Intensity of Free Radical Processes and Regulation of Cytoplasmic NADP-Isocitrate Dehydrogenase in Rat Cardiomyocytes under Normal and Ischemic Conditions

L. V. Medvedeva¹, T. N. Popova^1*, V. G. Artyukhov², L. V. Matasova¹, and M. A. A. Pinheiro de Carvalho³

¹Department of Analytical and Medical Biochemistry and Microbiology, School of Biology, Voronezh State University, Universitetskaya pl. 1, Voronezh, 394006 Russia; fax: (0732) 789-755; E-mail: tatiana@po.vsu.ru

²Department of Biophysics and Biotechnology, School of Biology, Voronezh State University, Universitetskaya pl. 1, Voronezh, 394006 Russia; fax: (0732) 789-755

³Department of Biochemistry and Biotechnology, Research Center of Biological and Geological Sciences, School of Biology, Madeira University, Funchal, 9000-081, Portugal

^* To whom correspondence should be addressed.

Received May 10, 2001; Revision received January 2, 2002

---

The intensity of free radical processes and the regulation of NADP-isocitrate dehydrogenase (EC 1.1.1.42; NADP-IDH) activity have been studied in the cytoplasmic fraction of normal and ischemized rat myocardium. Chemiluminescence parameters, such as the light sum (S) of slow flash and the tangent of the kinetic curve slope angle (tanalpha₁), which characterize the intensity of free radical processes, were increased in ischemia 2.1- and 20.0-fold, respectively. The slow flash intensity (I_max) was increased 22-fold. The contents of lipid peroxidation products--diene conjugates and malonic dialdehyde--were increased 11.9- and 4.7-fold, respectively, suggesting pronounced oxidative stress. Using homogenous enzyme preparations of NADP-IDH isolated from the normal and experimentally ischemized rat myocardium, a number of catalytic properties of the enzyme were characterized for normal and pathologic conditions. NADP-IDH from the normal and ischemized myocardium had the same electrophoretic mobility and was regulated similarly by Fe²⁺, Cu²⁺, Zn²⁺, and also with succinate and fumarate. However, under normal and pathologic conditions NADP-IDH was different in the affinity for substrates and in the sensitivity to inhibitory effects of hydrogen peroxide, reduced glutathione, and of Ca²⁺. The degree of synergy in the enzyme inhibition with Fe²⁺ and H₂O₂ was less pronounced in ischemia. The inhibitory effect of the reaction product 2-oxoglutarate was higher under normal conditions than in ischemia (the K_i values were 0.22 and 0.75 mM, respectively). The specific features of the NADP-IDH regulation in ischemia are suggested to promote the stimulation of the enzyme functioning during increased level of free radical processes, and this seems to be important for NADPH supplying for the glutathione reductase/glutathione peroxidase antioxidant system of cardiomyocytes.

---

KEY WORDS: rat heart, normal and ischemic conditions, free radical processes, NADP-isocitrate dehydrogenase, properties, activity regulation

---