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.
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism* / Q000494:pharmacology; D066298:In Vitro Techniques; D008214:Lymphocytes / Q000201:enzymology; D051381:Rats; D012794:Sialic Acids / Q000378:metabolism*; D012799:Sialyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D013154:Spleen / Q000201:enzymology; D013347:Subcellular Fractions / Q000201:enzymology; D014166:Transferases / Q000302:isolation & purification*
D012794:Sialic Acids; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D014166:Transferases; D012799:Sialyltransferases