RESUMEN

Previously, we demonstrated that autoantibodies (AAb) in multiple sclerosis (MS) reveal site-specific binding and cleavage toward myelin basic protein (MBP) epitope library. We have found several fragments of MBP immunodominant in terms of AAb binding. Here, we applied these peptides to DA rats with induced protracted relapsing experimental allergic encephalomyelitis (EAE) most closely related to MS. DA rats with EAE induced by syngenic spinal cord homogenate in complete Freund's adjuvant were treated by nasal route with human MBP 46-62, 81-102, 124-139, 147-170, and Copaxone. MBP 124-139 and 147-170 displayed only mild therapeutic effects but MBP 46-62 significantly reduced EAE, reflected by lower clinical scores and shorter EAE duration compared to controls.

Asunto(s)

Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Inmunosupresores/uso terapéutico , Proteínas del Tejido Nervioso/uso terapéutico , Factores de Transcripción/uso terapéutico , Animales , Anticuerpos Catalíticos/sangre , Anticuerpos Catalíticos/inmunología , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Autoantígenos/sangre , Autoantígenos/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Acetato de Glatiramer , Humanos , Proteína Básica de Mielina , Proteínas del Tejido Nervioso/inmunología , Péptidos/uso terapéutico , Ratas , Factores de Transcripción/inmunología

RESUMEN

The effect of immunocortin, an ACTH-like decapeptide VKKPGSSVKV corresponding to the 11-20 sequence of the variable part of the human IgG1 heavy chain on the content of 11-hydroxycorticosteroids (CS) in rat adrenal glands and blood serum in vivo was studied. An intramuscular injection of immunocortin at a dose of 10 microg/kg was found in an hour to induce a twofold decrease in CS content in the adrenal glands and a 1.8-fold increase in the blood serum CS content. At the same time, an immunocortin dose of 100 microg/kg exerted practically no effect on the CS content and its dose of 1000 microg/kg increased the CS content both in adrenal glands and in blood serum by 1.6 and 2.2 times, respectively. Four hours after the injection of any of the three doses of immunocortin, the CS content in adrenal glands did not differ from the control value, and after 24 h the content decreased threefold. Immunocortin was shown to be bound by the ACTH receptors in the membranes of the rat adrenal cortex with a high affinity and specificity (inhibiting the specific binding of 125I-labeled ACTH-(11-24) peptide with Ki of 1.2 nM).

Asunto(s)

11-Hidroxicorticoesteroides/metabolismo , Corteza Suprarrenal/metabolismo , Hormona Adrenocorticotrópica/química , Inmunoglobulina G/química , Inmunoglobulina G/farmacología , Región Variable de Inmunoglobulina/química , Fragmentos de Péptidos/farmacología , 11-Hidroxicorticoesteroides/sangre , Corteza Suprarrenal/citología , Corteza Suprarrenal/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Cosintropina/administración & dosificación , Cosintropina/farmacología , Inmunoglobulina G/administración & dosificación , Técnicas In Vitro , Radioisótopos de Yodo , Masculino , Datos de Secuencia Molecular , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/química , Ratas , Ratas Wistar , Receptores de Corticotropina/metabolismo

RESUMEN

We have synthesized two peptides, VKGFY and cyclo(VKGFY) (referred to as pentarphin (PNT) and cyclopentarphin (cPNT), respectively), and found that both peptides at 1 nM concentration increased the adhesion and spreading of murine peritoneal macrophages as well as their bactericidal activity in vitro, as shown by phagocytosis of Salmonella typhimurium virulent strain 415. PNT administered intraperitoneally at dose 20 microg/mouse on day 7, 3, and 1 prior to the isolation of macrophages also enhanced the macrophage adhesion and spreading. The receptor binding characteristics of PNT and cPNT were examined using 125I-labeled PNT. The binding of labeled PNT to peritoneal macrophages was high-affinity (K(d)=3.6 nM) and saturable. It was not inhibited by naloxone (NAL) or [Met(5)]enkephalin ([Met(5)]ENK) but completely inhibited by unlabeled cPNT (K(i)=2.6 nM), immunorphin (IMN, decapeptide SLTCLVKGFY, corresponding to the IgG heavy-chain sequence 364-373) (K(i)=3.2 nM) or beta-endorphin (beta-END) (K(i)=2.8 nM). Thus, the effects of PNT and cPNT on macrophages are mediated by NAL-insensitive receptors common for PNT, cPNT, IMN, and beta-END.

Asunto(s)

Activación de Macrófagos , Macrófagos/efectos de los fármacos , Oligopéptidos/farmacología , Péptidos Cíclicos/farmacología , Receptores Opioides/agonistas , Secuencia de Aminoácidos , Animales , Unión Competitiva , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Regiones Constantes de Inmunoglobulina , Cadenas gamma de Inmunoglobulina , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Oligopéptidos/química , Oligopéptidos/metabolismo , Fragmentos de Péptidos/metabolismo , Péptidos/química , Péptidos/farmacología , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Fagocitosis , Receptores Opioides/metabolismo , betaendorfina/metabolismo

RESUMEN

We synthesized linear and cyclic pentapeptides corresponding to the sequence 369-373 of human immunoglobulin G heavy chain--VKGFY (referred to as pentarphin and cyclopentarphin, respectively). The effect of pentarphin and cyclopentarphin on phagocytosis of Salmonella typhimurium virulent 415 strainbacteria by mouse peritoneal macrophages in vitro was studied. Control experiments showed that macrophages actively captured these bacteria, but did not digest them: the captured microbes were viable and continued to proliferate inside the phagocytes; within 12 h all macrophage monolayer was destroyed (incomplete phagocytosis). If 1 nM pentarphin or cyclopentarphin was added to the cultivation medium, macrophage bactericidal activity was significantly increased and they digested all captured microorganisms within 6 h (complete phagocytosis). To study the receptor binding properties of pentarphin and cyclopentarphin we prepared (125)I-labeled pentarphin (179 Ci/mmol specific activity). The binding of (125)I-labeled pentarphin to mouse peritoneal macrophages was high-affinity (K(d) = 3.6 +/- 0.3 nM) and saturable. Studies on binding specificity revealed that this binding was insensitive to naloxone and [Met(5)]enkephalin, but completely inhibited by unlabeled cyclopentarphin (K(i) = 2.6 +/- 0.3 nM), immunorphin (K(i) = 3.2 +/- 0.3 nM), and beta-endorphin (K(i) = 2.8 +/- 0.2 nM). Thus, the effects of pentarphin and cyclopentarphin on macrophages are mediated by naloxone-insensitive receptors common for pentarphin, cyclopentarphin, immunorphin, and beta-endorphin.

Asunto(s)

Activación de Macrófagos/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Oligopéptidos/farmacología , Péptidos Cíclicos/farmacología , Fagocitosis/efectos de los fármacos , Receptores Opioides/metabolismo , Animales , Isótopos de Yodo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Naloxona/farmacología , Antagonistas de Narcóticos , Oligopéptidos/antagonistas & inhibidores , Oligopéptidos/síntesis química , Oligopéptidos/metabolismo , Péptidos Cíclicos/antagonistas & inhibidores , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/metabolismo , Salmonella typhimurium/inmunología , Tuftsina/farmacología

RESUMEN

It has been found that beta-endorphin (beta-END) and a synthetic beta-END-like decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (termed immunorphin, IMN) corresponding to the sequence 364-373 of human IgG heavy chain stimulate Con A-induced proliferation of T lymphocytes from the blood of healthy donors. [Met(5)]enkephalin ([Met(5)]ENK) and an antagonist of opioid receptors naloxone (NAL) tested in parallel were not active. The stimulating effect of beta-END and IMN on T lymphocyte proliferation was not inhibited by NAL. Studies on receptor binding of (125)I-labeled IMN to T lymphocytes revealed that it binds with high affinity to NAL-insensitive binding sites (K(d) = 7.0 +/- 0.3 nM). Unlabeled beta-END completely inhibited the specific binding of (125)I-labeled IMN to NAL-insensitive binding sites on T lymphocytes (K(i) = 1.1 +/- 0.2 nM). Thus, beta-END and IMN bind to common NAL-insensitive binding sites on T lymphocytes and enhance Con A-induced proliferation of these cells.

Asunto(s)

Analgésicos no Narcóticos/agonistas , Oligopéptidos/metabolismo , Fragmentos de Péptidos/metabolismo , Receptores Opioides/agonistas , Linfocitos T/efectos de los fármacos , Secuencia de Aminoácidos , Unión Competitiva/efectos de los fármacos , Unión Competitiva/fisiología , División Celular/efectos de los fármacos , Concanavalina A/farmacología , Humanos , Regiones Constantes de Inmunoglobulina , Cadenas gamma de Inmunoglobulina , Radioisótopos de Yodo/química , Datos de Secuencia Molecular , Oligopéptidos/química , Oligopéptidos/farmacología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Receptores Opioides/metabolismo , Homología de Secuencia de Aminoácido , Linfocitos T/citología , Linfocitos T/metabolismo , betaendorfina/farmacología

RESUMEN

Beta-endorphin and the synthetic beta-endorphin-like decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (referred to as immunorphin), corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain, were shown to stimulate concanavalin A-induced proliferation of T lymphocytes from the blood of healthy donors. [Met(5)]Enkephalin and the antagonist of opioid receptors naloxone examined in parallel were inactive. The stimulating effect of beta-endorphin and immunorphin on T lymphocyte proliferation is not inhibited by naloxone. Studies on receptor binding of (125)I-labeled immunorphin to T lymphocytes revealed that it binds with high affinity to naloxone-insensitive receptors (K(d) = 7.0 +/- 0.3 nM). Unlabeled immunorphin completely inhibits (125)I-labeled beta-endorphin specific binding to naloxone-insensitive receptors on T lymphocytes (K(i) = 0.6 +/- 0.1 nM). Thus, beta-endorphin and immunorphin interact with common naloxone-insensitive receptors on T lymphocytes.

Asunto(s)

Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Linfocitos T/efectos de los fármacos , betaendorfina/farmacología , Unión Competitiva , División Celular/efectos de los fármacos , Transformación Celular Neoplásica , Concanavalina A/farmacología , Encefalina Metionina/farmacología , Humanos , Regiones Constantes de Inmunoglobulina , Inmunoglobulina G , Cadenas Pesadas de Inmunoglobulina , Cadenas gamma de Inmunoglobulina , Radioisótopos de Yodo/química , Activación de Linfocitos , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Oligopéptidos/síntesis química , Oligopéptidos/metabolismo , Receptores Opioides/metabolismo , Linfocitos T/metabolismo

RESUMEN

The synthetic peptide SLTCLVKGFY, corresponding to the 364-373 amino acid sequence of the human IgG heavy chain (Immunorphin), was found to compete with [125I] beta-endorphin for binding by high-affinity receptors on T lymphocytes isolated from the blood of healthy donors (Ki 0.6 nM). The fragments 3-10, 4-10, 5-10, and 6-10 of Immunorphin also inhibited the binding (Ki 2.2, 3.4, 8.0, and 15 nM, respectively). Specificity of these receptors was studied: they turned out to be insensitive to naloxone and, therefore, are not opioid. The Kd values of the specific binding of 125I-labeled Immunorphin and its 6-10 fragment to the receptor were found to be 7.4 and 36.3 nM, respectively.

Asunto(s)

Inmunoglobulina G/metabolismo , Cadenas Pesadas de Inmunoglobulina/metabolismo , Oligopéptidos/metabolismo , Fragmentos de Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Unión Competitiva , Humanos , Regiones Constantes de Inmunoglobulina , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas gamma de Inmunoglobulina , Oligopéptidos/genética , Oligopéptidos/inmunología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , betaendorfina/metabolismo

RESUMEN

The synthetic decapeptide H-SLTCLVKGFY-OH (termed immunorphin) corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain was found to compete with [125I]beta-endorphin for high-affinity receptors on T lymphocytes from the blood of healthy donors (K(i) = 0.6 nM). Besides immunorphin, its synthetic fragments H-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 15 nM), H-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 8.0 nM), H-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 3.4 nM), H-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 2.2 nM), H-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 1.0 nM) possessed the ability to inhibit specific binding of [125I]beta-endorphin to T lymphocytes. Tests of the specificity of the receptors revealed that they are not sensitive to naloxone and Met-enkephalin, i.e. they are not opioid receptors. K(d) values characterizing the specific binding of 125I- labeled immunorphin and its fragment H-Val-Lys-Gly-Phe-Tyr-OH to the receptors have been determined to be 7.4 nM and 36.3 nM, respectively.

Asunto(s)

Oligopéptidos/metabolismo , Fragmentos de Péptidos/metabolismo , Receptores Opioides/metabolismo , Linfocitos T/metabolismo , Secuencia de Aminoácidos , Humanos , Regiones Constantes de Inmunoglobulina , Cadenas gamma de Inmunoglobulina , Oligopéptidos/química , Fragmentos de Péptidos/química , betaendorfina

RESUMEN

The purpose of the present study was to investigate properties and mechanism of action of the synthetic adrenocorticotropin (ACTH)-like peptide VKKPGSSVKV, corresponding to the sequence 11-20 of the variable part of human immunoglobulin G1 (IgG1) heavy chain. The ACTH-like peptide was shown to act as an immunosuppressive agent in vitro: it inhibits the blast transformation of mouse thymocytes and reduces the spontaneous motility of mouse peritoneal macrophages as well as their bactericidal activity against Salmonella typhimurium 415 virulent strain bacteria. High affinity receptors for the ACTH-like peptide were found on thymocytes and macrophages and shown to be at the same time the receptors for ACTH. The kinetic characteristics of the ACTH-like peptide and 125I-labeled ACTH (13-24) (ACTH 'address segment') specific binding to the receptors were determined. It was found that the ACTH-like peptide binding to the receptors on target cells is accompanied by an increase in both adenylate cyclase activity and intracellular cAMP content.

Asunto(s)

Hormona Adrenocorticotrópica/farmacología , Inmunoglobulina G/farmacología , Activación de Linfocitos/inmunología , Activación de Macrófagos/inmunología , Macrófagos Peritoneales/inmunología , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacología , Linfocitos T/inmunología , Hormona Adrenocorticotrópica/inmunología , Animales , Humanos , Inmunoglobulina G/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Macrófagos/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Ratones Endogámicos CBA , Fragmentos de Péptidos/síntesis química , Biblioteca de Péptidos , Linfocitos T/efectos de los fármacos

RESUMEN

Influence of the ACTH-like peptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH corresponding to the sequence 11-20 of the variable part of human immunoglobulin G1 (IgG1) heavy chain on growth of MT-4 human T-lymphoblastoid cell line was investigated. It was found that the ACTH-like peptide at concentration range 10(-11) -10(-7) M inhibits the proliferation of MT-4 cells. Labeled ACTH 'address segment' [(125)I]ACTH (13-24) was used to establish that MT-4 cells express specific receptors for ACTH (K(d) = 97 pM). The ACTH-like peptide and human ACTH (but not IgG1 heavy chain) were shown to compete with [(125)I]ACTH (13-24) for binding to these receptors (K(i1) = 0.38 nM and K(i2) = 0.34 nM).

Asunto(s)

Hormona Adrenocorticotrópica/química , División Celular/efectos de los fármacos , Inmunoglobulina G/química , Inmunoglobulina G/farmacología , Fragmentos de Péptidos/farmacología , Hormona Adrenocorticotrópica/farmacocinética , Secuencia de Aminoácidos , Unión Competitiva , Línea Celular , Humanos , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/farmacología , Cinética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacocinética , Receptores de Corticotropina/fisiología , Linfocitos T/efectos de los fármacos

RESUMEN

The antiproliferative and immunosuppressive in vitro effects of immunocortin, a synthetic adrenocorticotropin-like (ACTH-like) decapeptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH, whose sequence corresponds to segment 11-20 of the variable part of the human IgG1 heavy chain, were studied. At concentrations of 10(-11)-10(-7) M, immunocortin was found to inhibit the growth of the human MT-4 T-lymphoblastoid cell line, to suppress the blast transformation of thymocytes, and to decrease the spontaneous mobility of peritoneal macrophages and their bactericidal action toward the virulent strain Salmonella typhimurium 415. By using a 125I-labeled "addressing" fragment of ACTH ¿[125I]ACTH-(13-24)¿, we showed that MT-4 cells express specific receptors for ACTH (Kd 97 pM). Immunocortin and human ACTH (but not the heavy chain of IgG1) competitively inhibited the binding of [125I]ACTH-(13-24) to these receptors with Ki1 of 0.38 and Ki2 of 0.34 nM, respectively. Specific receptors for ACTH (Kd 5.8 nM) on mouse thymocytes were detected and characterized. The unlabeled immunocortin was shown to complete with labeled ACTH-(13-24) for binding to these receptors (Ki = 1.8 nM) and this binding of immunocortin to receptors on thymocytes activates adenylate cyclase from these cells and increases the intracellular concentration of cAMP.

Asunto(s)

Hormona Adrenocorticotrópica/genética , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Oligopéptidos , Hormona Adrenocorticotrópica/metabolismo , Animales , Humanos , Activación de Macrófagos , Macrófagos/metabolismo , Ratones , Oligopéptidos/síntesis química , Oligopéptidos/química , Oligopéptidos/genética , Oligopéptidos/metabolismo , Receptores de Corticotropina/metabolismo , Salmonella typhimurium

Asunto(s)

Fluorocarburos/farmacología , Hipocampo/citología , Neuronas/ultraestructura , Sinapsis/ultraestructura , Animales , Astrocitos/efectos de los fármacos , Astrocitos/ultraestructura , Sustitutos Sanguíneos/farmacología , Capilares/efectos de los fármacos , Capilares/ultraestructura , Emulsiones , Hipocampo/irrigación sanguínea , Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , Sinapsis/efectos de los fármacos

RESUMEN

The synthetic ACTH-like decapeptide H-Val-Lys-Lys-Pro-Gly- Ser-Ser-Val-Lys-Val-OH, corresponding to amino acid residues 11-20 of the variable part of the human IgG1 heavy chain (referred to as immunocortin) was found to have an immunosuppressive effect on cells in vitro: it inhibits blast transformation of mouse thymocytes and reduces spontaneous motility of mouse peritoneal macrophages as well as their bactericidal activity against the virulent bacterial strain Salmonella typhimurium 415. Tritium-labeled immunocortin binds with high affinity to ACTH receptors on thymocytes and macrophages (Kd 2. 1 and 2.5 nM, respectively) and activates adenylate cyclase in these cells. Thus, the interaction of immunocortin with the target cell includes the following main steps: binding to the receptor, activation of adenylate cyclase, and elevation of the intracellular content of cAMP.

Asunto(s)

Hormona Adrenocorticotrópica/química , Inmunoglobulina G/química , Inmunoglobulina G/farmacología , Cadenas Pesadas de Inmunoglobulina/farmacología , Inmunosupresores/farmacología , Fragmentos de Péptidos/farmacología , Adenilil Ciclasas/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Humanos , Cadenas Pesadas de Inmunoglobulina/química , Inmunosupresores/química , Activación de Linfocitos/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Ratones , Ratones Endogámicos CBA , Fragmentos de Péptidos/química , Ratas , Timo/citología , Timo/efectos de los fármacos , Timo/inmunología

RESUMEN

It is known that in the blood of patients with some autoimmune diseases catalytically active antibodies hydrolyzing proteins, DNA, and RNA may be detected. In the present work homogeneous preparations of IgG antibodies (Ab) possessing high affinity for nucleic acids (NA) were obtained for the first time from blood and cerebrospinal fluid of patients with multiple sclerosis (MS). The fraction of IgG Ab as well as its Fab fragments and isolated light chains of both kappa- and lambda-types were shown to catalyze effectively the hydrolysis of DNA and RNA. It is shown by different methods that the capability for nucleic acid hydrolysis is an intrinsic property of the polyclonal Ab. NA-hydrolyzing Ab were detected in the blood of 69 of 72 and in the cerebrospinal fluid of 5 of 5 examined MS patients, while they were not detected in the blood of any of 50 healthy donors examined. Comparison of relative rates of RNA hydrolysis and of the substrate specificity in hydrolysis of various model RNAs--cCMP, poly(U), poly(A), and poly(C)--revealed pronounced differences of MS antibodies from ribonucleases of human blood, ribonuclease A, and all earlier described abzymes. The abzymes are usually characterized by relatively low specific activities in comparison with that of normal enzymes catalyzing analogous reactions. Ab from the blood of MS patients are the first example of autoabzymes whose specific activity in RNA hydrolysis is comparable or even higher than that of pancreatic ribonuclease A--one of the most active RNA-hydrolyzing enzymes.

Asunto(s)

Anticuerpos Catalíticos/líquido cefalorraquídeo , ADN/metabolismo , Inmunoglobulina G/líquido cefalorraquídeo , Esclerosis Múltiple/inmunología , ARN/metabolismo , Anticuerpos Catalíticos/sangre , Anticuerpos Catalíticos/aislamiento & purificación , Cromatografía en Gel , Humanos , Hidrólisis , Inmunoglobulina G/sangre , Inmunoglobulina G/aislamiento & purificación , Cinética , Esclerosis Múltiple/sangre , Esclerosis Múltiple/líquido cefalorraquídeo , Valores de Referencia , Especificidad por Sustrato

RESUMEN

By means of small angle X-ray scattering, an aggregation of beef pancreas Trp-tRNA synthetase (EC 6.1.1.2) was observed at physiological temperatures. A Trp-tRNA synthetase preparation which is homogeneous after PAGE in beta-ME-SDS was found to be heterogeneous in particle sizes even at low (4-8 degrees C) temperature. At heating up to 30-45 degrees C, the oligomer sizes increased as well as its proportion depending on the incubation time and temperature; very large aggregates were observed 10 times exceeding the sizes of initial particles. Cooling to 20 degrees C caused no disaggregation due to disulphide bond formation between associated subunits of Trp-tRNA synthetase. A hypothesis is proposed that the aggregation of bovine Trp-tRNA synthetase evaluated in vitro and not observed earlier with any aminoacyl-tRNA synthetases of unicellular organisms might serve as one of the mechanisms of its compartmentation in pancreas.

Asunto(s)

Triptófano-ARNt Ligasa/química , Animales , Bovinos , Disulfuros/química , Electroforesis en Gel de Poliacrilamida , Temperatura , Difracción de Rayos X

RESUMEN

Among clostripain hydrolysate peptides of beef pancreas tryptophanyl-tRNA synthetase the peptide Ile-Ser-Phe-Pro-Ala-Ile-Asn-Gln-Phe-Ala-Ala-Pro-Ser-Gln-Phe-Ser-Ile-Arg was revealed which contains the continuous antigenic determinant for monoclonal antibody Am1. This antibody specifically cross-reacts with tryptophanyl-tRNA synthetases of procaryotes, eucaryotes and archebacteriae. The synthetic peptide with identical amino acid sequence plus N-terminal Arg residue (S-peptide), being immobilized on enzyme immunoassay (EIA) microtitration plate, also binds with Am1. Am1 affinity constant (M-1) measured by non-competitive EIA was (3.0 +/- 0.3).10(7) for S peptide and (1.4 +/- 0.3).10(9) for the native enzyme. The sequence of immunoreactive peptide adopts with high probability the secondary structure including beta-turn(s) and antiparallel beta-sheet composed of inverted repeats. At the same time, the analysis of circular dichroism spectrum (in the far UV) of the peptide dissolved in water comes closest to 16% beta-turn and only 8% beta-sheet. The binding of Am1 with peptide was not observed in aqueous solution.

Asunto(s)

Epítopos/inmunología , Péptidos/genética , Triptófano-ARNt Ligasa/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Bovinos , Dicroismo Circular , Epítopos/genética , Técnicas para Inmunoenzimas , Datos de Secuencia Molecular , Conformación Proteica , Espectrofotometría Ultravioleta

RESUMEN

The polypeptide with a mobility of the tryptophanyl-tRNA-synthetase subunit can be labeled in bovine pancreas extracts from [gamma-32P]ATP. Immunoprecipitation analysis with monospecific polyclonal antibodies against the enzyme as well as identification of [32P]phosphoamino acids in the immunoprecipitate revealed that in bovine pancreas extracts tryptophanyl-tRNA-synthetase undergoes phosphorylation at serine residues. The level of phosphorylation does not change in the presence of activity modulators of cAMP-, cGMP- and Ca2(+)-dependent protein kinases, decreases after addition of phosphoseryl/phosphothreonyl-protein phosphatase inhibitors and increases in the presence of their activators. It was supposed that phosphorylation of tryptophanyl-tRNA-synthetase catalyzed by seryl/threonyl-specific protein kinase depends on the activity of phosphoseryl/phosphothreonyl-phosphatase.

Asunto(s)

Páncreas/enzimología , Extractos Pancreáticos/química , Triptófano-ARNt Ligasa/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Catálisis , Bovinos , Polipéptido Pancreático/biosíntesis , Fosforilación , Pruebas de Precipitina , Serina/química

RESUMEN

Localization of tryptophanyl-tRNA synthetase (TRS) was studied on ultrathin (UT) sections of Escherichia coli cells and of rat fibroblasts fixed with glutaraldehyde and embedded in "Lowicryl K4M" resin at -35 degrees C. The UT sections were treated with the complexes of monoclonal and/or polyclonal antibodies against TRS with colloidal gold 15 and 8 nm in size. In both types of the cells cytoplasm was the most intensely labelled. In fibroblast cytoplasm, zones with a greater amount of ribosomes were mainly labelled, the gold particles being found over both the cysternae of granular endoplasmic reticulum and the areas of localization of free ribosomes. In the zones of microfilament localization TRS was not detected. A great amount of TRS was found in mitochondria and in the fibroblast nuclei. In the latter case, the label was concentrated over the diffuse chromatin localization regions, a minimal binding being observed over compact chromatin. The number of particles observed over diffuse chromatin equals to 50-80% against the label in fibroblast cytoplasm. In contrast, the label used to be absent over the E. coli nucleoid. The presence of TRS in the fibroblast nucleus may evidence in favour of a possible regulatory role of TRS in eukaryots.

Asunto(s)

Aminoacil-ARNt Sintetasas/análisis , Escherichia coli/enzimología , Fibroblastos/enzimología , Triptófano-ARNt Ligasa/análisis , Animales , Anticuerpos Monoclonales , Núcleo Celular/enzimología , Núcleo Celular/ultraestructura , Escherichia coli/ultraestructura , Fibroblastos/ultraestructura , Microscopía Electrónica , Ratas , Especificidad de la Especie

RESUMEN

Monoclonal antibodies referred to as Am1, Am2 and Am3 against highly purified bovine tryptophanyl-tRNA synthetase were prepared. Am2 antibodies inhibit the Trp-tRNA synthetase activity and interact with the active truncated enzyme forms (dimers of either 40-kDa or 51-kDa fragments) produced by limited proteolysis. Am1 and Am3 antibodies exert no effect on the Trp-tRNA synthetase activity; epitopes recognized by them are mapped close to one another and reside at the dispensable part of the Trp-tRNA synthetase molecule. Am1 cross-reacts with Trp-tRNA synthetases of eukaryotic, prokaryotic and archaebacterial species, as revealed by immunoblot analysis. A rapid two-step technique was developed for isolating electrophoretically homogeneous Trp-tRNA synthetase from Escherichia coli. The purified enzyme interacted with Am1, but not with Am2 and Am3 antibodies taken at the same concentrations. As in the case of eukaryotic Trp-tRNA synthetase, Am1 did not influence the activity of Trp-tRNA synthetase from E. coli. From the aforementioned results it follows that: (a) the conservation of part of the Trp-tRNA synthetase structure which is not directly involved in the formation of the catalytic centre of prokaryotic and eukaryotic Trp-tRNA synthetases suggests that the dispensable part of the molecule might be involved in some additional biological function(s) of Trp-tRNA synthetase besides tRNA(Trp) charging; (b) the common antigenic determinant in Trp-tRNA synthetase of eukaryotes, prokaryotes and archaebacteria indicates that this enzyme was presumably present in the common ancestor of the above organisms.

Asunto(s)

Aminoacil-ARNt Sintetasas/inmunología , Anticuerpos Monoclonales , Archaea/enzimología , Bacterias/enzimología , Epítopos/análisis , Triptófano-ARNt Ligasa/inmunología , Animales , Evolución Biológica , Bovinos , Escherichia coli/enzimología , Ratones , Ratones Endogámicos BALB C , Mapeo Peptídico , Triptófano-ARNt Ligasa/análisis , Triptófano-ARNt Ligasa/aislamiento & purificación

RESUMEN

Method of isolation of the bovine pancreas tryptophanyl-tRNA synthetase is improved and a protein with greater than or equal to 99% purity, according to PAGE-SDS, is obtained. The pure enzyme is digested with clostripain and the hydrolysate is separated by FPLC anion-exchange chromatography followed by reversed phase HPLC. Amino acid sequences of 6 individual peptides, including C-terminal one, were determined by the automated Edman degradation. A peptide is also revealed which is encoded with the low degeneracy.

Asunto(s)

Aminoacil-ARNt Sintetasas/metabolismo , Páncreas/enzimología , Péptidos/genética , Triptófano-ARNt Ligasa/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Hidrólisis , Datos de Secuencia Molecular , Péptidos/aislamiento & purificación , Conformación Proteica , Triptófano-ARNt Ligasa/aislamiento & purificación