Affinity chromatography of thymidine kinase from a rat colon adenocarcinoma.
Thymidine kinase from a transplantable colon adenocarcinoma, induced by 1,2-dimethylhydrazine and maintained in CDF rats, was purified by affinity chromatography using thymidine-3'(4-aminophenylphosphate) coupled to carboxyhexyl-Sepharose. Most of the contaminating protein passed through the column; non-specifically adsorbed protein was washed from the column by 0.1 M KC1 in 0.01 M Tris-HC1, 7.5. Thymidine kinase was eluted with 0.1 mM thymidine, 0.1 M KC1 in 0.01 M Tris-HC1, pH 7.5. The purified enzyme accounted for about 26% of the applied activity; the specific activity of the purified material (peak fraction) was 3,500 moles TMP formed per mg protein per 10 min., a 1,800-fold purification of the applied extract. The preparation is free of nucleoside phosphotransferase, but contains other protein impurities. Purification was completed in less than 1 hour, making this a useful procedure for isolation of this unstable enzyme.
D000230:Adenocarcinoma / Q000201:enzymology*; D000818:Animals; D002846:Chromatography, Affinity; D003110:Colonic Neoplasms / Q000201:enzymology*; D004789:Enzyme Activation; D008274:Magnesium / Q000494:pharmacology; D009374:Neoplasms, Experimental / Q000201:enzymology; D051381:Rats; D011919:Rats, Inbred Strains; D013937:Thymidine Kinase / Q000302:isolation & purification* / Q000378:metabolism
D013937:Thymidine Kinase; D008274:Magnesium