Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1974 Jul;141(1):173–178. doi: 10.1042/bj1410173

Studies on the thiol group of lactose synthetase A protein from human milk and on the binding of uridine diphosphate galactose to the enzyme

B J Kitchen 1, P Andrews 1
PMCID: PMC1168064  PMID: 4375968

Abstract

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 α-lactalbumin was relatively unaffected. UDP-galactose appears to be bound to the enzyme mainly through its pyrophosphate group with Mn2+ 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.

Full text

PDF
173

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andrews P. Estimation of molecular size and molecular weights of biological compounds by gel filtration. Methods Biochem Anal. 1970;18:1–53. [PubMed] [Google Scholar]
  2. Andrews P., Kitchen B. J., Winzor D. J. Use of affinity chromatography for the quantitative study of acceptor-ligand interactions: The lactose synthetase system. Biochem J. 1973 Dec;135(4):897–900. doi: 10.1042/bj1350897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Andrews P. Purification of lactose synthetase a protein from human milk and demonstration of its interaction with alpha-lactalbumin. FEBS Lett. 1970 Aug 31;9(5):297–300. doi: 10.1016/0014-5793(70)80382-9. [DOI] [PubMed] [Google Scholar]
  4. Babad H., Hassid W. Z. Soluble uridine diphosphate D-galactose: D-glucose beta-4-D-galactosyltransferase from bovine milk. J Biol Chem. 1966 Jun 10;241(11):2672–2678. [PubMed] [Google Scholar]
  5. Barker R., Olsen K. W., Shaper J. H., Hill R. L. Agarose derivatives of uridine diphosphate and N-acetylglucosamine for the purification of a galactosyltransferase. J Biol Chem. 1972 Nov 25;247(22):7135–7147. [PubMed] [Google Scholar]
  6. Barman T. E. Purification and properties of bovine milk glyco-alpha-lactalbumin. Biochim Biophys Acta. 1970 Jul 27;214(1):242–244. doi: 10.1016/0005-2795(70)90094-2. [DOI] [PubMed] [Google Scholar]
  7. Brew K. Lactose synthetase: evolutionary origins, structure and control. Essays Biochem. 1970;6:93–118. [PubMed] [Google Scholar]
  8. Brew K., Vanaman T. C., Hill R. L. The role of alpha-lactalbumin and the A protein in lactose synthetase: a unique mechanism for the control of a biological reaction. Proc Natl Acad Sci U S A. 1968 Feb;59(2):491–497. doi: 10.1073/pnas.59.2.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brodbeck U., Denton W. L., Tanahashi N., Ebner K. E. The isolation and identification of the B protein of lactose synthetase as alpha-lactalbumin. J Biol Chem. 1967 Apr 10;242(7):1391–1397. [PubMed] [Google Scholar]
  10. Brodbeck U., Ebner K. E. Resolution of a soluble lactose synthetase into two protein components and solubilization of microsomal lactose synthetase. J Biol Chem. 1966 Feb 10;241(3):762–764. [PubMed] [Google Scholar]
  11. COHN M., HUGHES T. R., Jr Nuclear magnetic resonance spectra of adenosine di- and triphosphate. II. Effect of complexing with divalent metal ions. J Biol Chem. 1962 Jan;237:176–181. [PubMed] [Google Scholar]
  12. ELLMAN G. L. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959 May;82(1):70–77. doi: 10.1016/0003-9861(59)90090-6. [DOI] [PubMed] [Google Scholar]
  13. Fitzgerald D. K., Brodbeck U., Kiyosawa I., Mawal R., Colvin B., Ebner K. E. Alpha-lactalbumin and the lactose synthetase reaction. J Biol Chem. 1970 Apr 25;245(8):2103–2108. [PubMed] [Google Scholar]
  14. Fitzgerald D. K., McKenzie L., Ebner K. E. Galactosyl transferase activity in a variety of sources. Biochim Biophys Acta. 1971 Jun 16;235(3):425–428. doi: 10.1016/0005-2744(71)90282-8. [DOI] [PubMed] [Google Scholar]
  15. Klee W. A., Klee C. B. The role of alpha-lactalbumin in lactose synthetase. Biochem Biophys Res Commun. 1970 Jun 5;39(5):833–841. doi: 10.1016/0006-291x(70)90398-0. [DOI] [PubMed] [Google Scholar]
  16. Morrison J. F., Ebner K. E. Studies on galactosyltransferase. Kinetic investigations with N-acetylglucosamine as the galactosyl group acceptor. J Biol Chem. 1971 Jun 25;246(12):3977–3984. [PubMed] [Google Scholar]
  17. Morrison J. F., Ebner K. E. Studies on galactosyltransferase. Kinetic investigations with glucose as the galactosyl group acceptor. J Biol Chem. 1971 Jun 25;246(12):3985–3991. [PubMed] [Google Scholar]
  18. Porath J., Axen R., Ernback S. Chemical coupling of proteins to agarose. Nature. 1967 Sep 30;215(5109):1491–1492. doi: 10.1038/2151491a0. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES