RESUMEN
Application of genetic complementation procedure for evaluation of heterogeneity and development of a number of human inherited impairments, particularly, lysosomal storage diseases, are reviewed. The genetic complementation procedure is involved in cytobiochemical diagnosis of a number of enzymopathies as well as in studies of subunit containing enzymes reconstruction and their function.
Asunto(s)
Prueba de Complementación Genética , Errores Innatos del Metabolismo/genética , Animales , Humanos , Células Híbridas , Errores Innatos del Metabolismo/diagnóstico , Enfermedad de Sandhoff/diagnóstico , Enfermedad de Sandhoff/genética , Enfermedad de Tay-Sachs/diagnóstico , Enfermedad de Tay-Sachs/genéticaRESUMEN
It was shown that human alpha-D-galactosidase is represented by multiple forms, only one of which can also split alpha-D-fucoside. Fabry's disease was found to be associated not only with the deficiency of the alpha-D-galactosidase total activity but also with the deficiency of the alpha-D-fucosidase activity. The decrease in the alpha-D-galactosidase activity is due to the lack of two enzyme forms, while the profile of alpha-D-fucosidase multiple forms during isoelectric focusing of human enzyme preparations is modified very little in comparison with the normal one. The deficiency of both enzymes was expressed in most degree in leukocytes as compared to other tissues. The residual activities of alpha-D-galactosidase and alpha-D-fucosidase in leukocytes were equal to 3.5 and 21%, respectively. Since the decrease in the alpha-D-fucosidase activity was not so noticeable as in the alpha-D-galactosidase activity, it may be expected that the determination of the alpha-D-fucosidase activity can no longer be regarded as a reliable test for the diagnosis of Fabry's disease. The data obtained suggest that alpha-D-galactoside and alpha-D-fucoside are split by the same enzyme, the multiple forms of which are characterized by selective specificity towards these substrates.
Asunto(s)
Enfermedad de Fabry/enzimología , Galactosidasas/metabolismo , Isoenzimas/metabolismo , alfa-Galactosidasa/metabolismo , alfa-L-Fucosidasa/metabolismo , Glicósidos/síntesis química , Humanos , Himecromona/análogos & derivados , Himecromona/síntesis química , Focalización Isoeléctrica , Riñón/enzimología , Leucocitos/enzimología , Hígado/enzimología , Bazo/enzimología , Especificidad por SustratoRESUMEN
This method is based on Coomassie Brilliant Blue G-250 binding with proteins. The mechanism of protein molecule binding with the stain is described. The authors emphasize high sensitivity of this simple and rapid method and discuss its advantages and shortcomings vs. Lowry's method. The range of laboratory application of the method is considered.
Asunto(s)
Proteínas/análisis , Humanos , Métodos , Coloración y EtiquetadoRESUMEN
Three fractions of glycolipids--monohexosylceramide, dihexosylceramide (DHC) and trihexosylceramide (THC) were isolated from kidney of patient with Fabry disease. As compared with normal state amount of DHC and THC was increased in the patient kidney 9-19-fold and 15-26-fold, respectively. Gas liquid chromatography showed that the DHC fraction consisted in digalactosylceramide, while the THC fraction--a mixture of digalactosylglucosylceramide (90%) and trigalactosylceramide (10%). Presence of the latter glycolipid was not early found in human body both in normal state and in Fabry disease. Accumulation of DHC and THC was also detected in urine precipitates of the patients using thin-layer chromatography, whereas these substances were not found in urine of one of the patients daughter, who was heterozygote gene carrier of Fabry disease. The data obtained corroborate the Fabry disease presence, which have been predetermined by means of clinical diagnosis as well as basing on deficiency of alpha-D-galactosidase in blood plasma and leukocytes.