Isolation, Purification, and Characterization of a Neutral Mg²⁺-Dependent Deoxyribonuclease of the Colorado Potato Beetle Leptinotarsa decemlineata Say

S. A. Borodin^1,2 and A. S. Konichev¹

¹Department of Organic and Biological Chemistry, Moscow State Pedagogical University, ul. Kibal'chicha 6/5, Moscow, 129278 Russia; fax: (095) 283-1202.

²To whom correspondence should be addressed.

Submitted April 15, 1997.

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A neutral Mg²⁺-dependent deoxyribonuclease from the Colorado potato beetle was isolated and characterized in physicochemical terms. An electrophoretically homogeneous preparation of the enzyme was obtained using salt fractionation, Sephadex G-100 gel filtration, and subsequent preparative isoelectrofocusing in an Ultrodex layer. The molecular weight of the purified DNase preparation (with a purification degree of 104) and its isoelectric point were 100 kD and 9.1, respectively. The enzyme activity was maximal at pH 7.2 and 46°C in the presence of 10 mM Mg²⁺. The DNase of the Colorado beetle preferentially hydrolysed denatured DNA via the endonuclease pathway, degrading the substrate to oligonucleoside-3´-phosphates. As far as the physical and chemical properties are concerned, this Colorado beetle DNase seems different from previously investigated DNases of other insect species.

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KEY WORDS: deoxyribonuclease (DNase), enzyme properties, Colorado potato beetle.

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