Control of gluconeogenesis by isocitrate lyase in endosperm of germinatingcastor bean seedlings

The aim of this work was to quantify the contribution of isocitrate lyase t o the control of gluconeogenesis in endosperm from 4-day-old castor bean se edlings. The approach was based on metabolic control analysis following sel ective inhibition of enzyme activity. Both 3-nitropropionate and itaconate decreased the proportion of either [1-C-14]acetate or [2-C-14]acetate conve rted to sucrose, and increased the proportion metabolized through the trica rboxylic acid cycle. Kinetic analysis of isocitrate lyase activity from end osperm revealed that itaconate is a pure uncompetitive inhibitor (K-i' = 11 .9 +/- 0.98 mu M) with respect to isocitrate. In contrast, 3-nitropropionat e is a slow, tight-binding inhibitor. The half-time for inhibition of isoci trate lyase by 3-nitropropionate was 5-10 min, whereas the half-time for re activation was in excess of 10h. Incubating endosperm in 3-nitropropionate resulted in a concentration-dependent decrease in isocitrate lyase activity that remained stable in tissue extracts for at least 4h. From a comparison of the extent of in situ inactivation of isocitrate lyase by 3-nitropropio nate and the effect of this compound on the rate of sucrose production from [2-14C]acetate, the flux control coefficient of isocitrate lyase on glucon eogenesis from acetate in castor bean endosperm was calculated to be 0.66+/ -0.09. It is concluded that isocitrate lyase activity is quantitatively imp ortant in the control of gluconeogenic flux, and suggested that development al changes in the amount of this enzyme may be an important factor in deter mining the conversion of lipid to sugar in young castor bean seedlings.