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1.
Methods Mol Biol ; 347: 321-30, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-17072020

RESUMEN

This chapter describes in detail a practical procedure for the preparation of radiolabeled galactocerebroside and its use in the assay of galactocerebrosidase (GalCase), the enzyme deficient in globoid cell leukodystrophy (Krabbe disease). The reference range for leukocytes and fibroblasts is 0.9-4.4 and 8-36 nmoles substrate hydrolyzed per hour per milligram of protein, respectively. Because of its low abundance this enzyme is difficult to assay in certain situations, such as prenatal diagnosis by chorionic villus sampling. To obviate this a modified assay is used where only the radiolabeled substrate is included in the incubation. This provides a clear separation between affected samples and unaffected controls. The methods detailed here should be reproducible in any laboratory.


Asunto(s)
Galactosilceramidasa/análisis , Galactosilceramidas/metabolismo , Leucodistrofia de Células Globoides/diagnóstico , Células Cultivadas , Femenino , Fibroblastos , Galactosa Oxidasa/química , Galactosa Oxidasa/metabolismo , Galactosilceramidas/química , Humanos , Leucodistrofia de Células Globoides/metabolismo , Oxidación-Reducción , Embarazo , Diagnóstico Prenatal/métodos , Tritio
2.
Biochim Biophys Acta ; 1588(3): 247-53, 2002 Dec 12.
Artículo en Inglés | MEDLINE | ID: mdl-12393180

RESUMEN

Primary deficiency of beta-galactosidase results in GM1 gangliosidosis and Morquio B disease. Of the more than 40 disease-causing mutations described in the Gal gene to date, about 75% are of the missense type and are scattered along the length of the gene. No single, major common mutation has been associated with GM1 gangliosidosis. However, a Trp 273 Leu mutation has been commonly found in the majority of patients with Morquio B disease defined genotypically to date. We now report three new mutations in three Morquio B patients where the Trp 273 Leu mutation is absent. Two of the mutations, C1502G (Asn 484 Lys) and A1548G (Thr 500 Ala), were found in twins (one male, one female) who display a mild form of Morquio B disease and keratan sulfate in the urine. In their fibroblasts, residual activity was 1.9% and 2.1% of controls. On Western blots, the 84-kDa precursor and the 64-kDa mature protein were barely detectable. The occurrence of a 45-kDa degradation product indicates that the mutated protein reached the lysosome but was abnormally processed. In the third case, we identified only a G1363A (Gly 438 Glu) mutation (a major deletion on the second allele has not been ruled out). This female patient too displays a very mild form of the disease with a residual activity of 5.7% of control values. In fibroblasts from this case, the 84-kDa precursor and the 45-kDa degradation product were present, while the mature 64-kDa form was barely detectable. The occurrence of these three mutations in the same area of the protein may define a domain involved in keratan sulfate degradation.


Asunto(s)
Enfermedades en Gemelos/genética , Mucopolisacaridosis IV/genética , Mutación , beta-Galactosidasa/genética , Niño , Femenino , Fibroblastos/enzimología , Humanos , Leupeptinas , Masculino , Mucopolisacaridosis IV/metabolismo , Mucopolisacaridosis IV/patología , Reacción en Cadena de la Polimerasa , Gemelos Dicigóticos/genética , beta-Galactosidasa/análisis
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