Alterations in glycosyltransferase activities have been correlated with certain pathological conditions and diseases in animals. The current technology for assaying these enzymes is not readily adaptable for large scale clinical screening and diagnostics. In the studies proposed a new and general method will be developed for assaying glycosyltransferases. This new technique is rapid, 3 to 6 orders of magnitude more sensitive than existing assays and does not rely on the use of radioisotopes. This new technology, designated Enzyme Luminescence Assay (ELA), utilizes the remarkable sensitivity of the recombinant, bioluminescent protein aequorin and the high affinity interactions of streptavidin and biotin. ELA has been used to determine the activity of UDPGal:beta-D- GlcNAc(beta 1,4)galactosyltransferase. The two specific aims of the proposed studies are: (i) develop ELA's for additional glycosyltransferases, including UDPGal:Gal beta 1,4Glc(NAc) (alpha 1,3) galactosyltransferase and CMPNeuAc:Gal beta 1,3(4)GlcNAc (alpha 2,3) sialyltransferase; and (ii) adapt ELA's to a microtiter plate assay. The long term goal of this research is to develop ELA's for a wide variety of glycosyltransferases to allow clinical evaluation of glycosyltransferase activities as diagnostic markers.
