A method for galactose-1-phosphate uridyltransferase assay and the separation of its isozymes by DEAE-cellulose column chromatography.
A simplified radioactive assay for galactose-1-phosphate uridyltransferase (EC 2.7.7.12) using a small DEAE-cellulose column for the identification of the endproduct (uridine diphosphate galactose) is described. The enzyme activities in red blood cell hemolysates of normal subjects are in the range of 24 to 33 (average 30.3) mumol UDPgalactose produced per g hemoglobin per h and in fibroblasts 0.39 and 1.39 (average 0.71) nmol per mg protein per min. Furthermore, different isozymes of red blood cell galactose-1-phosphate uridyltransferase were separated on DEAE-cellulose columns. In the case of a normal genotype, most of the enzyme activity is eluted at the earlier fractions with the low molar phosphate buffer, whereas the Duarte variant appeared at later fractions with higher molar phosphate buffer.
D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D004912:Erythrocytes / Q000201:enzymology; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010375:Pedigree; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase / Q000302:isolation & purification / Q000378:metabolism*
D007527:Isoenzymes; D009713:Nucleotidyltransferases; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase