PURIFICATION AND CHARACTERIZATION OF ACONITASE ISOFORMS FROM ETIOLATED PUMPKIN COTYLEDONS

Although aconitase (EC 4.2.1.3) is involved in the glyoxylate cycle, w hich is localized in glyoxysomes, we detected very low aconitase activ ity in glyoxysomal fractions after sucrose gradient centrifugation of extracts prepared from etiolated pumpkin (Cucurbita sp.) cotyledons. T wo aconitase isoforms were purified to homogeneity, albeit in low yiel d, by hydrophobic interaction, hydroxylapatite and anion exchange chro matography. They were designated Aco I and Aco II; both were shown to be monomeric proteins of M(r) 100000 or 98000 by gel filtration and SD S-PAGE analysis, respectively; isoelectric points were 5.0 and 4.8, re spectively. Kinetic studies revealed similarities between Aco I and Ac o II. A third aconitase isoform (Aco III) was revealed but not purifie d to homogeneity.