Intracellular location of thymidylate synthase and its state of phosphorylation.
Thymidylate synthase (TS), an enzyme that is essential for DNA synthesis, was found to be associated mainly with the nucleolar region of H35 rat hepatoma cells, as determined both by immunogold electron microscopy and by autoradiography. In the latter case, the location of TS was established through the use of [6-3H]5-fluorodeoxyuridine, which forms a tight ternary complex of TS with 5-fluorodeoxyuridylate (FdUMP) and 5, 10-methylenetetrahydrofolylpolyglutamate within the cell. However, with H35 cells containing 50-100-fold greater amounts of TS than unmodified H35 cells, the enzyme, although still in the nucleus, was located primarily in the cytoplasm as shown by autoradiography and immunohistochemistry. In addition, TS was also present in mitochondrial extracts of both cell lines, as determined by enzyme activity measurements and by ternary complex formation with [32P]FdUMP and 5,10-methylenetetrahydrofolate. Another unique observation is that the enzyme appears to be a phosphoprotein, similar to that found for other proteins associated with cell division and signal transduction. The significance of these findings relative to the role of TS in cell division remains to be determined, but suggest that this enzyme's contribution to the cell cycle may be more complex than believed previously.
Samsonoff
W A
WA
Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, New York 12201-0509, USA.
Reston
J
J
McKee
M
M
O'Connor
B
B
Galivan
J
J
Maley
G
G
Maley
F
F
D000818:Animals; D001345:Autoradiography; D002455:Cell Division; D002467:Cell Nucleus / Q000201:enzymology*; D007150:Immunohistochemistry; D008114:Liver Neoplasms, Experimental / Q000201:enzymology / Q000473:pathology; D010766:Phosphorylation; D051381:Rats; D013940:Thymidylate Synthase / Q000032:analysis* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D013940:Thymidylate Synthase