Patent ID: 6500632
Filing Date: 2002-12-31
Classification: C12Q,G01N

Abstract:
A multiscreening assay for identifying inhibitors of a plant C4 acid cycle enzyme selected from the group consisting of pyruvate orthophosphate dikinase, phosphoenolpyruvate carboxylase and both enzymes, the multiscreening assay comprising at least three screens:a) a first screen for testing for inhibition of at least one of pyruvate orthophosphate dikinase, phosphoenolpyruvate carboxylase and malate dehydrogenase by: i) including a sample containing the potential inhibitor in a test mixture comprising pyruvate orthophosphate dikinase and substrates thereof, phosphoenolpyruvate carboxylase and the substrate bicarbonate, and malate dehydrogenase and the substrate NADH; ii) incubating said test mixture under conditions appropriate for the conversion of pyruvate to malate with oxidation of NADH; and iii) detecting inhibition of at least one of said pyruvate orthophosphate dikinase, phosphoenolpyruvate carboxylase and malate dehydrogenase by comparing the level of NADH or NAD+ in said test mixture with the level NADH or NAD+ in a control mixture incubated under the same conditions as in a)(ii); b) a second screen for testing for inhibition of phosphoenolpyruvate carboxylase or malate dehydrogenase with any sample which contains an inhibitor from said first screen by: i) including said sample in a test mixture containing phosphoenolpyruvate carboxylase and substrates thereof, and malate dehydrogenase and the substrate NADH; ii) incubating said test mixture under conditions appropriate for the conversion of phosphoenolpyruvate to malate with oxidation of NADH; and iii) detecting inhibition of said phosphoenolpyruvate carboxylase or malate dehydrogenase by comparing the level of NADH or NAD+ in said test mixture with the level of NADH or NAD+ in a control mixture incubated under the same conditions as in b)(ii); and c) a third screen for testing for inhibition of malate dehydrogenase with any sample which contains an inhibitor from said second screen by: i) including said sample in a test mixture containing malate dehydrogenase, oxaloacetate and NADH or including oxaloacetate in said test mixture from a)(ii) or b)(ii); ii) incubating said test mixture under conditions appropriate for the conversion of oxaloacetate to malate with oxidation of NADH; and iii) detecting inhibition of said malate dehydrogenase by comparing the level of NADH or NAD+ in said test mixture with the level of NADH or NAD+ in a control mixture incubated under the same conditions as in c)(ii), wherein said inhibitor is identified by detecting inhibition at least in a)(iii) or b)(iii), but substantially no inhibition detected in c)(iii).