Purification of normal and inactive galactosemic galactose-1-phosphate uridylyltransferase from human red cells.
A rapid method is described for the purification of galactose-1-phosphate uridylyltransferase (EC 2.7.7.12) from human red blood cells by the use of DEAE-cellulose and two steps of affinity chromatography on a "uridine-aminohexyl" agarose column. The enzyme consists of two identical subunits of 31,000 molecular weight as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Chromatography on Sephadex G-200 chromatography gave a molecular weight of 69,000 for the native enzyme, it being eluted from the column with bovine serum albumin. Cross-linking of the enzyme with dimethylsuberimidate followed by analysis of the products by sodium dodecyl sulfate polyacrylamide gel electrophoresis caused the near-disappearance of the 31,000 molecular weight subunit and the appearance of a protein with a molecular weight of about 65,000 without the appearance of higher polymers.
D000596:Amino Acids / Q000032:analysis; D004120:Dimethyl Suberimidate; D004912:Erythrocytes / Q000201:enzymology*; D005693:Galactosemias / Q000201:enzymology*; D006801:Humans; D005779:Immunodiffusion; D046911:Macromolecular Substances; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000097:blood*; D010446:Peptide Fragments / Q000032:analysis; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification
D000596:Amino Acids; D046911:Macromolecular Substances; D010446:Peptide Fragments; D004120:Dimethyl Suberimidate; D009713:Nucleotidyltransferases; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase