Lactose Synthesis in the Mammary Gland: Lactose Synthase and the Work of Robert L. Hill
Year: 2006
Type: article
Abstract: The Complete Amino Acid Sequence of Bovine α-LactalbuminJournal of Biological ChemistryVol. 245Issue 17PreviewThe complete amino acid sequence of bovine α-lactalbumin has been established. The unique sequence was deduced by characterization of the tryptic, chymotryptic, and peptic peptides isolated from enzymic hydrolysates of the two unique fragments obtained on cleavage of S-aminoethyl-α-lactalbumin with cyanogen bromide as described earlier. Important overlapping peptides were also obtained by sequence analysis of peptic and chymotryptic peptides isolated from unmodified or S-carboxymethyl-α-lactalbumin. Full-Text PDF Open AccessThe Disulfide Bonds of Bovine α-LactalbuminJournal of Biological ChemistryVol. 245Issue 17PreviewThe four disulfide bonds formed by the 8 half-cystinyl residues in α-lactalbumin have been established by sequence analysis of peptides isolated from enzymic digests. Two unique disulfide-containing peptides were isolated by chromatography from peptic digests of α-lactalbumin. A third peptic peptide contained 4 half-cystinyl residues. Further degradation of this peptide with thermolysin gave two additional, unique disulfide-containing peptides that were readily separated by column chromatography. Full-Text PDF Open AccessThe Purification and Properties of the A Protein of Lactose SynthetaseJournal of Biological ChemistryVol. 246Issue 21PreviewThe A protein of lactose synthetase has been purified from bovine skim milk. After chromatography on DEAE-Sephadex and cellulose phosphate, the active fractions were purified to constant specific activity by chromatography on columns of α-lactalbumin attached covalently to Sepharose. The A protein was bound specifically to this column in buffers containing glucose and was eluted by omitting glucose from the developing buffer. These highly purified preparations are inactivated easily but can be stabilized by N-acetylglucosamine or by bovine serum albumin. Full-Text PDF Open Access The Complete Amino Acid Sequence of α-Lactalbumin (Brew, K., Castellino, F. J., Vanaman, T. C., and Hill, R. L. (1970) J. Biol. Chem. 245, 4570–4582) The Disulfide Bonds of Bovine α-Lactalbumin (Vanaman, T. C., Brew, K., and Hill, R. L. (1970) J. Biol. Chem. 245, 4583–4590) The Purification and Properties of the A Protein of Lactose Synthetase (Trayer, I. P., and Hill, R. L. (1971) J. Biol. Chem. 246, 6666–6675) Robert L. Hill was born in Kansas City, Missouri in 1928. He earned a B.A. in chemistry in 1949 and a Ph.D. in biochemistry in 1954 from the University of Kansas. After receiving his Ph.D., he went to the University of Utah as a National Institutes of Health (NIH) postdoctoral fellow, where he worked with Emil L. Smith, who was featured in two Journal of Biological Chemistry (JBC) Classics (1Smith E.L. Bergmann M. JBC ClassicsJ. Biol. Chem. 1944; 153: 627-651http://www.jbc.org/cgi/content/full/279/47/e6Abstract Full Text PDF Google Scholar, 2DeLange R.J. Fambrough D.M. Smith E.L. Bonner J.J. JBC ClassicsJ. Biol. Chem. 1969; 244: 5669-5679http://www.jbc.org/cgi/content/full/280/36/e33Abstract Full Text PDF PubMed Google Scholar). Although Hill's Ph.D. thesis research concerned bacterial metabolism, his postdoctoral studies introduced him to protein and enzyme chemistry, research areas he pursued during his subsequent career. At Utah, he published papers on proteolytic enzymes and human hemoglobin and myoglobin. Hill remained at the University of Utah as a faculty member of the Department of Biochemistry until 1961. He then joined the faculty of the Department of Biochemistry at Duke University School of Medicine, where he remains today. At Duke, Hill continued his work on human hemoglobins, including several abnormal variants, but soon turned his attention to the structure-function relationships of other proteins and enzymes, including human fibrinogen and other blood coagulation factors, immunoglobulins, egg white lysozyme, bacterial acyl carrier protein, and lactose synthase. In subsequent years he studied glycosyltransferases and worked in several areas of glycobiology. Hill's work on lactose synthase is the subject of the three JBC Classics reprinted here. Lactose synthase consists of a catalytic galactosyltransferase in the endoplasmic reticulum of the mammary gland and a regulatory protein, α-lactalbumin, secreted in milk. Without α-lactalbumin, galactosyltransferase cannot synthesize lactose and instead catalyzes the attachment of galactose to N-acetylglucosamine units on glycoproteins. The presence of α-lactalbumin changes the specificity of galactosyltransferase so that it can transfer galactose to glucose. In the first JBC Classic, Hill, along with Keith Brew, Francis J. Castellino, and Thomas C. Vanaman, reports the complete amino acid sequence of bovine α-lactalbumin. They deduced the sequence by characterizing the tryptic, chymotryptic, and peptic peptides of α-lactalbumin cleaved with cyanogen bromide. Because the amino acid sequence of α-lactalbumin is very similar to that of egg white lysozyme, Hill and his colleagues aligned the two sequences and found that 49 of the residues were identical and 23 were conservative replacements. Thus, they concluded that the three-dimensional structures of the two proteins were probably very similar (3Browne W.J. North A.C. Phillips D.C. Brew K. Vanaman T.C. Hill R.L. A possible three-dimensional structure of bovine α-lactalbumin based on that of hen's egg-white lysozyme.J. Mol. Biol. 1969; 42: 65-86Crossref PubMed Scopus (411) Google Scholar) and that they most likely arose from a common ancestral gene. Hill, Brew, and Vanaman confirmed this structural similarity in the second Classic in which they describe the locations of the four disulfide bonds in α-lactalbumin. They found that the disulfide bonds are arranged in a manner similar to those in egg white lysozyme and proposed a three-dimensional structure for α-lactalbumin based on the three-dimensional structure of lysozyme, as shown in Fig. 2. Later, Hill, Brew, and Vanaman showed how α-lactalbumin serves as a regulatory protein for lactose synthase and permits the enzyme to synthesize lactose in the mammary gland. Fig. 2View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the final Classic reprinted here, Hill and Ian P. Trayer describe the purification of the galactosyltransferase component of lactose synthase or what they refer to as the “A protein.” A key step to their purification was the use of columns of α-lactalbumin attached covalently to Sepharose. In the presence of glucose, galactosyltransferase would bind to the column and could then be removed by omission of glucose from the elution buffer. Using this method, Hill and Trayer were able to purify galactosyltransferase from bovine milk about 12,000-fold. In 1969 Hill became the chairman of the Department of Biochemistry, and in 1974, the James B. Duke Professor. He served as chairman until 1993. He was President (1976) and Secretary (1972–1975) of the American Society of Biological Chemistry and was also on the Editorial Board (1965–1970, 1972–1977) and an Associate Editor (1988-present) of the JBC. Hill served on the FASEB Board (1972–1978), was General Secretary of the International Union of Biochemistry (1982–1991), and chair of the Organizing Committee of the 17th International Congress of Biochemistry and Molecular Biology in San Francisco in 1997. He was elected to the National Academy of Sciences in 1975, the Institute of Medicine in 1978, and the American Academy of Arts and Sciences in 1974. Hill received the Rose Award from the American Society for Biochemistry and Molecular Biology in 1991, the North Carolina Gold Medal (Science-1985), and the Karl Meyer Award from the Society for Glycobiology (2001). Each of the coauthors on these Classic papers was a student or postdoctoral fellow in Hill's laboratory, and all have had productive, independent careers in biochemistry. Thomas Vanaman was a Ph.D. student who subsequently became Professor of Biochemistry and Chair at the University of Kentucky. The others were postdoctoral fellows. Keith Brew went to the University of Leeds (UK) before returning to the U. S. where he became Professor of Biochemistry at the University of Miami. He is now at Florida Atlantic University. Francis J. Castellino is currently Professor of Biochemistry at the University of Notre Dame, and Ian P. Trayer was Professor and Head of the School of Chemistry at the University of Birmingham (UK). Brew and Castellino served on the JBC editorial board. Vanaman also served on the JBC board and currently is an Associate Editor.
Source: Journal of Biological Chemistry
Authors: Nicole Kresge, Robert D. Simoni
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W1504187046