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Query: EC:2.4.99.6 (
sialyltransferase
)
1,546
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
Two novel assay systems for the determination of
sialyltransferase
activity using a tetramethylrhodamine-labeled disaccharide Galbeta1-4GlcNAc (2) as the acceptor are described. The TMR-labeled disaccharide 2 was synthesized by directly coupling Galbeta1-4GlcNAc-O-(CH(2))(6)NH(2) (1) with 5-tetramethylrhodamine
N-hydroxysuccinimide ester
. The K(m) value for compound 2 obtained with alpha-2,6-
sialyltransferase
from rat liver (EC 2.4.99.1) was 160 +/- 20 microM. After incubation of compound 2 with
sialyltransferase
the product and the unreacted acceptor substrate were separated either by thin-layer chromatography (TLC) on C-18 silica gel plates or by polyacrylamide gel electrophoresis (PAGE). The density of the spots on the TLC plates and the fluorescence of the bands on the gel were quantified. The assay conditions were optimized using crude bovine colostrum extract and also alpha-2, 6-
sialyltransferase
from rat liver. The detection limits for the TLC and PAGE assays were 1 and 0.4 microU of the rat liver enzyme, respectively. Either assay allows the parallel investigation of several samples at a time and is useful for the testing of fractions during enzyme purification.
...
PMID:Thin-layer chromatography and polyacrylamide gel electrophoresis-based assays for sialyltransferases using tetramethylrhodamine-labeled acceptors. 1099 67
Ganglioside GM1 and its seven potential catabolic products: asialo-GM1, GM2, asialo-GM2, GM3, Lac-Cer, Glc-Cer and Cer, were labeled with tetramethylrhodamine (TMR) to permit ultra-sensitive analysis using laser-induced fluorescence (LIF) detection. The preparation involved acylation of the homogenous C(18)lyso-forms of GM1, Lac-Cer, Glc-Cer and Cer with the
N-hydroxysuccinimide ester
of a beta-alanine-tethered 6-TMR derivative, followed by conversion of these labeled products using galactosidase, sialidase, and
sialyltransferase
enzymes. The TMR-glycolipid analogs produced are detectable on TLC down to the 1 ng level by the naked eye. All eight compounds could be separated within 4 min in capillary electrophoresis where they could be detected at the zeptomole (ca. 1000 molecule) level using LIF.
...
PMID:Synthesis of reference standards to enable single cell metabolomic studies of tetramethylrhodamine-labeled ganglioside GM1. 1706 78
