Biochemical Genetics

, Volume 1, Issue 1, pp 11–24 | Cite as

UDP-glucose: α-D-galactose-1-phosphate uridylyltransferase activity in cultured human fibroblasts

  • James D. Russell
  • Robert DeMars
Article

Abstract

Galactose-1-phosphate uridyl transferase activity of normal, heterozygous and galactosemic strains is determined throughout the culture cycle of human fibroblasts using a new direct method of assay. The enzyme activities of high-density, stationary-phase cultures define three nonoverlapping classes, which correspond to the genotypes of the donors. During rapid growth, however, galactosemic strains show near-normal transferase activity. The incorporation of 14C from 14C1-galactose by living cells is measured. While heterozygous strains do not appear to differ from normal controls, homozygous mutant cells incorporate 14C at about one-half the normal rate throughout the culture cycle. Variables affecting the assay are investigated and the implications of our results for further genetic studies of mutations affecting transferase are discussed.

Keywords

Enzyme Activity Living Cell Normal Control Normal Rate Genetic Study 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Anderson, E. P., Kalckar, H. M., Kurahashi, K., and Isselbacher, K. J. (1957). A specific enzymatic assay for the diagnosis of congenital galactosemia. 1. The consumption test. J. Lab. Clin. Med. 50 469–477.Google Scholar
  2. Beutler, E., and Baluda, M. C. (1966). Biochemical properties of human red cell galactose-1-phosphate uridyl transferase (UDP-glucose: α-d-galactose-1-phosphate uridyltransferase EC 2.7.7.12) from normal and mutant subjects. J. Lab. Clin. Med. 67 947–954.Google Scholar
  3. Bretthauer, R. K., Hansen, R. G., Donnell, G., and Bergren, W. A. (1959). A procedure for detecting carriers of galactosemia. Proc. Nat. Acad. Sci. U.S. 45 328–331.Google Scholar
  4. Cuatrecasas, P., and Segal, S. (1966). Galactose conversion to d-xylulose: An alternate route of galactose metabolism. Science 153 549–551.Google Scholar
  5. DeMars, R. (1964). Some studies of enzymes in cultivated human cells, Nat. Cancer Inst. Monograph 13 181–193.Google Scholar
  6. DeMars, R., and Leroy, J. G. (1966). The remarkable cells cultured from a human with Hurler's syndrome: An approach to visual selection for in vitro genetic studies. In Vitro 2: in press.Google Scholar
  7. DeMars, R., and Nance, W. E. (1964). Electrophoretic variants of glucose-6-phosphate dehydrogenase and the single-active X in cultivated human cells. The Wistar Institute Monograph 1 35–48.Google Scholar
  8. De Moss, J. A., and Bonner D. M. (1959). Studies with normal and genetically altered tryptophan synthetase from Neurospora crassa. Proc. Nat. Acad. Sci. U.S. 45 1405–1412.Google Scholar
  9. Donnell, G. N., Bergren, W. R., Bretthauer, R. K., and Hansen, R. G. (1960). The enzymatic expression of heterozygosity in families of children with galactosemia. Pediatrics 25 572–581.Google Scholar
  10. Ham, R., and Puck, T. T. (1961). Quantitative mammalian cell culture. In: Methods in Enzymology, Vol. V, Academic Press, New York.Google Scholar
  11. Holzel, A., and Komrower, G. M. (1955). A study of the genetics of galactosemia. Arch. Disease Childhood, 30 155–159.Google Scholar
  12. Hsia, D. Y., Huang, I., and Driscoll, S. G. (1958). Heterozygous carrier in galactosemia. Nature 182 1389–1390.Google Scholar
  13. Hugh-Jones, K., Newcomb, A. L., and Hsia, D. Y. (1960). The genetic mechanisms of galactosemia. Arch. Disease Childhood 35 521–528.Google Scholar
  14. Isselbacher, K. J. (1958). A mammalian uridine-diphosphate galactose pyrophosphorylase. J. Biol. Chem. 232 429–444.Google Scholar
  15. Isselbacher, K. J., Anderson, E. P., Kurahashi, K., and Kalckar, H. M. (1956). Congenital galactosemia, a single enzymatic block in galactose metabolism. Science 123 635–636.Google Scholar
  16. Kalckar, H. M., Anderson, E. P., and Isselbacher, K. J. (1956). Galactosemia, a congenital defect in a nucleotide transferase: A preliminary report. Proc. Nat. Acad. Sci. U.S. 42 49–51.Google Scholar
  17. Kirkman, H. N., and Bynum, E. (1959). Enzymatic evidence of a galactosemic trait in parents of galactosemic children. Ann. Human Gen. 23 117–126.Google Scholar
  18. Krooth, R. S. (1966). Some properties of diploid cell strains developed from the tissues of patients with inherited biochemical disorders. In Vitro 2: in press.Google Scholar
  19. Krooth, R. S. (1965) The future of mammalian cell genetics. Birth Defects (Original article series 1 (2): 21–56Google Scholar
  20. Krooth, R. S. (1964). Characterization of genetically marked human diploid cell strains. Proceedings of the Symposium on the Characterization and Uses of Diploid Cell Strains. Opatija 1963. Zagreb: International Association of Microbiological Societies, 743 pp (pages 79–95).Google Scholar
  21. Krooth, R. S. (1964). Properties of diploid cell strains developed from patients with an inherited abnormality of uridine biosynthesis. Cold Spring Harbor Symposia on Quantitative Biology 29 189–212.Google Scholar
  22. Krooth, R. S., and Weinberg, A. N. (1961). Studies on cell lines developed from the tissues of patients with galactosemia. J. Exp. Med. 113 1155–1172.Google Scholar
  23. Mathai, C. K., and Beutler, E. (1966). Electrophoretic variation of galactose-1-phosphate uridyltransferase. Science 154 1179–1180.Google Scholar
  24. Neilands, J. B., Stumpf, P. K., and Staier, R. Y. (1955). Outlines of Enzyme Chemistry. John Wiley and Sons Inc., New York p. 93.Google Scholar
  25. Ng, Won Gin, Bergren, W. R., and Donnell, G. N. (1964). Galactose-1-phosphate uridyltransferase assay by use of radioactive galactose-1-phosphate. Clin. Chim. Acta. 10 337–343.Google Scholar
  26. Oyama, V. L., and Eagle, H. (1956). Measurement of cell growth in tissue culture with a phenol reagent (Folin-Ciocalteau). Proc. Soc. Exp. Biol. Med. 91 305–307.Google Scholar
  27. Robinson, A. (1963). The assay of galactokinase and galactose-1-phosphate uridyl transferase activity in human erythrocytes: A presumed test for heterozygous carriers of the galactosemic defect. J. Exp. Med. 118 359–370.Google Scholar
  28. Schwarz, V., Goldberg, L., Komrower, G. H., and Holzel, A. (1956). Some disturbances of erythrocyte metabolism in galactosemia. Biochem. J. 62 34–50.Google Scholar
  29. Suskind, S. R., and Kurek, L. J. (1957). Enzyme-inhibitor complex in a tryptophan-requiring mutant of Neurospora crassa. Science 126 1068–1069.Google Scholar
  30. Weinberg, A. N. (1961). Detection of congenital galactosemia and the carrier state using galactose C14 and blood cells. Metabolism 10 728–734.Google Scholar
  31. Weinberg, A. N., and Segal, S. (1960) Effect of galactose-1-phosphate on glucose oxidation by normal and galactosemic leukocytes. Science 132 1015–1016.Google Scholar

Copyright information

© Plenum Publishing Corporation 1967

Authors and Affiliations

  • James D. Russell
  • Robert DeMars

There are no affiliations available

Personalised recommendations