PURIFICATION AND PROPERTIES OF ISOCITRATE LYASE AND MALATE SYNTHASE FROM LIVER OF STARVING RATS

The activities of key enzymes of the glyoxylate cycle, isocitrate lyas e and malate synthase, were determined in liver of starving rats. Dete ctable activities were observed on the third day after deprivation of food and reached maxima on the fifth day. The specific activities of i socitrate lyase and malate synthase were 0.06 and 0.03 unit/mg protein , respectively. Isocitrate lyase was isolated and purified using ammon ium sulfate fractionation, chromatography on DEAE-cellulose, and gel f iltration on Toyopearl HW-65. The resulting enzyme preparation (yield 8.1%) had specific activity 9.0 units/mg protein. Gel filtration revea led molecular mass of isocitrate lyase of 145 kD. The enzyme follows M ichaelis-Menten kinetics (K-m for isocitrate 0.07 mM). It is competiti vely inhibited by glucose-l-phosphate (K-i = 1.1 mM), glucose-6-phosph ate (K-i 1.9 mM) and is activated by ADP. The enzyme has pH optimum of 7.4. Malate synthase was partially purified using ammonium sulfate fr actionation and gel filtration on Sephadex G-25. The resulting enzyme preparation (yield 45%) possessed specific activity 0.15 unit/mg prote in. The malate synthase has pH optimum of 7.6 and K-m for acetyl-CoA a nd glyoxylate of 0.2 and 3.0 mM, respectively.