Direct assay of glycosphingolipid glycosyltransferase activities on thin-layer chromatogram.
Anal Biochem
; 185(1): 151-5, 1990 Feb 15.
Article
em En
| MEDLINE
| ID: mdl-2344039
ABSTRACT
A modified method for the determination of glycosphingolipid glycosyltransferase activity using high-performance thin-layer chromatographic (HPTLC) plates has been developed. An acceptor glycosphingolipid was chromatographed on an HPTLC plate and was incubated with an enzyme mixture and an appropriate radioactive sugar nucleotide. After incubation, the plate was washed with phosphate buffer and 2% Tween 80. The radiolabeled reaction product was scrapped off the plate and the radioactivity determined using a liquid scintillation counter or, alternatively, the plate was exposed to an X-ray film to reveal the radioactive product. We have used this assay method to determine the activities of rat brain cytidine 5'-monophosphate-N-acetylneuraminic acid LacCer-, GM3-, GM1-, or GD3-sialyltransferases. This method is sensitive, fast, and reliable and is capable of assaying simultaneously the activities of glycosyltransferases with multiple acceptor specificity. It should be useful in monitoring the enzyme activities present in various column fractions during chromatographic fractionation of glycosyltransferases with different substrate specificities.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Sialiltransferases
/
Glicoesfingolipídeos
/
Cromatografia em Camada Fina
Limite:
Animals
Idioma:
En
Revista:
Anal Biochem
Ano de publicação:
1990
Tipo de documento:
Article