Studies on the thiol group of lactose synthetase A protein from human milk and on the binding of uridine diphosphate galactose to the enzyme.
The lactose synthetase activity of A protein from human milk was much decreased but not abolished by reaction with thiol-group reagents. Protection experiments indicated that a free thiol group on the enzyme is situated near the UDP-galactose binding site and inactivation of the enzyme with p-hydroxymercuribenzoate was probably due to prevention of UDP-galactose binding. Affinity chromatography showed that the mercuribenzoate substituent also decreased the affinity of A protein for N-acetylglucosamine but complex-formation between A protein-N-acetylglucosamine and alpha-lactalbumin was relatively unaffected. UDP-galactose appears to be bound to the enzyme mainly through its pyrophosphate group with Mn(2+) ion and through the cis hydroxyls of ribose, whereas its hexose moiety has little if any affinity for the enzyme. Lactose synthetase activity remaining after the reaction with thiol-group reagents indicates that a free thiol group is not an essential part of the A protein active site.
D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D011756:Diphosphates / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D005944:Glucosamine / Q000378:metabolism; D006801:Humans; D007768:Lactalbumin / Q000378:metabolism; D007788:Lactose Synthase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008626:Mercuribenzoates; D008895:Milk, Human / Q000201:enzymology*; D011485:Protein Binding; D012266:Ribose / Q000378:metabolism; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*
D011756:Diphosphates; D008626:Mercuribenzoates; D013438:Sulfhydryl Compounds; D014539:Uridine Diphosphate Sugars; D008345:Manganese; D012266:Ribose; D007768:Lactalbumin; D007788:Lactose Synthase; D005944:Glucosamine; D005690:Galactose