RESUMEN
4-Pentafluoroethylumbelliferyl-beta-D-glucoside is proposed as an efficient substrate for human leukocyte acid beta-glucosidase. Its synthesis is described. This substrate was compared directly with 4-trifluoromethylumbelliferyl-beta-D-glucoside synthesized by us earlier and with 4-methylumbelliferyl-beta-D-glucoside which is commonly used for acid beta-glucosidase activity assay. The specific activity of acid beta-glucosidase with 4-pentafluoroethylumbelliferyl-beta-D-glucoside was 3- and 8-fold higher than it was with the substrates mentioned above. The kinetic parameters KM and VMAX for human leukocyte acid beta-glucosidase with the three substrates was determined. One possible application of the newly synthesized substrate is its use in the diagnosis of acid beta-glucosidase hereditary deficiency (Gaucher's disease).
Asunto(s)
Colorantes Fluorescentes , Glucósidos/metabolismo , Umbeliferonas/metabolismo , beta-Glucosidasa/metabolismo , Enfermedad de Gaucher/enzimología , Glucósidos/síntesis química , Humanos , Concentración de Iones de Hidrógeno , Himecromona/química , Cinética , Leucocitos/enzimología , Espectroscopía de Resonancia Magnética , Estructura Molecular , Espectrofotometría , Umbeliferonas/síntesis químicaRESUMEN
A new fluorogenic substrate, 4-methylumbelliferyl beta-D-glucosaminide, was used for the assay of acetyl CoA:glucosaminide N-acetyltransferase in chorionic villi, cultured villus cells, and amniocytes. Optimal conditions for the assay and the ranges of enzyme activity were established for the various types of fetal cells. This simple fluorometric assay provides a reliable method for early prenatal diagnosis of Sanfilippo disease type C which is more convenient than current methods using radiolabelled substrates. The method was applied to amniotic fluid cells and fetal fibroblasts from an at-risk pregnancy in which an affected fetus was diagnosed by two-dimensional electrophoresis of glycosaminoglycans in the amniotic fluid.
Asunto(s)
Acetiltransferasas/análisis , Colorantes Fluorescentes , Mucopolisacaridosis III/diagnóstico , Diagnóstico Prenatal , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Acetiltransferasas/metabolismo , Líquido Amniótico/citología , Líquido Amniótico/enzimología , Células Cultivadas , Vellosidades Coriónicas/enzimología , Femenino , Fibroblastos/enzimología , Humanos , Himecromona/análogos & derivados , Himecromona/metabolismo , Mucopolisacaridosis III/enzimología , EmbarazoRESUMEN
Both the alpha- and beta-anomers of 4-methylumbelliferyl-D-glucosaminide were synthesized and shown to be substrates for the lysosomal acetyl-CoA:glucosaminide N-acetyltransferase. Using the beta-anomer, fibroblasts and leukocytes from 11 different Sanfilippo C patients showed < 1% of mean normal N-acetyltransferase activity. Heterozygotes showed intermediate activities. The enzymatic liberation of the fluorochrome from 4-methylumbelliferyl-beta-D-glucosaminide requires the sequential action of the N-acetyltransferase and beta-hexosaminidase. Normal beta-hexosaminidase activity caused complete hydrolysis of the reaction intermediate 4-methylumbelliferyl-beta-D-N-acetylglucosaminide formed by the N-acetyltransferase. In cell extracts with a beta-hexosaminidase deficiency, however, a second incubation in the presence of excess beta-hexosaminidase is needed to avoid underestimation of the N-acetyltransferase activity.
Asunto(s)
Acetiltransferasas/metabolismo , Mucopolisacaridosis III/diagnóstico , Acetiltransferasas/deficiencia , Células Cultivadas , Fibroblastos/enzimología , Fluorometría , Heterocigoto , Humanos , Himecromona/análogos & derivados , Himecromona/metabolismo , Leucocitos/enzimología , Mucopolisacaridosis III/enzimología , Valores de ReferenciaRESUMEN
The following glycosides of 4-trifluoromethylumbelliferone: alpha-D-mannopyranoside, alpha-L-fucopyranoside, alpha-D-glucopyranoside, beta-D-glucopyranoside, alpha-D-galactopyranoside, beta-D-galactopyranoside, alpha-L-iduronide and beta-D-glucuronide were studied. 4-Trifluoromethylumbelliferyl glycosides were shown to be substrates for glycosidases. Some of them were cleaved even better than the corresponding methylumbelliferyl glycosides. 4-Trifluoromethylumbelliferyl glycosides were applied for revealing the corresponding enzyme deficiencies upon diagnosis of Gaucher and Hurler diseases as well as GM1 gangliosidosis and alpha-mannosidosis. 4-Trifluoromethylumbelliferone released after enzymatic hydrolysis of 4-trifluoromethylumbelliferyl glycosides exhibits more contrast yellow fluorescence in UV-light than the blue one of methylumbelliferone upon exposure of enzyme activity on solid supports. Therefore 4-trifluoromethylumbelliferyl glycosides are convenient substrates for revealing glycosidase activity directly in tissue samples, e.g. in placenta, and thus for fast prenatal diagnosis of lysosomal diseases.