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.
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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.
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.
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.
Cuatrecasas, P., and Segal, S. (1966). Galactose conversion to d-xylulose: An alternate route of galactose metabolism. Science 153 549–551.
DeMars, R. (1964). Some studies of enzymes in cultivated human cells, Nat. Cancer Inst. Monograph 13 181–193.
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.
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.
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.
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.
Ham, R., and Puck, T. T. (1961). Quantitative mammalian cell culture. In: Methods in Enzymology, Vol. V, Academic Press, New York.
Holzel, A., and Komrower, G. M. (1955). A study of the genetics of galactosemia. Arch. Disease Childhood, 30 155–159.
Hsia, D. Y., Huang, I., and Driscoll, S. G. (1958). Heterozygous carrier in galactosemia. Nature 182 1389–1390.
Hugh-Jones, K., Newcomb, A. L., and Hsia, D. Y. (1960). The genetic mechanisms of galactosemia. Arch. Disease Childhood 35 521–528.
Isselbacher, K. J. (1958). A mammalian uridine-diphosphate galactose pyrophosphorylase. J. Biol. Chem. 232 429–444.
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.
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.
Kirkman, H. N., and Bynum, E. (1959). Enzymatic evidence of a galactosemic trait in parents of galactosemic children. Ann. Human Gen. 23 117–126.
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.
Krooth, R. S. (1965) The future of mammalian cell genetics. Birth Defects (Original article series 1 (2): 21–56
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).
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.
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.
Mathai, C. K., and Beutler, E. (1966). Electrophoretic variation of galactose-1-phosphate uridyltransferase. Science 154 1179–1180.
Neilands, J. B., Stumpf, P. K., and Staier, R. Y. (1955). Outlines of Enzyme Chemistry. John Wiley and Sons Inc., New York p. 93.
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.
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.
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.
Schwarz, V., Goldberg, L., Komrower, G. H., and Holzel, A. (1956). Some disturbances of erythrocyte metabolism in galactosemia. Biochem. J. 62 34–50.
Suskind, S. R., and Kurek, L. J. (1957). Enzyme-inhibitor complex in a tryptophan-requiring mutant of Neurospora crassa. Science 126 1068–1069.
Weinberg, A. N. (1961). Detection of congenital galactosemia and the carrier state using galactose C14 and blood cells. Metabolism 10 728–734.
Weinberg, A. N., and Segal, S. (1960) Effect of galactose-1-phosphate on glucose oxidation by normal and galactosemic leukocytes. Science 132 1015–1016.
Additional information
Paper # 1105 from the Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin. This work was supported by the National Institutes of Health (Grants # GM-08217, # GM-398, and # GM-06983).
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Russell, J.D., DeMars, R. UDP-glucose: α-D-galactose-1-phosphate uridylyltransferase activity in cultured human fibroblasts. Biochem Genet 1, 11–24 (1967). https://doi.org/10.1007/BF00487732
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DOI: https://doi.org/10.1007/BF00487732