Comparison of characteristics of thymidylate synthesis isolated from eukaryotic and prokaryotic cells

Abstract

The preparations of thymidylate synthase (TDS) from calf thymus, Gardner mouse lymphosarcoma and E. coli were investigated by affinity chromatography based on the formation of a ternary complex of the enzyme with tetrahydromethotrexate and deoxyuridine-5'-monophosphate (dUMP). The E. coli enzyme was a homogeneous protein of mol.wt. 70 000 and it was shown by reductive denaturation to contain one single chain of mol.wt. 35 000. The preparation isolated from the Gardner lymphosarcoma was a homogeneous protein of mol.wt. 72 000 containing a single subunit of mol.wt. 36 000. The enzyme from both sources yielded after treatment with 5-fluorodeoxyuridine-5'-monophosphate (FdUMP) and N5-10-methylenetetrahydrofolate a negatively charged complex which was separated electrophoretically. Three proteins of mol.wt 35 000 (component 1), 70 000 (component 2), and more than 250 000 (component 3), were isolated by affinity chromatography from calf thymus and separated by electrophoresis and gel filtration. Component 2 only was enzymatically active and yielded a complex when treated with 5-fluorodeoxyuridine-5'-monophosphate and N5-10-methylenetetrahydrofolate. Components 1 and 3 were enzymatically inactive yet were immunologically related. The antiserum against component 3 precipitated in addition to component 3 also the active enzyme (component 2) and component 1. The antiserum against the active enzyme precipitated proteins 1 and 3. All three components were found to contain one chain of mol.wt. 35 000 after reductive denaturation. Components 1 and 2 contained this chain only whereas other, heavier chains were found in component 3.