IMMUNOLOGICAL ANALYSIS OF ACONITASE IN PUMPKIN COTYLEDONS - THE ABSENCE OF ACONITASE IN GLYOXYSOMES

Aconitase (EC 4.2.1.3) was purified by column chromatography and SDS-P AGE. Specific antibodies for aconitase were prepared after affinity pu rification of the antiserum with purified aconitase. The antibodies re acted with purified pumpkin aconitase, and with the 98 kDa protein ban d after electrophoretic fractionation of extracts of pumpkin cotyledon s. Immunoblot analysis revealed a protein with similar molecular mass in extracts of several plants. The intensity of the 98 kDa band increa sed as pumpkin cotyledons developed in darkness, and decreased thereaf ter upon illumination. Aconitase activity showed a similar pattern. An ion exchange chromatography of a homogenate of pumpkin cotyledons, fol lowed by western blotting, displayed the presence of immunoreactive pr otein bands only in fractions showing aconitase activity. The results indicate that the antibodies were specific for aconitase. When we inve stigated the presence of immunoreactive bands after sucrose gradient f ractionation, aconitase was detected in the supernatant fractions and in mitochondria, while a very low amount was found in glyoxysomes. The se data provide additional proof that aconitase is not localized in gl yoxysomes.