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Malonic Acid Biosynthesis in Bush Bean Roots. II. Purification and Properties of Enzyme Catalyzing Oxidative Decarboxylation of Oxaloacetate

1963; Oxford University Press; Volume: 38; Issue: 6 Linguagem: Inglês

10.1104/pp.38.6.691

1532-2548

Leland M. Shannon, Jean de Vellis, J. Lew,

Plant pathogens and resistance mechanisms

In the preceding paper (4) evidence was presented that a soluble enzyme preparation from bush bean roots was capable of forming malonate from oxalo- acetate (OAA).Carbons 3 and 4 of OAA were equally incorporated into malonate, suggesting that OAA undergoes an a-decarboxylation.In the pres- ence of the crude enzyme preparation, Mn +2 ap- peared to be the only supplemental cofactor required for malonate formation.The experiments reported in the present paper describe the partial purification and characterization of the enzyme(s) catalyzing the conversion of OAA to malonate. Experimental ProcedureMaterials.Plant materials and C14 labeled sub- strates were prepared as described in the previous paper (4).Enzynme Assay.The basic procedure involved incubating OAA-4-C14 with enzyme and determi- nation of the radioactive malonate formed.The complete incubation medium contained: 60 pmoles of acetate buffer, pH 5.4; 1 Amole of MnCl2, 0.5 /mole of OAA-4-C14, S.A. 140,000 cpm/ttmole; 0.1 ml of boiled root extract; and enzyme to make a final volume of 1.0 ml.The reaction mixture was preincubated for 15 minutes in the absence of OAA-4-C14, and incubated for 15 minutes in the presence of OAA- 4-C14.Preincubation and incubation temperatures were 30°.The reaction was stopped by addition of 0.1 ml of 12 N formic acid.The unreacted OAA-4- C14 was converted to unlabeled pyruvate and C14"O by addition of 0.1 ml of freshly prepared 1: 1 (V: V) mixture of 50 % citric acid and aniline (19).The reaction mixture was permitted to stand at room temperature for 30 minutes to effect complete de- carboxylation.A 0.1 ml aliquot was then trans- ferred to a glass planchet and the radioactivity meas- ured using a Nuclear D-47 gas flow detector.As- cending chromatography of the reaction mixture us- ing ether: acetic acid: H,O (5: 2: 1) as solvent showed nmalonate to be the only radioactive com- 1