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J. Biol. Chem., Vol. 263, Issue 30, 15423-15428, Oct, 1988
PR Maycox, T Deckwerth, JW Hell and R Jahn
The dependence of glutamate uptake on ATP-generated proton electrochemical
potential was studied in a highly purified preparation of synaptic vesicles
from rat brain. At low chloride concentration (4 mM), the proton pump
present in synaptic vesicles generated a large membrane potential
(inside-positive), associated with only minor acidification. Under these
conditions, the rate of L-[3H]glutamate uptake was maximal. In addition,
L-glutamate induced acidification of the vesicle interior. D-Glutamate
produced only 40% of the effect, and L-aspartate or gamma-aminobutyric acid
produced less than 5%. The initial rate of glutamate-induced acidification
increased with increasing glutamate concentration. It was saturable and
showed first- order kinetics (KM = 0.32 mM). Correspondingly, L-glutamate
induced a small reduction in the membrane potential. The rate of ATP
hydrolysis was unaffected. In comparison, glutamate had no effect on
acidification or membrane potential in resealed membranes of chromaffin
granules. At high chloride concentration (150 mM), the vesicular proton
pump generated a large pH difference, associated with a small change in
membrane potential. Under these conditions, uptake of L-[3H]glutamate by
synaptic vesicles was low. For reconstitution, vesicle proteins were
solubilized with the detergent sodium cholate, supplemented with brain
phospholipids, and incorporated into liposomes. Proton pump and glutamate
uptake activities of the proteoliposomes showed properties similar to those
of intact vesicles indicating that the carrier was reconstituted in a
functionally active form. It is concluded that glutamate uptake by synaptic
vesicles is dependent on the membrane potential and that all components
required for uptake are integral parts of the vesicle membrane.
Glutamate uptake by brain synaptic vesicles. Energy dependence of transport and functional reconstitution in proteoliposomes
Department of Neurochemistry, Max-Planck-Institute for Psychiatry, Martinsried, Federal Republic of Germany.
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