TY - JOUR
T1 - Direct assay of glycosphingolipid glycosyltransferase activities on thin-layer chromatogram
AU - Gu, Xinbin
AU - Gu, Tianjue
AU - Yu, Robert K.
N1 - Copyright:
Copyright 2014 Elsevier B.V., All rights reserved.
PY - 1990/2/15
Y1 - 1990/2/15
N2 - A modified method for the determination of glycosphingolipid glycosyltransferase activity using high-performance thin-layer chromatographic (HPTLC) plates has been developed. An acceptor glycosphingo-lipid 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.
AB - A modified method for the determination of glycosphingolipid glycosyltransferase activity using high-performance thin-layer chromatographic (HPTLC) plates has been developed. An acceptor glycosphingo-lipid 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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U2 - 10.1016/0003-2697(90)90271-A
DO - 10.1016/0003-2697(90)90271-A
M3 - Article
C2 - 2344039
AN - SCOPUS:0025165133
SN - 0003-2697
VL - 185
SP - 151
EP - 155
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -