RESUMO
We compared the effectiveness of immunomodulators used in the treatment of patients with chronic salpingitis and oophoritis with or without changes in succinate dehydrogenase (SDH) activity in blood lymphocytes at incubation with the drug. Diurnal variations in individual reaction of SDH in blood lymphocytes to thymalin or ridostin were revealed. In the groups of women receiving ridostin or thymalin during the reaction of lymphocyte SDH to it, improvement of clinical laboratory and immunological parameters was observed in the majority of the patients and no effect was found in a lesser group of patients than in the groups treated with drugs during the absence of lymphocyte SDH reaction thereto. The timing of the presence of SDH reaction to drugs in the immunocompetent cells makes it possible to set the optimal daily regime of their application and to select a drug that would be most effective in each particular case.
Assuntos
Cronofarmacoterapia , Fatores Imunológicos/administração & dosagem , Subpopulações de Linfócitos/efeitos dos fármacos , Ooforite/tratamento farmacológico , RNA de Cadeia Dupla/administração & dosagem , RNA Fúngico/administração & dosagem , Salpingite/tratamento farmacológico , Succinato Desidrogenase/sangue , Hormônios do Timo/administração & dosagem , Adolescente , Adulto , Antibacterianos/uso terapêutico , Doença Crônica , Terapia Combinada , Grânulos Citoplasmáticos/enzimologia , Quimioterapia Combinada , Feminino , Humanos , Fatores Imunológicos/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/enzimologia , Células Matadoras Naturais/imunologia , L-Lactato Desidrogenase/sangue , Subpopulações de Linfócitos/enzimologia , Subpopulações de Linfócitos/imunologia , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , Monócitos/imunologia , Ooforite/imunologia , Ooforite/terapia , Modalidades de Fisioterapia , Medicina de Precisão , RNA de Cadeia Dupla/farmacologia , RNA Fúngico/farmacologia , Salpingite/imunologia , Salpingite/terapia , Hormônios do Timo/farmacologia , Resultado do Tratamento , Vitaminas/uso terapêutico , Adulto JovemRESUMO
The cytochemical and functional characteristics of chloragocytes of both 'control' and cold-stressed Eisenia fetida were examined. Flow cytometry revealed the heterogeneity of chloragocytes: the first group was characterized by low, the second one by high acid phosphatase (AcP) content. In 'control' animals the former, in cold-stressed ones the latter type were the dominant form. The elevated AcP-activity correlated with the accumulation of autophagic vacuoles (AVs) in chloragocytes. Both AVs and all small chloragosomes showed high AcP activity, while most of the large chloragosomes did not display any. Most 'control' granules (0.75-1.25 µm) contained high amounts of Ca and P, with less and variable quantities of S, Cl, K, Fe and Zn. Small chloragosomes with low Ca and P concentrations were seldom found. In cold-stressed animals the number of small granules (0.25-0.75 µm) increased up to 40% of total population. Their Ca and P contents were significantly lower; S and Fe concentrations were higher than those of large chloragosomes (1.0-1.5 µm). Our results prove that the formation and elemental composition of chloragosomes can be influenced by environmental stressors and suggest that the mature chloragosomes are tertiary lysosomes and their formation is coupled to autophagocytosis.
Assuntos
Vasos Sanguíneos/citologia , Cálcio/metabolismo , Grânulos Citoplasmáticos/metabolismo , Oligoquetos/metabolismo , Fósforo/metabolismo , Estresse Fisiológico , Fosfatase Ácida/metabolismo , Animais , Temperatura Baixa , Grânulos Citoplasmáticos/enzimologia , Grânulos Citoplasmáticos/ultraestrutura , Sistema Digestório/irrigação sanguínea , Oligoquetos/citologia , Oligoquetos/fisiologia , Forma das OrganelasRESUMO
Helichrysum picardii Boiss. & Reuter is a Mediterranean vegetal species from the Asteraceae family. From the methanolic extract of the aerial flowering parts of this plant, a fraction of two pentacyclic triterpenes has been isolated. Gas chromatography revealed that the triterpene isomers ursolic and oleanolic acids comprised 69% and 29% respectively of the composition of this fraction. The triterpene isomeric fraction was tested in two phagocyte cell systems. It inhibited compound 48/80-induced histamine release from rat peritoneal mast cells in an approximately percentage of 45% at 100 microM and myeloperoxidase secretion from A23187-ionophore-stimulated rat peritoneal leukocytes in a significant manner at doses of 50 and 100 miroM. Furthermore, the triterpene isomers very significantly and dose-dependently inhibited generation of the cyclo-oxygenase metabolite prostaglandin E2 (41% inhibition at 50 miroM) and the 5-lipoxygenase metabolite leukotriene B4 (79% inhibition at 50 microM) from activated rat leukocytes. This anti-eicosanoid activity of the triterpene fraction was more potent than that produced by the pure triterpene oleanolic acid used for comparision, indicating a stronger action of the ursolic acid, the major compound of the isolated triterpene fraction. From these data, it can be suggested that the triterpene isomers oleanolic and ursolic acids present in the medicinal plant Helichrysum picardii contribute to the anti-inflammatory profile of this vegetal species.
Assuntos
Eicosanoides/antagonistas & inibidores , Helichrysum/química , Antagonistas dos Receptores Histamínicos/farmacologia , Ácido Oleanólico/farmacologia , Triterpenos/farmacologia , Animais , Calcimicina/farmacologia , Cromatografia em Camada Fina , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/enzimologia , Flores/química , Cromatografia Gasosa-Espectrometria de Massas , Liberação de Histamina/efeitos dos fármacos , Técnicas In Vitro , Indicadores e Reagentes , Ionóforos/farmacologia , Isomerismo , Leucócitos/efeitos dos fármacos , Leucócitos/enzimologia , Leucócitos/metabolismo , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Metanol , Peroxidase/metabolismo , Ratos , Ratos Wistar , Solventes , p-Metoxi-N-metilfenetilamina/farmacologia , Ácido UrsólicoRESUMO
The provision of glutamine in vivo has been observed to reduce to normal levels the neutrophilia observed after exhaustive exercise and to decrease the neutrophil chemoattractant, interleukin-8. Thus, the role for glutamine in the regulation of inflammatory mediators of human neutrophil activation was investigated. The study sought to establish whether glutamine supplementation in vitro affects neutrophil function at rest and whether glutaminase, the major enzyme that metabolizes glutamine, is present in human polymorphonuclear neutrophils (PMN). During in vitro studies, the addition of 2 mm glutamine increased the respiratory burst of human PMN stimulated with both phorbol myristate acetate (PMA) and formyl-methionyl-leucyl-phenylalanine. These observations were made using a highly sensitive, real time chemiluminescent probe, Pholasin. Glutamine alone did not stimulate the release of reactive oxygen species. In a novel finding using glutaminase-specific antibodies in combination with flow cytometry and confocal microscopy, glutaminase was shown to be present on the surface of human PMN. Subcellular fractionation revealed that the enzyme was enriched in the secondary granules and could be released into cell culture medium upon stimulation with PMA. In conclusion, human PMN appeared to utilize glutamine and possess the appropriate glutaminase enzyme for metabolizing glutamine. This may depress some pro-inflammatory factors that occur during prolonged, exhaustive exercise.
Assuntos
Grânulos Citoplasmáticos/enzimologia , Glutaminase/metabolismo , Glutamina/farmacocinética , Ativação de Neutrófilo/fisiologia , Neutrófilos/enzimologia , Humanos , Imuno-Histoquímica , Microscopia Confocal , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/metabolismo , Oxirredução , Explosão Respiratória/efeitos dos fármacos , Explosão Respiratória/fisiologiaRESUMO
Reductions in activity of SSIII, the major isoform of starch synthase responsible for amylopectin synthesis in the potato tuber, result in fissuring of the starch granules. To discover the causes of the fissuring, and thus to shed light on factors that influence starch granule morphology in general, SSIII antisense lines were compared with lines with reductions in the major granule-bound isoform of starch synthase (GBSS) and lines with reductions in activity of both SSIII and GBSS (SSIII/GBSS antisense lines). This revealed that fissuring resulted from the activity of GBSS in the SSIII antisense background. Control (untransformed) lines and GBSS and SSIII/GBSS antisense lines had unfissured granules. Starch analyses showed that granules from SSIII antisense tubers had a greater number of long glucan chains than did granules from the other lines, in the form of larger amylose molecules and a unique fraction of very long amylopectin chains. These are likely to result from increased flux through GBSS in SSIII antisense tubers, in response to the elevated content of ADP-glucose in these tubers. It is proposed that the long glucan chains disrupt organization of the semi-crystalline parts of the matrix, setting up stresses in the matrix that lead to fissuring.
Assuntos
Amilopectina/metabolismo , Grânulos Citoplasmáticos/enzimologia , Solanum tuberosum/metabolismo , Sintase do Amido/metabolismo , Amilopectina/química , Amilopectina/isolamento & purificação , Configuração de Carboidratos , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Grânulos Citoplasmáticos/ultraestrutura , Microscopia Eletrônica de Varredura , Espalhamento de Radiação , Solanum tuberosum/enzimologia , Sintase do Amido/isolamento & purificaçãoRESUMO
Multiple allelism in heterozygous autopolyploid species like potato not only occurs for genes that affect morphological characteristics but also for genes involved in metabolic pathways. Based on a combination of Southern and PCR analyses, at least eight alleles encoding granule-bound starch synthase I (GBSSI), which is responsible for amylose biosynthesis, have been identified in potato. These alleles were grouped into four classes, distinguishable by Southern analysis, and subdivided based on PCR. Despite the heterozygous and polyploid character of potato it was possible to assign variation in GBSSI activity to the allelic composition at the GBSSI loci within a large population of Solanum tuberosum cultivars and Solanum breeding lines. Moreover, the availability of an amf allele made it possible to reduce heterogeneity and enabled us to demonstrate an effect of GBSSI allelic composition on amylose content. The major difference between the alleles identified was the absence or presence of a 140-bp fragment at a site 0.5 kb upstream of the ATG start codon of the gene for GBSSI. The absence of this 140-bp fragment had a major effect on GBSSI activity and amylose content, while the presence of small deletions and simple sequence repeats had no obvious effect.
Assuntos
Regiões Promotoras Genéticas , Solanum tuberosum/genética , Sintase do Amido/genética , Sintase do Amido/metabolismo , Amido/biossíntese , Amido/genética , Amido/metabolismo , Algoritmos , Alelos , Amilose/biossíntese , Sequência de Bases , Southern Blotting , Códon/genética , Cruzamentos Genéticos , Grânulos Citoplasmáticos/enzimologia , Heterozigoto , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Poliploidia , Mapeamento por Restrição , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Solanum tuberosum/classificação , Solanum tuberosum/enzimologia , Amido/químicaRESUMO
In granules of hematopoetic cells, dipeptidyl peptidase I (DPPI) processes inactive proenzymes into active enzymes, e.g., lymphocyte progranzyme A. Our goal was to develop irreversible inhibitors of intracellular DPPI. First, we identified inhibitors with aqueous stability. Then we determined which inhibitors were nontoxic, could enter cells and inactivate intracellular DPPI. We screened nine dipeptide vinyl sulfone (VS) inhibitors (kobs/[I] > 72 M-1 s-1) and found six that were nontoxic. Four affected intracellular DPPI at < 25 microM. These compounds contained only uncharged amino acid residues; the two less reactive compounds contained charged Glu residues. The best one, Leu-Phe-VS-CH3, inactivated DPPI in cells with an ID50 of approximately 5 microM. This inhibitor was not the best inhibitor of purified DPPI. Longer aqueous stabilities were important predictors of cellular efficacy. Leu-Phe-VS-CH3 had a half life of 97 min at the pH of the extracellular medium (7.5) and 1302 min at pH 5.5 (the intracellular environment of DPPI). This VS had no direct effect on granzyme activities. In contrast, the diazomethyl ketone inhibitor Gly-Phe-CHN2 inhibited chymase activity. Several good intracellular DPPI VS inhibitors lacked reactivity with cathepsins B, H and L. In conclusion, we have identified DPPI inhibitors suitable for cellular applications.
Assuntos
Catepsina C/antagonistas & inibidores , Dipeptídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Sulfonas/farmacologia , Animais , Catepsinas/antagonistas & inibidores , Grânulos Citoplasmáticos/enzimologia , Dipeptídeos/química , Avaliação Pré-Clínica de Medicamentos , Estabilidade de Medicamentos , Técnicas In Vitro , Líquido Intracelular/enzimologia , Cinética , Ativação Linfocitária , Masculino , Ratos , Ratos Endogâmicos F344 , Sulfonas/química , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia , Linfócitos T/imunologia , Células Tumorais CultivadasRESUMO
SH-EP is a vacuolar cysteine proteinase from germinated seeds of Vigna mungo. The enzyme has a C-terminal propeptide of 1 kDa that contains an endoplasmic reticulum (ER) retention signal, KDEL. The KDEL-tail has been suggested to function to store SH-EP as a transient zymogen in the lumen of the ER, and the C-terminal propeptide was thought to be removed within the ER or immediately after exit from the ER. In the present study, a protease that may be involved in the post-translational processing of the C-terminal propeptide of SH-EP was isolated from the microsomes of cotyledons of V. muno seedlings. cDNA sequence for the protease indicated that the enzyme is a member of the papain superfamily. Immunocytochemistry and subcellular fractionation of cotyledon cells suggested that the protease was localized in both the ER and protein storage vacuoles as enzymatically active mature form. In addition, protein fractionations of the cotyledonary microsome and Sf9 cells expressing the recombinant protease indicated that the enzyme associates with the microsomal membrane on the luminal side. The protease was named membrane-associated cysteine protease, MCP. The possibility that a papain-type enzyme, MCP, exists as mature enzyme in both ER and protein storage vacuoles will be discussed.
Assuntos
Cisteína Endopeptidases/metabolismo , Grânulos Citoplasmáticos/enzimologia , Retículo Endoplasmático/enzimologia , Proteínas de Membrana/metabolismo , Vacúolos/enzimologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Centrifugação com Gradiente de Concentração , Clonagem Molecular , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/isolamento & purificação , Grânulos Citoplasmáticos/ultraestrutura , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Fabaceae/citologia , Fabaceae/enzimologia , Complexo de Golgi/enzimologia , Complexo de Golgi/ultraestrutura , Imuno-Histoquímica , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Microscopia Eletrônica , Dados de Sequência Molecular , Peso Molecular , Oligopeptídeos , Papaína/metabolismo , Filogenia , Plantas Medicinais , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Vacúolos/ultraestruturaRESUMO
The lipophilic aglycone 5,7-dihydroxy-3,8-dimethoxyflavone (gnaphalin) isolated from the aerial flowering parts of Helichrysum picardii Boiss. & Reuter (Asteraceae) was tested for interactions with the cyclo-oxygenase and 5-lipoxygenase pathways of arachidonate metabolism in stimulated rat peritoneal leukocytes, and for its effects on leukocyte granular enzyme release, cell viability and interactions with reactive oxygen species. Gnaphalin dose-dependently inhibited generation of the cyclo-oxygenase metabolite thromboxane B2 (IC50 = 39.9 +/- 3.9 microM), and of the 5-lipoxygenase metabolite leukotriene B4, although the potency was two-fold less (IC50 = 81.8 +/- 12.9 microM). At concentrations of 6 to 320 microM, gnaphalin did not affect secretion of the pro-inflammatory enzymes lysozyme, myeloperoxidase and beta-glucuronidase from the neutrophil secretory granules, and did not scavenge hydrogen peroxide or hypochlorous acid. However, gnaphalin effectively scavenged superoxide radicals generated in the hypoxanthine/xanthine oxidase system (IC50 = 40 microM) and by PMA-stimulated leukocytes (> 60% at 500 microM), directly inhibited xanthine oxidase (85% at 395 microM) and inhibited Fe(3+)-ascorbate-induced liposomal peroxidation (IC50 = 215 microM). Thus, like some other flavonoids found in medicinal herbs, gnaphalin possesses an array of potentially beneficial anti-eicosanoid and free-radical scavenging properties which may alongside other constituents contribute to the claimed medicinal properties of the plant from which it is derived.
Assuntos
Inibidores de Ciclo-Oxigenase/farmacologia , Eicosanoides/metabolismo , Flavonoides/farmacologia , Sequestradores de Radicais Livres/farmacologia , Leucócitos/fisiologia , Inibidores de Lipoxigenase/farmacologia , Plantas Medicinais , Animais , Inibidores de Ciclo-Oxigenase/química , Inibidores de Ciclo-Oxigenase/isolamento & purificação , Grânulos Citoplasmáticos/enzimologia , Flavonoides/química , Flavonoides/isolamento & purificação , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/isolamento & purificação , Glucuronidase/metabolismo , Leucócitos/efeitos dos fármacos , Inibidores de Lipoxigenase/química , Inibidores de Lipoxigenase/isolamento & purificação , Muramidase/metabolismo , Peroxidase/metabolismo , Ratos , Acetato de Tetradecanoilforbol/farmacologia , Xantina Oxidase/metabolismoRESUMO
Synapsin I is a synaptic vesicle-associated protein involved in neurotransmitter release. The functions of this protein are apparently regulated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). We reported evidence for CaM kinase II and a synapsin I-like protein present in mouse insulinoma MIN6 cells (Matsumoto, K., Fukunaga, K., Miyazaki, J., Shichiri, M., and Miyamoto, E. (1995) Endocrinology 136, 3784-3793). Phosphorylation of the synapsin I-like protein in these cells correlated with the activation of CaM kinase II and insulin secretion. In the present study, we screened the MIN6 cDNA library with the full-length cDNA probe of rat brain synapsin Ia and obtained seven positive clones; the largest one was then sequenced. The largest open reading frame deduced from the cDNA sequence of 3695 base pairs encoded a polypeptide of 670 amino acids, which exhibited significant sequence similarity to rat synapsin Ib. The cDNA contained the same sequence as the first exon of the mouse synapsin I gene. These results indicate that synapsin Ib is present in MIN6 cells. Synapsin I was expressed in normal rat islets, as determined by reverse transcriptase-polymerase chain reaction analysis. Immunoblot analysis after subcellular fractionation of MIN6 cells demonstrated that synapsin Ib and delta subunit of CaM kinase II co-localized with insulin secretory granules. By analogy concerning regulation of neurotransmitter release, our results suggest that phosphorylation of synapsin I by CaM kinase II may induce the release of insulin from islet cells.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Insulina/metabolismo , Insulinoma/metabolismo , Sinapsinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Clonagem Molecular , Grânulos Citoplasmáticos/enzimologia , DNA Complementar , Secreção de Insulina , Insulinoma/enzimologia , Insulinoma/patologia , Camundongos , Dados de Sequência Molecular , Ratos , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Culture selected and expanded osteoblastic cells may be able to be reintroduced into massive skeletal defects to accelerate cell mediated regeneration of skeletal tissues, especially in bone ingrowth in total joint replacement, fracture healing, and osteoporosis. In vitro osteogenic cell culture is a useful model in studying the mechanism of bone metabolism under direct current stimulation. In this study, an osteoblastlike cell line was isolated from newborn rat calvaria. The osteogenic processes of the in vitro cultured cell line were studied by cytochemical, electron microscopic, and energy dispersive x-ray analysis techniques that resembled those observed in membrane bone ossification centers in vivo. Direct current stimulation of 100 microA/cm2 accelerated greatly the proliferation and calcification of the in vitro cultured cells. Intracellular free calcium ion metabolism was measured with an Adherent Cell Analysis and Sorting Machine. Under direct current stimulation, intracellular free calcium ion concentration increased an average of 2.3 times of the original level, which may play a key role in regulating osteogenesis and bone metabolism.
Assuntos
Cálcio/fisiologia , Estimulação Elétrica , Osteoblastos/fisiologia , Osteogênese/fisiologia , Fosfatase Alcalina/análise , Animais , Animais Recém-Nascidos , Artroplastia de Substituição , Doenças Ósseas/patologia , Doenças Ósseas/terapia , Regeneração Óssea , Calcificação Fisiológica/fisiologia , Cálcio/análise , Cálcio/metabolismo , Linhagem Celular , Células Cultivadas , Citoplasma/metabolismo , Grânulos Citoplasmáticos/enzimologia , Grânulos Citoplasmáticos/ultraestrutura , Microanálise por Sonda Eletrônica , Consolidação da Fratura/fisiologia , Histocitoquímica , Microscopia Eletrônica , Osteoblastos/metabolismo , Osteoblastos/transplante , Osteoblastos/ultraestrutura , Osteoporose/terapia , Fósforo/análise , Ratos , Ratos Sprague-Dawley , Crânio , Vacúolos/enzimologia , Vacúolos/ultraestruturaRESUMO
An insulin granule membrane protein-tyrosine phosphatase (PTP) homologue, phogrin, was cloned by expression screening of a rat insulinoma cDNA library. The 3723-base pair cDNA encoded a transmembrane glycoprotein of 1004 amino acids (Mr 111876) that underwent post-translational proteolysis to 60-64-kDa products after a 30-min delay. The kinetics of proteolytic conversion (t1/2 = 45 min) and turnover (t1/2 = 12 h) were consistent with sorting and conversion in a late compartment of the secretory pathway. Studies on the native beta-cell protein suggested that the COOH-terminal PTP domain was on the cytosolic face of the secretory granule. The lumenal segment was comprised of a protease-resistant globular domain of around 25 kDa. Its localization and topology is thus consistent with a transmembrane receptor function related to granule biogenesis, exocytosis, or subsequent membrane recovery, and it should prove to be a useful cell biological marker for the granule membrane. High expression of the mRNA (5.4 kilobases) and protein was evident in islets, pancreatic alpha- and beta-cell tumor lines, brain cells, and other cells of neuroendocrine lineage. It is closely related to the diabetic autoantigen ICA512 (IA-2) (42% identity overall; 80% in the 260-amino acid PTP domain) and thus a potential target of autoimmunity in diabetes mellitus.
Assuntos
Compartimento Celular , Grânulos Citoplasmáticos/enzimologia , Membranas Intracelulares/enzimologia , Ilhotas Pancreáticas/enzimologia , Glicoproteínas de Membrana/genética , Proteínas de Neoplasias/genética , Sequência de Aminoácidos , Animais , Autoantígenos , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Insulinoma , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/análise , Ratos , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
The insulin-regulated glucose transporter isotype GlutT4 expressed only in muscle and adipose cells is sequestered in a specific secretory vesicle. These vesicles harbor another major protein, referred to as vp165 (for vesicle protein of 165 kDa), that like GluT4 redistributes to the plasma membrane in response to insulin. We describe here the cloning of vp165 and show that it is a novel member of the family of zinc-dependent membrane aminopeptidases, with the typical large extracellular catalytic domain and single transmembrane domain but with a unique extended cytoplasmic domain. The latter contains two dileucine motifs, which may be critical for the specific trafficking of vp165, since this has been shown to be the case for this motif in GluT4. However, the tissue distribution of vp165 is much wider than that of GluT4; consequently, vp165 may also function in processes unrelated to insulin action and may serve as a ubiquitous marker for a specialized regulated secretory vesicle.
Assuntos
Aminopeptidases/genética , Aminopeptidases/metabolismo , Insulina/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Tecido Adiposo/enzimologia , Tecido Adiposo/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Membrana Celular/enzimologia , Clonagem Molecular , Cistinil Aminopeptidase , Grânulos Citoplasmáticos/enzimologia , Grânulos Citoplasmáticos/metabolismo , Primers do DNA/genética , DNA Complementar/genética , Transportador de Glucose Tipo 4 , Humanos , Insulina/farmacologia , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Distribuição TecidualRESUMO
The expression of the glutaminyl cyclase (QC), an enzyme responsible for the post-translational modification of N-terminal pyroglutamyl residues of neuropeptide precursors, was examined in bovine/porcine hypothalamic and pituitary tissue by means of immunocytochemistry and in situ hybridization. In the anterior pituitary a distinct pattern of QC immunoreactivity and mRNA expression was found associated exclusively with somatotrophs. In corticotrophs of the pars intermedia QC expression was undetectable, whereas a small portion of putative pars tuberalis cells in the rostral part of the pars distalis were heavily labelled. The neurointermediate lobe was devoid of signals for QC mRNA, but showed significant QC-immunoreactivity on secretory granules of axonal nerve endings. Also nuclei of the hypothalamus were found to be positive for QC-immunoreactivity. Intense labelling was observed in the nucleus supraopticus and nucleus paraventricularis. Staining of the nucleus periventricularis was found to be moderate, whereas no labelling of perikarya in the nucleus arcuatus, the preoptic area and the nucleus suprachiasmaticus was detectable. Moreover, varicose fibers stained positive for QC-immunoreactivity and could be identified in the main transport route from the hypothalamus to the pars neuronalis (tractus hypothalamo hypophysialis). These results suggest that the enzyme is transported via the same routes as its substrate/product to the median eminence or the neural lobe. Furthermore, the mapping of the cellular QC distribution reveals a strikingly distinctive expression pattern, that should be useful for the identification of yet undiscovered places of peptide synthesis and processing.
Assuntos
Aciltransferases/análise , Aminoaciltransferases , Bovinos/metabolismo , Hipotálamo/enzimologia , Hipófise/enzimologia , Suínos/metabolismo , Aciltransferases/genética , Animais , Axônios/enzimologia , Grânulos Citoplasmáticos/enzimologia , Hipotálamo/ultraestrutura , Imuno-Histoquímica , Hibridização In Situ , Fibras Nervosas/enzimologia , Núcleo Hipotalâmico Paraventricular/enzimologia , Hipófise/ultraestrutura , RNA Mensageiro/análise , Núcleo Supraóptico/enzimologia , Distribuição TecidualRESUMO
To yield biologically active hormones, prohomones are processed by cleavages at paired basic amino acid residues. In this study we report that a chymotrypsin-like activity has been colocalized with trypsin-like and carboxypeptidase-B-like proteases in neurosecretory granules, the site of intracellular processing of prohomones. Using a peptide of 11 amino acids as a simple model system for the study of prohomone processing, we have identified in neurosecretory granule membranes a novel protease that specifically recognizes and cleaves peptide bonds at aromatic residues. Studies were also performed with the fluorogenic peptide substrate N-succinyl-leucyl-leucyl-valyl-tyrosine-7-amino-4-methyl-coumarine . The identified protease activity is inactivated by a cloromethyl ketone derivative (Tos-Phe-CH2-Cl) by Trasylol, and markedly by phenylmethylsulfonylfluoride and diisopropylfluorophosphate. This activity is colocalized with other prohomone-processing enzymes in neurosecretory granules, which indicates that this activity is involved in the prohomone processing. Alternatively, this activity may function in the degradation of homones and neuropeptides when they are released in synapses.
Assuntos
Quimotripsina/metabolismo , Grânulos Citoplasmáticos/enzimologia , Hormônios/metabolismo , Hipotálamo/enzimologia , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Carboxipeptidase B , Carboxipeptidases/metabolismo , Fracionamento Celular , Centrifugação com Gradiente de Concentração , Quimotripsina/antagonistas & inibidores , Cumarínicos/metabolismo , Hipotálamo/ultraestrutura , Membranas Intracelulares/enzimologia , Cinética , Masculino , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Inibidores de Proteases/farmacologia , Ratos , Ratos Endogâmicos , Tripsina/metabolismoRESUMO
Peptide-hormones are synthesized as higher-molecular-weight precursor proteins which must undergo numerous posttranslational modifications to yield the bioactive peptide(s) which may include limited endoproteolysis, limited exopeptidase digestion, and C-terminal amidation. Three different enzymes which are likely to be the physiologically relevant processing enzymes of bovine pro-gonadotropin-releasing hormone (pro-GnRH) precursor protein have been colocalized to, and purified from, hypothalamic neurosecretory granules. Gonadotropin-releasing-hormone-associated-peptide-releasing enzyme initiates processing by endoproteolysis of the pro-hormone exclusively at the Arg 13-Asp 14 bond in the sequence, -Gly6-Leu-Arg-Pro-Gly 10-Gly-Lys 12-Arg 13-Asp-, which overlaps the sequence for GnRH (1-10) and GAP(14-69) within the pro-protein. Hypothalamic carboxypeptidase E then sequentially removes the -Lys12-Arg13- doublet from the newly formed peptide before peptidyl glycine alpha-amidating monooxygenase catalyzes the formation of amidated GnRH. Carboxypeptidase E activity is stimulated in vitro by cobalt ion and removes the Lys and Arg residues with equal facility. The residue which acts as the amide nitrogen donor for the alpha-amidating enzyme must be present as the free C-terminal residue of a substrate; the enzyme does not recognize peptide substrates with C-terminal extensions. Based on the mandatory ordered events for processing pro-GnRH and the relative pH profiles displayed by these enzymes, our results are consistent with the idea that endoproteolysis of the pro-hormone occurs upon formation of the secretory granule at the Golgi apparatus and that the processed peptides are the storage form within the secretory vesicles.
Assuntos
Grânulos Citoplasmáticos/enzimologia , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/ultraestrutura , Complexos Multienzimáticos , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Carboxipeptidase H , Carboxipeptidases/isolamento & purificação , Carboxipeptidases/metabolismo , Bovinos , Fracionamento Celular , Endopeptidases/metabolismo , Feminino , Oxigenases de Função Mista/isolamento & purificação , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismoRESUMO
The secretory granules of rat serosal mast cells are able efficiently to degrade the apolipoprotein B component of low density lipoproteins (LDL) Kokkonen, J. O., and Kovanen, P. T. (1985) J. Biol. Chem. 260, 14756-14763). The granules are known to contain two neutral proteases with complementary specificities: a chymotrypsin-like endopeptidase called chymase, and an exopeptidase, the granule carboxypeptidase A. The role of this enzyme pair in the proteolytic degradation of LDL was studied with the aid of specific enzyme inhibitors. Incubation of LDL with intact granules (both enzymes active) led to the formation of numerous low molecular weight peptides and the liberation of free amino acids, most of which (95%) were aromatic (Phe, Tyr, Trp) or branched-chain aliphatic (Leu, Ile, Val). Selective inhibition of granule carboxypeptidase A (leaving chymase active) blocked the liberation of free amino acids, but left the formation of peptides uninhibited. On the other hand, selective inhibition of granule chymase (leaving carboxypeptidase A active) totally abolished the proteolytic degradation of LDL. The results are consistent with a model according to which the proteolytic degradation of LDL by mast cell granules results from coordinated action of the two granule-bound enzymes, whereby the chymase first cleaves peptides from the apolipoprotein B of LDL, and thereafter the carboxypeptidase A cleaves amino acids from the peptides formed.
Assuntos
Apolipoproteínas B/metabolismo , Carboxipeptidases/fisiologia , Grânulos Citoplasmáticos/metabolismo , Endopeptidases/fisiologia , Lipoproteínas LDL/metabolismo , Mastócitos/metabolismo , Serina Endopeptidases , Aminoácidos/análise , Animais , Carboxipeptidases/análise , Carboxipeptidases A , Quimases , Grânulos Citoplasmáticos/enzimologia , Endopeptidases/análise , Técnicas In Vitro , Lipoproteínas LDL/análise , Masculino , Peptídeos/análise , Ratos , Ratos EndogâmicosRESUMO
A new membrane bound protease has been identified in bovine hypothalamic neurosecretory granules using synthetic substrates that we prepared based on the sequence in pro-gonadotropin-releasing hormone protein that overlaps gonadotropin-releasing hormone and gonadotropin-associated peptide (thought to be prolactin-releasing hormone-inhibiting hormone). The enzyme was solubilized from neurosecretory granules using the detergent Triton X-100 and was further purified by high-performance gel permeation liquid chromatography. The enzyme hydrolyzes the Arg-2-naphthylamide (NA) bond of benzoyl(Bz)-Gly-Leu-Arg-Pro-Gly-Gly-Lys-Arg-2-NA which contains two likely processing sites, Arg-Pro and Lys-Arg. On the basis of the ratio of Vmax to Km as a measure of substrate specificity, Bz-Gly-Leu-Arg-Pro-Gly-Gly-Lys-Arg-2-NA is about 50-fold better than Bz-Gly-Gly-Lys-Arg-2-NA. Bz-Leu-Arg-2-NA and Bz-Gly-Leu-Arg-Pro-Gly-Gly are not hydrolyzed. The pH optimum for hydrolysis is 7.2 (Bz-Gly-Gly-Lys-Arg-2-NA substrate). As determined by gel permeation chromatography, the apparent molecular weight of the enzyme depends on the chromatography conditions; in the absence of NaCl, the Mr is approximately equal to 160,000 but is approximately equal to 80,000 if NaCl is included in the eluting buffer. After high-performance gel permeation liquid chromatography, the peak fraction containing the enzyme was lyophilized and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis; silver staining revealed a single protein band, Mr approximately equal to 70,000.
Assuntos
Grânulos Citoplasmáticos/enzimologia , Hipotálamo/enzimologia , Peptídeo Hidrolases/isolamento & purificação , Hormônios Liberadores de Hormônios Hipofisários/biossíntese , Precursores de Proteínas/metabolismo , Animais , Bovinos , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Feminino , Hidrólise , Peptídeo Hidrolases/metabolismo , Especificidade por SubstratoRESUMO
An Arg-Lys esteropeptidase that converts somatostatin-28 in vitro into somatostatin-14 was previously characterized in extracts of rat cerebral cortex. Both the octacosapeptide somatostatin-28 and a synthetic undecapeptide containing the sequence around the Arg-Lys site, i.e. Peptide I: Pro-Arg-Glu-Arg-Lys-Ala-Gly-Ala-Lys-Asn-125 I-Tyr (NH2), were used as substrates. We demonstrate that the converting activity is associated with neurosecretory granule fractions prepared from both cortical and hypothalamic tissue. This activity co-sediments with ghosts obtained from intact vesicles by osmotic shock. After solubilization either by mild ionic strength or sonication of vesicle membranes, the converting activity appears to possess properties indistinguishable from the convertase prepared directly from unfractionated tissue. It cleaves Peptide I to Ala-Gly-Ala-Lys-Asn-125I-Tyr (NH2) (Peptide II) and generates both the NH2- and COOH-terminal fragments of somatostatin-28, i.e. somatostatin-28 (1-12) and somatostatin-14, when the octacosapeptide is used as substrate. The selectivity appears to be strict and to depend upon the sequence around the Arg-Lys pair, as inferred from competition studies conducted with structural analogs possessing either an Arg-Lys or Arg-Arg doublet. It is concluded that this convertase could represent the enzyme system involved in the in vivo production of both the dodeca and tetradeca peptides from their common somatostatin-28 precursor.
Assuntos
Córtex Cerebral/enzimologia , Grânulos Citoplasmáticos/enzimologia , Endopeptidases/isolamento & purificação , Membranas Intracelulares/enzimologia , Sequência de Aminoácidos , Animais , Fracionamento Celular , Grânulos Citoplasmáticos/ultraestrutura , Endopeptidases/metabolismo , Hipotálamo/enzimologia , Membranas Intracelulares/ultraestrutura , Cinética , Masculino , Oligopeptídeos/farmacologia , Ratos , Ratos Endogâmicos , Relação Estrutura-AtividadeRESUMO
Human neutrophils (PMN), when stimulated with such chemotaxins as phorbol myristate acetate (PMA), destroy erythrocytes and other targets. Cytotoxicity depends on PMN-generated reactive oxygen metabolites, yet the exact toxic specie and its mode of production is a matter of some dispute. Using 51Cr-labeled erythrocytes as targets, we compared various reactive-O2 generating systems for their abilities to lyse erythrocytes as well as to oxidize hemoglobin to methemoglobin. PMA-activated PMNs or xanthine oxidase plus acetaldehyde were added to target erythrocytes in amounts that provided similar levels of superoxide. PMNs lysed 68.3 +/- 2.9% (SEM) of targets, whereas the xanthine oxidase system was virtually impotent (2.3 +/- 0.8%). In contrast, methemoglobin formation by xanthine oxidase plus acetaldehyde was significantly greater than that caused by stimulated PMNs (P less than 0.001). A similar dichotomy was noted with added reagent H2O2 or the H2O2-generating system, glucose plus glucose oxidase; neither of these caused 51Cr release, but induced 10-70% methemoglobin formation. Thus, although O2- and H2O2 can cross the erythrocyte membrane and rapidly oxidize hemoglobin, they do so evidently without damaging the cell membrane. That a granule constituent of PMNs is required to promote target cell lysis was suggested by the fact that agranular PMN cytoplasts (neutroplasts), although added to generate equal amounts of O2- as intact PMNs, were significantly less lytic to target erythrocytes (P less than 0.01). Iron was shown to be directly involved in lytic efficiency by supplementation studies with 2 microM iron citrate; such supplementation increased PMN cytotoxicity by approximately 30%, but had much less effect on erythrocyte lysis by neutroplasts (approximately 3% increase), and no effect on lysis in the enzymatic oxygen radical-generating systems. These results suggest a critical role for an iron-liganding moiety that is abundantly present in PMN, marginally so in neutroplasts, and not at all in purified enzymatic systems--a moiety that we presume catalyzes very toxic O2 specie generation in the vicinity of juxtaposed erythrocyte targets. The obvious candidate is lactoferrin (LF), and indeed, antilactoferrin IgG, but not nonspecific IgG, reduced PMN cytotoxicity by greater than 85%. Re-adding 10(-8) M pure LF to neutroplasts increased their ability to promote hemolysis by 48.4 +/- 0.9%--to a level near that of intact PMNs. We conclude that O-2 and H2O2 are not sufficient to mediate target cell lysis, but require iron bound to LF, which, in turn, probably generates and focuses toxic O2 radicals, such as OH, to target membrane sites.