonstrate that the P. pastoris expression system has similar power to the baculovirus expression system in high-level production of two G-protein-coupled receptors, the mouse 5HT5A 5-hydroxtryptamine receptor and the human beta2-adrenergic receptor. Different expression plasmids were constructed in which the cDNAs of the two receptors were cloned under the transcriptional control of the highly inducible promoter of the P. pastoris alcohol oxidase 1 (AOX1) gene. In three expression plasmids, the receptors were fused to the Saccharomyces cerevisiae alpha-factor prepropeptide and also to the c-myc tag or the FLAG tag to permit immunological detection of the receptors. After transformation into P. pastoris strains KM71 and SMD 1163, recombinant clones were selected and tested for the production of the 5HT5A receptor and the beta2-adrenergic receptor by radioligand binding using [N-methyl-3H]lysergic acid diethylamide and [5,7-3H](-)CGP-12177 respectively. The production level of the 5HT5A receptor was improved by a factor of three by fusion with the alpha-factor prepropeptide. Also, the higher gene dosage resulting from multiple insertions of the expression cassette led to an improvement in production by a factor of two for both receptors. The addition of the adrenergic antagonist alprenolol to the culture medium had a positive effect on the number of specific binding sites detectable in clones producing the beta2-adrenergic receptor. For the 5HT5A receptor the addition of yohimbine resulted in a similar but smaller effect. Binding assays revealed that approx. 25 pmol of beta2-adrenergic receptor and approx. 40 pmol of 5HT5A receptor per mg of membrane protein in crude membrane preparations were produced. The pharmacological profiles for the heterologously produced receptors, estimated by ligand-displacement analysis using certain adrenergic and serotoninergic agonists and antagonists, were comparable with those reported for the receptors expressed in mammalian systems. Immunoblot analysis of the 5HT5A receptor revealed an apparent molecular mass about 20 kDa higher than expected from the amino acid sequence. Here, the Kex2 endopeptidase failed to process the alpha-factor leader correctly. Blocking glycosylation in vivo by tunicamycin or in vitro deglycosylation of membranes by endoglycosidase H resulted in correct processing. In contrast, the beta2-adrenergic receptor fusion to the alpha-factor leader was correctly processed by the internal Kex2 endopeptidase. The Kex2-processed beta2-adrenergic receptor was not glycosylated. In conclusion, the high-level production of the two receptors in P. pastoris will allow their purification in quantities sufficient for subsequent biophysical and structural studies.

Adrenergic beta-Agonists↗

PubMed1998

Prevalence of monoclonal immunoglobulins after liver transplantation: relationship with posttransplant lymphoproliferative disorders.

BACKGROUND: A high incidence of serum monoclonal immunoglobulins (mIgs) has been described after solid organ transplantation. For transplant recipients, the prevalence of posttransplant lymphoproliferative disorders (PTLDs) has been reported to be between 2% and 6%. The relationship between the finding of serum mIg in transplant recipients and the subsequent development of PTLDs is not clearly documented. METHODS: We retrospectively analyzed all cases of mIg and PTLD that occurred in 86 liver transplant recipients who survived more than 3 months. Patients were characterized by protein electrophoresis, immunofixation electrophoresis, pre- and post-liver transplantation Epstein-Barr virus (EBV) serology, EBV presence in lymphoproliferative tissues by in situ hybridization, type of infection episodes, rejection episodes, and immunosuppressive treatment. RESULTS: Thirty-eight patients (44%) had abnormal immunofixation electrophoresis with an electrophoretic Ig peak. Twelve patients had a polyclonal Ig peak, and 26 patients had mIgs (30%). These 26 patients were divided into two groups: 13 patients had a transient mIg peak with a mean delay for normalization of electrophoresis of 2 months, and 13 patients had a permanent mIg peak. No correlation could be demonstrated between the appearance of abnormal banding and indications for transplantation, age of patients, and acute rejection rate. There was a strong correlation between occurrence of viral infections and presence of permanent mIg. Three patients with permanent mIg (23%) developed PTLD and died. CONCLUSIONS: We concluded that the prevalence of mIg after liver transplantation was 30%. Viral infections increase the risk of developing mIg. Persistence of mIg beyond 7 months may be regarded as prelymphomas necessitating a careful follow-up in these patients.

Adolescent↗

PubMed1998

The coupling of electron transfer and proton translocation: electrostatic calculations on Paracoccus denitrificans cytochrome c oxidase.

We have calculated the electrostatic potential and interaction energies of ionizable groups and analyzed the response of the protein environment to redox changes in Paracoccus denitrificans cytochrome c oxidase by using a continuum dielectric model and finite difference technique. Subsequent Monte Carlo sampling of protonation states enabled us to calculate the titration curves of all protonatable groups in the enzyme complex. Inclusion of a model membrane allowed us to restrict the calculations to the functionally essential subunits I and II. Some residues were calculated to have complex titration curves, as a result of strong electrostatic coupling, desolvation, and dipolar interactions. Around the heme a3-CuB binuclear center, we have identified a cluster of 18 strongly interacting residues that account for most of the proton uptake linked to electron transfer. This was calculated to be between 0.7 and 1.1 H+ per electron, depending on the redox transition considered. A hydroxide ion bound to CuB was determined to become protonated to form water upon transfer of the first electron to the binuclear site. The bulk of the protonation changes linked to further reduction of the heme a3-CuB center was calculated to be due to proton uptake by the interacting cluster and Glu(II-78). Upon formation of the three-electron reduced state (P1), His325, modeled in an alternative orientation away from CuB, was determined to become protonated. The agreement of these results with experiment and their relevance in the light of possible mechanisms of redox-coupled proton transfer are discussed.

Catalysis↗

PubMed1998

Prevalence of antiphospholipid antibodies in patients with chronic liver disease related to alcohol or hepatitis C virus: correlation with liver injury.

Antiphospholipid antibodies (APAs) have been reported in various clinical conditions. However, the pathogenesis and clinical significance of these antibodies are still unclear. The objective of this study was to assess the prevalence of APAs in patients with chronic alcohol- or hepatitis C virus (HCV)-related liver disease and to evaluate their relation to the underlying liver disease. We prospectively studied 201 patients referred to an hepato-gastroenterology department, including 77 patients with a history of alcohol abuse (group I) and 124 with chronic HCV infection (group II), and 107 healthy subjects (control population). Liver biopsy was performed in all patients. In cirrhotic patients, the severity of the liver disease was assessed with the use of Child's classification, as modified by Pugh. Several biologic parameters, including lupus anticoagulant and anticardiolipin antibodies, were determined. Forty-eight percent of patients in group I and 33% of those in group II had APAs. Among cirrhotic patients, APAs were more frequent in patients with Child grade B or C than in those with grade A severity. In patients with chronic HCV-related liver disease, a correlation was found between APA levels and liver fibrosis (P = 0.009); no relation was found between APA levels and histologic liver disease activity (P = 0.25). In the control group, one subject was APA-positive. None had lupus anticoagulant. APAs seem to be frequently associated with chronic liver disease of various causes. These results suggest further investigations on the potential role of these antibodies in fibrosis or liver injury.

Adult↗

PubMed1998

The role of electrostatic interactions for cytochrome c oxidase function.

In recent years, the enormous increase in high-resolution three-dimensional structures of proteins together with the development of powerful theoretical techniques have provided the basis for a more detailed examination of the role of electrostatics in determining the midpoint potentials of redox-active metal centers and in influencing the protonation behavior of titratable groups in proteins. Based on the coordinates of the Paracoccus denitrificans cytochrome c oxidase, we have determined the electrostatic potential in and around the protein, calculated the titration curves for all ionizable residues in the protein, and analyzed the response of the protein environment to redox changes at the metal centers. The results of this study provide insight into how charged groups can be stabilized within a low-dielectric environment and how the range of their electrostatic effects can be modulated by the protein. A cluster of 18 titratable groups around the heme a3-CuB binuclear center, including a hydroxide ion bound to the copper, was identified that accounts for most of the proton uptake associated with redox changes at the binuclear site. Predicted changes in net protonation were in reasonable agreement with experimentally determined values. The relevance of these findings in the light of possible mechanisms of redox-coupled proton movement is discussed.

Electron Transport↗

PubMed1998

Characterization of the human dopamine transporter heterologously expressed in BHK-21 cells.

1. cDNA of the human dopamine transporter (hDAT) was cloned into a cloning vector based on the Semliki Forest virus. Electroporation of in vitro transcribed mRNA from this plasmid into BHK-21 cells resulted in production of the transporter as measured by [3H]dopamine uptake (Km = 2.0 +/- 0.4 microM), which was specifically inhibited in the presence of cocaine. 2. The recombinant transporter protein exhibited an apparent molecular mass of 56 kDa, which was reduced to 50 kDa after tunicamycin treatment of the producing BHK-21 cells. Tunicamycin treatment of the electroporated cells also resulted in a decrease in transport activity with no change in the Km value (2.1 +/- 0.4 microM). 3. The localization of the heterologously produced transporter in the BHK cells either with or without tunicamycin treatment was studied by electron microscopic immunogold staining. The glycosylated transporter was found to be localized at the plasma membrane, whereas in the case of the unglycosylated transporter, transport to the plasma membrane was blocked.

Animals↗

PubMed1998

Characterization of the human gonadotropin-releasing hormone receptor heterologously produced using the baculovirus/insect cell and the Semliki Forest virus systems.

1. Two eukaryotic viral systems, the baculovirus/insect cell and the Semliki Forest virus systems, were tested for heterologous expression of human gonadotropin-releasing hormone receptor (GnRHR) cDNA. 2. An unmodified as well as a c-myc epitope-tagged human GnRH receptor was produced in two insect cell lines (Spodoptera frugiperda, Trichoplusia ni) after infection with the respective recombinant baculoviruses. In both insect cell lines, the receptor was identified by immunoblot analysis as a triplet of bands between 35 and 40 kDa. After deglycosylation of the receptor the molecular mass decreased to 35 kDa. The GnRH receptor was localized in membrane compartments within the infected insect cells. However, only in membranes of infected Trichoplusia ni insect cells could approximately 2000 receptors per cell be detected. 3. Production of the GnRH receptor in BHK cells using the Semliki Forest virus system resulted in approximately 50,000 receptors per cell. A maximal yield of 0.42 pmol/mg membrane protein was obtained 24 hr after electroporation of BHK cells with in vitro synthesized RNA. Binding of the antagonist [125I]Cetrorelix was saturable with a KD of 1.3 nM. The receptor produced in the BHK cells was further characterized by ligand displacement studies. The rank order of agonist and antagonist affinities was Cetrorelix > Triptorelin > Antide > GnRH.

Amino Acid Sequence↗

PubMed1998

Effects of interferon-alpha on cytochrome P-450 isoforms 1A2 and 3A activities in patients with chronic hepatitis C.

BACKGROUND/AIM: The risk of adverse drug interactions with interferon-alpha has been poorly assessed. The aim of our study was to establish whether administration of interferon-alpha at therapeutic doses in patients with chronic hepatitis C may have significant inhibitory effects on other drug metabolism. The study was focused on cytochromes P-450 1A2 and 3A, two major isoforms involved in the metabolism of numerous substrates. METHODS: Eighteen patients with chronic active hepatitis C requiring an interferon-alpha treatment were studied. Cytochrome P-450 1A2 activity was determined on the basis of an in vivo caffeine metabolism study. Cytochrome P-450 3A activity was determined according to in vivo cortisol metabolism into 6-beta-hydroxycortisol. Both activities were determined 1 month before, at initiation and 1 month after interferon-alpha therapy (3 x 10(6) units, three times a week). RESULTS: There were no significant differences in the caffeine index (CYP 1A2) and in the 6-beta-hydroxycortisol/free cortisol urinary ratio (CYP 3A) before and after alpha interferon treatment CONCLUSION: Chronic administration of interferon-alpha at therapeutic doses does not change in vivo cytochrome P-450 1A2 and 3A activities. These results support the suggestion that drugs metabolized by these isoenzymes may be used together with interferon-alpha in patients with chronic hepatitis C without significant risks of drug interactions.

Adult↗