GLYCOSYLTRANSFERASE LUMINESCENCE ASSAY: A NEW TECHNOLOGY

ALTERATIONS IN GLYCOSYLTRANSFERASE ACTIVITIES HAVE BEEN CORRELATED WITH CERTAIN PATHOLOGICAL CONDITIONS AND DISEASESIN ANIMALS. THE CURRENT TECHNOLOGY FOR ASSAYING THESE ENZYMES IS NOT READILY ADAPTABLE FOR LARGE-SCALE CLINICAL SCREENING AND DIAGNOSTICS. IN THESE STUDIES, A NEW AND GENERAL METHOD WILL BE DEVELOPED FOR ASSAYING GLYCOSYLTRANSFERASES. THIS NEW TECHNIQUE IS RAPID, 3 TO 6 ORDERS OF MAGNITUDE MORESENSITIVE THAN EXISTING ASSAYS, AND DOES NOT RELY ON THE USEOF 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(BETA1,4) GALACTOSYLTRANSFERASE. THE SPECIFIC AIMS OF THESE STUDIES ARE: (1) TO DEVELOP ELA'S FOR ADDITIONAL GLYCOSYLTRANSFERASES, INCLUDING UDPGAL:GALBETA1,4GLC(NAC)(ALPHA1,3)GALACTOSYLTRANSFERASE AND CMPNEUAC:GALBETA1,3(4)GLCNAC(ALPHA2,3)SIALYLTRANSFERASE;AND (2) TO ADAPT ELA'S TO A MICROTITER PLATE ASSAY. THE LONG-TERM GOAL IS TO DEVELOP ELA'S FOR A WIDE VARIETY OF GLYCOSYLTRANSFERASES TO ALLOW CLINICAL EVALUATION OF GLYCOSYLTRANSFERASE ACTIVITIES AS DIAGNOSTIC MARKERS.