GALACTOSE-1-PHOSPHATE URIDYLTRANSFERASE IN CULTURED-CELLS

A method to measure the enzyme galactose-1-phosphate [Gal-1-P] uridyltransferase [EC 2.7.7.12] in cultured cells is described. The optimun pH was 8.7 and no enzyme activity was found without preincubation with dithiothreitol. The Km values for Gal-1-P and UDPG [UDP glucose] for the wild type enzyme were 0.2 mM and 0.08 mM, respectively. Values for the Duarte variant were identical. No significant change in enzyme activity with time after subculture was found in either cultured skin fibroblasts or cultured amniotic fluid cells. Different transferase genotypes were clearly distinguished and reference range established. Transferase levels found in normal cultured amniotic fluid cells were the same as in normal cultured skin fibroblasts. The result of a prenatal diagnosis was obtained within 3 wk of amniocentesis.