Using antibody prepared against pure
uridine kinase from Ehrlich
ascites cells, we have measured the expression of
enzyme protein by the Western blot technique. Variations were observed in the Mr of the
enzyme subunit for
uridine kinase from different species: 32,000 (mouse Ehrlich
ascites cells), 30,000 (normal human lymphocytes), 28,000 (mouse tissues), 27,500 (rat tissues). For different normal tissues from the same species, there was no significant variation in the subunit size. Transformed human and mouse cell lines, selected for a deficiency of
uridine kinase activity in the presence of inhibitors activated by this
enzyme, expressed two cross-reacting
proteins, one with a normal (30,000) and one with a smaller (21,000) subunit molecular weight than was found in the parental cell line (human
lymphoma), or only a smaller
protein of Mr 25,000 (mouse
lymphoma). Our results show that selection protocols using metabolite inhibitors do not always repress the expression of the
enzyme but instead may lead to selection of those cells that have a mutation in the
uridine kinase gene, resulting in the expression of an inactive
enzyme. The expression of
uridine kinase protein changes when cells are stimulated to divide. For both mouse fibroblasts and human lymphocytes, expression of
uridine kinase protein as well as activity clearly increased after cells were stimulated to grow. In fibroblasts, increases are seen by 3 hr after stimulation, and plateau after 9 hr at a sevenfold increase. In lymphocytes, no change is seen until 12 hr after stimulation, and a plateau is not reached until 72 hr, with a total increase of approximately 50-fold. There has been considerable interest in the possibility of
uridine kinase isozymes. Except for cells that have been mutagenized, the present results show that, as judged by subunit molecular weight, there appears to be only one
enzyme form in normal and neoplastic cells or in cells in which
uridine kinase activity is induced.