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1.
Neuropharmacology ; 55(7): 1165-71, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18687344

RESUMO

Voltage-gated Na channels and AMPA receptors play key roles in neuronal physiology. Moreover, both channels have been implicated in the pathophysiology of both grey and white matter in a variety of conditions. Dissecting out the roles of these channels requires specific pharmacological tools. In this study we examined the potential non-specific effects on Na(v)1.6 channels of five widely used AMPA receptor blockers. Using whole-cell patch clamp electrophysiology, we identified a TTX-sensitive persistent Na channel current in HEK cells stably expressing the Na(v)1.6 channel. From a holding potential of -120 mV, slow ramp depolarization to +75 mV generated an inward current that peaked at approximately -15 mV. Superfusion of purportedly specific AMPA antagonists, 1-naphthylacetyl spermine, SYM2206, CP465022, GYKI52466, blocked Na(v)1.6-mediated persistent currents in a dose-dependent manner. Each of these AMPA receptor blockers significantly inhibited (to approximately 70% of control levels) the persistent Na current at concentrations routinely used to selectively block AMPA receptors. The AMPA/kainate blocker, NBQX, did not significantly affect persistent Na channel currents. Furthermore, peak transient current was insensitive to NBQX, but was reversibly inhibited by SYM2206, CP465022 and GYKI52466. These results indicate that many commonly used AMPA receptor antagonists have modest but significant blocking effects on the persistent components of Na(v)1.6 channel activity; therefore caution should be exercised when ascribing actions to AMPA receptors based on use of these inhibitors.


Assuntos
Antagonistas de Aminoácidos Excitatórios/farmacologia , Proteínas do Tecido Nervoso/fisiologia , Receptores de AMPA/antagonistas & inibidores , Bloqueadores dos Canais de Sódio , Canais de Sódio/fisiologia , Anestésicos Locais/farmacologia , Linhagem Celular , Interpretação Estatística de Dados , Relação Dose-Resposta a Droga , Eletrofisiologia , Humanos , Canal de Sódio Disparado por Voltagem NAV1.6 , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Técnicas de Patch-Clamp , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/genética , Tetrodotoxina/farmacologia
2.
Biophys J ; 80(6): 2678-93, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11371444

RESUMO

Mechanosensitive (MS) ion channels are ubiquitous in eukaryotic cell types but baffling because of their contentious physiologies and diverse molecular identities. In some cellular contexts mechanically responsive ion channels are undoubtedly mechanosensory transducers, but it does not follow that all MS channels are mechanotransducers. Here we demonstrate, for an archetypical voltage-gated channel (Shaker-IR; inactivation-removed), robust MS channel behavior. In oocyte patches subjected to stretch, Shaker-IR exhibits both stretch-activation (SA) and stretch-inactivation (SI). SA is seen when prestretch P(open) (set by voltage) is low, and SI is seen when it is high. The stretch effects occur in cell-attached and excised patches at both macroscopic and single-channel levels. Were one ignorant of this particular MS channel's identity, one might propose it had been designed as a sophisticated reporter of bilayer tension. Knowing Shaker-IR's provenance and biology, however, such a suggestion would be absurd. We argue that the MS responses of Shaker-IR reflect not overlooked "mechano-gating" specializations of Shaker, but a common property of multiconformation membrane proteins: inherent susceptibility to bilayer tension. The molecular diversity of MS channels indicates that susceptibility to bilayer tension is hard to design out of dynamic membrane proteins. Presumably the cost of being insusceptible to bilayer tension often outweighs the benefits, especially where the in situ milieu of channels can provide mechanoprotection.


Assuntos
Ativação do Canal Iônico , Canais de Potássio/metabolismo , Animais , Artefatos , Pressão Atmosférica , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Elasticidade , Condutividade Elétrica , Gadolínio/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/química , Canais de Potássio/genética , Desnaturação Proteica , Renaturação Proteica , Superfamília Shaker de Canais de Potássio , Xenopus laevis
3.
Biochim Biophys Acta ; 1512(1): 111-24, 2001 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-11334629

RESUMO

All cells contain mechanosensitive ion channels, yet the molecular identities of most are unknown. The purpose of our study was to determine what encodes the Xenopus oocyte's mechanosensitive cation channel. Based on the idea that homologues to known channels might contribute to the stretch channels, we screened a Xenopus oocyte cDNA library with cation channel probes. Whereas other screens were negative, P2X probes identified six isoforms of the P2X4 subtype of ATP-gated channels. From RNase protection assays and RT-PCR, we demonstrated that Xenopus oocytes express P2X4 mRNA. In expression studies, four isoforms produced functional ATP-gated ion channels; however, one, xP2X4c, had a conserved cysteine replaced by a tyrosine and failed to give rise to functional channels. By changing the tyrosine to a cysteine, we showed that this cysteine was crucial for function. We raised antibodies against a Xenopus P2X4 C-terminal peptide to investigate xP2X4 protein expression. This affinity purified anti-xP2X4 antibody recognized a 56 kDa glycosylated Xenopus P2X4 protein expressed in stably transfected HEK-293 cells and in P2X4 cDNA injected oocytes overexpressing the cloned P2X4 channels; however, it failed to recognize proteins in control, uninjected oocytes. This suggests that P2X4 channels and mechanosensitive cation channels are not linked. Instead, oocyte P2X4 mRNA may be part of the stored pool of stable maternal mRNA that remains untranslated until later developmental stages.


Assuntos
Receptores Purinérgicos P2/genética , Xenopus laevis/genética , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/química , Biblioteca Gênica , Dados de Sequência Molecular , Oócitos/metabolismo , Técnicas de Patch-Clamp , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Antagonistas do Receptor Purinérgico P2 , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2X4 , Alinhamento de Sequência , Transfecção , Xenopus laevis/embriologia , Xenopus laevis/metabolismo
4.
J Muscle Res Cell Motil ; 21(1): 67-77, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10813636

RESUMO

As a cell's shape and volume change. its surface area must re-adjust. How is the plasma membrane's spectrin skeleton implicated? For erythrocytes, cells of fixed surface area, spectrin responses to mechanical disturbances have been studied, but for more typical cells with changeable surface areas, they have not. In rapidly shrinking cells, surface membrane at an adherent substratum invaginates, forming transient vacuole-like dilations (VLDs). We exploited this readily inducible surface area perturbation to pose a simple question: is newly invaginated plasma membrane naked or is it supported by a spectrin skeleton? The spectrin skeleton was examined immunocytochemically in L6 cells (rat skeletal muscle) before and after VLD formation, using fixation in cold methanol and 4112, an antibody against beta-fodrin and beta-spectrin. 4112 was visualized by confocal fluorescence microscopy, while paired phase contrast images independently located the VLDs. To generate VLDs, cells were hypotonically swelled then reshrunk in isotonic medium. Swollen L6 cells maintained their plasma membrane (sarcolemma) spectrin skeleton. Within minutes of subsequent shrinkage, VLDs of 1-2 microm diameter invaginated at the substratum surface of myotubes. Both sarcolemma and VLDs were lined by a relatively uniform spectrin skeleton. Z-series suggested that some of the spectrin skeleton-lined sarcolemma became internalized as vacuoles.


Assuntos
Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Espectrina/metabolismo , Espectrina/ultraestrutura , Vacúolos/metabolismo , Vacúolos/ultraestrutura , Animais , Western Blotting , Tamanho Celular/fisiologia , Células Cultivadas , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Imunofluorescência , Microscopia Confocal , Microscopia de Contraste de Fase , Ratos , Fatores de Tempo
5.
Biophys J ; 77(2): 758-74, 1999 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10423424

RESUMO

The alpha subunit of the human skeletal muscle Na(+) channel recorded from cell-attached patches yielded, as expected for Xenopus oocytes, two current components that were stable for tens of minutes during 0.2 Hz stimulation. Within seconds of applying sustained stretch, however, the slower component began decreasing and, depending on stretch intensity, disappeared in 1-3 min. Simultaneously, the faster current increased. The resulting fast current kinetics and voltage sensitivity were indistinguishable from the fast components 1) left after 10 Hz depolarizations, and 2) that dominated when alpha subunit was co-expressed with human beta1 subunit. Although high frequency depolarization-induced loss of slow current was reversible, the stretch-induced slow-to-fast conversion was irreversible. The conclusion that stretch converted a single population of alpha subunits from an abnormal slow to a bona fide fast gating mode was confirmed by using gigaohm seals formed without suction, in which fast gating was originally absent. For brain Na(+) channels, co-expressing G proteins with the channel alpha subunit yields slow gating. Because both stretch and beta1 subunits induced the fast gating mode, perhaps they do so by minimizing alpha subunit interactions with G proteins or with other regulatory molecules available in oocyte membrane. Because of the possible involvement of oocyte molecules, it remains to be determined whether the Na(+) channel alpha subunit was directly or secondarily susceptible to bilayer tension.


Assuntos
Músculo Esquelético/metabolismo , Canais de Sódio/metabolismo , Sequência de Aminoácidos , Animais , Fenômenos Biofísicos , Biofísica , Feminino , Humanos , Técnicas In Vitro , Ativação do Canal Iônico , Cinética , Potenciais da Membrana , Modelos Biológicos , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Técnicas de Patch-Clamp , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canais de Sódio/química , Canais de Sódio/genética , Estresse Mecânico , Xenopus
6.
Am J Physiol ; 276(2): C318-27, 1999 02.
Artigo em Inglês | MEDLINE | ID: mdl-9950759

RESUMO

Mechanosensitive (MS) channels, ones whose open probability varies with membrane tension in patch recordings, are diverse and ubiquitous, yet many are remarkably insensitive to mechanical stimuli in situ. Failure to elicit mechanocurrents from cells with abundant MS channels suggests that, in situ, the channels are protected from mechanical stimuli. To establish what conditions affect MS channel gating, we monitored Lymnaea neuron stretch-activated K (SAK) channels in cell-attached patches after diverse treatments. Mechanosensitivity was gauged by rapidity of onset and extent of channel activation during a step pressure applied to a "naive" patch. The following treatments enhanced mechanosensitivity: actin depolymerization (cytochalasin B), N-ethylmaleimide, an inhibitor of ATPases including myosin, elevated Ca (using A-23187), and osmotic swelling (acutely and after 24 h). Osmotic shrinking decreased mechanosensitivity. A unifying interpretation is that traumatized cortical cytoskeleton cannot prevent transmission of mechanical stimuli to plasma membrane channels. Mechanoprotection and capricious mechanosensitivity are impediments to cloning efforts with MS channels. We demonstrate a potpourri of endogenous MS currents from L-M(TK-) fibroblasts; others had reported these cells to be MS current null and hence to be suitable for expressing putative MS channels.


Assuntos
Fibroblastos/fisiologia , Canais Iônicos/fisiologia , Mecanorreceptores/fisiologia , Neurônios/fisiologia , Animais , Cálcio/metabolismo , Membrana Celular/fisiologia , Células Cultivadas , Citocalasina B/farmacologia , Condutividade Elétrica , Etilmaleimida/farmacologia , Membranas Intracelulares/metabolismo , Canais Iônicos/efeitos dos fármacos , Lymnaea , Mecanorreceptores/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Concentração Osmolar , Técnicas de Patch-Clamp , Estimulação Física , Cloreto de Sódio/farmacologia , Reagentes de Sulfidrila/farmacologia
7.
Nucleic Acids Res ; 23(5): 803-10, 1995 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-7535923

RESUMO

We have analyzed 154 single amino acid replacement mutants within a 40 amino acid region (residues 164-203) of the reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1). This region consists of two antiparallel beta-strands (strands 9 and 10) flanked by two alpha helices (E and F). The structure of this region of the 'palm' subdomain is conserved in a variety of DNA and RNA polymerases, indicating a critical role in enzyme structure and function. Functional assays were performed by screening RT activity of mutants expressed in E. coli. A functionally important region corresponding closely to beta-strands 9 and 10 and the loop joining them was revealed by its mutational sensitivity. Structural analysis of mutants was performed by using Western blots to assay correct folding, which is required for processing to produce the mature p66 and p51 RT species. This analysis indicates that beta-strand 10 is a structurally important region. Combined analysis of these two assays revealed diagnostic patterns of mutational sensitivity which identify key positions in the RT sequence at which a specific amino acid side chain is critical, either for structure or function, as well as residues which are external to the RT structure. This work illustrates the utility of large-scale mutagenesis in relating primary sequence to significant features of protein structure and function.


Assuntos
HIV-1/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Sequência de Aminoácidos , Catálise , Cristalografia por Raios X , Escherichia coli/genética , Transcriptase Reversa do HIV , Dados de Sequência Molecular , Mutação , Conformação Proteica , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética
8.
J Biol Chem ; 267(6): 3764-70, 1992 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-1371277

RESUMO

The prokaryotic hlyB gene product is a member of a superfamily of ATP-binding transport proteins that include the eukaryotic multidrug-resistance P-glycoprotein, the yeast STE6, and the cystic fibrosis CFTR gene products (Juranka, P. F., Zastawny, R. L., and Ling, V. (1989) FASEB J. 3, 2583-2592). Previous genetic studies have indicated that HlyB is involved in the transport of the 107-kDa HlyA protein from Escherichia coli; however, the HlyB protein has not been purified for biochemical studies due to its low abundance. In this study, we have engineered a monoclonal antibody epitope into the C-terminal end of HlyB that did not destroy its function. This has allowed us to use immunological methods to identify and localize various molecular forms of the HlyB protein present in vivo.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias , Proteínas de Transporte/genética , Epitopos/genética , Proteínas Hemolisinas/genética , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Sequência de Bases , Transporte Biológico , Western Blotting , Proteínas de Transporte/metabolismo , DNA Bacteriano , Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Plasmídeos
9.
FASEB J ; 3(14): 2583-92, 1989 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-2574119

RESUMO

The study of multidrug resistance (MDR) in tumor cell lines has led to the discovery of the plasma membrane P-glycoprotein (Pgp) molecule. This protein functions as an energy-dependent pump for the efflux of diverse anticancer drugs from MDR cells. It now appears that Pgp-mediated MDR tumor cells do occur in human cancers, and that they are likely to play a role in the ultimate response of patients to chemotherapy. Chemosensitizers, compounds able to reverse the MDR phenotype, have been identified and offer the exciting possibility of improving efficacy for some nonresponsive malignancies. Surprisingly, Pgp-like molecules can be found in evolutionarily distant species among both eukaryotes and prokaryotes. As a group, these proteins form a superfamily of ATP-dependent transport proteins. This finding has broad implications and provides new insights into how living organisms use this fundamental transport system to regulate the trafficking of diverse molecules across biological membranes.


Assuntos
Resistência a Medicamentos , Glicoproteínas de Membrana/fisiologia , Família Multigênica , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Animais , Proteínas de Transporte/genética , Resistência a Medicamentos/genética , Regulação da Expressão Gênica , Humanos , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Estrutura Molecular , Neoplasias/tratamento farmacológico
10.
Somat Cell Mol Genet ; 15(2): 101-11, 1989 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-2538926

RESUMO

Human DNA was used to transform adenosine kinase (AK)-deficient BHK cells followed by selection of AK+ cells in medium containing alanosine, adenosine, and uridine (AAU medium). Twenty AAUr isolates were analyzed, and none of them contained AK activity. Several purine salvage enzymes were, however, found to be affected in these cells. The levels of hypoxanthine-guanine phosphoribosyltransferase and adenylosuccinate synthetase activities were elevated, while the adenylosuccinase activity was reduced. AAU-resistance may be explained by elevated activity of adenylosuccinate synthetase to overcome the alanosine block; thus AAUr cells were able to convert exogenous adenosine----inosine----hypoxanthine----IMP----AMPS----AMP. Moreover, these AAUr cells required exogenous purines for growth. HPLC analyses of endogenous nucleotide pools of AAUr cells showed that the levels of adenine nucleotides have diminished to less than 10% of the parental levels. These results suggest that the AAU-resistant mutation, which elicits pleiotropic phenotypes in BHK cells, affects an important component in the regulation of adenine nucleotide synthesis. By including erthyro-9-(2-hydroxy-3-nonyl)adenine in the AAU medium (renamed as AAUE medium) to block deamination of adenosine, AK+ BHK cells were isolated.


Assuntos
Nucleotídeos de Adenina/metabolismo , Adenosina Quinase/metabolismo , Adenosina/metabolismo , Fosfotransferases/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Adenosina Quinase/biossíntese , Adenosina Quinase/genética , Adenilossuccinato Liase/metabolismo , Adenilossuccinato Sintase/metabolismo , Alanina/análogos & derivados , Alanina/metabolismo , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Cricetinae , Meios de Cultura/metabolismo , Humanos , Hipoxantina Fosforribosiltransferase/metabolismo , Mutação , Purinas/metabolismo , Seleção Genética , Transformação Genética , Uridina/metabolismo
12.
Mol Cell Biol ; 7(11): 4075-81, 1987 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-2893255

RESUMO

Overexpression of P-glycoprotein is characteristic of multidrug-resistant cells. We analyzed four P-glycoprotein transcripts that are simultaneously expressed in a drug-sensitive Chinese hamster ovary cell line. We concluded that these transcripts are encoded by two distinct members of a P-glycoprotein multigene family, each of which has two alternative polyadenylation sites. A comparison of the two hamster sequences with the single reported human and mouse P-glycoprotein cDNA sequences demonstrates that P-glycoprotein is a highly conserved protein, that the hamster multigene family is undergoing concerted evolution, and that differences between gene family members are maintained across species. These conserved differences suggest that there may be functional differences between P-glycoprotein molecules.


Assuntos
Genes , Glicoproteínas de Membrana/genética , Transcrição Gênica , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Cricetinae , Cricetulus , DNA/isolamento & purificação , Resistência a Medicamentos , Feminino , Humanos , Dados de Sequência Molecular , Ovário , Homologia de Sequência do Ácido Nucleico
13.
Nature ; 324(6096): 485-9, 1986.
Artigo em Inglês | MEDLINE | ID: mdl-2878368

RESUMO

Increased expression of P-glycoprotein, a plasma membrane glycoprotein of relative molecular mass (Mr) 170,000 (170K), occurs in a wide variety of cell lines that exhibit pleiotropic resistance to unrelated drugs. The presence of P-glycoprotein in human cancers refractory to chemotherapy suggests that tumour cells with multidrug resistance can arise during malignant progression. We have discovered striking homology between P-glycoprotein and the HlyB protein, a 66K Escherichia coli membrane protein required for the export of haemolysin (protein of Mr 107K). P-glycoprotein can be viewed as a tandem duplication of the HlyB protein. The hydropathy profiles of the two proteins are similar and reveal an extensive transmembrane region resembling those found in pore-forming plasma membrane proteins. The C-terminal region of P-glycoprotein and the HlyB protein contain sequences homologous to the nucleotide-binding domains of a group of closely related bacterial ATP-binding proteins. We propose a model for multidrug resistance in which P-glycoprotein functions as an energy-dependent export pump to reduce intracellular levels of anticancer drugs.


Assuntos
Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Transporte/genética , Resistência a Medicamentos , Glicoproteínas/genética , Proteínas Hemolisinas/metabolismo , Homologia de Sequência do Ácido Nucleico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Escherichia coli/genética , Genes , Genes Bacterianos , Humanos , Modelos Biológicos
14.
J Cell Physiol ; 124(2): 226-32, 1985 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-3900094

RESUMO

Analysis of the response of baby hamster kidney cells to adenosine in the presence of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine has revealed two distinct mechanisms of toxicity. The first is apparent at low concentrations of adenosine (less than 5 microM) and is dependent upon the presence of a functional adenosine kinase. The initial toxicity is abolished by uridine, is unrelated to the inhibition of ribonucleotide reductase, and is accompanied by a decrease in the size of the pyrimidine nucleotide pool. Toxicity at higher concentrations of adenosine is adenosine kinase independent and is potentiated by homocysteine thiolactone. An elevation in the intracellular level of S-adenosylhomocysteine, which was observed following treatment with higher concentrations of adenosine (greater than 10 microM), is believed to mediate toxicity at these levels. Interestingly, BHK cells were resistant to intermediate levels of adenosine. The mechanism of resistance is currently unknown, but appears unrelated to a lack of inhibition of adenosine deaminase. It is proposed that substrate inhibition of adenosine kinase may be a determinant of this property.


Assuntos
Adenosina/toxicidade , Rim/efeitos dos fármacos , Adenina/análogos & derivados , Adenina/farmacologia , Inibidores de Adenosina Desaminase , Adenosina Quinase/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Coformicina/análogos & derivados , Coformicina/farmacologia , Resistência a Medicamentos , Sinergismo Farmacológico , Cobaias , Homocisteína/análogos & derivados , Homocisteína/farmacologia , Nucleotídeos/metabolismo , Pentostatina , Ribonucleotídeo Redutases/metabolismo , S-Adenosil-Homocisteína/metabolismo , Uridina/toxicidade
15.
J Biol Chem ; 260(12): 7738-43, 1985 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-2987266

RESUMO

The adenosine kinase enzymes from arabinofuranosyladenine-resistant (araAr) mutants of the baby hamster kidney cell line were analyzed using adenosine kinase-specific antibody probes purified by adenosine kinase-Sepharose column chromatography. Wild-type baby hamster kidney cells were shown to produce two adenosine kinase polypeptides of Mr 43,000 and 40,000. The class I araAr mutants that have no detectable adenosine kinase activity are completely deficient in the two adenosine kinase polypeptides. As expected, the class II araAr mutants, which had been shown to have an altered ribonucleotide diphosphate reductase activity, produce a wild-type level of the two adenosine kinase polypeptides. The five class III araAr mutants which are adenosine-sensitive (AdoS) have various levels of adenosine kinase activity and produced two adenosine kinase polypeptides with similar Mr as that of wild-type cells. The adenosine kinase proteins synthesized by two of the AdoS mutants, ara-19a and ara-74b, differed from wild type in their isoelectric points. These results plus the observations that the AdoS mutants produce adenosine kinase enzymes with altered kinetic properties suggest a point mutation in the adenosine kinase gene. An araAr mutant, ara-60a, with intermediate adenosine sensitivity, was shown to have two truncated adenosine kinase polypeptides. This observation strongly supports the genetic data which suggests that there is only one functional adenosine kinase allele in baby hamster kidney cells and that the two adenosine kinase polypeptides are due to posttranscriptional modification.


Assuntos
Adenosina Quinase/genética , Mutação , Fosfotransferases/genética , Adenosina Quinase/isolamento & purificação , Animais , Anticorpos , Complexo Antígeno-Anticorpo , Linhagem Celular , Cricetinae , Citarabina/farmacologia , Resistência a Medicamentos , Rim , Cinética , Fígado/enzimologia , Mesocricetus
16.
Mutat Res ; 129(3): 397-402, 1984 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-6096704

RESUMO

A class of arabinosyladenine (araA)-resistant mutants of baby hamster kidney (BHK 21/C13) cells exhibits multiple phenotypes: resistance to araA and deoxyadenosine, extreme sensitivity to adenosine (Ado) and varying degrees of deficiency in adenosine kinase (AK) activity. One of these Ados/araAr strains, ara-S10d, was isolated without mutagenesis and was shown to possess about 59% level of the wild-type AK activity. The AK from ara-S10d had an altered Km and pH optimum and was stimulated by K+ cations. A number of Ados to Ador revertants were isolated from ara-S10d, and in all of the 7 examined, the AK activity was reduced to a nondetectable level. The altered kinetic parameters of the AK enzyme in ara-S10d cells suggest a mutation of the AK gene that leads to the synthesis of an altered enzyme. The loss of AK activity in the Ador revertants suggests an association of the enhanced Ado sensitivity to the AK mutation.


Assuntos
Adenosina Quinase/genética , Adenosina/farmacologia , Mutação , Fosfotransferases/genética , Animais , Cátions Monovalentes , Linhagem Celular , Cricetinae , Citarabina/farmacologia , Desoxiadenosinas/farmacologia , Rim
17.
Mol Cell Biol ; 1(6): 568-71, 1981 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-6965110

RESUMO

Two mutator genes of mammalian cells were demonstrated. One was associated with the ribonucleoside diphosphate reductase, and the other was associated with an extreme adenosine sensitivity.


Assuntos
Mutação , Adenosina/farmacologia , Animais , Linhagem Celular , Cricetinae , Resistência a Medicamentos , Genes , Ribonucleosídeo Difosfato Redutase/genética , Tioguanina/farmacologia , Vidarabina/farmacologia
18.
Somatic Cell Genet ; 7(2): 147-60, 1981 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-6974904

RESUMO

A large number of 9-beta-D-arabinofuranosyladenine (araA) -resistant mutants of baby hamster kidney cells (BHK 21/Cl3) were isolated. These mutants can be grouped into three mechanistically distinct classes. All the mutants showed cross-resistance to deoxyadenosine (dAdo). The mechanism of resistance to araA and dAdo in the class I mutants can be attributed to a mutation to adenosine kinase (AK) deficiency. The class II mutants have normal levels of AK, adenosine deaminase, and deoxyadenosine kinase. These mutants also show resistance to 1-beta-D-arabinofuranosylcytosine (araC), and the mechanism of resistance is probably due to a mutation in the ribonucleotide reductase gene producing an enzyme that has an increased resistance to the inhibition by 9-beta-D-arabinofuranosyladenine 5'-triphosphate (araATP) and 2'-deoxyadenosine 5'-triphosphate (dATP). The class III mutants, unlike those of classes I and II, show extreme adenosine (Ado) sensitivity. The Ados/araAr/dAdor phenotypic properties can be attributed to a single mutation. Classes II and III are novel araA-resistant mutants.


Assuntos
Resistência a Medicamentos , Mutação , Vidarabina/farmacologia , Animais , Linhagem Celular , Cricetinae , Cricetulus , Reações Cruzadas , Desoxiadenosinas/farmacologia , Rim
19.
Cancer Res ; 40(11): 4123-6, 1980 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-6162544

RESUMO

The relative cytotoxicity of 9-beta-D-arabinofuranosyladenine and 9-beta-D-arabinofuranosyladenine 5'-monophosphate (ara-AMP) were compared using wild-type and adenosine kinase (AK)-deficient baby hamster kidney somatic cell mutants. The cytotoxicity of ara-AMP to baby hamster kidney cells was dependent on the presence of AK activity since AK-deficient mutants were resistant to ara-AMP. On an equimolar basis, ara-AMP was consistently less cytotoxic than was 9-beta-D-arabinofuranosyladenine to wild-type and AK-deficient baby hamster kidney mutant cells. These findings are consistent with the common view that ara-AMP molecules do not enter mammalian cells as an intact nucleotide. Presumably, ara-AMP molecules were hydrolyzed by the nonspecific phosphatases and 5'-nucleotidase found in the serum or by the ecto-5'-nucleotidase on the outer surface of the membrane and only enter the mammalian cells as 9-beta-D-arabinofuranosyladenine.


Assuntos
Arabinonucleotídeos/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Fosfato de Vidarabina/toxicidade , Vidarabina/toxicidade , Adenosina Quinase/deficiência , Animais , Linhagem Celular , Cricetinae , Relação Dose-Resposta a Droga , Rim , Nucleotidases/metabolismo
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