Purification and Properties of Isocitrate Lyase from Pupas of the Butterfly Papilio machaon L.

A. T. Eprintsev, M. Yu. Shevchenko^*, and V. N. Popov

Faculty of Biology, Voronezh State University, Universitetskaya pl. 1, Voronezh 394693, Russia; fax: (0732) 78-9755; E-mail: bsbc366@main.vsu.ru

^* To whom correspondence should be addressed.

Received April 7, 2003; Revision received May 20, 2003

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Key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, were identified in pupas of the butterfly Papilio machaon L. The activities of these enzymes in pupas were 0.056 and 0.108 unit per mg protein, respectively. Isocitrate lyase was purified by a combination of various chromatographic steps including ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Toyopearl, and gel filtration. The specific activity of the purified enzyme was 5.5 units per mg protein, which corresponded to 98-fold purification and 6% yield. The enzyme followed Michaelis-Menten kinetics (K_m for isocitrate, 1.4 mM) and was competitively inhibited by succinate (K_i = 1.8 mM) and malate (K_i = 1 mM). The study of physicochemical properties of the enzyme showed that it is a homodimer with a subunit molecular weight of 68 ± 2 kD and a pH optimum of 7.5 (in Tris-HCl buffer).

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KEY WORDS: glyoxylate cycle, isocitrate lyase, malate synthase, kinetic characteristics, diapause

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