RESUMEN
Anglerfish islets were homogenized in 0.25 M sucrose and separated into seven separate subcellular fractions by differential and discontinuous density gradient centrifugation. The objective was to isolate microsomes and secretory granules in a highly purified state. The fractions were characterized by electron microscopy and chemical analyses. Each fraction was assayed for its content of protein, RNA, DNA, immunoreactive insulin (IRI), and immunoreactive glucagon (IRG). Ultrastructural examination showed that two of the seven subcellular fractions contain primarily mitochondria, and that two others consist almost exclusively of secretory granules. A fifth fraction contains rough and smooth microsomal vesicles. The remaining two fractions are the cell supernate and the nuclei and cell debris. The content of DNA and RNA in all fractions is consistent with the observed ultrastructure. More than 82 percent of the total cellular IRI and 89(percent) of the total cellular IRG are found in the fractions of secretory granules. The combined fractions of secretory granules and microsomes consistently yield >93 percent of the total IRG. These results indicate that the fractionation procedure employed yields fractions of microsomes and secretory granules that contain nearly all the immunoassayable insulin and glucagons found in whole islet tissue. These fractions are thus considered suitable for study of proinsulin and proglucagon biosynthesis and their metabolic conversion at the subcellular level.
Asunto(s)
Fraccionamiento Celular/métodos , Glucagón/biosíntesis , Islotes Pancreáticos/metabolismo , Proinsulina/biosíntesis , Animales , Gránulos Citoplasmáticos/metabolismo , ADN/análisis , Peces , Islotes Pancreáticos/análisis , Islotes Pancreáticos/ultraestructura , Microsomas Hepáticos/metabolismo , ARN/análisis , Fracciones Subcelulares/análisis , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/ultraestructuraRESUMEN
Anglerfish proinsulin and insulin were selectively labeled with [(14)C]isoleucine, while proglucagon, conversion intermediate(s), and glucagon were selectively labeled with[(3)H]tryptophan. After various periods of continuous or pulse-chase incubation, islet tissue was subjected to subcellular fractionation. Fraction extracts were analyzed by gel filtration for their content of precursor, conversion intermediate(s), and product peptides. Of the seven subcellular fractions prepared after each incubation, only the microsome and secretory granule fractions yielded significant amounts of labeled insulin-related and glucagon-related peptides. After short-pulse incubations, levels of both [(14)C]proinsulin and [(3)H]proglucagon (mol wt approximately 12,000) were highest in the microsome fraction. This fraction is therefore identified as the site of synthesis. With increasing duration of continuous incubation or during chase incubation in the absence of isotopes, proinsulin, proglucagon, and conversion intermediate(s) are transported to secretory granules. Conversion of proinsulin to insulin and proglucagon to a approximately 4,900 mol wt conversion intermediate and 3,500 mol wt glucagon occurs in the secretory granules. Converting activity also was observed in the microsome fraction. The recovery of most of the incorporated radioactivity in microsome and secretory granule fractions indicates that the newly synthesized islet peptides are relegated to a membrane-bound state soon after synthesis at the RER is completed. This finding supports the concept of intracisternal sequestration and intragranular maintenance of peptides synthesized for export from the cell of origin.
Asunto(s)
Glucagón/biosíntesis , Insulina/biosíntesis , Islotes Pancreáticos/metabolismo , Proinsulina/biosíntesis , Animales , Radioisótopos de Carbono , Fraccionamiento Celular , Gránulos Citoplasmáticos/metabolismo , Peces , Isoleucina , Microsomas Hepáticos/metabolismo , Biosíntesis de Péptidos , Proinsulina/metabolismo , Precursores de Proteínas/metabolismo , Fracciones Subcelulares/metabolismo , Tritio , TriptófanoRESUMEN
A cytochemical technique for localizing cytochrome oxidase activity, based upon the oxidative polymerization of 3,3-diaminobenzidine (DAB) to an osmiophilic reaction product, has been employed to test the possibility of extramitochondrial cytochrome oxidase in gastric mucosa. Deposition of reaction product was found to be exclusively localized within mitochondris; in particular, no reaction product was observed at the apical plasma membrane. Measurements of the effect of DAB on acid secretion revealed a biphasic action consisting of an initial stimulation followed by inhibition. The stimulation of secretion of DAB is interpreted to indicate that DAB is oxidized via a pathway which is linked to the secretory process. The combined cytochemical and physiological measurements provide evidence that the metabolic energy supply for acid secreation is derived from mitochondrial reactions. The results are discussed in relation to current models for the coupling between acid secreation and oxidative metabolism.
Asunto(s)
Complejo IV de Transporte de Electrones/análisis , Mucosa Gástrica/enzimología , Animales , Bencidinas/farmacología , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/ultraestructura , Histocitoquímica , Humanos , Microscopía Electrónica , Mitocondrias/ultraestructura , Consumo de Oxígeno , Rana catesbeiana , Coloración y EtiquetadoRESUMEN
Several cases of arterial thrombosis associated with inflammatory enterocolitis affections have been reported. In all but one case reported in the literature the outcome was favorable. A 40 year old woman developed thrombosis of the radial and ulnar arteries during an acute episode of ulcerative colitis. The course of the thrombotic lesions was irreversible and all fingers of the right hand had to be amputated. The corticotherapy as a possible predisposing factor for these thromboses is discussed, but their onset during an inflammatory episode suggests a role for the inflammatory enterocolitis. A thrombocytosis (807,000 platelets/mm3) could have predisposed to their onset, and in this case the hemostatic disturbances and fibrinolysis associated with the ulcerative colitis could have played a major role.