RESUMEN
Up to now, only glucocorticoids were thought to act on the renal proximal Na+/H+ exchanger. Using fluorimetric techniques we studied the kinetics of Na+/H+ exchange in brush border vesicles from ADX rats treated with increasing doses of corticosterone (B) and 18-hydroxycorticosterone (18OHB). Significant linear correlations were obtained when the Vmax of each treatment were plotted against log doses. 18OHB exhibits a slightly higher sensitivity than B and log-dose responses were steeper for 18OHB than for B treated rats. Differences between both treatments were highly significant at the 4.8 microg/100 g level, corresponding to the physiological blood level of 18OHB. Physiological doses of both steroids elicited equal Na+/H+ exchange-responses. 18OHB is not a glucocorticoid since even 88 microg/100 g did not promote hepatic glycogen deposition while the same dose of B increases glycogen deposits 3.5-fold. These results demonstrate the importance of the Na+/H+ exchanger as a mediator between corticoid action and H+ transport and that of the non-glucocorticoid 18OHB in this process.
Asunto(s)
18-Hidroxicorticosterona/farmacología , Corticosterona/farmacología , Túbulos Renales Proximales/metabolismo , Adrenalectomía , Animales , Proteínas Portadoras/metabolismo , Relación Dosis-Respuesta a Droga , Glucógeno/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Hígado/metabolismo , Masculino , Microvellosidades/metabolismo , Ratas , Ratas Sprague-Dawley , Simportadores de Cloruro de Sodio-PotasioRESUMEN
A slab gel electrophoresis apparatus with the ability to operate over a pressure range of 10(-3) to 2 kbar is described. The system presented here is an improvement of a previous apparatus (A. A. Paladini, J. L. Silva, and G. Weber, Anal. Biochem. 161, 358-364, 1987). It consists of a flat bed gel, with a significantly enlarged buffer reservoir, which eliminates the requirement of high concentrations of running buffers, and at the same time allows shorter runs, leading to enhanced resolution and reproducibility. The application of the method to the dissociation of the tetramer glycogen phosphorylase a as a function of hydrostatic pressure is described. The flat geometry of the apparatus allows for the first time the analysis of the stability of oligomers and their constituent subunits to chemical denaturation by urea gradient electrophoresis gels at high pressure. Dimeric hexokinase shows a reversible cooperative unfolding transition with a midpoint at 3.8 M urea. In contrast, the monomers unfold at very low urea concentration (< 1.0 M). The observed differences in stability validates oligomerization as an important stabilizing element of the protein structure.
Asunto(s)
Oligopéptidos/química , Conformación Proteica , Pliegue de Proteína , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/análisis , Proteínas Fúngicas/química , Hexoquinasa/análisis , Hexoquinasa/química , Sustancias Macromoleculares , Oligopéptidos/análisis , Fosforilasa a/análisis , Fosforilasa a/química , Fosforilasas/análisis , Fosforilasas/química , Presión , Desnaturalización Proteica , Saccharomyces cerevisiae/química , UreaRESUMEN
This work was aimed at elucidating the effect of thyroid function on the physiology and biochemistry of the islet-cell population within the endocrine pancreas. To this end, we performed a comparative study of the physiochemical properties of islet-cell membranes and of the dynamics of glucose-induced insulin secretion in isolated pancreatic islets prepared from euthyroid i.e. control (C), hypothyroid (H), and thyroxin-supplemented hypothyroid (HT) rats. H rats were obtained by injecting normal rats with 131iodine, while HT rats consisted of H rats treated with thyroxin (T4). Insulin secretion was studied in isolated islets perifused with 3.3 and 16.6 mM glucose. Physicochemical properties of the partially purified islet plasma membranes were assessed by measurements of fluorescence polarization with the fluorophore 1,6-diphenyl-1,3,5-hexatriene (DPH) as a lipidic molecular probe. Insulin output during either the first or second phase of insulin secretion in H islets was significantly lower than in C islets. The slope of the curve in the second phase of insulin secretion was also lesser in H than in C islets, suggesting an additional defect in their velocity of hormone release. T4 administration of H rats reversed the decrease in insulin output to the range found in C islets but was incapable of correcting the defect in the hormone-secretion velocity. Several changes were found in the physicochemical properties of the membranes obtained from H islets as compared to C islets.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Membrana Celular/fisiología , Hipotiroidismo/fisiopatología , Insulina/metabolismo , Islotes Pancreáticos/fisiopatología , Animales , Membrana Celular/química , Fenómenos Químicos , Química Física , Difenilhexatrieno , Polarización de Fluorescencia , Colorantes Fluorescentes , Secreción de Insulina , Masculino , Ratas , Ratas Wistar , Termodinámica , Tiroxina/farmacología , ViscosidadRESUMEN
A method is presented for wavelength calibration of spectrofluorometer monochromators. It is based on the distortion that the characteristic absorption bands of glass filters (holmium or didymium oxide), commonly used for calibration of spectrophotometers, introduce in the emitted fluorescence of fluorophores like indole, diphenyl hexatriene, xylene or rhodamine 6G. Those filters or a well characterized absorber with sharp bands like benzene vapor can be used for the same purpose. The wavelength calibration accuracy obtained with this method is better than 0.1 nm, and requires no modification in the geometry of the spectrofluorometer sample compartment.
Asunto(s)
Calibración , Espectrometría de Fluorescencia/instrumentación , Pesos y MedidasRESUMEN
Cyst(e)ine residues of bovine white-matter proteolipid proteins were characterized in a highly purified preparation. From a total of 10.6 cyst(e)ine residues/molecule of protein, as determined by performic acid oxidation, 2.5-3 thiol groups were freely accessible to iodoacetamide, iodoacetic acid and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), when the proteins were solubilized in chloroform/methanol (C/M) (2:1, v/v). The presence of lipids had no effect on thiol-group exposure. One thiol group available to DTNB in C/M could not be detected when proteolipids were solubilized in the more polar solvent n-butanol. In a C/M solution of purified proteolipid proteins, SDS did not increase the number of reactive thiol groups, but the cleavage of one disulphide bridge made it possible to alkylate six more groups. C.d. and fluorescence studies showed that rupture of this disulphide bond changed the protein conformation, which was reflected in partial loss of helical structure and in a greater exposure to the solvent of at least one tryptophan residue. Cyst(e)ine residues were also characterized in the different components [PLP (principal proteolipid protein), DM20 and LMW (low-Mr proteins)] of the proteolipid preparation. Although the numbers of cyst(e)ine residues in PLP and DM20 were similar, in LMW fewer residues were alkylated under four different experimental conditions. The differences, however, are not simply related to differences in Mr.