Detection of ectosiallyltransferase activity using whole cells. Correction of misleading results due to the release of intracellular CMP-N-acetylneuraminic acid.

An inhibitory effect due to broken cells is observed when sialyltransferase (CMP-N-acetylneuraminate:D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) is measured with mixture of intact and homogenized lymphocytes. This intracellular inhibitory factor ib purified and characterized as CMP-N-acetylneuraminic acid (CMP-NeuNAc) by its behavior in various chromatographic and electrophoretic systems and by its susceptibility to CMP-NeuNAc hydrolase. This endogenous CMP-NeuNAc leads to an isotopic dilution of the exogenous labelled CMP-NeuNAc explaining the apparently lower activity of homogenate when compared to whole cells. Consequently, the radioactivity bound to acceptors may not be related to a known number of sialyl residues transferred, calling into question the validity of comparing the incorporation of [14C]NeuNAc by homogenate and whole cells in order to assign sialyltransferase activity to ectoenzyme. A new approach is developed to detect ectoglycosyltransferases with whole cells, taking into account that both intracellular enzymes and endogenous precursor may be introduced by the small percentage of broken cells.