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Characteristics and Regulatory Properties of the H + ‐ATPase in a Plasma Membrane Fraction Purified from Arabidopsis thaliana
Author(s) -
Olivari C.,
Pugliarello Maria Chiara,
RasiCaldogno Franca,
Michelis Maria Ida
Publication year - 1993
Publication title -
botanica acta
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.871
H-Index - 87
eISSN - 1438-8677
pISSN - 0932-8629
DOI - 10.1111/j.1438-8677.1993.tb00332.x
Subject(s) - arabidopsis thaliana , arabidopsis , membrane , fraction (chemistry) , atpase , chemistry , gene , biochemistry , enzyme , chromatography , mutant
The aqueous two‐phase partitioning technique was utilized to isolate a plasma membrane (PM) fraction from etiolated seedlings of Arabidopsis thaliana . The purification procedure adopted yielded a fraction highly enriched in PM as compared to inner membranes, with a recovery of about 30%, as judged from the activities of PM markers such as vanadate‐sensitive ATPase, FC binding and UDP‐glucose sterol glucosyltransferase. The purified PM fraction displayed vanadate‐sensitive H + pumping activity. Its purity was confirmed by the biochemical characteristics of its ATPase activity assayed in the absence of Ca 2+ : sensitivity to vanadate (IC 50 ca. 1 μM), Mg 2+ ‐dependence, insensitivity to molybdate, oligomycin and nitrate, pH optimum at 6.6. The PM H + ‐ATPase activity was stimulated by fusicoccin and by a controlled treatment of the PM with trypsin. In both cases stimulation was much stronger on the activity assayed at pH 7.5 than on the activity at pH 6.6. Moreover, neither fusicoccin nor the treatment with trypsin stimulated the portion of activity (30 to 40% at pH 7.5) which decayed upon preincubation of the PM in assay medium without ATP.