Independent characterization of thymidine transport and subsequent metabolism in Hymenolepis diminuta--II. Purification and preliminary analysis of thymidine kinase.
1. An affinity column for the purification of thymidine kinase (TK) from the cestode Hymenolepis diminuta is described. Using an epoxy-activated Sepharose 6B affinity column containing thymidine as a ligand, a 698-fold purification of thymidine kinase was obtained. 2. Thymidine kinase eluted from this affinity column was partially characterized as having an apparent Km value of 3.94 microM thymidine. This value is very similar to those observed in mammalian systems. 3. Thymidine kinase appears to be an extremely active and ubiquitous enzyme, whose primary function is to rapidly phosphorylate incoming thymidine and thus "trap" it for the cell's use, reducing efflux to a minimum. 4. The apparent Km for TK is two orders of magnitude lower than the Kt for thymidine transport. Thus, theories postulating that long-term (2 min) uptake kinetics for thymidine actually represent subsequent metabolism must look further along the thymidine phosphorylating pathway, beyond TK and its very active role.
Insler
G D
GD
Department of Biology, Adelphi University, Garden City, NY 11530.
Halikias
F J
FJ
D000645:Ammonium Sulfate; D000818:Animals; D001692:Biological Transport; D002846:Chromatography, Affinity; D005590:Fractional Precipitation; D006926:Hymenolepis / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D010766:Phosphorylation; D051381:Rats; D013936:Thymidine / Q000378:metabolism*; D013937:Thymidine Kinase / Q000302:isolation & purification / Q000378:metabolism*
D013937:Thymidine Kinase; D000645:Ammonium Sulfate; D013936:Thymidine