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Purification and properties of deoxy thymidine kinase ec 2.7.1.21 induced by bacterio phage t 4 infection


Journal of Biochemistry (Tokyo) 82(5): 1347-1360
Purification and properties of deoxy thymidine kinase ec 2.7.1.21 induced by bacterio phage t 4 infection
Deoxythymidine kinase [EC 2.7.1.21] activity in Escherichia coli increased in response to infection with T even phages but not with T odd phages. This was due to the formation of a new enzyme distinct from the deoxythymidine kinase of E. coli. Among the phages tested, T4 am N82 (gene 44) induced the enzyme most strikingly. Phage-induced deoxythymiding kinase was purified 440-fold from a crude extract of T4 am N82-infected E. coli KY896 (tdk-: deoxythymidine kinase deficient) by streptomycin treatment, (NH4)2SO4 fractionation, and DEAE- and phosphocellulose chromatographies. The MW of the enzyme was about 86,000 by Sephadex G-200 gel filtration. The enzyme seemed to be composed of subunits with a MW of aobut 28,000. The partially purified enzyme had a pH optimum at 6.8 and was stimulated several-fold by Mg2+, Mn2+ and Co2+. Deoxythymidine served as a good phosphate acceptor but other deoxynucleosides and 5-halogenated deoxyuridines did not. As phosphate donors, dATP and dGTP were about 50% as active as ATP. dTTP was a potent inhibitor of the enzyme, and the inhibition was highly dependent on pH. No remarkable activation was found with various nucleotides. The saturation curve for ATP was sigmoidal. Only 1 Km value for deoxythymidine (7.0 .times. 10-5 M) was obtained from Lineweaver-Burk plots with an ATP concentration of 1.43 mM, but 2 Km values (3.3 .times. 10-% M and 2.4 .times. 10-5 M) were found with 0.46 mM ATP. When dTTP was present, 2 Km values (7.0 .times. 10-5 M and 4.2 .times. 10-4 M) were found even at 1.43 mM ATP. Unlike the E. coli enzyme, T4 deoxythymidine kinase was inactivated quickly above 50.degree. C but was stable at moderate temperatures, and no drastic conformational change was detected even though in the presence of regulatory nucleotides.


Accession: 006229589



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