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PLANT PHYSIOLOGY , Vol 101, Issue 3 931-937, Copyright © 1993 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Monoclonal Antibodies to the [alpha]- and [beta]-Subunits of the Plant Mitochondrial F1-ATPase
M. H. Luethy, A. Horak and T. E. Elthon
School of Biological Sciences and the Center for Biotechnology, University of Nebraska, Lincoln, Nebraska, 68588-0118 (M.H.L., T.E.E.)
We have generated nine monoclonal antibodies against subunits of the maize
(Zea mays L.) mitochondrial F1-ATPase. These monoclonal antibodies were
generated by immunizing mice against maize mitochondrial fractions and
randomly collecting useful hybridomas. To prove that these monoclonal
antibodies were directed against ATPase subunits, we tested their
cross-reactivity with purified F1-ATPase from pea cotyledon mitochondria.
One of the antibodies ([alpha]-ATPaseD) cross-reacted with the pea
F1-ATPase [alpha]-subunit and two ([beta]-ATPaseD and [beta]-ATPaseE)
cross-reacted with the pea F1-ATPase [beta]-subunit. This established that,
of the nine antibodies, four react with the maize [alpha]-ATPase subunit
and the other five react with the maize [beta]-ATPase subunit. Most of the
monoclonal antibodies cross-react with the F1-ATPase from a wide range of
plant species. Each of the four monoclonal antibodies raised against the
[alpha]-subunit recognizes a different epitope. Of the five [beta]-subunit
antibodies, at least three different epitopes are recognized. Direct
incubation of the monoclonal antibodies with the F1-ATPase failed to
inhibit the ATPase activity. The monoclonal antibodies [alpha]-ATPaseD and
[beta]-ATPaseD were bound to epoxide-glass QuantAffinity beads and
incubated with a purified preparation of pea F1-ATPase. The ATPase activity
was not inhibited when the antibodies bound the ATPase. The antibodies were
used to help map the pea F1-ATPase subunits on a two-dimensional map of
whole pea cotyledon mitochondrial protein. In addition, the antibodies have
revealed antigenic similarities between various isoforms observed for the
[alpha]- and [beta]-subunits of the purified F1-ATPase. The specificity of
these monoclonal antibodies, along with their cross-species recognition and
their ability to bind the F1-ATPase without inhibiting enzymic function,
makes these antibodies useful and invaluable tools for the further
purification and characterization of plant mitochondrial F1-ATPases.
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