PLANT PHYSIOLOGY , Vol 109, Issue 1 285-292, Copyright © 1995 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
The Tonoplast H+-ATPase of Acer pseudoplatanus Is a Vacuolar-Type ATPase That Operates with a Phosphoenzyme Intermediate
T. Magnin, A. Fraichard, C. Trossat and A. Pugin
Laboratoire de Biochimie et Biologie Moleculaire, Unite de Formations et de Recherches des Sciences et Techniques, 16, Route de Gray, 25 030 Besancon Cedex, France (T.M., A.F., C.T.)
The tonoplast H+-ATPase of Acer pseudoplatanus has been purified from
isolated vacuoles. After solubilization, the purification procedure
included size-exclusion and ion-exchange chromatography. The H+-ATPase
consists of at least eight subunits, of 95, 66, 56, 54, 40, 38, 31, and 16
kD, that did not cross-react with polyclonal antibodies raised to the
plasmalemma ATPase of Arabidopsis thaliana. The 66-kD polypeptide
cross-reacted with monoclonal antibodies raised to the 70-kD subunit of the
vacuolar H+-ATPase of oat roots. The functional molecular size of the
tonoplast H+-ATPase, analyzed in situ by radiation inactivation, was found
to be around 400 kD. The 66-kD subunit of the tonoplast H+-ATPase was
rapidly phosphorylated by [[gamma]-32P]ATP in vitro. The complete loss of
radio-activity in the 66-kD subunit after a short pulse-chase experiment
with unlabeled ATP reflected a rapid turnover, which characterizes a
phosphorylated intermediate. Phosphoenzyme formed from ATP is an
acylphosphate-type compound as shown by its sensitivity to hydroxylamine
and alkaline pH. These results lead us to suggest that the tonoplast
H+-ATPase of A. pseudoplatanus is a vacuolar-type ATPase that could operate
with a plasmalemma-type ATPase catalytic mechanism.
