RESUMO
Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, and Xenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes. However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.
Assuntos
Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares , Sequência de Aminoácidos , Animais , Western Blotting , Galinhas , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Proteínas de Choque Térmico/química , Humanos , Dados de Sequência Molecular , Especificidade de Órgãos , Coelhos , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , XenopusRESUMO
Percutaneous balloon valvuloplasty was performed on a patient with pulmonary stenosis. Right to left shunting through a patent foramen ovale during balloon inflation was documented by contrast two-dimensional echocardiography. Right and left ventricular pressures recorded during balloon inflation showed a decrease in left ventricular end-diastolic pressure and equilibration with right ventricular end-diastolic pressure. Systemic hypotension was minimal during balloon inflation, possibly due to persistent filling of the left ventricle via the patent foramen ovale. Persistent right ventricular systolic hypertension immediately after valvuloplasty may have been due to infundibular narrowing and resolved on restudy 2 weeks later.
Assuntos
Cateterismo/métodos , Comunicação Interatrial/complicações , Estenose da Valva Pulmonar/terapia , Adulto , Pressão Sanguínea , Cateterismo Cardíaco , Dilatação , Ecocardiografia , Comunicação Interatrial/fisiopatologia , Ventrículos do Coração/fisiopatologia , Humanos , Hipotensão/etiologia , Hipotensão/prevenção & controle , Masculino , Contração Miocárdica , Valva Pulmonar/fisiopatologia , Estenose da Valva Pulmonar/complicações , Estenose da Valva Pulmonar/fisiopatologiaRESUMO
Male-limited gonadotropin-independent precocious puberty (MPP) is frequently associated with mutations of the human LH/CG receptor (hLHR) that result in constitutively active hLHRs. Many such activating mutations have been identified in transmembrane 6 of the hLHR, with the substitution of Asp-578 being the most frequently observed mutation. Mutagenesis of a transmembrane helix of a G protein-coupled receptor can cause local alterations in the conformation near the mutated residue, allosteric changes elsewhere in the protein, and/or changes in the interhelical packing of the receptor. Therefore, while it has been hypothesized that activation of the receptor by mutations of Asp-578 may arise via alterations in the interactions of helix 6 with other transmembrane helices and/or by allosterically altering the conformation of the third intracellular loop, it has not been possible to ascertain the role of the sixth transmembrane helix per se in activating Gs in the mutated full-length receptor. Recently, however, we have shown that a peptide KMAILIFT, corresponding to the juxtacytoplasmic portion of helix 6 of the hLHR, is capable of activating Gs. These results suggest that helix 6 itself can directly interact with Gs. Importantly, the KMAILIFT peptide did not include Asp-578, which lies just C-terminal to this sequence. We show herein that a peptide extended to include Asp-578 (KMAILIFTDFT) is a poor activator of Gs. However, if the peptide is synthesized with the aspartate replaced with either a glycine or tyrosine, substitutions that are found in some patients with MPP, these peptides have Gs-stimulating activity. Additionally, a transmembrane 6 peptide with the substitution of Ile-575 with leucine, another mutation found in MPP, mimicked the activating effects of this mutation in the full-length receptor. The ability of peptides in which Asp-578 or Ile-575 is substituted to mimic the activating effects of these mutations in the full-length receptor suggests that the sixth transmembrane helix represents a site for direct interaction with Gs. In addition to the stimulatory effects of transmembrane 6 peptides, peptides corresponding to the juxtacytoplasmic portions of the fourth, fifth, and seventh helices were also able to stimulate Gs. These results are consistent with the hypothesis that the transmembrane helices may form a pocket for interaction with Gs and that constitutive activation of the hLHR may involve the opening of the pocket formed by these helices, thus exposing Gs-binding sites on these helices.
Assuntos
Membrana Celular/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Mutação , Estrutura Secundária de Proteína , Receptores do LH/genética , Receptores do LH/metabolismo , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Membrana Celular/enzimologia , Embrião de Mamíferos , Humanos , Rim , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Receptores do LH/química , Relação Estrutura-AtividadeRESUMO
The amino acid sequences of human inhibin alpha-, beta A- and beta B-subunits were analyzed for hydrophilicity and chain flexibility to predict regions that are on the surface of the subunits and, therefore, are potential antigenic sites. Based on these analyses, a total of nine peptides were synthesized, and rabbit antisera against the peptides were prepared. Peptides of the N-terminus (residues 1-16 and 13-24) and region 109-123 of the alpha-subunit produced high titer antibodies. Regions 69-79 and 93-105 of the beta A-subunit and region 93-104 of the beta B-subunit were also immunogenic. Immunoblotting of an inhibin preparation with anti-alpha-peptide antiserum revealed that a 32K band (inhibin) and an 18K band (alpha-subunit) were stained. Immunoblotting with anti-beta-peptide antiserum detected a 32K band (inhibin), a 24K band (activin), and a 14K band (beta-subunit). Injection (iv) of these antisera into rats induced dramatic elevation of serum FSH in 6-12 h and suggested immunoneutralization of endogenous inhibin. RIAs for each subunit were developed using radioiodinated peptides as tracers. Competition binding assays indicated crossreactivity with human follicular fluid, semen, serum, plasma, and crude inhibin preparations. Parallel dilution curves were obtained. Antisera against beta A- and beta B-subunit peptide cross-reacted with each other. In immunocytochemical studies, these antisera were used in conjunction with gold-labeled goat antirabbit immunoglobulin G to localize inhibin in cells of the rat testis. Specific staining of inhibin was localized within the Sertoli cells of some tubules in adult rat testis. Positive staining could be blocked by preadsorbing the sera with the appropriate synthetic peptide. These results suggest that antibodies against synthetic inhibin peptides are useful in elucidating the roles of inhibin and activin.
Assuntos
Inibinas/análise , Fragmentos de Peptídeos/análise , Animais , Anticorpos/imunologia , Immunoblotting , Imuno-Histoquímica , Inibinas/metabolismo , Inibinas/fisiologia , Masculino , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/fisiologia , Radioimunoensaio , Ratos , Testículo/citologia , Testículo/metabolismoRESUMO
A comprehensive series of overlapping synthetic peptides have been used to study the relationship between the primary structure of the ovarian receptor for LH/human CG (hCG) and hormone binding. Twenty-four consecutive, overlap peptides that replicate the entire extracellular domain of the rat luteal receptor have been synthesized by standard solid-phase techniques on an automated synthesizer. Eight additional peptides from the extracellular domain and three peptides replicating the putative extracellular loop regions have also been synthesized. Each peptide was evaluated in RRAs for interaction with hCG by measuring its ability to competitively inhibit binding of 125I-hCG to membrane receptor. Twelve peptides were found to be potent in RRAs and caused a reduction of half-maximal binding of 125I-hCG at concentrations of 10-250 x 10(-6) M. The 12 active peptides (and adjacent inactive peptides) defined at least 4 independent receptor regions that can interact with hormone. One site near the NH2-terminus was localized to receptor residues Arg21-Pro38. Two more sites of hormone interaction were identified by peptides replicating residues Arg102-Thr115 and Tyr253-Phe272. A fourth binding region was identified in the third putative extracellular loop, replicated by rat luteal receptor peptide Lys573-Lys583. The amino acid sequences of the four active rat LH/hCG receptor regions were aligned and compared with published sequences for other glycoprotein hormone receptors. Three regions (Arg102-Thr115, Tyr253-Phe272, and Lys573-Lys583) showed high sequence homology with the human LH/hCG receptor, human TSH receptor, and rat FSH receptor and may represent contact sites for the alpha-subunit of hormone. The other binding region, Arg21-Pro38 had low sequence homology with the other glycoprotein hormone receptors and is postulated to be a binding determinant for beta-hCG/LH. This report demonstrates that synthetic overlap peptides of confirmed sequence can be used to successively identify hormone interaction sites of glycoprotein hormone receptors.
Assuntos
Fragmentos de Peptídeos/química , Receptores do LH/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Gonadotropina Coriônica/metabolismo , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Ratos , Receptores do LH/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
Twenty-seven synthetic peptides, representing the entire structure of the human glycoprotein hormone alpha-subunit were used to map the antigenic structure of the alpha-subunit. Solution phase and solid phase assays were performed with these peptides and a panel of eight monoclonal antibodies (MAb). Two dominant regions were localized between residues 22-37 and 70-87. All eight antibodies recognized these regions, but differed somewhat with respect to whether they saw the more N-terminal, middle, or C-terminal portions of these regions. The sequence of residues 13-22 was recognized by three MAbs. The C-terminal region from residues 84-92 was recognized by three MAbs. All MAbs recognized conformational epitopes in that they reacted with two or more regions. Three MAbs (two against free alpha and one against human CG) have linear amino acid sequences as part of their conformational epitope.
Assuntos
Antígenos/imunologia , Glicoproteínas/imunologia , Hormônios/imunologia , Mapeamento de Peptídeos/métodos , Anticorpos Monoclonais , Ensaio de Imunoadsorção Enzimática , Glicoproteínas/química , Hormônios/química , Humanos , Peptídeos/síntese química , Radioimunoensaio/métodosRESUMO
Synthetic peptides of the alpha-subunit of human glycoprotein hormones have been shown previously to inhibit binding of [125I]iodo-hCG to ovarian membranes, thus indicating the importance of the alpha-subunit in the structure-function relationships of the gonadotropic hormone. These same synthetic alpha-subunit peptides, the sequences of which are common to all human glycoprotein hormones, were found to inhibit the binding of [125I]iodo-TSH to human thyroid membrane preparations and FRTL-5 rat thyroid cells. The active portions of the subunit were represented in synthetic peptides alpha 21-35, alpha 31-45, alpha 26-46, and alpha 81-92, indicating that 2 separate sites within the alpha-subunit have binding activity for TSH. Peptides alpha 26-46 and alpha 31-45 were also found to potently inhibit the stimulation of adenylate cyclase activity by bovine TSH in TSH bioassay using FRTL-5 cells. Seven other synthetic peptides, including the remainder of the 92-amino acid sequence of the alpha-subunit, demonstrated little or no ability to inhibit binding of the tracer or inhibit the bioactivity of intact TSH. The findings were very similar to those of previous studies involving hCG binding, except that the two active sites appeared to be somewhat shifted towards the COOH-terminal end of the subunit. These studies support the concept of the importance of the alpha-subunit in receptor binding of all glycoprotein hormones and demonstrate the utility of the overlapping synthetic peptide strategy in investigations of protein structure-function relationships.
Assuntos
Gonadotropina Coriônica/farmacologia , Doença de Graves/metabolismo , Fragmentos de Peptídeos/farmacologia , Receptores da Tireotropina/metabolismo , Glândula Tireoide/metabolismo , Tireotropina/metabolismo , Sequência de Aminoácidos , Ligação Competitiva , Humanos , Cinética , Substâncias Macromoleculares , Receptores da Tireotropina/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
The most highly conserved feature of steroid receptor primary structure, the conserved cysteine region, corresponds to the DNA-binding domain of steroid receptors. This domain of chick progesterone receptor (PR) and other receptors was immunochemically characterized using site-directed antibodies. Eight peptides were synthesized corresponding to portion of the conserved cysteine region from PR, glucocorticoid, or estrogen receptor proteins. Polyclonal antibodies were obtained by immunizing rabbits with peptide conjugated to a protein carrier. The cross-reactivity of each antiserum was tested by Western blotting against chick and human PR, human glucocorticoid receptor, and human estrogen receptor. Of the several antisera positive by Western blots, only one cross-reacted with native chick PR. It was found that antibody bound to 4S transformed receptor, but not to 8S nontransformed receptor. At 50 mM KCl, antibody bound preferentially to transformed receptor form A, but at 400 mM KCl antibody bound equally well to either receptor form A or B. Binding of PR to DNA-cellulose was partially inhibited by the presence of cross-reacting peptide antibody. Thus, we have obtained at least one site-directed antibody against steroid receptors which is a useful structural probe to the DNA-binding functional domain of these gene regulatory proteins.
Assuntos
Anticorpos/imunologia , Cisteína , Receptores de Estrogênio/imunologia , Receptores de Glucocorticoides/imunologia , Receptores de Progesterona/imunologia , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Celulose/análogos & derivados , Celulose/metabolismo , Centrifugação com Gradiente de Concentração , Galinhas , DNA/análogos & derivados , DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Epitopos/imunologia , Feminino , Células HeLa/análise , Humanos , Soros Imunes/imunologia , Imunoensaio , Dados de Sequência Molecular , Concentração Osmolar , Coelhos , Útero/análiseRESUMO
The glycoprotein hormones CG, LH, FSH, and TSH are composed of two noncovalently linked subunits, alpha and beta. The beta-subunit confers hormone specificity, while the alpha-subunit is homologous within a species. To help in determining the antigenic structure of the common alpha-subunit, six monoclonal antibodies (mAbs) to the free or heterodimeric alpha-subunit of human (h) gonadotropic hormones have been prepared and, along with two previously isolated mAbs, have been characterized for binding specificity to alpha- and beta-subunits and the human glycoprotein hormones, CG, LH, FSH, and TSH. Each mAb was derived from hybidomas of FO myeloma cells fused with spleen cells from mice immunized with free alpha-subunit, hCG or hFSH. mAbs A101, A102, and E512 were specific for the alpha-subunit but showed the highest affinity for the intact hormone; K2.18, K94.6, E501, E502, and E511 were specific for free alpha. All of the antibodies inhibited binding of 125I-hCG to luteal membrane receptor, and 125I-labeled mAbs did not recognize hCG/receptor complex. Characterization by two-site binding assays using alpha, hCG, or hFSH as antigen revealed that all the mAbs bind to unique sites on alpha which may be overlapping, and which are modified in the intact hormone. The antigenic sites for mAbs E502, E511, and K2.18 are at least partially linear because they bind to reduced, carboxymethylated alpha.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/análise , Subunidade alfa de Hormônios Glicoproteicos/imunologia , Animais , Gonadotropina Coriônica/imunologia , Gonadotropina Coriônica/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Hormônio Foliculoestimulante/imunologia , Subunidade alfa de Hormônios Glicoproteicos/análise , Hormônio Luteinizante/imunologia , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Ovário/metabolismo , Radioimunoensaio , Ensaio Radioligante , Ratos , Receptores da Gonadotropina/metabolismo , Tireotropina/imunologiaRESUMO
Eight monoclonal antibodies, specific for the glycoprotein hormone alpha-subunit, were raised against human free alpha-subunit, human FSH, or human CG. All of these antihuman monoclonal antibodies were tested for cross-reactivity with alpha-subunits derived from bovine, porcine, equine, bull frog, sea turtle, turkey, and ostrich glycoprotein hormones. All showed cross-reactivity with affinities ranging from 10(-4) to 10(-8) depending upon the antibody and the species of alpha-subunit. Cyanogen bromide fragments of bovine and equine alpha, when tested with selected antibodies indicated that antigenic determinants could be localized in two regions: alpha 9-33 and alpha 76-92. Comparison of amino acid sequences, and relative potencies, suggest that major antigenic determinants involve residues 21, 22, and 23 (F-F-S in human alpha) and 76-85 (G-G-F-K-V-E-N-H-T-A in human alpha). As part of this study the N-terminal amino acid sequences of bull frog, sea turtle, turkey, and ostrich alpha-subunits were determined and reported for the first time.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/análise , Subunidade alfa de Hormônios Glicoproteicos/imunologia , Sequência de Aminoácidos , Animais , Reações Cruzadas , Subunidade alfa de Hormônios Glicoproteicos/análise , Subunidade alfa de Hormônios Glicoproteicos/genética , Humanos , Dados de Sequência Molecular , Radioimunoensaio , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
The glycoprotein hormones (LH, hCG, FSH, and TSH) have a common 92-amino acid alpha-subunit which is noncovalently linked to a hormone-specific beta-subunit. Synthetic peptides of the alpha-subunit have been shown to inhibit binding of [125I]iodo-hCG to rat ovarian membrane and [125I]iodo-TSH to human thyroid membrane preparations. Synthetic overlapping peptides of the alpha-subunit of hCG were prepared by solid phase techniques and tested in a standard in vitro rat Leydig cell bioassay. Three regions in the alpha-subunit (alpha 1-15, alpha 30-45, and alpha 71-85) were found to stimulate testosterone production. All three regions correlate with inhibition of hCG binding to ovarian receptors, but subtle differences exist between the binding sites and effector sites. These data indicate that the glycoprotein alpha-subunit has intrinsic bioactivity.
Assuntos
Subunidade alfa de Hormônios Glicoproteicos/farmacologia , Células Intersticiais do Testículo/metabolismo , Fragmentos de Peptídeos/farmacologia , Testosterona/biossíntese , Sequência de Aminoácidos , Animais , Bioensaio , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Gonadotropina Coriônica/metabolismo , Dicroísmo Circular , Feminino , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Dados de Sequência Molecular , Ovário/efeitos dos fármacos , Ovário/metabolismo , Conformação Proteica , Ratos , Ratos Endogâmicos , Relação Estrutura-AtividadeRESUMO
Synthetic peptides, representing specific portions of the alpha-subunit of the human glycoprotein hormones, can inhibit both the binding of labeled TSH to thyroid membranes and adenylate cyclase stimulation by TSH in vitro. The same synthetic peptides (alpha 26-46 and alpha 31-45) significantly (P less than 0.05) inhibited the adenylate cyclase-stimulating activity of thyroid-stimulating immunoglobulins (TSI) from 10 patients with hyperthyroid Graves' disease. Peptide alpha 26-46 was the most potent, resulting in 79.1 +/- 8.8% (+/- SE) inhibition at 133 micrograms/mL, while peptide alpha 31-45 inhibited TSI activity by 36.3 +/- 5.2%. Peptides alpha 61-75 and alpha 81-92, that had only minimal ability to inhibit TSH-mediated cAMP generation, did not significantly inhibit TSI activity. The inhibitory action of alpha 26-46 was dose dependent, and a significant negative correlation was found between the maximum TSI activity of the serum sample and the inhibition achieved by the synthetic peptide, suggesting that differences in TSI affinity and/or titer may account for the variable inhibitory activity of the peptides. These results suggest that TSI interact with the TSH receptor at the site that recognizes the portion of the TSH alpha-subunit represented by the synthetic peptide alpha 26-46 and, thus, support the concept that the TSH-binding site of the TSH receptor is the site of antigen binding between TSI and the thyroid cell.
Assuntos
Doença de Graves/imunologia , Imunoglobulina G/fisiologia , Hormônios Adeno-Hipofisários/farmacologia , AMP Cíclico/biossíntese , Feminino , Subunidade alfa de Hormônios Glicoproteicos , Humanos , Imunoglobulina G/antagonistas & inibidores , Imunoglobulina G/metabolismo , Imunoglobulinas Estimuladoras da Glândula Tireoide , Masculino , Receptores da Tireotropina/imunologiaRESUMO
Peptide nucleic acids (PNAs) are DNA analogs that can hybridize to complementary sequences with high affinity and stability. Here, we report the first evidence of intracellular delivery of PNAs in vivo. Two CNS receptors, an opioid (mu) and a neurotensin (NTR-1), were targeted independently by repeated microinjection of PNAs into the periaqueductal gray. Behavioral responses to neurotensin (antinociception and hypothermia) and morphine (antinociception) were lost in a specific manner. Binding studies confirmed a large reduction in receptor sites. The loss of behavioral responses was long lasting but did fully recover. The implications of specifically and readily turning off gene expression in vivo are profound.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Neurônios/metabolismo , Oligodesoxirribonucleotídeos/metabolismo , Peptídeos/metabolismo , Animais , Masculino , Morfina/metabolismo , Morfina/farmacologia , Neurotensina/metabolismo , Neurotensina/farmacologia , Oligodesoxirribonucleotídeos/farmacologia , Peptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Neurotensina/genética , Receptores Opioides/genéticaRESUMO
The diagnosis of acquired myasthenia gravis (MG) in apparently seronegative individuals is aided by finding immunoglobulin complexed to acetylcholine receptors (AChR) and a reduction in the number of binding sites for alpha-bungarotoxin (alpha-BTx) in nerve-muscle biopsies. In this study, we found that anti-AChR antibodies in extracellular fluids can complex with cytoplasmic epitopes of AChR in the process of muscle extraction. When normal muscle was briefly exposed to antibodies (greater than or equal to 0.3 nmol/l) in the initial step of tissue homogenization (before detergent extraction), membranous AChR became complexed with IgG. This was so even with a nonmyasthenogenic monoclonal antibody specific for the alpha-subunit's presumptive cytoplasmic segment 366-389. We also found that antibodies reactive with AChR's alpha-BTx binding region can significantly lower apparent yields of alpha-BTx binding sites extracted from muscle. Thus, the finding of IgG complexed to AChR extracted from biopsied muscle does not necessarily reflect in vivo binding but, nevertheless, is a sensitive indicator of AChR seropositivity in patients suspected to have MG.
Assuntos
Imunoglobulina G/metabolismo , Músculos/metabolismo , Receptores Colinérgicos/metabolismo , Animais , Anticorpos Monoclonais , Sítios de Ligação , Bungarotoxinas/metabolismo , Eletromiografia , Eletrofisiologia , Feminino , Placa Motora/fisiopatologia , Miastenia Gravis/diagnóstico , Miastenia Gravis/fisiopatologia , Ratos , Ratos Endogâmicos Lew , Testes SorológicosRESUMO
synthetic peptides corresponding to residues 63-78 of the first domain of the beta chain of murine I-A/I-E class II antigens were used in the unconjugated rather than the traditional protein-conjugated form to immunize (129J X B6)F1 mice. The sequences made represented the four haplotypes; Ak beta, Ad beta, Abm-12 beta and Ed beta. These sequences were selected on the basis of computer algorithms used to predict surface accessibility and main-chain flexibility profiles, and by reported hypervariability and site-directed mutagenesis experiments of these regions. Factors such as the use of complete Freund's adjuvant, a continuous immunization regime, and the sex of the mice used were found to influence the amount of anti-peptide antibody produced when unconjugated peptide was used as the immunogen. Antibodies produced were shown by FACS analysis to react with I-A/I-E class II antigens expressed on the lymphocyte surface. These findings indicate that the use of unconjugated synthetic peptides representing sequences of proteins which are in close proximity to disulfide bonds may be an advantage over conventional methods of peptide conjugation to proteins.
Assuntos
Sequência de Aminoácidos , Soro Antilinfocitário/biossíntese , Antígenos de Histocompatibilidade Classe II/imunologia , Fragmentos de Peptídeos/imunologia , Animais , Feminino , Haplótipos , Antígenos de Histocompatibilidade Classe II/administração & dosagem , Antígenos de Histocompatibilidade Classe II/análise , Masculino , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/síntese química , Caracteres SexuaisRESUMO
The simple chemical method described for detecting synthetic peptides bound to polystyrene should facilitate interpretation of studies involving interactions of antibodies or solubilized major histocompatibility complex (MHC) molecules with immobilized peptide antigens. After its application to an ELISA plate, the peptide is biotinylated in situ and avidin-biotinylated horseradish peroxidase complex and substrate are added sequentially. For 36 of 43 peptides tested (11-27 residues long), a colored reaction product confirmed that the peptide was bound. In three of the seven instances of a negative result, peptides were positively detected by binding of antibody. Four instances remained in which it could not be determined whether the peptide did not bind to the plate or whether it was not biotinylated. On the other hand, the biotin-ABC assay positively detected peptide in 18 of 22 instances without evidence of antibody binding, implying seronegativity or a loss of antigenic conformation in the bound peptide. This general method should be applicable to assays of microbial antigens, autoantigens and allergens. Modification of the technique by use of biotin hydrazide should enable monitoring of the binding to polystyrene of carbohydrates, glycolipids or DNA.
Assuntos
Peptídeos/análise , Animais , Avidina , Biotina , Ensaio de Imunoadsorção Enzimática , Feminino , Antígenos de Histocompatibilidade/análise , Poliestirenos , Ratos , Ratos Endogâmicos LewRESUMO
Animal studies with neurotensin (NT) directly injected into brain suggest that it has pharmacological properties similar to those of antipsychotic drugs. Here, we present radioligand binding data for some novel hexapeptide analogs of NT(8-13) at the molecularly cloned rat and human neurotensin receptors (NTR-1), along with behavioral and physiological effects of several of these peptides after intraperitoneal (i.p.) administration in rats. One unique analog, NT66L, which had high affinity (0.85 nM) for the molecularly cloned rat neurotensin receptor (NTR-1), caused a drop in body temperature and antinociception at doses as low as 0.1 mg/kg after i.p. injection. At 30 min post-injection, the ED50 for NT66L-induced hypothermia (rectal temperature) and antinociception (hot plate test) was 0.5 and 0.07 mg/kg, respectively. At a dose of 1 mg/kg i.p., NT66L caused 100% of the maximum possible effect for antinociception for up to 2 h after administration. At this dose body temperature lowering was greater than -2.5 degrees C from 20 to 120 min after i.p. administration. These results in animals suggest that NT66L has agonist properties at NTR-1 in vivo after extracranial administration and provide support for its further study in behavioral tests predictive of neuroleptic activity.
Assuntos
Barreira Hematoencefálica , Sistema Nervoso Central/efeitos dos fármacos , Neurotensina/agonistas , Neurotensina/farmacocinética , Fragmentos de Peptídeos/farmacocinética , Animais , Sítios de Ligação , Sistema Nervoso Central/metabolismo , Humanos , Masculino , Neurotensina/análogos & derivados , Neurotensina/síntese química , Neurotensina/química , Fragmentos de Peptídeos/síntese química , Ratos , Ratos Sprague-DawleyRESUMO
Synthetic peptides corresponding to amino acid sequences in eosinophil granule major basic protein (MBP) were evaluated for cytotoxic activity toward K562 cells and for ability to stimulate basophil mediator release. Results obtained using 14 peptides spanning the 117-amino acid sequence of MBP in overlapping fashion indicated that the activities mapped to peptide sequences near the amino and carboxy termini of MBP. The activity of these regions was confirmed using two peptides corresponding to MBP residues 18-45 and 89-117. A 20-h incubation with 5 microM peptide 18-45 or peptide 89-117 caused approximately the same levels (>60%) of cytotoxicity in K562 cells as 5 microM MBP. Similarly, a 30-min incubation with peptides 18-44 and 89-117 stimulated basophil histamine release in a concentration-dependent manner over the range of 5-20 microM. The level of release stimulated by 20 microM peptide 89-117 approached that stimulated by 2 microM MBP. A 20 microM concentration of peptide 89-117 also stimulated leukotriene C4 (LTC4) production by the basophils. Neither peptide 18-45 nor peptide 89-117 was cytotoxic for basophils under the experimental conditions for histamine and LTC4 release, as determined by 51Cr release. These results indicate that two MBP peptide sequences, including one (89-117) that contains a unique carbohydrate-binding region, share the biologic activities of MBP.
Assuntos
Proteínas Sanguíneas/análise , Proteínas Sanguíneas/fisiologia , Eosinófilos/imunologia , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/fisiologia , Mapeamento de Peptídeos , Ribonucleases , Análise de Sequência de Proteína , Sequência de Aminoácidos , Basófilos/metabolismo , Citotoxicidade Imunológica , Proteínas Granulares de Eosinófilos , Histamina/metabolismo , Humanos , Células K562 , Leucotrieno C4/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Mapeamento de Peptídeos/métodos , Análise de Sequência de Proteína/métodos , Células Tumorais CultivadasRESUMO
The neurotensin (NT) receptor, subtype 1 (NTR1), is a 7-transmembrane-spanning receptor, forming 3 extracellular and 3 intracellular loops. Previously, we showed that the third outer loop (E3) is the binding site for NT and its analogs, several of which bind with higher affinity to rat NTR1 (rNTR1) than to human NTR1 (hNTR1). In particular, NT34 [3,1'-naphthyl-l-Ala(11)]NT(8-13) has greater than 60-fold higher affinity for rNTR1 (46 and 60 pM for transiently- and stably-transfected cells, respectively) than for hNTR1 (2.8 and 5.8 nM for transiently- and stably-transfected cells, respectively) isolated from transfected cell membranes. Previously, our molecular modeling studies of rNTR1 and hNTR1 showed that the binding pocket in the human receptor for NT34 is smaller in volume from the bulky residue Tyr(339) in the pocket center, as compared with the corresponding residue Phe(344) in the rat binding pocket. Therefore, with site-directed mutagenesis, we derived mutant forms of rNTR1(F344Y) and hNTR1(Y339F). Examination of the mutant receptors from membranal preparations of transfected cells in radioligand binding assays and with intact cells in functional assays (phosphatidyl-4,5-bisphosphate turnover) showed that the human-like rat receptor and the rat-like human receptor bound NT34 with a predicted reverse of binding compared with its binding to the wild-type receptors. These results strongly affirm our molecular modeling studies and demonstrate the importance of the study of even minor structural variations in proteins to determine the basis of significantly different drug responses, an area of focus for pharmacological research in the 21st century.
Assuntos
Neurotensina/farmacologia , Fragmentos de Peptídeos/farmacologia , Receptores de Neurotensina/agonistas , Substituição de Aminoácidos , Animais , Células CHO , Células Cultivadas , Cricetinae , Humanos , Mutagênese Sítio-Dirigida , Neurotensina/química , Fragmentos de Peptídeos/química , Ensaio Radioligante , Ratos , Receptores de Neurotensina/química , Receptores de Neurotensina/genética , TransfecçãoRESUMO
Neurotensin(8-13), the carboxyl-terminal portion of neurotensin, is 4-50 times more potent than native neurotensin in binding to intact neuroblastoma N1E-115 cells and human brain tissue and in stimulation of intracellular cyclic GMP production and inositol phospholipid hydrolysis in clone N1E-115 (Gilbert JA and Richelson E, Eur J Pharmacol 99: 245-246, 1984; Gilbert JA et al., Biochem Pharmacol 35: 391-397, 1986; Kanba KS et al., J Neurochem 46: 946-952, 1986; and Kanba KS and Richelson E, Biochem Pharmacol 36: 869-874, 1987). A series of novel analogs of neurotensin (8-13) was synthesized, and a structure-activity study was done comparing the abilities of these peptides to stimulate intracellular cyclic GMP production in intact neuroblastoma clone N1E-115 and to inhibit the binding of [3H]neurotensin to these cells and to membranal preparations from human brain. A direct correlation was found for each analog between its EC50 for biochemical activity and its KD for binding ability in studies with clone N1E-115. Furthermore, a strong correlation existed for each peptide between its KD for binding to neurotensin receptors on these cells and its KD for binding to neurotensin receptors in human brain tissue. In this study, the residues that were important to the biochemical and binding activities of neurotensin (8-13) proved to be identical to the amino acids that are necessary for the functional integrity of native neurotensin (Gilbert JA et al., Biochem Pharmacol 35: 391-397, 1986.