Regulation by thymidine monophosphate and other nucleotides of thymidine kinase activity in extracts from primary rabbit kidney cells infected by HSV type 1 and 2

In an attempt to differentiate b tween thymidine kinase (EC. 2.7. I. 2i) induced by herpesvirus hominis type I (TK r) and type 2 (TK2), the different susceptibilities to the modifying effects of some thymidine analogues proved to be useful criteria: 0) 2'-deoxythymidine-5'-triphosphate (dThd-5'-PPP) inhibits TK 2 at a concentra-tion of o'I25 mM by 9o ~, whereas TK I is inhibited at 4"o3 mM by 5o ~. (2) 2'-deoxythymidine-5'-monophosphate (dThd-5'-P) competitively inhibits TK 2 at all concentrations tested. On the other hand, the direction of its effect on TK I is concentration dependent: at 5oo/~M it stimulates and at 8 mM inhibits TK I activity. During enzyme kinetic studies, TK I displays ubstrate inhibition which is reversed by dThd-5'-P. This result explains the stimulating effect of dThd-5'-P at 5oo #M. This phenomenon suggests the existence on the enzyme molecule of a second binding site for dThd which mediates substrate inhibition and which can be occupied also by dThd-5'-P. After polyacrylamide g l electrophoresis of TK I, the stimulation by dThd-5'-P disappears, suggesting the separation of the second binding site from the catalytic entre