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Immunological analysis of aconitase in pumpkin cotyledons: the absence of aconitase in glyoxysomes
Author(s) -
Bellis Luigi,
Hayashi Makoto,
Biagi Pier Paolo,
HaraNishimura Ikuko,
Alpi Amedeo,
Nishimura Mikio
Publication year - 1994
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1994.tb02534.x
Subject(s) - aconitase , biochemistry , affinity chromatography , chemistry , antiserum , fractionation , molecular mass , chromatography , antibody , biology , mitochondrion , enzyme , immunology
Aconitase (EC 4.2.1.3) was purified by column chromatography and SDS‐PAGE. Specific antibodies for aconitase were prepared after affinity purification of the antiserum with purified aconitase. The antibodies reacted with purified pumpkin aconitase, and with the 98 kDa protein band after electrophoretic fractionation of extracts of pumpkin cotyledons. Immunoblot analysis revealed a protein with similar molecular mass in extracts of several plants. The intensity of the 98 kDa band increased as pumpkin cotyledons developed in darkness, and decreased thereafter upon illumination. Aconitase activity showed a similar pattern. Anion exchange chromatography of a homogenate of pumpkin cotyledons, followed by western blotting, displayed the presence of immunoreactive protein bands only in fractions showing aconitase activity. The results indicate that the antibodies were specific for aconitase. When we investigated the presence of immunoreactive bands after sucrose gradient fractionation, aconitase was detected in the supernatant fractions and in mitochondria, while a very low amount was found in glyoxysomes. These data provide additional proof that aconitase is not localized in glyoxysomes.