RESUMO
The remarkable conservation of the primary structures and anatomical location of dogfish α-melanocyte-stimulating hormone (MSH), corticotrophin-like intermediate lobe peptide (CLIP) and adrenocorticotrophic hormone (ACTH) compared with mammals reinforced the tissue-specific processing hypothesis of ACTH peptides in the pituitary gland. The cloning of dogfish pro-opiomelanocortin (POMC) led to the identification of δ-MSH and simultaneously revealed the high conservation of the γ-MSH sequence during evolution. These studies have also shown that ß-MSH is much less conserved during evolution and in some species is not even processed from ß-LPH. Human pro-γ-MSH potentiates the corticosteroidogenic activity of ACTH and peptides generated from its N-terminal, in particular big-γ-MSH, appear to have adrenal mitogenic activity. Human big-γ-MSH (from the zona intermedia) may also cause the adrenache. The review finishes with a cautionary note with regard to the misdiagnosis of the ectopic ACTH syndrome in which partial processing of ACTH can result in large concentrations of α-MSH and CLIP, which can interfere in the performance of two-site immunoassays, and the problem of the correct disulphide bridge arrangement in synthetic N-POMC peptides is also discussed.
Assuntos
Hormônio Adrenocorticotrópico/isolamento & purificação , Hormônios Estimuladores de Melanócitos/isolamento & purificação , Pró-Opiomelanocortina/isolamento & purificação , Síndrome de ACTH Ectópico/sangue , Síndrome de ACTH Ectópico/metabolismo , Glândulas Suprarrenais/metabolismo , Hormônio Adrenocorticotrópico/sangue , Hormônio Adrenocorticotrópico/química , Hormônio Adrenocorticotrópico/genética , Animais , Peptídeo da Parte Intermédia da Adeno-Hipófise Semelhante à Corticotropina/química , Peptídeo da Parte Intermédia da Adeno-Hipófise Semelhante à Corticotropina/genética , Peptídeo da Parte Intermédia da Adeno-Hipófise Semelhante à Corticotropina/isolamento & purificação , História do Século XX , Humanos , Hormônios Estimuladores de Melanócitos/sangue , Hormônios Estimuladores de Melanócitos/química , Hormônios Estimuladores de Melanócitos/genética , Hipersecreção Hipofisária de ACTH/sangue , Hipersecreção Hipofisária de ACTH/metabolismo , Hipófise/metabolismo , Pró-Opiomelanocortina/química , Pró-Opiomelanocortina/genética , Pró-Opiomelanocortina/história , Isoformas de Proteínas , alfa-MSH/química , alfa-MSH/genética , alfa-MSH/isolamento & purificação , beta-Endorfina/química , beta-Endorfina/genética , beta-Endorfina/isolamento & purificaçãoRESUMO
Several studies have described changes in beta-endorphin-like immunoreactivity (beta-ELI) in the rat brain in response to pain and stress stimuli. In order to ascertain the components of beta-ELI, brain samples of rats experiencing acute prolonged (tonic) pain were evaluated for their beta-ELI and later submitted to a chromatographic purification allowing the measurement of beta-endorphin (beta-EP) and acetyl beta-EP. The chromatographic analysis of both ventromedial hypothalamus (VMH) and periaqueductal grey (PAG) homogenates indicates that beta-ELI is distributed in several fractions including shortened forms of beta-EP and their respective acetylated compounds. Quantitatively, while beta-ELI in formalin-injected animals was increased by 48% in VMH and 45% in PAG in respect to controls, the net increase of purified beta-EP was 1100% and 470%, respectively, for VMH and PAG. Moreover, the maximal increase of beta-ELI was evident at 120 min, in both tissues. In contrast, the beta-EP peak was reached at 30 min in VMH and at 60 min in PAG. Acetyl beta-EP was unchanged by treatment in both central areas. No correlation of beta-ELI and beta-EP was found in VMH. These data demonstrate that the evaluation of beta-ELI gives a poor estimate of beta-EP changes, due to several components of the endorphin family.
Assuntos
Encéfalo/metabolismo , Dor/metabolismo , beta-Endorfina/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Masculino , Substância Cinzenta Periaquedutal/metabolismo , Radioimunoensaio , Ratos , Ratos Endogâmicos , beta-Endorfina/análogos & derivados , beta-Endorfina/isolamento & purificaçãoRESUMO
The pituitary of the Australian lungfish, Neoceratodus forsteri, was screened immunohistochemically with heterologous antisera specific for either the C-terminal of mammalian beta-endorphin or the acetylated N-terminal of beta-endorphin. Immunopositive cells were only detected with the N-terminal specific antiserum; these cells were restricted to the intermediate pituitary. Acid extracts of the intermediate pituitary were fractionated by Sephadex gel filtration chromatography, CM cation exchange chromatography and reverse phase HPLC. Fractions were analyzed by radioimmunoassay (RIA) with a N-acetyl specific beta-endorphin RIA and by radioreceptor assay for the presence of opiate active forms of beta-endorphin. Both immunoreactive and opiate active forms of beta-endorphin were detected. Of the total beta-endorphin-related material isolated from the intermediate pituitary, approximately 97% was detected with the N-terminal specific RIA and approximately 3% was detected by the radioreceptor assay. The N-acetylated immunoreactive beta-endorphin could be separated into two forms. The major form had an apparent molecular weight of 3.2 Kda. This material had a net charge at pH 2.5 of +5. The minor form of immunoreactive beta-endorphin had an apparent molecular weight of 1.4 Kda and a net charge at pH 2.5 of +1. Neither immunoreactive form exhibited receptor binding activity in the radioreceptor assay. A single peak of opiate active beta-endorphin was detected. This material had an apparent molecular weight of 3.5 Kda and a net charge at pH 2.5 of +7.
Assuntos
Peixes/fisiologia , Hipófise/análise , beta-Endorfina/isolamento & purificação , Animais , Encéfalo/citologia , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Hipófise/citologia , Radioimunoensaio , Ensaio RadioliganteRESUMO
Acid extracts of the posterior pituitary of the amphibian, Xenopus laevis, were analyzed with two heterologous region specific beta-endorphin RIAs. Following gel filtration chromatography and cation exchange chromatography four peaks of immunoreactivity were detected. All four peaks were detected with a N-acetyl specific beta-endorphin RIA. Peak I represented 92% of the total immunoreactivity isolated following cation exchange chromatography. This peak had a net positive charge at pH 2.5 of +1 and an apparent molecular weight of 1.4 Kd. Following reverse phase HPLC, Peak I fractionated into two peaks: Peak Ia and Peak Ib. Both peaks were detected with the N-acetyl specific beta-endorphin RIA and a Met-enkephalin RIA, however, neither peak co-migrated with either Met-enkephalin or N-acetyl-beta-endorphin(1-16). At present it is not clear whether Peak I is derived from pro-opiomelanocortin or one of the other opioid polyproteins. Peaks II, III, and IV represented 8% of the total immunoreactivity recovered following cation exchange chromatography. These peaks had net positive charges of +3, +4, and +5, respectively, and apparent molecular weights of 2.8, 3.2, and 3.5 Kd, respectively. These apparently N-acetylated beta-endorphin-sized forms are minor end products of the pro-opiomelanocortin biosynthetic pathway.
Assuntos
Encefalina Metionina/isolamento & purificação , Neuro-Hipófise/análise , beta-Endorfina/isolamento & purificação , Animais , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Encefalina Metionina/imunologia , Peso Molecular , Radioimunoensaio , Xenopus laevis , beta-Endorfina/imunologiaRESUMO
Recent demonstration of immunoreactive (IR) atrial natriuretic factor (ANF) and beta-endorphin (beta-EP) in the thymus prompted a reexamination of the distribution and cellular localization of the two peptides within that tissue. Double labeling immunohistochemistry was carried out on gelatin-embedded cryostat thymic sections of adult male Sprague-Dawley rats. Cells stained positive with antiserum (S118), raised against rANF(1-28), were colocalized in > 95% of cases with immunofluorescent staining of IR-beta-EP(1-31). The cells were found sparsely distributed along the corticomedullary junction and in subcapsular regions. In 1- or 5-day monolayer cultures of adherent thymic cells, 15-20% of the cells stained positive for either IR-ANF or IR-beta-EP. Under these conditions, > 95% of IR-ANF or IR-beta-EP positive cells were also fluorescence stained for the rat macrophage marker ED-1. Thus, taken together with previous reports, our present findings suggest that, in the rat thymus, both ANF and beta-EP are produced by the same population of macrophages. To further investigate their presence in the thymus, the contents and molecular species of the two peptides were compared over the developmental period of the animal using well-characterized radioimmunoassays (RIA). Both peptides significantly increased their contents between day 2 and day 60. However, in terms of concentration, IR-ANF at day 2 was approximately 50% higher than day 16 and five times greater that at day 60; in comparison the concentration of IR-beta-EP remained relatively constant and the only significant difference from day 2 being a slight increase in the day 16 animals.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Fator Natriurético Atrial/isolamento & purificação , Macrófagos/química , Timo/química , beta-Endorfina/isolamento & purificação , Envelhecimento , Animais , Fator Natriurético Atrial/imunologia , Células Cultivadas , Secções Congeladas , Imuno-Histoquímica , Masculino , Radioimunoensaio , Ratos , Ratos Sprague-Dawley , Timo/citologia , beta-Endorfina/imunologiaRESUMO
Fractionation of an acid extract of 15 B. marinus intermediate pituitaries by a combination of gel filtration chromatography and cation exchange chromatography revealed one major and five minor forms of beta-endorphin in this tissue. Based on reversed-phase HPLC and immunological properties, as well as amino acid composition and primary sequence analysis, it was deduced that the sequence of the major form of B. marinus beta-endorphin is N-acetyl-YGGFMTPE. Overall, the steady-state analyses of the minor forms of beta-endorphin indicated that the posttranslational processing of beta-endorphin in the toad intermediate pituitary includes endoproteolytic cleavage at both paired basic and monobasic cleavage sites.
Assuntos
Endopeptidases/metabolismo , Hipófise/metabolismo , Processamento de Proteína Pós-Traducional , beta-Endorfina/metabolismo , Sequência de Aminoácidos , Animais , Bufo marinus , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Peso Molecular , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , beta-Endorfina/química , beta-Endorfina/isolamento & purificaçãoRESUMO
We evaluated protocols for the extraction of calcitonin gene-related peptide, neuropeptide Y, substance P, peptide YY and beta-endorphin from rat lung tissue for subsequent radioimmunoassay. The effects of varying acidity of the extraction solution and repeating extraction on the recovery of peptide immunoreactivity and non-specific tracer-binding were compared by analysis of variance. Moreover, variability of immunoreactivity was quantified for comparison. Considering all three criteria, the optimal acidity for extraction was: 0.1 M or 1 M acetic acid for CGRP and beta-endorphin, 0.1 M acetic acid for NPY, 1 M acetic acid for substance P and phosphate buffer for peptide YY. Double or combined extraction unambiguously improved assay results only for substance P. Reversed-phase high-performance liquid chromatography of CGRP-, NPY- and SP-immunoreactivity obtained from selected extracts suggested that differences in recovery of these peptides are not explainable by differential peptide fragmentation during extraction.
Assuntos
Pulmão/química , Neuropeptídeos/isolamento & purificação , Animais , Peptídeo Relacionado com Gene de Calcitonina/análise , Peptídeo Relacionado com Gene de Calcitonina/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Concentração de Íons de Hidrogênio , Masculino , Neuropeptídeo Y/análise , Neuropeptídeo Y/isolamento & purificação , Neuropeptídeos/análise , Peptídeo YY/análise , Peptídeo YY/isolamento & purificação , Radioimunoensaio , Ratos , Ratos Sprague-Dawley , Substância P/análise , Substância P/isolamento & purificação , beta-Endorfina/análise , beta-Endorfina/isolamento & purificaçãoRESUMO
In the intermediate pituitary of the anuran amphibian, Bufo marinus, the N-acetylation of ACTH(1-13)-NH2 to yield alpha-MSH occurs as a cosecretory processing event, whereas the N-acetylation of beta-endorphin occurs as a posttranslational processing event. To understand how these two N-acetylation reactions are segregated, B. marinus intermediate pituitary cells were analyzed by immunogold labeling electron microscopy, and by using an ultracentrifugation procedure. The immunogold labeling studies indicated that ACTH(1-13)-NH2-related immunoreactivity was colocalized with N-acetylated beta-endorphin-related immunoreactivity in secretory granules. Furthermore, ACTH(1-13)-NH2-related immunoreactivity was not detected in either the ER or the Golgi. N-Acetylated beta-endorphin-related immunoreactivity, however, was detected in the Golgi. Ultracentrifugation analysis revealed that in an ER/microsomal fraction, beta-LPH-sized and nonacetylated beta-endorphin-sized immunoreactive material were present in a molar ratio of 1:2. No N-acetylated forms of beta-endorphin were detected in the ER/microsomal fraction. In a Golgi/secretory granule fraction, the molar ratio of beta-LPH to beta-endorphin was 1:9 with 58% of the beta-endorphin being N-acetylated. Collectively, these data support the following hypotheses. The proteolytic cleavage of ACTH (1-39) to yield ACTH (1-13)-NH2 is a late processing event occurring in secretory granules. The cleavage of beta-LPH to yield nonacetylated beta-endorphin is an early processing event that may occur in the ER or the Golgi. Because N-acetylated beta-endorphin and nonacetylated ACTH(1-13)-NH2 are colocalized in secretory granules, it appears, therefore, that the N-acetylation of beta-endorphin is completed prior to loading into secretory granules. Thus, there is a spatial and temporal separation of the posttranslational processing events associated with the beta-LPH portion and ACTH portion of the POMC biosynthetic pathway in amphibian intermediate pituitary cells.
Assuntos
Bufo marinus/metabolismo , Citoplasma/química , Fragmentos de Peptídeos/isolamento & purificação , Hipófise/metabolismo , Pró-Opiomelanocortina/metabolismo , alfa-MSH/análogos & derivados , beta-Endorfina/análogos & derivados , Acetilação , Animais , Citoplasma/ultraestrutura , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/ultraestrutura , Complexo de Golgi/química , Complexo de Golgi/ultraestrutura , Microscopia Imunoeletrônica , Hipófise/química , Processamento de Proteína Pós-Traducional , Frações Subcelulares/química , alfa-MSH/isolamento & purificação , beta-Endorfina/isolamento & purificaçãoRESUMO
Substance P (SP), a neuropeptide widely distributed in the organism, has been shown to stimulate lymphocyte proliferation and immunoglobulin synthesis. However, the effect of SP on specific lymphokines is unknown. Therefore we investigated the influence of SP on mitogen-induced interferon-gamma (IFN-gamma) production in vitro. Peripheral blood mononuclear cells (PBMC) of healthy donors were isolated by density gradient centrifugation and cultured in supplemented RPMI 1640 medium with phytohemagglutinin (PHA) or pokeweed mitogen (PWM), 0.125 and 0.25 mg/liter each, and varying concentrations of SP (10(-12) to 10(-6) M). After 24 and 48 h, IFN-gamma was measured in the supernatant using radioimmunoassay. Results were expressed as percent change of controls. SP alone had no relevant IFN-gamma inducing properties. It enhanced the IFN-gamma production of PWM-stimulated cells significantly up to 18%. The maximal effect was observed at 10(-8) M. PHA-stimulated cells also increased their IFN-gamma production after addition of SP. However, due to great interindividual variations this effect did not attain statistical significance. Stimulation of IFN-gamma production by SP might be of physiological importance, since the effect was seen at concentrations comparable to those found in the body. Our data lend further support to the immunoregulatory functions of SP.
Assuntos
Interferon gama/sangue , Leucócitos Mononucleares/metabolismo , Fragmentos de Peptídeos/isolamento & purificação , Substância P/fisiologia , beta-Endorfina/isolamento & purificação , Células Cultivadas , Humanos , Interferon gama/biossíntese , Leucócitos Mononucleares/efeitos dos fármacos , Mitógenos/farmacologiaRESUMO
The intermediate lobe of the pituitary gland synthesizes the multifactorial precursor molecule pro-opiomelanocortin (POMC), from which, through a process of post-translational enzymatic processing, beta-endorphin-(1-31) (beta E) and a variety of N alpha-acetylated and C-terminally shortened forms of this peptide are generated. Using an in vitro superfusion system, the release of these endorphins from intact rat neurointermediate lobes (NILs) was investigated under basal and isoproterenol (ISO) stimulated conditions. Superfusion of NILs with the beta-adrenergic agonist ISO (30 min pulse) resulted in a rapid, sustained and concentration-dependent stimulation of the release of beta E-like immunoreactivity (beta E-IR) over basal as determined with an antiserum directed against the C-terminus of the beta E- (1-31) sequence (10(-6) M: + 145%; 10(-7) M: + 73%; 10(-8) m: + 41%). The release of N(alpha)-acetylated-endorphin-like immunoreactivity (AcE-IR) was stimulated to a similar extent. These effects of ISO were antagonized by the competitive alpha-adrenoceptor antagonist propranolol in a concentration-dependent manner, indicating the involvement of alpha-adrenoceptors. The beta-related peptides released from the NILs under basal and ISO-stimulated conditions were further characterized, based on their retention times in a reversed-phase HPLC system and their reactivity with specific antisera recognizing respectively the midportion of beta E, the N-terminus of acetylated endorphins, the C-terminus of tau-endorphin (beta E-(1-17); tau E), or the C-terminus of alpha-endorphin (beta E-(1-16); alpha E). In HPLC fractionated superfusates 10 peaks were resolved that reacted with the midportion beta E antiserum. In superfusates collected under basal conditions, three major peaks possessed chromatographical and immunological characteristics of Ac beta E-(1-26), Ac beta E- (1-27) Ac beta E-(1-31). In addition, a prominent peak was found eluting around the retention time of beta E-(1-31), that contained both acetylated and non-acetylated material. Six smaller peaks were observed, with the characteristics of beta E-(1-26) and beta E-(1-27) (these peptides were not resolved with the HPLC system used), Ac tau E, tau E, Aa alpha E, and des-tyrosine-alpha E (DT alpha E), respectively. In superfusates collected during superfusion of NILs with ISO (10(-6) M) all peaks were increased. However, those eluting as beta E-(1-31), beta E-(1-26)/beta E-(1-27), Ac beta E-(1-26) and Ac tau E appeared to be preferentially stimulated.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Isoproterenol/farmacologia , Neuro-Hipófise/metabolismo , beta-Endorfina/metabolismo , Acetilação , Animais , Cromatografia Líquida de Alta Pressão , Cinética , Masculino , Neuro-Hipófise/efeitos dos fármacos , Ratos , Ratos Endogâmicos , beta-Endorfina/isolamento & purificaçãoRESUMO
The opioid activity of immunoreactive beta-endorphin-like peptide extracted from pork pancreas duplicates the effects of morphine and synthetic beta-endorphin when measured by inhibition of isolated guinea pig ileal muscle response to electro-stimulation in vitro and by morphine-like analgesia following intravenous injection in the mouse. These responses are reversed by the opiate antagonist naloxone, indicating that a potent opioid mu receptor binding ligand is present in pancreatic extract. These findings imply a pancreatic source of plasma immunoreactive beta-endorphin that may explain a number of physiological and behavioral effects generally attributed to hypophyseal beta-endorphin alone.
Assuntos
Analgésicos , Contração Muscular/efeitos dos fármacos , Animais , Cobaias , Íleo/efeitos dos fármacos , Técnicas In Vitro , Camundongos , Naloxona/farmacologia , Medição da Dor , Hormônios Pancreáticos/isolamento & purificação , Hormônios Pancreáticos/metabolismo , Receptores Opioides/metabolismo , beta-Endorfina/isolamento & purificação , beta-Endorfina/metabolismoAssuntos
Cação (Peixe)/genética , Hormônios Estimuladores de Melanócitos/fisiologia , Neuroimunomodulação , Hipófise/fisiologia , Pró-Opiomelanocortina/fisiologia , Hormônio Adrenocorticotrópico/isolamento & purificação , Animais , Carpas , Hormônios Estimuladores de Melanócitos/genética , Hormônios Estimuladores de Melanócitos/farmacologia , Fagócitos/efeitos dos fármacos , Fagócitos/fisiologia , Pró-Opiomelanocortina/genética , alfa-MSH/isolamento & purificação , beta-Endorfina/isolamento & purificação , beta-MSH/isolamento & purificação , gama-MSH/isolamento & purificaçãoRESUMO
Des-enkephalin-gamma-endorphin is a neuroleptic endogenous peptide that is active in the central nervous system in extremely low concentrations. The pharmacokinetics of this peptide could not be studied in detail as a bioanalytical method for determining endogenous levels of this peptide in biological matrices was not available. Liquid chromatography with fluorescence reaction detection in principle offers sufficient sensitivity for this application, provided that a selective sample pretreatment can be performed. The development of a pretreatment method for plasma samples is described. After protein precipitation with trichloroacetic acid, high-molecular-weight compounds are removed using on-line continuous-flow dialysis. After dialysis, polar low-molecular-weight compounds, including those containing amino functions, are removed by solid-phase isolation, while simultaneously the analyte is concentrated. By means of valve switching the pretreatment system is coupled on-line to the liquid chromatographic system. With the developed system it is possible to determine desenkephalin-gamma-endorphin in plasma in the range 10-100 ng/ml.
Assuntos
beta-Endorfina/análise , Acetonitrilas , Proteínas Sanguíneas/isolamento & purificação , Soluções Tampão , Diálise , Humanos , Concentração de Íons de Hidrogênio , Peso Molecular , Fosfatos , Espectrometria de Fluorescência , Ácido Tricloroacético , beta-Endorfina/sangue , beta-Endorfina/isolamento & purificaçãoRESUMO
In order to simulate the distribution and elimination of radioiodinated human beta-endorphin (125I-beta-EP) after iv bolus injection in rats, we proposed a physiologically based pharmacokinetic model incorporating diffusional transport of 125I-beta-EP across the capillary membrane. This model assumes that the distribution of 125I-beta-EP is restricted only within the blood and the tissue interstitial fluid, and that a diffusional barrier across the capillary membrane exists in each tissue except the liver. The tissue-to-blood partition coefficients were estimated from the ratios of the concentration in tissues to that in arterial plasma at the terminal (pseudoequilibrium) phase. The total body plasma clearance (9.0 ml/min/kg) was appropriately assigned to the liver and kidney. The transcapillary diffusion clearances of 125I-beta-EP were also estimated and shown to correlate linearly with that of inulin in several tissues. Numerically solving the mass-balance differential equations as to plasma and each tissue simultaneously, simulated concentration curves of 125I-beta-EP corresponded well with the observed data. It was suggested by the simulation that the initial rapid disappearance of 125I-beta-EP from plasma after iv injection could be attributed in part to the transcapillary diffusion of the peptide.
Assuntos
beta-Endorfina/farmacocinética , Animais , Capilares/metabolismo , Cromatografia em Gel , Difusão , Meia-Vida , Radioisótopos do Iodo , Masculino , Taxa de Depuração Metabólica , Modelos Biológicos , Ratos , Ratos Endogâmicos , Distribuição Tecidual , beta-Endorfina/isolamento & purificaçãoRESUMO
The simultaneous purification and concentration of synthetic human beta-endorphin from plasma is described, which when used together with an appropriate isocratic high-performance liquid chromatographic-electrochemical detection (HPLC-ED) system allows the determination of elevated physiological levels of beta-endorphin. Purification of plasma was gained by flash-freezing in liquid nitrogen, acidifying with 100 microliters of trifluoroacetic acid (10%, v/v) per ml of plasma, thawing at 4 degrees C and centrifuging to remove any precipitate. Solid-phase extraction with silica sorbent was utilised, which allowed further isolation of the analyte, a method of concentration and a procedure whereby beta-endorphin could be transferred to the HPLC mobile phase. Silica sorbent demonstrated greater selectivity than C18 for synthetic human beta-endorphin and, in addition, provided improved recovery of this analyte when utilising elution volumes of 500 microliters or less. Proteolytic degradation and heparin-induced high-affinity binding in plasma were shown not to effect the recovery of beta-endorphin if blood was rapidly chilled and plasma quickly obtained, frozen and acidified. Validation of this purification/concentration method using [125I]beta-endorphin demonstrated a recovery of 85.6% which was not jeopardised when concentrating the sample twenty-fold. This provided an increase in the sensitivity of detection, when used in conjunction with HPLC-ED, from 5 ng/ml to 250 pg/ml.
Assuntos
beta-Endorfina/sangue , Aprotinina , Cromatografia Líquida de Alta Pressão , Eletroquímica , Encefalina Leucina/sangue , Encefalina Leucina/isolamento & purificação , Encefalina Metionina/sangue , Encefalina Metionina/isolamento & purificação , Heparina , Humanos , Reprodutibilidade dos Testes , Temperatura , beta-Endorfina/isolamento & purificaçãoRESUMO
Snake (Ptyas mucosa) brains (400 g) were extracted with a mixture of acetone, water, and hydrochloric acid. The precipitate (5.6 g) that formed upon addition of five volumes of acetone to the extract, designated acid-acetone powder, was subjected to gel filtration on Sephadex G-25. A large unretarded peak (SB-1) with molecular weight greater than 5000 and a small retarded peak (SB-2) with molecular weight smaller than 5000 were obtained. They were then separately subjected to ion-exchange chromatography on CM-cellulose. Adrenocorticotropic activity was detected in the fractions by their ability to stimulate isolated rat adrenal cells to produce corticosterone. Opiate activity was detected in the fractions by their ability to inhibit the binding of (D-Ala2,D-Leu5)-[tyrosyl-3,5-3H]enkephalin to rat brain membranes and their cross-reactivity in a beta-endorphin radioimmunoassay. Adrenocorticotropic and opiate activities were found to be concentrated in fractions strongly adsorbed on CM-cellulose, which were eluted by combined pH and ammonium acetate concentration gradients. There appeared to be a separation between adrenocorticotropic and opiate activities, suggesting that they were due to separate molecular entities.
Assuntos
Hormônio Adrenocorticotrópico/isolamento & purificação , Química Encefálica , Serpentes/metabolismo , beta-Endorfina/isolamento & purificação , Animais , Corticosterona/biossíntese , Masculino , Radioimunoensaio , Ensaio Radioligante , Ratos , Ratos Endogâmicos , Receptores Opioides/metabolismoRESUMO
The effects of bile salts on the release of beta-endorphin-like immunoreactivity (beta-END-LI) were investigated in men using a specific radioimmunoassay developed by the authors. Plasma beta-END-LI was determined after extraction by the acid-acetone method (recovery: 73 +/- 5%). Oral administration of 400 mg of sodium taurocholate caused a rise in plasma beta-END-LI from 9.9 +/- 0.5 pmol/liter to 21.3 +/- 1.2 pmol/liter after 30 min and 18.1 +/- 0.5 pmol/liter after 60 min, with return to the initial value after 90 min. Oral administration of chenodeoxycholic acid (CDCA) also increased plasma beta-END-LI from a basal level of 8.4 +/- 0.7 pmol/liter to 18.7 +/- 0.8 pmol/liter after 30 min. Oral administration of ursodeoxycholic acid (UDCA) increased plasma beta-END-LI from 7.3 +/- 0.3 pmol/liter to 30.6 +/- 0.2 pmol/liter after 30 min. In gel chromatography, the beta-END-LI released after UDCA administration separated into two components, which eluted in the same positions as human beta-lipotropin and human beta-endorphin, respectively. These results suggested that bile salts may participate the release of beta-END-LI.
Assuntos
Reações Antígeno-Anticorpo/efeitos dos fármacos , Ácidos e Sais Biliares/farmacologia , beta-Endorfina/sangue , Administração Oral , Adulto , Cromatografia em Gel , Humanos , Masculino , Radioimunoensaio , Fatores de Tempo , beta-Endorfina/isolamento & purificaçãoRESUMO
Analysis of lipotropin from the pars intermedia of bovine pituitary showed that it contained the full COOH-terminal sequence of beta-endorphin. In contrast the beta-endorphin related peptides exhibited a high degree of C-terminal proteolysis. The implications of these findings are discussed with reference to the cellular compartments where the processing reactions take place.
Assuntos
Hipófise/metabolismo , beta-Endorfina/biossíntese , Animais , Bovinos , Adeno-Hipófise/metabolismo , Processamento de Proteína Pós-Traducional , beta-Endorfina/genética , beta-Endorfina/isolamento & purificação , beta-Lipotropina/metabolismoRESUMO
1. Turtle heart and intestine acetone powders were extracted with an acetone-water-HCl mixture. An acid acetone powder resulted by adding a copious volume of acetone to the extract. The powder was subjected to gel filtration on Sephadex G-25 and ion exchange chromatography on CM-cellulose. 2. In turtle heart, corticotropin-like bioactivity was distributed among chromatographic fractions (derived from material unretarded on Sephadex G-25) unadsorbed and adsorbed on CM-cellulose. The highest opiate receptor binding activity and beta-endorphin-like immunoreactivity were adsorbed on CM-cellulose. 3. In turtle intestine, corticotropin-like bioactivity was absent. Opiate receptor binding activity was present in fractions unretarded as well as in fractions retarded on Sephadex G-25, indicating a molecular weight of greater and smaller than 5000 respectively. 4. The highest opiate receptor binding activity and beta-endorphin-like immunoreactivity were found in a fraction adsorbed on CM-cellulose.