Lactose synthetase catalyzes the final step in the biosynthesis of lactose in the mammary gland by the reaction UDP-D-galactose + D-glucose --lactose + UDP.(1) A soluble, partially purified form of the enzyme, found in bovine milk, can be separated by gel filtration into two protein components, designated the A and B proteins. Neither component will catalyze reaction (1) separately, but when they are combined, lactose synthesis is obtained. More recently, the B protein has been found to be identical with the familiar milk protein, a-lactalbumin.2 3 Work in our laboratory has shown that the amino acid sequence of bovine a-LA is very similar to that of hen egg-white lysozyme.4 This homology in primary structure suggests that the structural genes for lysozyme and a-LA have evolved from a relatively recent, common ancestor. The structural similarities also suggest that a-LA may have a conformation quite similar to that established for lysozyme.A It is possible to fit the side chains of bovine a-LA to the lysozyme polypeptide backbone, and thereby generate a structure which retains the major structural features of the lysozyme molecule.6The studies reported here have been designed to define the roles of the A protein and a-LA in lactose synthetase. The separated A protein has been found to be a UDP-galactose: N-acetylglucosamine galactosyltransferase which catalyzes the following reaction: UDP-galactose + N-acetyl-D-glucosamine-N-acetyllactosamine + UDP. (2) Under normal assay conditions, a-LA inhibits this reaction and allows synthesis of lactose in the presence of glucose by reaction (1). Thus, the a-LA modifies the substrate (acceptor) specificity of a galactosyltransferase from NAG to glucose. This appears to be a role hitherto not ascribed to a protein and a-LA may be termed a "specifier" protein. The mechanism by which it effects a change in substrate specificity appears to be complex, but it is possible that it represents a new type of molecular control of a biological reaction. Experimental Methods.-Assay of UDP-galactose: glucose (31--*4 galactosyltransferase (lactose synthetase) and UDP-galactose: N-acetylglucosamine (1--*4 galactosyltransferase (N-acetyllactosamine [NAL]synthetase): Assays were performed by a modification of the method of Babad and Hassid.' NAL synthetase was determined by incubating 100-jiliter mixtures containing 5 Mmoles Tris-HCl, pH 7.4, 4 Mmoles MnC12, 2 /Amoles NAG, 63 m/Amoles [C14] UDP-galactose (10,000 cpm) (Calbiochem), and enzyme for 5-20 min at 37CC. Appropriate controls were included to correct for the small amount of nonspecific hydrolysis of UDP-galactose.
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