[Purification and properties of isocitrate lyase and malate synthase from fasting rat liver]

Key enzymes of glyoxylate cycle, isocitrate lyase and malate synthase, are active in the fasting rat liver. The enzymes were synthesized on day 3 after food deprivation and their activities were maximal on day 5 of fasting. Specific activity of isocitrate lyase was 0.06 units/mg protein and specific activity of malate synthase was 0.03 units/mg protein. Isocitrate lyase was isolated and purified by ammonium sulfate fractionation, DEAE-cellulose chromatography and Toyopearl HW-65 gel filtration. Enzyme was purified to specific activity of 9.0 units/mg protein with 8.2% yield. Molecular mass of isocitrate lyase was 145 kD according to gel filtration. Catalytic characteristics of isocitrate lyase indicate that the enzyme follows Michaelis-Menten kinetics (Km for isocitrate is 0.07 mM), is competitively inhibited by glucose-I-phosphate (Ki = 1.1 mM) and glucose-6-phosphate (Ki = 1.9 mM), and is activate by ADP; optimal pH is 7.4. Malate synthase was partially purified by ammonium sulfate fractionation and Sephadex G-25 gel filtration. Enzyme was purified to specific activity of 0.15 units/mg protein with 45% yield. Km of malate synthase for acetyl-CoA was 0.2 mM and Km for glyoxylate was 0.3 mM; optimal pH was 7.6.