-transparent.png){kind=logo}
The interaction of 5-fluorodeoxyuridylate (FdUMP) with thymidylate synthetase to form a binary complex has been widely reported, yet previous attempts to detect this complex by nitrocellulose filtration have failed. In contrast, a nitrocellulose-filter-binding assay utilizing [6-3H]FdUMP which measures the interaction of the enzyme with the nucleotide is reported. Extensive washing of the nitrocellulose-filtered complex between FdUMP and the enzyme resulted in no loss of bound ligand. Following denaturation with trichloroacetic acid, intact complex was detected by nitrocellulose filtration. No binding was observed between 5-fluorodeoxyuridine and the enzyme or between FdUMP and the N-ethylmaleimide-modified enzyme. As measured by the nitrocellulose filtration method, at least a 600-fold excess of FdUMP to enzyme was required to achieve saturation. The stoichiometry of FdUMP bound to the enzyme detected at saturation was 0.5-0.6 for native samples. When identical samples were subjected to denaturation prior to filtration, the stoichiometry of nucleotide binding was 0.3-0.4.
F Ahmed, and
M A Moore, and
R B Dunlap
August 1981,
Archives of biochemistry and biophysics,
F Ahmed, and
M A Moore, and
R B Dunlap
August 1978,
Biochemical and biophysical research communications,
F Ahmed, and
M A Moore, and
R B Dunlap
January 1980,
Biochemistry,
F Ahmed, and
M A Moore, and
R B Dunlap
July 1981,
The Journal of biological chemistry,
F Ahmed, and
M A Moore, and
R B Dunlap
January 1974,
Biochemistry,
F Ahmed, and
M A Moore, and
R B Dunlap
January 1971,
Biochemistry,
F Ahmed, and
M A Moore, and
R B Dunlap
November 1980,
Biochemical and biophysical research communications,
F Ahmed, and
M A Moore, and
R B Dunlap
January 1971,
Preparative biochemistry,
F Ahmed, and
M A Moore, and
R B Dunlap
October 1970,
The Journal of biological chemistry,
F Ahmed, and
M A Moore, and
R B Dunlap
January 1979,
Cancer biochemistry biophysics,
