An improved quantitative assay of galactose-1-phosphate uridyltransferase activity in erythrocytes based on the determination of glucose 1-phosphate generation.
An improved quantitative method for measuring galactose-1-phosphate uridyltransferase (EC 2.7.7.12, Gal-PUT) activity in erythrocytes was developed based on the detection of glucose 1-phosphate generated under the catalytic influence of the enzyme. This is achieved by incubating the enzyme with galactose 1-phosphate and uridyldiphosphoglucose during 15 min, followed by deproteinisation. The glucose 1-phosphate generated is quantitated subsequently by measuring NADPH formation from added NADP+ in a second incubation step with added phosphoglucomutase (EC 2.7.5.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Gal-PUT activity is calculated from the increment in absorption at 340 nm. Because it is technically a simple assay that is sensitive, specific and not affected by UDPgalactose-4-epimerase (EC 5.1.3.2) activity in the erythrocytal lysates it is suggested to be the method of choice for measuring Gal-PUT activity. Activities in erythrocytes of controls varied from 264 to 556 U/kg hemoglobin; in obligate heterozygotes from 53 to 190 U/kg Hb and in homozygous deficient patients less than 5 U/kg Hb was measured at 37 degrees C.
D004912:Erythrocytes / Q000201:enzymology*; D005693:Galactosemias / Q000201:enzymology; D005958:Glucosephosphates / Q000097:blood*; D006801:Humans; D008722:Methods; D009713:Nucleotidyltransferases / Q000097:blood*; D013056:Spectrophotometry, Ultraviolet; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase / Q000097:blood*
D005958:Glucosephosphates; D009713:Nucleotidyltransferases; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase