TY  - CHAP
A1  - Axelrod, V. D.
A1  - Gorboulev, Valentin G.
A1  - Kutateladze, T. V.
A1  - Barciszewski, J.
A1  - Bayev, A. A.
T1  - The new approach to tRNA primary structure determination : the primary structure of valine tRNA\(^{Val}_{2b}\)
N2  - The new combination of TLC and high voltage electrophoresis on cooling plate is described.We have applied this technique to study of primary structure of tRNA.Preliminary sequence of baker's yeast tRNA^Val_2b is described. New approach to preparation of large tRNA fragments is demonstrated.
KW  - RNS
Y1  - 1976
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-50920
ER  - 
TY  - JOUR
A1  - Gorboulev, Valentin G.
A1  - Kutateladze, Tamara V.
A1  - Barciszewski, Jan
A1  - Axelrod, Vladimir D.
T1  - The separation of  oligonucleotides of baker's yeast valine transfer ribonucleic acid 2b by high-voltage electrophoresis on DEAE-paper and by thin-layer chromatography
N2  - No abstract available
Y1  - 1977
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-32536
ER  - 
TY  - JOUR
A1  - Gorboulev, Valentin G.
A1  - Axelrod, Vladimir D.
A1  - Bayev, Alexander A.
T1  - Primary structure of baker's yeast valine tRNA\(^{Val}_{2b}\)
N2  - The minor form of vallne tBNA from baker's yeaat - tRNA\(^{Val}_{2b}\) - purified by column chromatography was completely digesteft with guanylo-BNase and pancreatic ENase. The products of these digestions were separated by a combination of thin-layer chromatography on cellulose and high voltage electrophoresis on DEAE-paper and then identified. The halves of tRNA Val 2b were prepared by partial digestion with pancreatic Mass and their complete guanylo-BNase and pancreatic ENase, digests were analysed. Basing on the obtained data the primary structure of baker1s yeast tRNA\(^{Val}_{2b}\) was reconstructed.
Y1  - 1977
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-32546
ER  - 
TY  - JOUR
A1  - Kutateladze, T. V.
A1  - Axelrod, V. D.
A1  - Gorboulev, Valentin G.
A1  - Belzhelarskaya, S. N.
A1  - Vartikyan, R. V.
T1  - New procedure of high-voltage electrophoresis in polyacrylamide gel and its application to the sequencing  of nucleic acids
N2  - Fractionation of nucleic acids and their fragments with polyacrylamide gel has been widely applied in sequencing of nucleic acids. Although the conditions of electrophoresis for this purpose have previously been suggested. we have found that polyacrylamide gel electrophoresis at 5000 V (100 V/cm) is possible and effective. An apparatus consisting of a horizontal thermostated plate is used to remove the heat which was formed during the electrophoretic process. The techniques for loading samples on the horizontal thin gel and the procedure for high-voltage gel electrophoresis are described and illustrated by the fractionation of the spleen phosphodiesterase partial digest of tRNA¥~1 as well as by the RNA synthesis by RNA polymerase from E. coli with poly[d(A- T)j as template in the presence of "terminator," 3'-O-methyluridine 5'-triphosphate. This same technique was used for electrophoresis of oligonucleotides on acetylcellulose and was incorporated into a two-dimensional system which was demonstrated by fingerprinting of the guanylo-RNase digest of tRNAT'P from baker's yeast. In the third part of the article a simple technique for the electric trapping of nucleic acids or their fragments from a slab gel on a DEAE-paper sheet is presented.
Y1  - 1979
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-46927
ER  - 
TY  - CHAP
A1  - Viviani, A.
A1  - Lutz, Werner K.
T1  - Modulation of the in vivo covalent binding of the carcinogen benzo(a)pyrene to rat liver DNA by selective induction of microsomal and nuclear aryl hydrocarbon hydroxylase activity
N2  - The influence of microsomal (mAHH) and nuclear (nAHH) aryl hydrocarbon hydroxylase activity on the covalent binding of t:titiated benzo(a)pyrene to rat liver DNA was evaluated in vivo. Induction ofmAHH was obtained after phenobarbitone treatment (180% of control), which increased DNA binding to 210%, but left the nAHH unchanged. mAHH and nAHH were slightly indilced with dieldrin (130% and 120%), but the binding remairred unchanged. The increasing effect of mAHlt as weil as the possibly decreasing effect of nAHH induction on the binding became obvious when the data of 11 individual rats were used to solve the equation Binding = aX(mAHH) + bX(nAHH) + c. Multiple linear regression analysis resulted in positive values for a and c, a negative value for b, and a multiple correlation coefficient R = 0.82. An influence of other enzymes involved in the metabolism of benzo(a)pyrene cannot be excluded. The Study shows clearly that the binding of a foreign compound to DNA in vivo is not only dependent on microsomal enzyme activities but also on nuclear activities even if the latter are considerably lower than those of mic'rosomes.
KW  - DNA
KW  - Benzo(a)pyrene
KW  - DNA-Binding
KW  - Carcinogen
KW  - Enzyme
KW  - Induction
KW  - Aryl Hydrocarbon Hydroxylase
KW  - Rats
KW  - Phenobarbitone
KW  - Dieldrin
Y1  - 1979
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-80132
ER  - 
TY  - JOUR
A1  - Agranovsky, A. A.
A1  - Dolya, V. V.
A1  - Gorboulev, Valentin G.
A1  - Kozlov, J. V.
A1  - Atabekov, J. G.
T1  - Aminoacylation of barley stripe mosaic virus RNA: polyadenylate-containing RNA has a 3'-terminal tyrosine-accepting structure
N2  - Barley stripe mosaic virus (BSMV) RNA which was previously reported to contain poly(A) sequences (Agranovsky et al., 1978) can be specifically esterified with tyrosine in vitro in the presence of an aminoacyl-tRNA synthetase fraction from wheat embryos. All the three RNA components of the BSMV strain with a three-component genome (Norwich) and both RNA components of a two-component strain (Russian) can be tyrosylated. The poly(A)-containing (bound to oligo(dT)-cellulose) and poly(A)-deficient(not bound to oligo(dT)-cellulose) fractions of BSMV RNA display a similar amino acidaccepting ability. The nucleotide sequence which accepts tyrosine is coupled with the intact genomic polyadenylated BSMV RNA. The viral RNA isolated after sucrose density gradient centrifugation under drastic denaturing conditions retains its aminoacylating activity, which suggests that this activity is not due to the presence in a BSMV RNA preparation of a tyrosine tRNA associated with BSMV RNA. Inhibition of aminoacylation of the 3’-oxidized (treated with sodium metaperiodate) BSMV RNA suggests that the tyrosine-accepting structure is localized at the 3’ terminus of BSMV RNA molecules. It is shown that segments of different lengths obtained upon random fragmentation can be tyrosylated. The 3’-terminal (tyrosine-accepting) poly(A)+ segments can be isolated. The shortest segments of viral RNA capable of being aminoacylated [i.e., containing both tRNA-like structure and poly(A)] consists of approximately 150-200 nucleotides. The analysis of the oligonucleotides derived from individual BSMV RNA components labeled with 32P at the 3’ end revealed two types of 3’-terminal sequences different from poly(A). It is suggested that a poly(A) sequence is intercalated between a 3’-terminal tyrosineaccepting structure and the 5’-terminal portion of poly(A)+ BSMV RNA.
Y1  - 1981
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-32566
ER  - 
TY  - JOUR
A1  - Kozlov, J. V.
A1  - Gorboulev, Valentin G.
A1  - Kurmanova, A. G.
A1  - Bayev, Alexander A.
A1  - Shilov, A. A.
A1  - Zhdanov, V. M.
T1  - On the origin of the H1N1 (A/USSR/90/77) influenza virus
N2  - The influenza virus H1N1 (the A/USSR/90/77 strain) that reappeared in 1977 after the H1N1 influenza viruses had disappeared from the human population, is compared with the A/FM/1/47 and the A/FW/1/50 influenza viruses by the method of oligonucleotide mapping of individual segments of the viral RNAs. Seven genes of the A/USSR/90/77 virus appear to be very similar to the corresponding genes of the A/FW/1/50 virus, whereas the gene coding for the M protein displays considerable homology to the corresponding gene of the A/FM/1/47 virus. The data demonstrate that the A/USSR/90/77 strain is a recombinant virus.
KW  - influenza virus ; virion RNA segments ; oligonucleotide mapping ; gene reassortment
Y1  - 1981
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-32556
ER  - 
TY  - JOUR
A1  - Artyukova, E. V.
A1  - Gorboulev, Valentin G.
A1  - Rodionova, N. P.
A1  - Krylov, A. V.
A1  - Atabekov, J. G.
T1  - Comparative study of structural peculiarities and translation of potexvirus RNAs
N2  - Structural peculiarities of the S'-end segments of genomic RNA were studied in F potato virus (F-PV) and white clover mosaic virus (WCMV). The methods of affinity chromatography on oligo(dT) cellulose and oligonucleotide mapping revealed a prolonged (up to 210 nucleotides) polyadenyl sequence at the 3'-end of F-PV RNA. A polyadenyl sequence is missing at the 3'end of WCMV RNA. A study of the translation products of WCMV and F-PV RNAs in a oe11-free protein-synthesizing system derived from rabbit reticulocytes showed that polypeptides electrophoretically comigrating with a structural protein of either virus were synthesized alongside high-molecular-weight polypeptides (M\(_r\)\(\approx\) 180-150 kdaltons).
Y1  - 1985
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-46985
ER  - 
TY  - JOUR
A1  - Rubtsov, P. M.
A1  - Chernov, V. G.
A1  - Gorboulev, Valentin G.
A1  - Parsadanyan, A. S.
A1  - Sverdlova, P. S.
A1  - Chupeeva, V. V.
A1  - Golova, Yu. B.
A1  - Batchikova, N. V.
A1  - Zvirblis, G. S.
A1  - Skryabin, K. G.
A1  - Bayev, A. A.
T1  - Genetic engineering of peptide hormones
N2  - Peptide and polypeptide hormones represent an extensive group of biologically active compounds of important significance for medicine and agriculture. In recent years genetic engineering methods have been used to create strains of microorganisms synthesizing eukaryotic proteins, including hormones and their precursors. The first stage of such developments is the isolation of DNA coding the des~red product. We have accomplished the cloning of the cDNA of a number of polypeptide and peptide hormones of the pituitary of man and domestic animals. The model gene of human calcitonin has also been synthesized and cloned. The obtained genes are being used to develop methods for the microbiological synthesis of human and animal-hormones.
Y1  - 1985
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-46964
ER  - 
TY  - JOUR
A1  - Boriskin, Yu S.
A1  - Desyatskova, R. G.
A1  - Bogomolova, N. N.
A1  - Gorboulev, Valentin G.
T1  - Stability of rubella virus after long-term persistence in human cell line
N2  - Primary infection of HEp-2 cells with rubella virus resulted in non-cytophatic longterm persistent infection. During four years of persistence the virus was produced in sufficient quantities (up to 6 logs PFU/ml) and did not differ from the parental variant in its pathogenicity for BHK-21 or RK-13 cells, or hemagglutinating activity, but formed smaller plaques. Persistent virus preserved the original antigenicity as judged from reciprocal hemagglutination-inhibition or plaque reduction-neutralization tests with polyclonal antisera. Both original and persistent rubella viruses were thermoresistant (T 56° C) and sligthly temperature-sensitive. Clonal analysis revealed presence of ts-mutants among both original and persistent virus clones with different degrees of plating efficiency at 40°/34° C. RNA fingerprinting showed only minor changes in persistent rubella virus.
KW  - Rubella virus
KW  - persistant infection
KW  - cell culture
Y1  - 1986
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-46944
ER  - 
TY  - JOUR
A1  - Zvirblis, G. S.
A1  - Gorboulev, Valentin G.
A1  - Rubtsov, P. M.
A1  - Chernov, B. K.
A1  - Golova, Yu. B.
A1  - Pozmogova, G. E.
A1  - Skryabin, K. G.
A1  - Bayev, A. A.
T1  - Genetic engineering of peptide hormones : III. Cloning of cDNA of porcine growth hormone and construction of gene for expression of hormone in bacteria
N2  - Results are presented of cloning cDNA of procine growth hormone, analysis of its primary structure, and creation of a construction capable of expression of this cDNA in Esqheriahia coti cells. It is shown that in the population of mRNA coding porcine growth hormone, heterogeneity is noted which is manifested not only at the level of the nucleotide sequence, but also is reflected in the amino acid sequence of the mature hormone.
Y1  - 1988
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-46958
ER  - 
TY  - JOUR
A1  - Rubtsov, P. M.
A1  - Oganessyan, R. G.
A1  - Gorboulev, Valentin G.
A1  - Skryabin, K. G.
A1  - Bayev, A. A.
T1  - Genetic engineering of peptide hormones : II. Possible polymorphism of preprolactin in cattle. Data of molecular cloning
N2  - Primary structure is determined of an insertion of a clone isolated from the library of hypophyseal cDNA of cattle by hybridization with a probe specific for prolactin. Analysis of nucleotide sequences showed that in the process of cloning, reorganization occurred in structure of preprolactin cDNA, including an inversion of the 5'-terminal and deletion of the central section of cDNA. Nevertheless, from structure of cDNA, nucleotide sequences can be deduced of extended 5'- and 3'-terminal sections of preprolactin mRNA in cattle with lengths of 257 and 551 nucleotide residues, respectively. When these sequences are compared to those established previously, some differences were found in primary structure. The most important of them is the presence of an additional codon which codes alanine at the position (-22) of the signal peptide. It is suggested that heterogeneity of preprolactin mRNA of cattle in the section coding the signal peptide is the result of alternative splicing, as was shown for preprolactin mRNA in rats.
Y1  - 1988
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-46975
ER  - 
TY  - JOUR
A1  - Tsfasman, I. M.
A1  - Nesmeyanova, M. A.
A1  - Gorboulev, Valentin G.
A1  - Rubtsov, P. M.
A1  - Skryabin, K. G.
T1  - Biosynthesis and secretion of bovine growth hormone in Escherichia coli under the control of the secretory vector containing a promoter and signal sequence of alkaline phosphatase gene
N2  - A recombinant plasmid was constructed containing the gene for bovine growth hormone joinea with the regulatory region and the region coding the signal sequence of the Escherichia coli alkaline phosphatase gene. In conditions of phosphorus starvation, which c~s derepression of alkaline phosphatase, expression was shown of the gene for bovine growth hormone, in addition to partial processing and secretion of protein into periplasm.
Y1  - 1989
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-46932
ER  - 
TY  - JOUR
A1  - Gorboulev, Valentin
A1  - Akhundova, Aida
A1  - Luzius, Heike
A1  - Fahrenholz, Falk
T1  - Molecular cloning of substance P receptor cDNA from guinea-pig uterus
N2  - 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 Iigand affinity in the order: SP >> neurokinin A > neurokinin B.
KW  - Biologie
KW  - Substance P receptor
KW  - G-protein-coupled receptor
KW  - Guinea-pig uterus
KW  - COS cell expression
Y1  - 1992
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59293
ER  - 
TY  - JOUR
A1  - Gorboulev, Valentin
A1  - Akhundova, Aida
A1  - Büchner, Hubert
A1  - Fahrenholz, Falk
T1  - Molecular cloning of a new bombesin receptor subtype expressed in uterus during pregnancy
N2  - The homology screening approach has been used to clone a new member of the guanine-nucleotidebinding-protein-coupled receptor superfamily from guinea pig uterus. The cloned cDNA encodes a 399-amino-acid protein and shows the highest amino acid similarity to members of the bombesin receptor family; 52% and 47% similarity to the gastrin-releasing-peptide (GRP) receptor and the neuromedin-B receptor, respectively. Bindingexperiments with the stably transfected LLC-PK<sub>1</sub> cell line expressing the new receptor protein confmned the bombesin-like nature of the cloned receptor. The relative order ofligand affinity, GRP = neuromedin C >> neuromedin B, suggests that the cloned cDNA represents the GRP subtype rather than the neuromedin-B subtype of bombesin receptors. Northern-blot analysis of mRNA species from several guinea-pig tissues showed that the mRNA for the new bombesin receptor subtype is expressed mainly in uteri of pregnant animals.
KW  - Biologie
Y1  - 1992
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59304
ER  - 
TY  - JOUR
A1  - Gorboulev, Valentin
A1  - Büchner, Hubert
A1  - Akhundova, Aida
A1  - Fahrenholz, Falk
T1  - Molecular cloning and functional characterization of V<sub>2</sub> [8-Iysine] vasopressin and oxytocin receptors from a pig kidney cell line (LLC-PK1)
N2  - No abstract available
KW  - Biologie
Y1  - 1993
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59311
ER  - 
TY  - JOUR
A1  - Gründemann, Dirk
A1  - Gorboulev, Valentin
A1  - Gambaryan, Stepan
A1  - Veyhl, Maike
A1  - Koepsell, Hermann
T1  - Drug excretion mediated by a new prototype of polyspecific transporter
N2  - CATIO~IC drugs of different types and structures (antihistaminics, antiarrhythmics, sedatives, opiates, cytostatics and antibiotics, for example) are excreted in mammals by epithelial cells of the renal proximal tubules and by hepatocytes in the liver<sup>1-4</sup>. In the proximal tubules, two functionally disparate transport systems are involved which are localized in the basolateral and luminal plasma membrane and are different from the previously identified neuronal monoamine transporters and A TP-dependent multidrug exporting proteins<sup>1-3,5-12</sup>. Here we report the isolation of a complementary DNA from rat kidney that encodes a 556-amino-acid membrane protein, OCT1, which has the functional characteristics of organic cation uptake over the basolateral membrane of renal proximal tubules and of organic cation uptake into hepatocytes. OCTl is not homologous to any other known protein and is found in kidney, liver and intestine. As OCTl translocates hydrophobic and hydrophilic organic cations of different structures, it is considered to be a new prolotype of polyspecific transporters that are important for drug elimination.
KW  - Biologie
Y1  - 1994
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-59327
ER  - 
TY  - JOUR
A1  - Gorboulev, Valentin G.
A1  - Akhundova, Aida
A1  - Grzeschik, K.-H.
A1  - Fahrenholz, Falk
T1  - Organization and chromosomal localization of the gene for the human bombesin receptor subtype expressed in pregnant uterus
N2  - The gene encoding the human homologue of the guinea pig uterine bombesm receptor [( 1992) Eur. J. Biochem. 208,405] was isolated from a genomic lambda library by the PCR/homology screening approach. The gene spans more than 4 kb and consists of 3 exons and 2 introns. The deduced amino acid sequence shows about 86% identity to that of guinea pig bombesin receptor. This subtype of bombesin receptor is expressed in the pregnant uterus and in two human tumour cell lines, T47D (ductal breast carcinoma) and A431 (epidermal carcinoma). PCR analysis of genomic DNA from human-mouse cell hybrids allows the cloned gene to be localized to the region q26q28 on chromosome X.
KW  - Bombesin ; Bombesin receptor ; Chromosomal localization
Y1  - 1994
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-32572
ER  - 
TY  - THES
A1  - Arndt, Petra
T1  - Klonierung und funktionelle Charakterisierung von organischen Kationentransportern aus der Rattenniere
T1  - Cloning and functional characterization of organic cation transporters from rat kidney
N2  - Der organische Kationentransport im proximalen Tubulus der Niere spielt eine wichtige Rolle bei der Aufrechterhaltung der Homöostase der Körperflüssigkeiten und der Ausschleusung von toxischen organischen Kationen. Der Transport von organischen Kationen wird an der Bürstensaummembran durch den H+/organische Kationen-Austauscher vermittelt, während bei dem Transport von organischen Kationen an der basolateralen Membran das nach innen gerichtete negative Membranpotential eine treibende Kraft darstellt. Durch Expressionsklonierung wurde der erste organische Kationentransporter, rOCT1, aus der Rattenniere isoliert. Kurz darauf wurde im Rahmen dieser Arbeit ein zweiter organischer Kationentransporter ebenfalls aus der Ratenniere kloniert. rOCT2 besteht aus 593 Aminosäuren und besitzt 12 putative Transmembrandomänen. Zum funktionellen Vergleich zwischen rOCT1 und rOCT2 wurde das Oozytenexpressionssystem verwendet. In der vorliegenden Arbeit wurde ein pharmakologisches Profil von rOCT2 erstellt. Das Substratsprektrum von rOCT2 ist dem von rOCT1 sehr ähnlich. Die Affinitäten von rOCT2 gegenüber verschiedenen Substanzen wurden direkt mit denen von rOCT1 verglichen. Einerseits fanden wir bei einigen Substraten Unterschiede in den Km- und Vmax-Werten, aber andererseits auch viele Ähnlichkeiten zwischen beiden Transportern. Anionen (z. B. p-Aminohippurat) wurden als neue Gruppe von Inhibitoren für den durch rOCT1- und rOCT2-vermittelten Transport identifiziert. Die Potentialdifferenz ist die treibende Kraft des rOCT1- und rOCT2-vermittelten Transportes. Wir konnten potentialabhängige Veränderungen der Km-Werte von Cholin-induzierten Einwärtsströmen zeigen. Bei dem Austausch von Na+-Ionen gegen K+-Ionen im Reaktionspuffer wurde die Aufnahme von Cholin und MPP durch rOCT2 erniedrigt. Der bidirektionale Transport von MPP wurde gezeigt und trans-Stimulationsexperimente für MPP-Influx und MPP-Efflux durchgeführt, um die Asymmetrie des Transporters zu studieren. Darüberhinaus wurde in der vorliegenden Arbeit die Interaktion von verschiedenen Substraten mit rOCT1 und rOCT2 untersucht und ein kompetitver und nicht-kompetitiver Hemmtyp bei der TEA-Aufnahme gefunden.
N2  - Organic cation transport in the renal tubule is an important physiological function for the maintenance of body fluid homeostasis and detoxification of harmful organic cations. In general, transport of organic cations in brush-border membranes is mediated by the H+/organic cation antiporter, whereas transport of organic cations in basolateral membranes is stimulated by the inside-negative membrane potential. By the expression cloning method, the organic cation transporter rOCT1, which is expressed in rat liver and kidney, was isolated. In 1996 another organic cation transporter from rat kidney, rOCT2, was isolated by homology cloning. rOCT2 was deduced to be a glycoprotein comprised of 593 amino acid residues with 12 putative transmembrane domains. To analyse the functional characteristics of rOCT2 in comparison with rOCT1 we utilized the Xenopus expression system. During this dissertation a pharmacological profile was made for rOCT2. The apparent substrate spectrum of rOCT2 was similar to that of rOCT1. Affinities of rOCT2 against several compounds were directly compared with those of rOCT1. We found differences in Km- and IC50-values for distinct substrates but also a lot of similarities between both transporters. Anions like p-aminohippuric acid were identified as a new group of inhibitors for rOCT1- and rOCT2-mediated transport. The potential difference is the driving force of transport mediated by rOCT1 and rOCT2. We showed the potential-dependent changes of Km-values of choline induced inward currents. Further when extracellular Na+ ions were replaced with K+ ions, the uptake of MPP and choline by rOCT2 was decreased. The bidirectional transport of MPP was shown and trans-sitmulation experiments for MPP influx and efflux were performed to study asymmetry of the transporter. The mechanism of interaction of several substrates with rOCT1 and rOCT2 were investigated and we found competitive and non-competitive inhibition of TEA uptake.
KW  - Ratte
KW  - Niere
KW  - Kation
KW  - Stofftransport <Biologie>
KW  - Molekularbiologie
KW  - rOCT1
KW  - rOCT2
KW  - proximaler Tubulus
KW  - Niere
KW  - Sekretion
KW  - Xenopus laevis
KW  - Oozyte
KW  - Transport
KW  - Inhibition
KW  - Homologieklonierung
KW  - rOCT1
KW  - rOCT2
KW  - proximal tubule
KW  - kidney
KW  - secretion
KW  - Xenopus laevis
KW  - oocyte
KW  - transport
KW  - inhibition
KW  - homology cloning
Y1  - 2000
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-793
ER  - 
TY  - THES
A1  - Kühlkamp, Thomas
T1  - Der plasmamembran assoziierte Transportregulator RS1 bindet Ubiquitin und gelangt in den Zellkern
T1  - The plasma membrane associated transport modifier RS1binds ubiquitin und migrates into the nucleus
N2  - Die vorliegende Arbeit liefert wichtige Erkenntnisse über die subzelluläre Verteilung und die Funktion des RS1-Proteins vom Schwein (pRS1), einem Regulator von Plasmamembran-transportern. Das grün fluoreszierende Protein (GFP) wurde mit pRS1 fusioniert und in LLC-PK1 Zellen exprimiert. Das GFP-pRS1 Fusionsprodukt (96 kD) konnte an der Plasmamembran, im Zytosol und im Zellkern entdeckt werden. Bei GFP-Fusion mit trunkierten pRS1-Proteinen zeigte sich, dass der C-Terminus die Kernlokalisierung beeinflusst. Dagegen wurde die Kernlokalisierung durch eine Trunkierung des N-Terminus nicht gestört. Im C-Terminus des pRS1 konnte von AS 579 bis 616 eine Ubiquitin associated domain (UBA) identifiziert werden, die auch in den anderen bisher bekannten RS1-Proteinen aus Mensch, Kaninchen und Maus konserviert vorliegt. Eine Ubiquitin-Affinitätschromatographie zeigte, dass das pRS1-Protein Ubiquitin auf nicht kovalente Weise bindet. Nach der Trunkierung der UBA-Domäne war keine Wechselwirkung des pRS1-Proteins mit Ubiquitin mehr feststellbar. Ein konserviertes Di-Leucin-Endozytose-Motiv (pRS1 AS 366/67) deutet eine Funktion des pRS1-Proteins bei der Internalisierung von Plasmamembranproteinen an. Deshalb wurde das Endozytoseverhalten von pRS1 überexprimierenden LLC-PK1 Zellen untersucht, wobei sich zeigte, dass diese Zellen eine deutlich höhere Aufnahme des Endozytosefarbstoffes RH 414 aufwiesen als Zellen, die pRS1 nicht überexprimierten. Die in dieser Arbeit gesammelten Daten zum RS1-Protein wurden zusammen mit früher erhobenen Ergebnissen zum RS1-Protein im Rahmen eines Modells zusammengefasst. In diesem hypothetischen Modell wird angenommen, dass RS1 ein Adapterprotein ist, welches die ubiquitinabhängige Endozytose von Plasmamembrantransportern vermittelt und als Signalmolekül in den Zellkern gelangen kann, wo es an der Transcriptionsrepression des SGLT1 beteiligt ist.
N2  - This work discribes investigations about the subcellular distribution and function of the plasma membrane-associated protein RS1, an regulator of plasma membrane transporters like the Na+-D-glucose cotransporter SGLT1 or the organic cation transporter OCT2 (Vehyl et al., 1993; Reinhardt et al., 1999; Valentin et al., 2000). The green fluorescent protein (GFP) was fused to RS1 from pig (pRS1) and expressed in LLC-PK1 cells. The GFP-pRS1 protein could be detected at the plasma membrane, in the cytoplasma and in the nucleus. Expression of various truncated forms of GFP-pRS1 showed that the N-terminal half of pRS1 (amino acids 1-328) is not necessary for the migration of pRS1 into the nucleus. In contrast, truncations of the C-terminus inhibited translocation into the nucleus. The C-terminus of pRS1 contains a conserved ubiquitin associated domain (UBA) at amino acids 579 to 616. Affinity chromatography with ubiquitin-conjungated Sepharose beads showed a noncovalent binding of pRS1 to immobilized ubiquitin, which was abolished in the presence of an excess of free ubiquitin. Further analysis schowed that the C-terminal 111 amino acids were indispensable for ubiquitin binding. A conserved di-leucine signal (pRS1 336/337) is a well known endocytosis motif and points to an involvement of pRS1 in the internalisation of plasma membrane proteins. This hypothesis was supported by the finding of an increased uptake of the intercalating membrane dye RH 414 in a pRS1-overexpressing LLC-PK1 cell line. Based on this findings together with previous data, a model for the physiological role of RS1 was proposed. In this model, RS1 serves as an adaptor which links ubiquitinated plasma membrane transporters such as SGLT1 to the endocytosis machinery. Moreover RS1 migrates into the nucleus and is involved in the transcriptional suppression of SGLT1.
KW  - Ubiquitin
KW  - Carrier-Proteine
KW  - Plasmamembran
KW  - Zellkern
KW  - RS1
KW  - SGLT1
KW  - UBA
KW  - Ubiquitin
KW  - Plasmamembrane
KW  - Zellkern
KW  - Endocytose
KW  - RS1
KW  - SGLT1
KW  - UBA
KW  - ubiquitin
KW  - plasma membrane
KW  - nucleus
KW  - endocytosis
Y1  - 2001
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-1179507
ER  -