RESUMEN
Apoptosis, or programmed cell death, occurs because of the activation of a protease cascade amplification circuit that includes the critical effector caspase-3. Previously, we identified the widely expressed actin modulatory protein gelsolin as a prominent substrate of caspase-3 and demonstrated that the N-terminal gelsolin cleavage product promotes apoptosis. Here we show that phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3, 4-bisphosphate in pure micelles or mixed vesicles prevent caspase-3 cleavage of gelsolin. Moreover, phosphatidylinositol 4, 5-bisphosphate-gelsolin strongly inhibits caspase-3 and -9 activity through the formation of a stable phosphatidylinositol 4, 5-bisphosphate-gelsolin-caspase complex. In addition, phosphatidylinositol 4,5-bisphosphate-gelsolin prevents apoptotic progression mediated by caspase-3 in a cell-free system, and phosphatidylinositol 4,5-bisphosphate-gelsolin-caspase-9 and phosphatidylinositol 4,5-bisphosphate-gelsolin-caspase-3 complexes form in mouse embryonic fibroblasts during apoptosis induction when stimulated with fibronectin, to delay cell death. The results suggest that gelsolin can act as both an effector and an inhibitor of caspase-3, the latter in concert with phosphatidylinositol 4, 5-bisphosphate, and other membrane phospholipids to regulate the onset and progression of apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Inhibidores de Caspasas , Gelsolina/farmacología , Fosfatidilinositol 4,5-Difosfato/farmacología , Animales , Caspasa 3 , Caspasa 9 , Caspasas/metabolismo , Inhibidores Enzimáticos/farmacología , Fibroblastos , Gelsolina/química , Humanos , Células Jurkat , Cinética , Hígado/enzimología , Ratones , Fosfatidilinositol 4,5-Difosfato/química , Fosfatos de Fosfatidilinositol/farmacologíaRESUMEN
The caspase-3 (CPP32, apopain, YAMA) family of cysteinyl proteases has been implicated as key mediators of apoptosis in mammalian cells. Gelsolin was identified as a substrate for caspase-3 by screening the translation products of small complementary DNA pools for sensitivity to cleavage by caspase-3. Gelsolin was cleaved in vivo in a caspase-dependent manner in cells stimulated by Fas. Caspase-cleaved gelsolin severed actin filaments in vitro in a Ca2+-independent manner. Expression of the gelsolin cleavage product in multiple cell types caused the cells to round up, detach from the plate, and undergo nuclear fragmentation. Neutrophils isolated from mice lacking gelsolin had delayed onset of both blebbing and DNA fragmentation, following apoptosis induction, compared with wild-type neutrophils. Thus, cleaved gelsolin may be one physiological effector of morphologic change during apoptosis.
Asunto(s)
Apoptosis , Caspasas , Tamaño de la Célula , Cisteína Endopeptidasas/metabolismo , Gelsolina/metabolismo , Actinas/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Animales , Caspasa 3 , Línea Celular , Cicloheximida/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Citoesqueleto/metabolismo , Fragmentación del ADN , Humanos , Ratones , Neutrófilos/citología , Neutrófilos/metabolismo , Proteínas Recombinantes/metabolismo , Transfección , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacología , Receptor fas/fisiologíaRESUMEN
M-CSF (CSF-1) can be produced in a variety of structural forms that may affect function in vivo. Truncated, nonglycosylated forms of recombinant M-CSF (rM-CSF) from E. coli have been refolded in vitro in high yield and shown to be functionally equivalent in vitro to glycosylated rM-CSF secreted from mammalian cells. An N-terminal domain of 149 amino acids is produced by all of the known M-CSF mRNA splice variants and is the region responsible for bioactivity observed in vitro. Heterodimeric rM-CSFs from different splice variants containing this domain were produced in pure form by refolding in vitro, and are fully active, but have yet to be observed in vivo. The circulating half-life of truncated M-CSF forms injected intravenously into rats increased with the MW of the M-CSF used. Large increases in half-life in vivo were observed following chemical addition of a single molecule of 10 kD polyethylene glycol to rM-CSF in vitro. The crystal structure of rM-CSF revealed that M-CSF is a member of a family of molecules related by having a distinctive four-helical-bundle structural core. Site-directed mutagenesis showed that residues in or near helix A and helix C are involved in receptor binding, as reflected by decreased bioactivity and receptor binding of certain mutants. A soluble form of the M-CSF receptor, c-fms, was produced in a baculovirus/Sf9 expression system and purified to homogeneity. The MW of rM-CSF saturated with this soluble receptor was determined by molecular sieve chromatography and light scattering. Each dimeric M-CSF molecule appears to bind two soluble receptor molecules in vitro, supporting the observation that M-CSF signaling is linked to receptor dimerization.
Asunto(s)
Factor Estimulante de Colonias de Macrófagos/química , Animales , Cristalografía por Rayos X , Semivida , Humanos , Factor Estimulante de Colonias de Macrófagos/farmacocinética , Factor Estimulante de Colonias de Macrófagos/fisiología , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica , Ratas , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Relación Estructura-ActividadRESUMEN
Galectin-3 is a beta-galactoside-specific lectin implicated in diverse processes involved in cellular interactions. Recently, the Mac-2-binding protein, a heavily N-glycosylated secreted protein with a subunit Mr of 97,000, was identified as its ligand. The present study characterizes the interaction between galectin-3 and Mac-2-binding protein in whole cells and measures their relative expression levels. Incubation of A375 cells with affinity-purified Mac-2-binding protein resulted in its binding to galectin-3 on the cell surface in a specific carbohydrate-dependent manner. Mac-2-binding protein also induced homotypic cell aggregation, which was inhibited by lactose or Fab' fragments of an anti-galectin-3 antibody. Northern blotting analysis revealed differences in the transcriptional regulation of galectin-3 and Mac-2-binding protein. These results provide the first direct evidence for a Mac-2-binding protein function and suggest that it may play a role in tumor cell embolization during metastasis through interaction with galectin-3.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Proteínas Portadoras/metabolismo , Adhesión Celular/fisiología , Glicoproteínas/metabolismo , Antígenos de Diferenciación/genética , Antígenos de Neoplasias , Biomarcadores de Tumor , Northern Blotting , Neoplasias de la Mama/patología , Proteínas Portadoras/genética , Agregación Celular , ADN Complementario/genética , Femenino , Galectina 3 , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/genética , Humanos , Melanoma/patología , Células Neoplásicas Circulantes , Unión ProteicaRESUMEN
The crystal structure of PR3, a serine proteinase from the azurophilic granules of human polymorphonuclear neutrophils, has been solved by molecular replacement using the human leukocyte elastase structure. The PR3 structure has been refined to an R-factor (= sigma parallel Fo magnitude of-Fc parallel/sigma magnitude of Fo) of 0.201 for all data in the range of 10.0 to 2.2 A resolution. The enzyme was crystallized in space group P21 with four molecules in the asymmetric unit (Vm approximately equal to 2.6 A/Da). The overall fold consists of two domains of beta-barrel structures typical of the chymotrypsin family of serine proteinases. In general, the substrate binding sites, S4 to S3', are more polar than comparable sites in the related proteinase, human leukocyte elastase. The experimentally observed preference of PR3 for small aliphatic residues at the P1 position of a substrate is explained by the Val to Ile substitution at position 190 when compared to the elastase structure. The substitution of Ala by Asp at position 213 at the back of S1 should not affect its specificity greatly, as the Asp side-chain points back into the interior of the protein. The PR3 structure includes a disaccharide unit (N-linked 2-acetamido-2-deoxy-beta-D-glucopyranose and 1,6-linked alpha-L-fucopyranose) covalently attached to Asn 159. The linear antigenic sites of PR3 reported to react with Wegener's granulomatosis autoantibodies occur in regions of the three-dimensional structure that may implicate the inactive pro-form of the enzyme in the pathogenesis of the disease.
Asunto(s)
Autoantígenos/química , Granulomatosis con Poliangitis/inmunología , Neutrófilos/inmunología , Serina Endopeptidasas/química , Secuencia de Aminoácidos , Sitios de Unión , Secuencia de Carbohidratos , Cristalografía por Rayos X , Glicosilación , Humanos , Elastasa de Leucocito , Modelos Moleculares , Datos de Secuencia Molecular , Mieloblastina , Elastasa Pancreática/química , Conformación Proteica , Proteínas Recombinantes/química , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Recombinant proteins that have survived the challenges of process development and clinical trials are becoming blockbuster medical products. Growth factors, enzymes and antibodies are being improved by mutational approaches, fused with other proteins, and even chemically modified in vitro. Drug development and testing approaches have advanced, and proteins produced in transgenic animals are new becoming available. Future protein products might include cancer vaccines and therapies for a variety of genetic diseases, but alternative treatments involving gene therapy or small synthetic compounds will provide competition.
Asunto(s)
Proteínas Recombinantes/uso terapéutico , Animales , Biotecnología , Clonación Molecular , Industria Farmacéutica , Expresión Génica , Humanos , Ingeniería de Proteínas , Pliegue de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/aislamiento & purificación , Vacunas Sintéticas/uso terapéuticoRESUMEN
Human macrophage colony-stimulating factor (M-CSF) is a homodimeric cytokine that is a member of a structurally related family of hormones defined by an unusual up-up-down-down alpha-helical bundle. To identify regions on the surface of M-CSF that might interact with the M-CSF receptor, single and double amino acid substitutions were introduced into a truncated form of human M-CSF alpha by site-directed mutagenesis, and the homodimeric M-CSF analogs were purified and characterized. Certain substitutions in the region before and in helix A and in helix C decreased specific bioactivity and correlated with an approximately equivalent reduction in M-CSF receptor affinity. The most dramatic change was observed in an analog in which residues His-9 and His-15 were replaced with alanines, resulting in a 9,100-fold decrease in specific bioactivity. X-ray crystallographic analysis of this M-CSF alpha H9A,H15A analog at a resolution of 2.5 A revealed no significant changes in structure other than the expected new side chains at residues 9 and 15. Analogs containing only one of these two histidine substitutions exhibited a decrease in specific bioactivity of 6- and 1200-fold for the H9A and H15A mutations, respectively. It appears that selected amino acids in the NH2-terminal region of M-CSF and possibly portions of the surface formed by helices A and C are significantly involved in interactions with the M-CSF receptor.
Asunto(s)
Factor Estimulante de Colonias de Macrófagos/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Factor Estimulante de Colonias de Macrófagos/química , Factor Estimulante de Colonias de Macrófagos/aislamiento & purificación , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Desnaturalización Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
We have purified and sequenced a secreted glycoprotein from both the human breast carcinoma cell line, SK-BR-3, and human breast milk. The native protein binds specifically to a human macrophage-associated lectin known as Mac-2. This Mac-2 binding protein (Mac-2-BP) has an apparent native molecular mass of several million daltons and contains subunits of 85-97 kDa that are very susceptible to proteolysis at a dibasic cleavage site. Western analysis suggests that Mac-2-BP is found in serum, semen, saliva, urine, and tears, in addition to breast milk. The gene encoding Mac-2-BP was cloned from a cDNA bank of a human monocytic cell line, using degenerate PCR primers based on the protein sequence. Recombinant Mac-2-BP was expressed in Cos cells and secreted as a high molecular weight complex. The cDNA clone encodes a mature protein of 567 amino acids, preceded by an 18-amino acid leader. The mature protein contains 16 cysteines and has seven potential N-linked glycosylation sites. The first 106 amino acids represent a domain that is highly similar to an ancient protein superfamily defined by the macrophage scavenger receptor cysteine-rich domain.
Asunto(s)
Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Cisteína , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Receptores de Superficie Celular/genética , Receptores Inmunológicos , Secuencia de Aminoácidos , Animales , Antígenos de Neoplasias , Secuencia de Bases , Biomarcadores de Tumor , Neoplasias de la Mama , Proteínas Portadoras/aislamiento & purificación , Línea Celular , Clonación Molecular/métodos , ADN , Femenino , Biblioteca de Genes , Glicoproteínas/aislamiento & purificación , Humanos , Leche Humana/metabolismo , Datos de Secuencia Molecular , Monocitos , Familia de Multigenes , Oligodesoxirribonucleótidos , Receptores Depuradores , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Homología de Secuencia de Aminoácido , Transfección , Células Tumorales CultivadasRESUMEN
Macrophage colony-stimulating factor (M-CSF) triggers the development of cells of the monocyte-macrophage lineage and has a variety of stimulatory effects on mature cells of this class. The biologically active form of M-CSF is a disulfide-linked dimer that activates an intrinsic tyrosine kinase activity on the M-CSF receptor by inducing dimerization of the receptor molecules. The structure of a recombinant human M-CSF dimer, determined at 2.5 angstroms by x-ray crystallography, contains two bundles of four alpha helices laid end-to-end, with an interchain disulfide bond. Individual monomers of M-CSF show a close structural similarity to the cytokines granulocyte-macrophage colony-stimulating factor and human growth hormone. Both of these cytokines are monomeric in their active form, and their specific receptors lack intrinsic tyrosine kinase activity. The similarity of these structures suggests that the receptor binding determinants for all three cytokines may be similar.
Asunto(s)
Factor Estimulante de Colonias de Macrófagos/ultraestructura , Cristalografía , Disulfuros , Factor Estimulante de Colonias de Granulocitos y Macrófagos/ultraestructura , Hormona del Crecimiento/química , Modelos Moleculares , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/ultraestructura , Homología de Secuencia de Aminoácido , Difracción de Rayos XRESUMEN
Using two enzymes purified from the white-rot fungus, Polyporus obtusus, 5% solutions of D-glucose have been quantitatively converted in vitro into D-arabino-hexos-2-ulose (D-glucosone) and subsequently into a compound having antimicrobial activity. The antibiotic has been shown by nuclear magnetic resonance and mass spectroscopy to be chemically identical to a previously described fungal metabolite known as cortalcerone. Based on kinetic analysis of the synthetic process, a pathway for the biosynthesis of cortalcerone is proposed, involving both chemical rearrangement and enzymically catalyzed steps. Two enzymes, pyranose 2-oxidase and a previously uncharacterized D-arabino-hexos-2-ulose-utilizing enzyme, may be sufficient for the biosynthesis of cortalcerone from glucose in vivo. The D-arabino-hexos-2-ulose-utilizing enzyme dehydrates certain aldosuloses and has been named aldos-2-ulose dehydratase. The enzyme, which appears to be a dimer of 95-kDa subunits, has been purified 450-fold. Additional properties of aldos-2-ulose dehydratase are described, including its apparent ability to catalyze two different steps in the proposed biosynthetic pathway for cortalcerone.
Asunto(s)
Antibacterianos/síntesis química , Deshidrogenasas de Carbohidratos/química , Glucosa/química , Polyporaceae/enzimología , Antibacterianos/química , Polyporaceae/química , Piranos/síntesis química , Piranos/química , PironasRESUMEN
Human ras GTPase-activating protein (GAP) is a cytoplasmic factor that stimulates the GTPase activity of normal N-ras p21 while having no stimulatory effect on the GTPase activity of oncogenic variants of N-ras p21. We have purified two forms of native ras GAP from human placental tissue. In addition to the Mr = 120,000 type I GAP reported previously (1), an equivalent amount of an Mr = 95,000 molecule with GAP activity was recovered and shown to have the N-terminal sequence expected for type II GAP. The two GAP forms in placental extracts were resolved by molecular sieve chromatography and appeared to have a monomeric native structure. Human recombinant type I GAP was produced intracellularly in Sf9 insect cells using a baculovirus expression vector, and 10-mg quantities were purified to homogeneity in three steps. Comparison of the purified native and recombinant GAP molecules revealed that all three displayed similar biological specific activities in an in vitro GAP assay. A polyclonal antibody to purified recombinant GAP was prepared and shown to neutralize the activity of both native and recombinant GAPs. The antibody was also highly specific for the detection of native GAP by Western blot. Type I and II GAP species were detected in approximately equal amounts in cytoplasmic extracts of human placenta, but only type I GAP was observed when other human tissues were examined.
Asunto(s)
Proteínas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Cromatografía por Intercambio Iónico , Clonación Molecular , Proteínas de Unión al GTP , Proteínas Activadoras de GTPasa , Humanos , Insectos , Datos de Secuencia Molecular , Placenta/química , Proteínas/química , Proteínas/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Activadoras de ras GTPasaRESUMEN
The single gene for human macrophage colony-stimulating factor (M-CSF, or CSF-1) generates multiple mRNA species that diverge within the coding region. We have characterized translation products of these mRNA species from native and recombinant sources. Immunoblots of reduced native M-CSF indicate that multiple glycosylated species ranging from 25 kd to 200 kd are secreted by human monocytes and cell lines. In contrast, CV-1 cells expressing a short M-CSF clone secrete only 24 kd recombinant M-CSF. Synthetic peptide antibodies were developed to distinguish between secreted recombinant M-CSF from long and short mRNA splicing variants. Immunoblot analysis indicates that alternative mRNA splicing generates some M-CSF protein heterogeneity. Most secreted MIA PaCa-2 M-CSF reacts with long-clone-specific antibody. Lectin affinity chromatography shows that variable glycosylation contributes significantly to MIA PaCa-2 M-CSF size heterogeneity. In addition, cell lysates also contain larger M-CSF species that apparently undergo proteolytic processing before secretion. The data indicate that M-CSF protein heterogeneity results from both pre- and post-translational processing.
Asunto(s)
Factores Estimulantes de Colonias/metabolismo , Macrófagos/metabolismo , Empalme del ARN , ARN Mensajero/metabolismo , Western Blotting , Cromatografía de Afinidad , Factores Estimulantes de Colonias/aislamiento & purificación , Glicosilación , Humanos , Hidrólisis , Radioinmunoensayo , Células Tumorales CultivadasRESUMEN
We have detected endogenous human macrophage colony-stimulating factor (M-CSF) in blood of normal individuals, using a novel RIA that accurately measures M-CSF concentrations as low as 60 U/ml (1.2 ng/ml) in the presence of serum proteins. The RIA uses an antibody to highly purified recombinant human M-CSF and is calibrated to a mouse bone marrow colony-forming assay. Ten samples of normal human blood plasma contained an average 118 +/- 9 U/ml of M-CSF, and similar concentrations were detected in serum prepared from the same individuals. RIA-positive samples contained biologically active M-CSF, as determined in a colony assay performed on mouse bone marrow cells. The M-CSF biological activity was removed by specific immune precipitation and inhibited by addition of M-CSF antibody. Physical characterization of plasma M-CSF was done by immunoblotting after partial purification on controlled pore glass and immunoaffinity chromatography. The major reduced protein species of plasma M-CSF had an apparent molecular weight of about 24 kd, and minor species of 30, 45, and 60-70 kd were also present. The RIA results on ten normal individuals suggest that endogenous circulating M-CSF is present at a low but detectable concentration. This RIA can be used to measure M-CSF in clinical samples that contain serum proteins and other growth factors that may interfere with accurate bioassay determinations.
Asunto(s)
Ensayo de Unidades Formadoras de Colonias , Factores Estimulantes de Colonias/sangre , Sustancias de Crecimiento/sangre , Immunoblotting , Radioinmunoensayo , Factores Estimulantes de Colonias/normas , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Sustancias de Crecimiento/normas , Humanos , Macrófagos/fisiología , Radioinmunoensayo/métodos , Radioinmunoensayo/normas , Proteínas Recombinantes/sangre , Proteínas Recombinantes/normas , Estándares de ReferenciaRESUMEN
The ras p21 GTPase-activating protein (GAP) was purified from human placental tissue. Internal amino acid sequence was obtained from this 120,000-dalton protein and, by means of this sequence, two types of complementary DNA clones were isolated and characterized. One type encoded GAP with a predicted molecular mass of 116,000 daltons and 96% identity with bovine GAP. The messenger RNA of this GAP was detected in human lung, brain, liver, leukocytes, and placenta. The second type appeared to be generated by a differential splicing mechanism and encoded a novel form of GAP with a predicted molecular mass of 100,400 daltons. This protein lacks the hydrophobic amino terminus characteristic of the larger species, but retains GAP activity. The messenger RNA of this type was abundantly expressed in placenta and in several human cell lines, but not in adult tissues.
Asunto(s)
Clonación Molecular , ADN/genética , Placenta/análisis , Proteínas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Química Encefálica , ADN/aislamiento & purificación , Femenino , Proteínas Activadoras de GTPasa , Regulación de la Expresión Génica , Humanos , Leucocitos/análisis , Hígado/análisis , Pulmón/análisis , Datos de Secuencia Molecular , Peso Molecular , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos , Embarazo , Proteínas/aislamiento & purificación , ARN Mensajero/análisis , ARN Mensajero/genética , Homología de Secuencia de Ácido Nucleico , Proteínas Activadoras de ras GTPasaRESUMEN
Current malaria vaccine strategies focus on subunit vaccines that contain one or a limited number of malaria Ag. However, there is widespread nonresponsiveness to many of these Ag probably resulting from Ir gene control. Using a congenic mouse model, we demonstrated that human rIL-2 (as an adjuvant) can overcome Ir gene controlled low immune responsiveness to peptide malaria Ag vaccine candidates [R32tet32, R32LR, and Th2R-NP (NANP)5NA] as determined by the antibody response, providing it is emulsified with the Ag during immunization. This effect is not caused by IL-2 merely acting as a foreign protein and stimulating noncognate help; it requires biologic activity of the IL-2, as determined by studying the effect of inactive rIL-2, which has minimal biologic activity but which has retained its antigenicity. IL-2 does not appear to be working by an effect on priming of specific Th, and IL-2 cannot overcome an Ir gene controlled low T cell proliferative response. IL-2 may have a role to play in human vaccine development where a high titer antibody response to a subunit vaccine is required.
Asunto(s)
Antígenos de Superficie/administración & dosificación , Genes MHC Clase II/efectos de los fármacos , Antígenos H-2/genética , Interleucina-2/farmacología , Plasmodium falciparum/inmunología , Proteínas Protozoarias , Proteínas Recombinantes/farmacología , Animales , Antígenos de Superficie/inmunología , Humanos , Inmunización Secundaria , Ratones , Linfocitos T Colaboradores-Inductores/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaAsunto(s)
Proteínas de la Membrana/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Pirofosfatasas , Transducción de Señal , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transformación Celular Neoplásica , Clonación Molecular , ADN/genética , Femenino , Proteínas Activadoras de GTPasa , Humanos , Cinética , Datos de Secuencia Molecular , Oocitos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Placenta/metabolismo , Embarazo , Proteínas/genética , Proteínas/aislamiento & purificación , Proteínas Proto-Oncogénicas p21(ras) , Xenopus , Proteínas Activadoras de ras GTPasaRESUMEN
Human recombinant interleukin-2 (IL-2) and related species have been characterized by chemical modifications, tryptic digestion, and cyanogen bromide digestion. The oxidation states of the cysteines and methionines in several IL-2 muteins have been determined. Reversed-phase high-performance liquid chromatography allowed us to distinguish the modifications in these muteins and to correlate retention behavior with their structure.
Asunto(s)
Interleucina-2/análisis , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Bromuro de Cianógeno , Cisteína/análisis , Humanos , Hidrólisis , Metionina/análisis , Oxidación-Reducción , Proteínas Recombinantes/análisis , TripsinaRESUMEN
Highly purified interleukin-2 (IL-2) induced proliferation of fresh human peripheral blood mononuclear leukocytes (PBML) in the absence of identifiable exogenous mitogenic or antigenic stimuli. Dose dependent proliferation was observed with three IL-2 preparations, including two preparations purified from natural sources and a preparation produced by recombinant DNA techniques. Both T and non-T cells proliferated. Purified helper/inducer and suppressor/cytotoxic T cells cultured in the absence of non-T cells proliferated only weakly; the proliferative response to IL-2 was restored by the addition of irradiated non-T cells. Proliferation to IL-2 was not blocked by the monoclonal antibody anti-Leu 4, which reacts with a component of the T-cell receptor complex for antigen and blocks mitogen and antigen-induced T-cell responses. Monoclonal antibody to HLA-DR also failed to significantly block the proliferation of resting cells to IL-2. The IL-2 induced proliferative response thus appears to be dependent on interactions between different subpopulations of PBML but probably does not simply reflect augmentation by IL-2 of antigen-driven or autoreactive processes.
Asunto(s)
Interleucina-2/inmunología , Activación de Linfocitos , Anticuerpos/inmunología , Antígenos de Superficie/inmunología , Unión Competitiva , Humanos , Técnicas In Vitro , Linfocitos/clasificación , Linfocitos/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis TumoralRESUMEN
The gene for interleukin-2 was isolated from the Jurkat cell line and from normal peripheral blood lymphocytes and, when inserted in Escherichia coli, was expressed at high concentrations. This interleukin-2 was purified to apparent homogeneity and tested for biological activity in a variety of assays in vitro and in vivo. The recombinant lymphokine supports the growth of murine and human interleukin-2 dependent cell lines, enhances the generation of murine and human cytolytic cells in vitro, and generates lymphokine activated killer cells from murine and human lymphocytes. It has a serum half-life of 2 to 3 minutes in the mouse and significantly enhances the generation of cytolytic cells in vivo after alloimmunization. No functional differences between native and the recombinant interleukin-2 molecules have been detected.