Assay of Rat Brain Sialyltransferase Activities on Thin-Layer Plate

Abstract

Abstract A modified method for measuring the activities of glycosyltransferases using high-performance thin-layer chromatographic (HPTLC) plates is described. In this method a mixture of glycosphingolipid acceptors were chromatographed on a silica gel coated plate and were incubated with the enzyme solution and an appropriate radio-active sugar nucleotide donor. Following incubation, the plate was washed with a buffer and the radiolabeled reaction products on the plate were scrapped off and the radioactivities determined using a liquid scintillation counter. Alternatively, the plate could be exposed to an X-ray film to reveal the radioactive products. This method was used to assay the activities of rat brain cytidine 5′-monophosphate-N-acetylneuraminic acid: LacCer-, GM3-, GM1-, or GD3-sialyltransferases. We found that this method was sensitive, fast and reliable and was capable of assaying However, we found that this kind of pretreatment could influence the sialylation reaction, especially when the subst...