PLANT PHYSIOLOGY , Vol 105, Issue 1 47-52, Copyright © 1994 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
The Phosphate Transporter from Pea Mitochondria (Isolation and Characterization in Proteolipid Vesicles)
C. A. McIntosh and D. J. Oliver
Department of Molecular Biology and Biochemistry, University of Idaho, Moscow, Idaho 83843
The phosphate transporter from mitochondria will exchange matrix phosphate
for cytosolic phosphate and facilitate either phosphate/proton symport or
phosphate/hydroxyl ion antiport. The phosphate transported into the matrix
by this carrier is either used for ATP synthesis or exchanges back out to
the cytosol on the dicarboxylate transporter, permitting entry of malate
and succinate into the matrix. The phosphate transporter was solubilized
from etiolated pea (Pisum sativum L. cv Alaska) mitochondrial membranes
with Triton X-114, purified approximately 500-fold by hydroxylapatite
chromatography, and reconstituted into azolectin vesicles that were
preloaded with 0.1 or 10 mM phosphate. Phosphate transport was measured as
the exchange of preloaded phosphate for external [32P]phosphate.
Phosphate/phosphate exchange occurred for over 40 min at room temperature
with an apparent K0.5 of 1.6 mM and a maximum velocity of over 700 nmol (mg
protein)-1 min-1. Diethyl pyrocarbonate was used as an inhibitor-stop
reagent. Transport was inhibited by p-hydroxyphenylglyoxal,
p-hydroxymercuribenzoate, pyridoxal 5-phosphate, and dansyl chloride but
was insensitive to sulfate, nitrate, and N-ethylmaleimide, the standard
inhibitor for the mammalian phosphate transporter. Phosphate/hydroxyl
exchange was stimulated when the proton gradient was collapsed with
carbonyl cyanide m-chlorophenylhydrazone, but phosphate/phosphate exchange
was unaffected by the uncoupler.
