Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Dermatite Atópica/tratamento farmacológico , Idoso , Anticorpos Monoclonais Humanizados/administração & dosagem , Asma/diagnóstico , Asma/imunologia , Dermatite Atópica/psicologia , Eczema/diagnóstico , Eczema/imunologia , Feminino , Humanos , Injeções Subcutâneas , Prurido/complicações , Qualidade de Vida , Índice de Gravidade de Doença , Transtornos do Sono-Vigília/etiologia , Resultado do TratamentoRESUMO
The S-100 is a group of low molecular weight (10-12 kD) calcium-binding proteins highly conserved among vertebrates. It is present in different tissues as dimers of homologous or different subunits (alpha, beta). In the nervous system, the S-100 exists as a mixture composed of beta beta and alpha beta dimers with the monomer beta represented more often. Its intracellular localisation is mainly restricted to the glial cytoplasmic compartment with a small fraction bound to membranes. In this compartment the S-100 acts as a potent inhibitor of phosphorylation on several substrates including the synaptosomal C-Kinase and Tau, a microtubule-associated protein. The S-100 in particular conditions, after binding with specific membrane sites (Kd = 0.2 microM; Bmax = 4.5 nM), is able to modify the activity of adenylate cyclase, probably via G-proteins. In addition, the Ca2+ homeostasis is also modulated by S-100 via an increase of specific membrane conductance and/or Ca2+ release from intracellular stores. "In vitro" and "in vivo" experiments showed that lower (nM) concentrations of extracellular S-100 beta act on glial and neuronal cells as a growth-differentiating factor. On the other hand, higher concentrations of the protein induce apoptosis of some cells such as the sympathetic-like PC12 line. Finally, data obtained from physiological (development, ageing) or pathological (dementia associated with Down's syndrome, Alzheimer's disease) conditions showed that a relationship could be established between the S-100 levels and some aspects of the statii.
Assuntos
Proteínas S100/fisiologia , Animais , Proteínas de Ligação ao Cálcio , Células Cultivadas , Peso Molecular , Fosforilação , Proteínas S100/genéticaRESUMO
The intracellular expression of antibodies in mammalian cells is a strategy to inhibit the in vivo function of selected molecules but is limited by the unpredictable behaviour of antibodies when intracellularly expressed. Recent advances in the field of antibody expression in Escherichia coli show that the introduction of mutations can improve the properties of some antibody domains, but the general applicability of this approach to intracellular antibodies remains to be proved. As a complement to rational approaches, we describe selection schemes in which antibodies are selected on the basis of their performance in vivo as intracellular antibodies.
Assuntos
Anticorpos Antibacterianos/química , Anticorpos Antibacterianos/isolamento & purificação , Biotecnologia/tendências , Escherichia coli/imunologia , Imunofenotipagem/tendências , Animais , Células COS/imunologia , Células COS/microbiologiaRESUMO
This review deals with the mechanism of nerve growth factor action. In view of the many and diversified effects of this growth factor, and since it could utilize different mechanism(s) in distinct types of cells, we have confined our analysis to the best characterized and more extensively studied target, the clonal cell line PC12. When exposed to NGF in vitro, these neoplastic cells recapitulate the last major steps of neuronal differentiation, i.e., the commitment to become a neuron and the acquisition of the neuronal phenotype. This is characterized by electrically excitable neurites, a display of a highly organized cytoskeleton, and the specific chemical and molecular neuronal properties. These effects are elicited upon the interaction of NGF with a receptor whose gene has been cloned and whose kinetic properties are now relatively well characterized. It is not yet clear, on the contrary, if and which of the several potential second messengers (cAMP, Ca, or phosphoinositides) that undergo marked fluctuations following NGF binding, transduce and amplify the NGF message. Among both the early and late effects of NGF is the modulation of expression of several genes. Some of the products of these genes are mainly restricted to nerve cells and others appear to play a crucial role in regulating the proper assembly of cytoskeletal elements. It is hypothesized that this complex array of chemical, molecular, and ultrastructural changes is triggered by NGF, not through activation of a single pathway, but more likely via combinatorial processes whereby several intracellular signals interplay before the irreversible commitment of becoming a neuron is undertaken.
Assuntos
Fatores de Crescimento Neural/farmacologia , Feocromocitoma , Receptores de Superfície Celular/fisiologia , Células Tumorais Cultivadas/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Ratos , Receptores de Fator de Crescimento Neural , Células Tumorais Cultivadas/citologiaRESUMO
Following the demonstration that intracellular expression of antibodies ('intracellular immunization') may be utilized to engineer new traits in mammalian cells, we undertook experiments to perturb the function of p21ras proteins, by engineering the intracellular expression of the anti-p21ras antibody Y13-259. The variable regions of this antibody have been cloned and, after verifying their antigen binding activity, expressed in general purpose vectors for the intracellular expression of antibodies. The results confirmed that the cloned antibody has been efficiently expressed both in the secretory and the intracellular forms. Thus, intracellular immunization of mammalian cells against p21ras, or any other antigen for which a monoclonal antibody is available, can now be performed.
Assuntos
Anticorpos Monoclonais/genética , Região Variável de Imunoglobulina/genética , Proteína Oncogênica p21(ras)/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Western Blotting , Linhagem Celular , Clonagem Molecular , Imunofluorescência , Hibridomas/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Dados de Sequência Molecular , Neoplasias Hipofisárias , Plasmídeos , Reação em Cadeia da Polimerase , RatosRESUMO
The anti-p21ras Y13-259 single-chain Fv fragment (scFv) neutralizes the activity of p21-ras when intracellularly expressed in different systems. We have studied the mode of action of this inhibition in 3T3 K-ras fibroblasts and demonstrated that (i) this antibody fragment is highly aggregating when cytoplasmically expressed and (ii) the p21-ras antigen is sequestered in these aggregates in an antibody-dependent manner. This co-segregation leads to an efficient inhibition of DNA synthesis. These results suggest that an antigen can be diverted from its normal location inside the cells in an antibody mediated way, prospecting a new mode of action for intracellular antibodies in vivo.
Assuntos
Anticorpos Antineoplásicos/imunologia , Fragmentos de Imunoglobulinas/imunologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Células 3T3 , Animais , Células COS , Divisão Celular , DNA/biossíntese , Imunofluorescência , Fragmentos de Imunoglobulinas/genética , Camundongos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , TransfecçãoRESUMO
We report the application of a strategy to inactivate cellular proteins in vertebrate cells based on the intracellular expression of immunoglobulin genes. We have selected, in this instance, the p21 protein, encoded by the ras proto-oncogene, as a target protein. The variable regions of the neutralizing anti-p21ras monoclonal antibody Y13-259 were cloned in vectors for the expression of either the whole antibody molecule or its single-chain Fv fragment (ScFv) derivative. In order to target the recombinant antibodies to the cytosol, their hydrophobic leader sequence for secretion was mutated or deleted. When these proteins are expressed in the cytosol of Xenopus laevis oocytes they colocalize with the endogenous p21ras protein in the cytoplasmic face of the oocyte plasma membrane, and they markedly inhibit the H1 kinase activity induced by insulin. Moreover, cytosolic anti-p21ras ScFv fragments block the ensuing meiotic maturation. Thus the intracellular expression of both whole antibodies and antibody domains can be used to block a biological function.
Assuntos
Citosol/imunologia , Imunização , Imunoglobulinas/genética , Oócitos/imunologia , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Animais , Especificidade de Anticorpos , Proteína Quinase CDC2/antagonistas & inibidores , Polaridade Celular/imunologia , Imunoglobulinas/imunologia , Insulina/farmacologia , Meiose/efeitos dos fármacos , Oócitos/citologia , Proteínas Recombinantes/imunologia , Xenopus laevisRESUMO
In this paper we have engineered the targeting of ScFv fragments to mitochondria and demonstrated that this can occur efficiently. This extends the range of subcellular compartments where antibody domains can be targeted in order to interfere with the action of the corresponding antigen. Moreover, we have compared the redox state of ScFv fragments targeted to the secretory compartment, the cytosol and the mitochondria, and demonstrated that cysteine residues in ScFv targeted to the secretory compartments and to the mitochondria are oxidized. On the contrary, cytosolic antibody domains are expressed in a reduced state, which is probably the reason for their lower expression levels. These pitfalls, however, do not prevent their successful utilization for intracellular immunization.
Assuntos
Citosol/imunologia , Retículo Endoplasmático/imunologia , Fragmentos de Imunoglobulinas/química , Mitocôndrias/imunologia , Sequência de Aminoácidos , Animais , Células COS , Cisteína/química , Dissulfetos/química , Ditiotreitol/química , Complexo IV da Cadeia de Transporte de Elétrons/genética , Imunofluorescência , Expressão Gênica , Fragmentos de Imunoglobulinas/genética , Dados de Sequência Molecular , Oxirredução , Sinais Direcionadores de Proteínas/química , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Proteínas Recombinantes de Fusão , Transfecção , Proteínas rasRESUMO
Binding and fate of the nerve growth factor (NGF) in pheochromocytoma cells (clone PC12) have been measured with the use of iodine-labeled ligand and with 125I-NGF antibodies. With such double approach it is possible to distinguish between surface bound and total NGF bound to PC12 cells. It is found that NGF-receptor complexes undergo down-regulation. This process is noticeable at low but not at high cell densities, and only in PC12 cells never exposed to NGF. Previous incubation with growth factor leads to the disappearance of down-regulation of NGF-receptor complexes. Assuming that this process is an indirect measure of NGF-receptor internalization, it is concluded that it is modulated by the cell density or by previous exposure to the factor. These findings are postulated to be relevant to the mechanism of action of NGF and to its multiple effects on target cells.
Assuntos
Neoplasias das Glândulas Suprarrenais/metabolismo , Fatores de Crescimento Neural/metabolismo , Feocromocitoma/metabolismo , Animais , Contagem de Células , Células Clonais/metabolismo , Concentração de Íons de Hidrogênio , Receptores de Superfície Celular/metabolismo , Receptores de Fator de Crescimento Neural , Fatores de TempoRESUMO
Diverting the antigen from its normal intracellular location to other compartments in an antibody-mediated way represents a mode of action for intracellular antibodies [Cardinale, A., Lener, M., Messina, S., Cattaneo, A. & Biocca, S. (1998) FEBS Lett., 439, 197-202; Lener, M., Horn, I.R., Cardinale, A., Messina, S., Nielsen, U.B., Rybak, S.M., Hoogenboom, H.R., Cattaneo, A. & Biocca, S. (2000) Eur J Biochem. 267, 1196-205]. In the case of p21Ras, the sequestration of the antigen in aggregated structures in the cytoplasm of transfected cells leads to the inhibition of its biological function. We have further investigated the intracellular fate of the antigen-antibody complex by analyzing the effect of proteasome inhibitors on the formation and the intracellular localization of the aggregates. Overexpression of anti-Ras scFv fragments or inhibition of proteasomes activity leads to the formation of large perinuclear aggresomes formed of ubiquitinated-scFv fragments in which p21Ras is sequestered and degraded in an antibody-mediated way. Disruption of microtubules by nocodazole completely abrogates the accumulation of scFv fragments in a single aggresome and induces the dispersion of these structures in the periphery of the cell. Cotransfection of the GFP-scFv with a myc-tagged ubiquitin and colocalization with specific anti-proteasome antibodies indicate the recruitment of exogenous ubiquitin and proteasomes to the newly formed aggresomes. Taken together these results suggest that the intracellular antigen-antibody complex is naturally addressed to the ubiquitin-proteasome pathway and that the mechanism of ubiquitination does not inhibit the antibody binding properties and the capacity to block the antigen function.
Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Fragmentos de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Cisteína Endopeptidases/metabolismo , Citoplasma/metabolismo , Citoplasma/efeitos da radiação , Citoplasma/ultraestrutura , Fragmentos de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Complexos Multienzimáticos/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Ubiquitinas/metabolismoRESUMO
Cellular extracts derived from pheochromocytoma cells (PC12-) inhibit the assembly of calf brain tubulin, while those derived from nerve growth factor-differentiated cells (PC12+) do not display this effect. Incubation with RNase abolishes the inhibition by PC12- extracts and reveals the presence of an activating effect exerted by PC12+ extracts. Activation of microtubule assembly is enhanced when extracts are prepared from PC12+ cells exposed for 1 day to 1.0 microM taxol and is abolished when PC12+ extracts are: (a) prepared from cells incubated for 1 day with 1 microM colchicine, (b) treated with the non-ionic detergent Nonidet P-40 or (c) centrifuged at 100 000 g instead of 80 000 g. 2D gel electrophoresis of the proteins of the 100 000 g pellet responsible for the activating effect (referred to as 100 K g pellet) reveals the presence of 100 K, 88 K and 32 K proteins which are markedly enriched in PC12+ extracts. The 88 K protein is further enriched in taxol-treated cells and markedly reduced in the same cells incubated with colchicine. A correlation between the differential protein composition of the 100 K g pellets and their effect on microtubule formation is postulated.
Assuntos
Neoplasias das Glândulas Suprarrenais/patologia , Microtúbulos/ultraestrutura , Fatores de Crescimento Neural/farmacologia , Feocromocitoma/patologia , Neoplasias das Glândulas Suprarrenais/análise , Humanos , Cinética , Peso Molecular , Feocromocitoma/análise , Proteínas/análiseRESUMO
Arrest of mitosis and neurite outgrowth induced by nerve growth factor (NGF) in rat pheochromocytoma cells (clone PC12) is accompanied by a progressive inhibition of the synthesis of a protein that binds to single-stranded but not to double-stranded DNA. Time course experiments show that this inhibition is already apparent after a 2-day incubation with NGF and is maximum (85-95%) upon achievement of complete PC12 cell differentiation. Inhibition of the synthesis of this single-stranded DNA binding protein after 48 hr of incubation with NGF is potentiated by concomitant treatment of PC12 cells with antimitotic drugs acting at different levels of DNA replication. Purification on a preparative scale of this protein and analysis of its major physicochemical properties show that: (i) it constitutes 0.5% of total soluble proteins of naive PC12 cells; (ii) its molecular weight measured by NaDodSO4/PAGE is Mr 34,000 (sucrose gradient centrifugation under nondenaturing conditions yields a sedimentation coefficient s20,w of 8.1 S, indicating that the native protein is an oligomer); (iii) amino acid analysis demonstrates a preponderance of acidic over basic residues, while electrofocusing experiments show that it has an isoelectric point around 8.0; (iv) approximately 15% of the protein is phosphorylated in vivo. It is postulated that control of the synthesis of this protein is connected with activation of a differentiative program triggered by NGF in the PC12 neoplastic cell line at some step(s) of DNA activity.
Assuntos
Neoplasias das Glândulas Suprarrenais/metabolismo , Proteínas de Ligação a DNA/biossíntese , Fatores de Crescimento Neural/toxicidade , Feocromocitoma/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Metionina/metabolismo , Peso Molecular , Fosfatos/metabolismo , Radioisótopos de Fósforo , Ratos , Radioisótopos de EnxofreRESUMO
The KDEL retention signal, when added at the C-terminal of the constant region of light and heavy chains of immunoglobulins is able to efficiently retain assembled immunoglobulins only in cells of nonlymphoid origin. In transfected myeloma cells the wild type and the KDEL-Ig mutants are secreted with the same efficiency. This phenomenon is not due to a proteolytic cleavage of the KDEL signal nor to a lack of intermolecular disulfide bond formation and is not due to an impaired recognition of the KDEL signal in myeloma cells. Thus, the constitutive secretion of assembled immunoglobulins, currently considered to follow a default process, appears to be regulated by a mechanism that is able to overcome an efficient ER retention system.
Assuntos
Retículo Endoplasmático/imunologia , Imunoglobulina G/metabolismo , Mieloma Múltiplo/imunologia , Sinais Direcionadores de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico Ativo , Células COS , Linhagem Celular , Primers do DNA/genética , Retículo Endoplasmático/metabolismo , Imunoglobulina G/química , Imunoglobulina G/genética , Camundongos , Modelos Biológicos , Mieloma Múltiplo/metabolismo , Sinais Direcionadores de Proteínas/genética , Ratos , Transfecção , Células Tumorais CultivadasRESUMO
Genes encoding the heavy and light chains of a hapten-specific IgM antibody were modified by site-directed mutagenesis to destroy the hydrophobic leader sequences and allow expression in the cytoplasm of non-lymphoid cells. The in situ assembly of the mutant heavy and light chains was tested in transfected cell lines by immunofluorescence using anti-idiotypic antibodies. A positive diffuse cytoplasmic staining was observed. This demonstrated that the antibody polypeptide chains could assemble in the cell cytoplasm and led us to ask whether antibodies could be further targeted to the nucleus. Mutations were therefore made in which the leader sequence of the light chain was replaced by the nuclear localization signal of the SV40 large T antigen. Transfectants in which the heavy chain lacking the hydrophobic leader was expressed together with a light chain carrying the nuclear localization signal were selected and a nuclear distribution of the assembled antibody was found. Thus, it should prove possible to target a specific antibody to the cell nucleus with the aim of interfering with the function of a nuclear antigen.
Assuntos
Expressão Gênica , Genes de Imunoglobulinas , Imunoglobulina M/genética , Sequência de Aminoácidos , Animais , Antígenos Virais de Tumores/genética , Sequência de Bases , Núcleo Celular/imunologia , Citoplasma/imunologia , Imunofluorescência , Haptenos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Camundongos , Dados de Sequência Molecular , Mutação , Plasmídeos , Sinais Direcionadores de Proteínas/genética , Ratos , Vírus 40 dos Símios/imunologia , Transfecção , Células Tumorais CultivadasRESUMO
We have previously demonstrated that synthesis of a 34 kD protein having specific, high affinity for single-stranded DNA (34kD-ssb protein), is markedly inhibited by nerve growth factor (NGF) in the neoplastic clonal cell line PC12. We report here that total content as well as mRNA for this protein are progressively reduced in PC12 cells undergoing mitotic arrest and morphological differentiation induced by NGF. It is also shown that binding of the 34K-ssb protein to ssDNA is fully inhibited by NADH but not by NAD+ or by several other nucleotides. Enzymatic tests on the possible NADH/NAD+-dependent dehydrogenase activity of the 34K-ssb protein have demonstrated that it has lactic dehydrogenase activity (LDH) with a specific activity comparable to that of rabbit muscle. Furthermore, the 34K-ssb protein has the same peptide mapping as LDH purified from rat muscle. Antibodies directed against the 34K-ssb protein cross-react with the rabbit muscle enzyme and, vice versa, antibodies raised against rabbit LDH cross-react with the 34K-ssb protein. It is concluded that the 34K-ssb protein is identifiable with the type M of LDH, although possible differences in primary structure of the two proteins may have escaped the present studies. We hypothesize that interaction of the PC12 lactic dehydrogenase with ssDNA occurs also in vivo, as indicated by the findings reported in the accompanying paper, and may be modulated by the cellular content of NADH which, in turn, is related to energy metabolism.
Assuntos
Proteínas de Ligação a DNA/biossíntese , L-Lactato Desidrogenase/metabolismo , Fatores de Crescimento Neural/farmacologia , Animais , Linhagem Celular , DNA/biossíntese , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Meia-Vida , L-Lactato Desidrogenase/análise , Peso Molecular , NAD/farmacologia , Nucleotídeos/farmacologia , Peptídeos/análise , Feocromocitoma , RNA Mensageiro/metabolismo , Ratos , Timidina/metabolismoRESUMO
In the preceding article [1] we identified the 34 kD single-stranded DNA-binding (ssb) protein, whose synthesis is inhibited in PC12 cells concomitantly with nerve growth factor (NGF)-induced mitotic arrest, with the enzyme lactic dehydrogenase (LDH-ssb protein). Localization studies performed with antibodies raised against the LDH-ssb protein demonstrate the presence of a pool of this protein in the nucleus of several cell types. The nuclear association of this protein is sensitive to DNase treatment of the cells and quantitative electron microscopy confirms that the LDH-ssb protein is located close to chromatin structures. These results point to a possible involvement of the LDH-ssb protein in some nuclear function(s).
Assuntos
Núcleo Celular/enzimologia , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/análise , L-Lactato Desidrogenase/análise , Animais , Astrócitos/enzimologia , Linhagem Celular , Cromatina/enzimologia , Citoplasma/enzimologia , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases/farmacologia , Imunofluorescência , L-Lactato Desidrogenase/metabolismo , Microscopia Eletrônica , Feocromocitoma , Codorniz , Ratos , Ribonucleases/farmacologiaRESUMO
The recombinant variable regions of the monoclonal antibody Y13-259, directed against the p21ras protein, have been engineered for expression as intracellular single chain Fv fragments. The activity of the plasmid was confirmed by in vitro and in vivo translation of mRNA showing that the intracellularly expressed single chain fragments are stably and efficiently expressed as cytosolic proteins. The expression of the anti-p21ras single chain antibodies in the cytoplasm of Xenopus laevis oocytes leads to the inhibition of the insulin-induced meiotic maturation. This finding represents the first successful application of the strategy of intracellular antibodies to block a complex biological process in the cytosol of vertebrate cells.
Assuntos
Anticorpos Monoclonais/metabolismo , Região Variável de Imunoglobulina/metabolismo , Meiose/fisiologia , Oócitos/citologia , Oócitos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Feminino , Região Variável de Imunoglobulina/biossíntese , Insulina/farmacologia , Meiose/efeitos dos fármacos , Microinjeções , Oócitos/efeitos dos fármacos , Biossíntese de Proteínas , RNA Mensageiro/administração & dosagem , RNA Mensageiro/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Reticulócitos/metabolismo , Transcrição Gênica , Vertebrados , Xenopus laevisRESUMO
We describe in this paper the binding and biochemical properties of two human antibody fragments directed against the human immunodeficiency virus type 1 reverse transcriptase (RT). These fragments were isolated from a synthetic combinatorial library of human Fab antibody fragments displayed on the surface of filamentous phage. The antibody fragments were selected by using recombinant heterodimeric human immunodeficiency virus type 1 RT purified from insect cells as a solid-phase selector. This procedure led to the isolation of two antibody fragments that completely neutralize the RNA-dependent DNA polymerase activity of RT at nanomolar concentrations. Both antibody fragments bind only to the enzymatically active form of the RT. The inhibitory activity of the anti-RT antibody fragments is competitive with respect to the template primer. The antibody fragments also neutralize the activities of RTs from avian and murine retroviruses and of DNA polymerases of prokaryotic origin as well as human DNA polymerase alpha. Thus, the antibody fragments selected and characterized in this study appear to recognize a structural fold that is common to the different DNA polymerases and necessary for their activity. The results provide an immunological experimental basis for a purely structural and evolutionary classification of the polymerase family.
Assuntos
Anticorpos Anti-HIV/imunologia , Transcriptase Reversa do HIV/imunologia , HIV-1/enzimologia , DNA Polimerase Dirigida por RNA/imunologia , Animais , Vírus da Mieloblastose Aviária/enzimologia , Linhagem Celular , Colífagos , Biblioteca Gênica , Transcriptase Reversa do HIV/genética , Humanos , Camundongos , Vírus da Leucemia Murina de Moloney/enzimologia , Testes de Neutralização , DNA Polimerase Dirigida por RNA/classificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Spodoptera/citologiaRESUMO
The binding of nerve growth factor (NGF) to its receptors in PC12 cells was studied in two experimental conditions: (a) cell fixation with paraformaldehyde followed by permeabilization of the plasma membrane with methanol and (b) metabolic poisoning of living cells with sodium azide. Paraformaldehyde fixation of PC12 cells causes a 60-70% reduction of NGF binding capacity; the original binding capacity is restored following permeabilization with methanol. A kinetic analysis of NGF binding under these conditions reveals a single homogeneous population of receptors at variance with experiments performed in living cells where two kinetically distinct types of NGF receptors were demonstrated [Landreth, G. E. and Shooter, E. M. (1980) Proc. Natl Acad. Sci. USA, 77, 4751-4755; Schechter, A. L. and Bothwell, M. A. (1981) Cell, 24, 867-874]. Our results suggest that a proportion of the NGF receptors in PC12 cells is hidden, i.e. not available for binding to the ligand, and in a dynamic equilibrium with exposed receptors. The existence of hidden receptors is confirmed by treatment of PC12 cells with sodium azide, which causes a 50% reduction in NGF binding capacity and protection from trypsin digestion of the remaining pool of hidden receptors. The latter become exposed at the cell surface following removal of sodium azide. Our data provide an interpretation for the as yet unsatisfactorily explained data on NGF receptors.