Biomedical subjects

F Fahrenholz

Publications and source records attributed to F Fahrenholz.

At least 73 records · Page 4Linked to original sources

PubMed1992

Cloning and nucleotide sequence of the psrA gene of Wolinella succinogenes polysulphide reductase.

The polysulphide reductase (formerly sulphur reductase) of Wolinella succinogenes is a component of the phosphorylative electron transport system with polysulphide as the terminal acceptor. Using an antiserum raised against the major subunit (PsrA, 85 kDa) of the enzyme, the corresponding gene (psrA) was cloned from a lambda-gene bank. The N-terminal amino acid sequence of PsrA mapped within the psrA gene product, which also contained an apparent signal peptide. Downstream of the psrA gene two more open reading frames (psrB and psrC) were found. The three genes may form a transcriptional unit with the transcription start site in front of psrA. The three genes were present only once on the genome. PsrA is a hydrophilic protein homologous to the largest subunits of six prokaryotic molybdoenzymes. PsrB is predicted to be hydrophilic, to contain ferredoxin-like cysteine clusters and to be homologous to the smaller hydrophilic subunits of four molybdoenzymes. PsrC is predicted to be a hydrophobic protein that could possibly serve as the membrane anchor of the enzyme.

Amino Acid Sequence↗

PubMed1992

The quinone-reactive Ni/Fe-hydrogenase of Wolinella succinogenes.

The hydrogenase (Hyd) isolated from the cytoplasmic membrane of Wolinella succinogenes consists of three polypeptides (HydA, HydB and HydC) and contains cytochrome b (6.4 mumol/g protein), which was reduced upon the addition of H2. The enzyme catalyzed the reduction of 2,3-dimethyl-1,4-naphthoquinone with H2, in contrast to an earlier preparation which was made up of HydA and HydB only and did not contain cytochrome b (Unden, G., Böcher, R., Knecht, J. & Kröger, A. (1982) FEBS Lett. 145, 230-234). This suggests that HydC is a cytochrome b which serves as a mediator in the electron transfer from H2 to the quinone. The hydrogenase genes were cloned, sequenced and identified by sequence comparison with the N-termini of the three subunits. The three genes were arranged in the order hydA, hydB, hydC, with the transcription start site in front of hydA, and were present only once on the genome. Separated by an intergene region of 69 nucleotides, hydC was followed by at least two more open reading frames of unknown function. The amino acid sequences derived from hydA, hydB and hydC were similar to those of the membrane Ni-hydrogenases of seven other bacteria. HydA and HydB also showed similarity to the small and the large subunits of periplasmic Ni-hydrogenases. HydC was predicted to contain four hydrophobic segments which might span the bacterial membrane. Two histidine residues located in hydrophobic segments are conserved in the corresponding sequences of the other membrane hydrogenases and might ligate the haem B.

Amino Acid Sequence↗

PubMed1992

Molecular cloning of substance P receptor cDNA from guinea-pig uterus.

A cDNA encoding guinea-pig uterine substance P (SP) receptor has been isolated using the homology screening approach. Northern blot analysis reveals that the corresponding mRNA, of approx. 4.8 kb, is expressed in all tissues tested, but predominantly in the uteri of non-pregnant animals; during pregnancy its expression is reduced. The guinea-pig SP receptor was expressed in COS-7 cells and demonstrated relative ligand affinity in the order: SP much greater than neurokinin A greater than neurokinin B.

Amino Acid Sequence↗

PubMed1992

Detection of a new hormone contact site within the insulin receptor ectodomain by the use of a novel photoreactive insulin.

We have used a preparation of soluble human insulin receptor ectodomain and a novel photoreactive, biotinylated derivative of insulin (4-azidosalicyloyl(B1-biocytinyl-B2-lysine)-insulin) to identify a new hormone contact site within the extracellular domain of the insulin receptor. The ectodomain was photoaffinity-labeled and digested to completion with trypsin, and the resulting tryptic fragment was purified by either HPLC or by streptavidin-affinity chromatography. The amino terminus of the fragment was identified as Gly390 within the alpha-subunit. These results suggest that residues that are carboxyl-terminal to the cysteine-rich domain, in addition to previously identified regions within the amino terminus of the alpha-subunit, contribute to the insulin binding site. The implications of these results for the de novo folding of the insulin receptor to constitute the hormone binding site are discussed.

Affinity Labels↗

PubMed1992

N-glycosylation plays a role in biosynthesis and internalization of the adenylate cyclase stimulating vasopressin V2-receptor of LLC-PK1 renal epithelial cells: an effect of concanavalin A on binding and expression.

The role of N-glycosylation in the function and biosynthesis of the vasopressin V2-receptor in LLC-PK1 renal epithelial cells was examined using various lectins and inhibitors operating at different steps of the glycosidic pathway. Tunicamycin, which blocks all N-glycosylation, and castanospermine, which inhibits glycosidase I and hence blocks formation of high-mannose-type N-glycosylated intermediates, resembled one another in affecting V2-receptor biosynthesis and internalization in a concentration-dependent manner. In contrast, swainsonine, an inhibitor of mannosidase II and hence of complex-type oligosaccharide formation, had no effect. Interestingly, the alpha-D-mannose/alpha-D-glucose-specific lectin concanavalin A, (Con A), in contrast to the beta-D-galactose-specific lectin ricin, had a marked effect on the V2-receptor in LLC-PK1 cells, increasing both receptor numbers up to twofold in vivo and specific [3H]AVP binding up to 50% in vitro in a concentration-dependent manner. The concentrations inducing half-maximal response were about 0.2 and 20 micrograms/ml for the in vivo and in vitro responses, respectively, implying distinct effects on V2-expression and ligand binding. That the in vitro effect on binding was due to a direct effect on the V2-receptor could be shown by the lack of a Con A effect on [3H]AVP binding in membranes prepared from LLC-PK1 cells down-regulated for the V2-receptor or from cells of the LLC-PK1 V2-receptor deficient mutant M18. All results were consistent with a functional role for N-glycosylation of the V2-receptor in LLC-PK1 cells.

Adenylyl Cyclases↗

PubMed1992

Generation of anti-idiotypic monoclonal antibodies recognizing vasopressin receptors in cultured cells and kidney sections.

To produce anti-idiotypic antibodies against receptors for the neurohypophyseal hormone vasopressin, an anti-vasopressin monoclonal antibody with a ligand specificity similar to that of vasopressin receptors was employed for immunization. Three anti-idiotypic monoclonal antibodies were obtained which induced, like vasopressin, plasminogen activator production in the renal epithelial cell line LLC-PK1 (expressing V2-receptors). Induction of plasminogen activator synthesis by the anti-idiotypic antibodies could be inhibited by coincubation with a vasopressin antagonist. In a fashion similar to that of vasopressin itself, the anti-idiotypic antibodies induced receptor down-regulation. The anti-idiotypic antibodies were employed to visualize vasopressin receptors on LLC-PK1 and A7r5 (V1-receptor-expressing) smooth muscle cells by immunofluorescence. Antibody-mediated fluorescence was not observed in receptor-deficient mutant cell lines or vasopressin-receptor-down-regulated cells. Furthermore, these antibodies were used for immunohistochemical localization of vasopressin receptors in rat and bovine kidney preparations. In accordance with earlier physiological and biochemical observations, vasopressin receptors were detected predominantly in collecting ducts in cortex and medulla. On the cellular level, a differential staining pattern was observed.

Animals↗

PubMed1992

Colocalization of the gene for nephrogenic diabetes insipidus (DIR) and the vasopressin type 2 receptor gene (AVPR2) in the Xq28 region.

The gene for nephrogenic diabetes insipidus (DIR) and the vasopressin type 2 receptor gene (AVPR2) have both been localized in the Xqter region by genetic mapping and functional expression studies, respectively. In this paper genetic evidence that the DIR locus is localized distal to the DXS305 locus and that the functional gene for the V2 receptor is localized between the markers DXS269 and F8 is presented. These further refinements in the localization of both genes strengthen the assumption that both genes are identical and provide a rationale for cloning the gene by reversed genetics strategies.

Animals↗

PubMed1992

A low affinity vasopressin V2-receptor in inherited nephrogenic diabetes insipidus.

Congenital nephrogenic diabetes insipidus (NDI) is an X-linked inherited disorder characterized by renal resistance to the antidiuretic hormonal action of vasopressin. This study describes the molecular basis of nephrogenic diabetes insipidus in a dog family. Kidney membranes prepared from NDI-affected male huskies were examined for vasopressin binding and response. Compared to membranes from unaffected canines, those from the kidney inner medulla of NDI-dogs possessed normal V2-receptor numbers, but with 10-fold lower affinity for [Arg8] vasopressin (AVP). Adenylate cyclase stimulation by AVP in contrast to that by forskolin or GTP-analogues was similarly reduced in a dose responsive manner. The NDI-affected dogs showed antidiuretic responses to very high doses of V2-specific agonists, consistent with their possessing V2-receptors of lower affinity. Prolonged treatment with V2-agonists, 1-deamino [D-Arg8] VP (dDAVP) and 1-deamino [Val4, Sar7] AVP (dVSAVP), rendered the NDI-affected dogs near normal in terms of water intake and urine osmolality.

Adenylyl Cyclases↗

PubMed1991

Identification and enzymatic deglycosylation of the myometrial oxytocin receptor using a radioiodinated photoreactive antagonist.

To identify and characterize oxytocin receptors, a 125I-labeled photoreactive oxytocin antagonist was synthesized. The specific oxytocin antagonist [1-(beta-mercapto-beta,beta- cyclopentamethylenepropionic acid), 2-O-methyltyrosine,4-threonine,8- ornithine,9-tyrosylamide]oxytocin ([Mca,Tyr(O-Me)2,Thr4,Orn8,Tyr9-NH2]oxytocin) described by Elands et al. (Elands, J., Barberis, C., Jard, S., Tribollet, E., Dreifuss, J.-J., Bankowski, K., Manning, M., and Sawyer, W. H. (1987) Eur. J. Pharmacol. 147, 192-207) bound to the guinea pig uterine oxytocin receptor with high affinity (apparent Kd = 0.74 nM). The introduction of a 4-azidophenylamidino group at Orn8 resulted in the photoreactive ligand [Mca1,Tyr(O-Me)2,Thr4,Orn(4-azidophenylamidino)8,Tyr9- NH2]oxytocin, which retained the high binding affinity (Kd = 0.69 nM) of the parent compound. The photoreactive antagonist monoiodinated at Tyr9 had approximately double (Kd = 0.39 nM) the affinity of the photoreactive antagonist and several times that of oxytocin (Kd = 2.6 nM) for the guinea pig uterine oxytocin receptor. In photo-affinity labeling experiments using myometrial membranes obtained from guinea pigs during late pregnancy, the 125I-labeled photoreactive antagonist specifically labeled a protein with an apparent molecular mass of between 68 and 80 kDa: the labeling of this protein was completely suppressed by a 100-fold molar excess of oxytocin and oxytocin receptor-specific agonists, but not by vasopressin analogues specific for V1 or V2 receptors or by other peptide hormones. The ability of oxytocin to suppress labeling was decreased in the presence of guanosine 5'-O-(thiotriphosphate) or in the absence of Mn2+. Digestion of the photolabeled oxytocin receptor with endoglycosidase F gave rise to a protein with an apparent molecular mass of 38 +/- 2 kDa. The endoglycosidase F effect and the lack of endoglycosidase H action show that the myometrial oxytocin receptor is highly glycosylated with asparagine-linked complex oligosaccharide chains. Our results suggest that the radioiodinated photoreactive oxytocin antagonist could be a helpful tool in the isolation and further characterization of the oxytocin receptor.

Affinity Labels↗

PubMed1991

Conformation of [8-arginine]vasopressin and V1 antagonists in dimethyl sulfoxide solution derived from two-dimensional NMR spectroscopy and molecular dynamics simulation.

Structural and dynamic properties of [8-arginine]vasopressin and a class of highly potent vasopressin V1 antagonists which contain 3-mercapto-3,3-cyclopentamethylene propionic acid (Mca) in position 1 of the vasopressin sequence have been determined. On the basis of two-dimensional NMR experiments in dimethyl sulfoxide solution, interproton distances were derived according to which model conformations were built and refined using molecular dynamics simulations. The conformation of vasopressin and the V1 antagonists differ mainly in the region of the mutated residue. The antagonistic property was found to be related to an inversed chirality of the disulfide bridge. In all investigated molecules, characteristic beta-turn structure elements were found for the backbone conformation of the endocyclic residues Tyr2-Asn5. For this portion of the peptide sequence, various conformational equilibria were detected which matched different time scales. For [Arg8]vasopressin, averaged NMR parameters were obtained which could be explained by rapid interconversion between different beta-turn geometries, whereas multiple slowly exchanging conformations were observed for the V1 antagonists. V1 antagonists containing sarcosine in position 7 exhibited multiple spectral patterns for the exocyclic part attributed to cis/trans isomerization. The X-ray structure of deamino-oxytocin [Wood, S. P., Tickle, I. J., Treharne, A. M., Pitts, J. E., Mascarenhas, Y., Li, J. Y., Husain, J., Cooper, S., Blundell, T. L., Hruby, V. J., Buku, A., Fischman, A. J. & Wyssbrod, H. R. (1986) Science 232, 633-636] was found to represent one sample of the conformational space covered by the multiple conformations found for [Mca1, Arg8]vasopressin.

Amino Acid Sequence↗

PubMed1991

Cloning and nucleotide sequence of the structural genes encoding the formate dehydrogenase of Wolinella succinogenes.

The formate dehydrogenase of Wolinella succinogenes is a membraneous molybdo-enzyme which is involved in phosphorylative electron transport. The gene (fdhA) encoding the largest subunit was isolated from a gene bank by immunological screening. The fdhA gene was located in an apparent transcriptional unit (fdhA,B,C,D) together with three more structural genes. The N-terminal sequences of three polypeptides present in the isolated enzyme were found to map within the fdhA, B and C structural genes. A polypeptide corresponding to fdhD was not detected in the enzyme preparation. This suggested that the functional formate dehydrogenase was made up of three or four different subunits. The genes fdhA and C encode larger preproteins which differ from the corresponding mature proteins by N-terminal signal peptides. The N-terminal half of the mature FdhA is homologous to the larger subunits of the formate dehydrogenases of E. coli (formate-hydrogenlyase linked) and Methanobacterium formicicum as well as to three bacterial reductases containing molybdenum. It harbours a conserved cysteine cluster and two more domains which may be involved in binding the molybdenum cofactor. FdhB may represent an iron-sulphur protein, twelve cysteine residues of which are arranged in two clusters which are typical of ligands of the iron-sulfur centers in ferredoxins. FdhC is a hydrophobic protein with four predicted transmembrane segments, which appears to be identical with the cytochrome b present in the isolated formate dehydrogenase. It may form the membrane anchor of the enzyme and react with the bacterial menaquinone.

Amino Acid Sequence↗

PubMed1991

The progesterone antagonist onapristone increases the effectiveness of oxytocin to produce delivery without changing the myometrial oxytocin receptor concentrations.

The progesterone antagonist onapristone was used in guinea pigs during late pregnancy (43 +/- 2 days after coitus) and before term (day 61 after coitus) to investigate the role of progesterone on uterine reactivity to exogenous oxytocin, concentration of oxytocin receptors, and gap junctions in the myometrium. Onapristone priming increased the ability of oxytocin to induce delivery during late pregnancy and before term by factors of greater than or equal to 30 and approximately 10, respectively. The intrauterine pressure recording on day 43 after coitus revealed phasic, laborlike contractions in response to oxytocin in onapristone-treated animals, in contrast to tonic reactions in controls. The increase in the oxytocin response in onapristone-treated animals was not associated with an increase in myometrial oxytocin receptor concentrations either during late pregnancy or before term. By contrast, treatment with onapristone significantly decreased the input resistance of myometrial cells in guinea pigs in late pregnancy (43 +/- 1 day after coitus) to the level of animals at term. This was associated with a marked increase in myometrial gap junctions stained with antibodies against connexin 43. These results indicate that progesterone may control myometrial reactivity to oxytocin in pregnant guinea pigs by effects on postreceptor events mainly by suppressing the gap junctions.

Animals↗

PubMed1991

Isolation and genetic characterization of a renal epithelial cell mutant defective in vasopressin (V2) receptor binding and function.

A novel mutant of the LLC-PK1 renal epithelial cell line, VPR1, was isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and selection using a photoactivatable vasopressin analogue [1-(3-mercapto)propionic acid, 8-(N6-4-azidophenylamidino)lysine] vasopressin. The VPR1 mutant cell line possessed less than 5% parental V2 receptor binding for vasopressin but exhibited normal calcitonin receptor binding. In contrast to LLC-PK1 cells (wild type), VPR1 cells exhibited no response to vasopressin in terms of in vitro adenylate cyclase activation, in vivo cAMP production, or urokinase-type plasminogen activator induction. The responses of VPR1 cells to other agents, such as calcitonin, the adenylate cyclase activator forskolin, the GTP analogue guanosine 5'-[beta, gamma-imino] triphosphate, 8-bromo adenosine-3',5'-monophosphate were comparable to those of the parental cell line. Somatic cell hybrids were derived from the cell lines LLC-PK1 and VPR1 and analyzed for the dominance/recessiveness of the VPR1 mutant phenotype. Hybrids were found to possess normal vasopressin binding activity as well as functional responses to the hormone, indicating that the mutation affecting the V2 receptor in VPR1 cells is recessive. The VPR1 cell line may thus have application as a recipient for the expression of the V2 receptor gene using DNA-transfer.

Adenylyl Cyclases↗

PubMed1991

Monensin-resistant LLC-PK1 cell mutants are affected in recycling of the adenylate cyclase-stimulating vasopressin V2-receptor.

The ionophore monensin was found to markedly reduce the rate of return of vasopressin V2-receptors to the membrane following down-regulation with [Arg8]vasopressin (AVP), as well as hormone dissociation (unloading) from cells following ligand binding and internalization in LLC-PK1 renal epithelial cells. Monensin-resistant LLC-PK1 mutants were isolated and characterized for V2-receptor recycling. Whilst the MN-41 mutant appeared to be impaired in [3H]AVP internalization, the MN-11 and MN-21 mutants exhibited parental V2-receptor binding and internalization, but markedly impaired receptor recycling subsequent to ligand-dependent receptor down-regulation. Unloading subsequent to ligand binding and internalization at 37 degrees C was also much slower in the mutants either at 37 degrees C or 23 degrees C. In contrast, unloading subsequent to binding at 23 degrees C, or to binding at 37 degrees C in the presence of NH4Cl, was comparable in LLC-PK1 and mutant cells implying the active nature of the recycling process impaired in the mutants. The mutations conferring resistance to monesin thus concomitantly impaired V2-receptor recycling in the mutants. Results argue for a monensin-sensitive endosomal/lysosomal pathway for the renal V2-receptor, representing the first such report for an adenylate cyclase stimulating receptor.

Adenylyl Cyclases↗

PubMed1991

A major Litomosoides carinii microfilarial sheath glycoprotein (gp22): amino terminal sequence and immunological studies with corresponding synthetic peptides.

The major glycoprotein of the sheath of Litomosoides carinii microfilariae (gp22) was analysed for its amino acid and amino sugar composition. It is rich in proline, glutamine/glutamic acid and glycine and contains (N-acetyl)galactosamine. The N-terminal amino acid sequence was determined up to position 37. It consists of a group of 6 repeats of the pentapeptide sequence methionine-glycine-proline-glutamine-proline with two minor modifications in repeats 3-6, while the first two repeats follow the general pattern more loosely. Identical N-terminal amino acid sequences were found in at least two other sheath polypeptides (33 kDa, 39 kDa). Antisera prepared against 3 overlapping synthetic peptides corresponding to the amino terminus of gp22 recognized different epitopes. They all reacted with identical patterns of sheath polypeptides. The antisera failed to recognize antigens of 4th-stage larvae of L. carinii. In contrast, cross-reacting epitopes were detected in other parasite stages. Antisera reacted with material surrounding embryos and microfilariae in the uterus of females, and caused patchy fluorescence on the sheath of blood-derived and in vitro-released microfilariae.

Amino Acid Sequence↗

PubMed1991

Vasopressin V2-receptor mobile fraction and ligand-dependent adenylate cyclase activity are directly correlated in LLC-PK1 renal epithelial cells.

The role of hormone receptor lateral mobility in signal transduction was studied using a cellular system in which the receptor mobile fraction could be reversibly modulated to largely varying extents. The G-protein-coupled vasopressin V2-type receptor was labeled in LLC-PK1 renal epithelial cells using a fluorescent analogue of vasopressin, and receptor lateral mobility measured using fluorescence microphotolysis (fluorescence photobleaching recovery). The receptor mobile fraction (f) was approximately 0.9 at 37 degrees C and less than 0.1 at 10 degrees C, in accordance with previous studies. When cells were incubated for 1 h at 4 degrees C without hormone, and then warmed up to 37 degrees C and labeled with the vasopressin analogue, f increased from approximately 0.4 to 0.8 over approximately 1 h. The apparent lateral diffusion coefficient was not markedly affected by temperature pretreatment. Studies with radiolabeled vasopressin indicated that temperature pretreatment influenced neither receptor number nor binding/internalization kinetics. F-actin staining revealed that temperature change resulted in reversible changes of cytoskeletal structure. The maximal rate of in vivo cAMP production at 37 degrees C in response to vasopressin, but not to forskolin (receptor-independent agonist), was also markedly influenced by preincubation of cells at 4 degrees C, thus paralleling the effects of temperature preincubation on f. A linear correlation between f and maximal cAMP production was observed, suggesting that the receptor mobile fraction is a key parameter in hormone signal transduction in vivo. We conclude that mobile receptors are required to activate G-proteins, and discuss the implications of this for signal transduction mechanisms.

Actins↗

PubMed1990

An inverse relationship between receptor internalization and the fraction of laterally mobile receptors for the vasopressin renal-type V2-receptor. An active role for receptor immobilization in down-regulation?

Lateral mobility of the vasopressin renal-type V2-receptor was investigated in LLC-PK1 porcine epithelial cells using the technique of fluorescence microphotolysis (photobleaching) and a rhodamine-labelled vasopressin analogue. At various times after ligand addition, cells were analyzed for both receptor lateral mobility and ligand internalization. The V2-receptor mobile fraction diminished from 0.9 to 0.43 over 60 min at 37 degrees C, whereas the apparent lateral diffusion coefficient remained essentially unchanged (2-3 X 10(-10) cm2/s). Interestingly, the fraction of immobile V2-receptors corresponded exactly with the fraction of internalized receptors, implying a functional relationship. These observations together with comparable results reported for other polypeptide hormone receptors indicate a possible mechanistic role for receptor immobilization in the desensitization of hormonal response.

Animals↗

PubMed1990

Differential inactivation of vasopressin receptor subtypes in isolated membranes and intact cells by N-ethylmaleimide.

Vasopressin receptors in plasma membranes and on cell monolayers were treated with sulfhydryl reagents. Specific binding of [3H]AVP to renal V2 receptors in membranes from bovine and porcine kidney and on LLC-PK1 cells was markedly (80-90%) reduced after treatment with NEM but that to V1 receptors on rat liver membranes and A7r5 smooth muscle cells only slightly (10-30%). Inactivation of receptors by NEM reduced the number of binding sites without altering the affinity of unmodified receptor molecules. High affinity ligands (agonists and antagonists), in complex with the V2 receptor, protected against its inactivation. The results suggest that one or more cysteine residues are located in the ligand-binding site of the V2 receptor, and are essential for hormone binding. Furthermore, it is possible to use NEM to differentiate between vasopressin isoreceptors.

4-Chloromercuribenzenesulfonate↗