Purification and Some Properties of Thymidilate Kinase from Sea Urchin

L. L. Terentyev^*, N. A. Terentyeva, and V. A. Rasskazov

Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences, pr. Stoletiya Vladivostoka 159, Vladivostok-22, 690022 Russia; E-mail: rasskaz@piboc.marine.su

^* To whom correspondence should be addressed.

Received March 2, 1998; Revision received October 5, 1998

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Thymidilate kinase (EC 2.7.4.9, ATP:dTMP phosphotransferase) was isolated from eggs of the sea urchin Strongylocentrotus intermedius. The enzyme preparation was purified by 1073-fold and was not contaminated with phosphatase or ATPase. The molecular weight of the sea urchin thymidilate kinase is 100 kD, and the pH optimum of its action is 8-8.5. The thymidilate kinase activity is maximal in the presence of 2-5 mM ATP and 10 mM MgCl₂. In the reaction of phosphorylation with dTMP, Mg²⁺ can be partially substituted by other bivalent metal ions whose efficiency decreases in the series: Mg²⁺ > Mn²⁺ > Ca²⁺ = Cd²⁺ = Co²⁺. In the presence of Zn²⁺, Fe²⁺, Cu²⁺, and Pb²⁺ the thymidilate kinase is inactive. The sea urchin thymidilate kinase can utilize ATP, dCTP, and dTTP as donors of the phosphate group. Either dTMP or dCMP can serve as the acceptor of phosphate. Addition of thymidine and other nucleosides to the reaction medium has virtually no effect on the rate of dTMP phosphorylation.

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KEY WORDS: sea urchin, thymidilate kinase, purification, properties

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