Mechanism of glutamate-aspartate translocation across the mitochondrial inner membrane

Marc E. Tischler, James Pachence, John R. Williamson, Kathryn F. La Noue

Research output: Contribution to journalArticle

44 Scopus citations

Abstract

In order to study the mechanism of the glutamate-aspartate translocator, rat liver mitochondria were loaded with either glutamate or aspartate. In the presence of ascorbate plus tetramethyl-p-phenylenediamine as an electron donor at the third energy conservation site, exchange of external glutamate for matrix aspartate is highly favored over the reverse exchange. In the absence of an energy source, although the asymmetry of the exchange rates is much smaller, it is still observable. Further studies have shown that the proton uptake accompanying influx of glutamate in exchange for aspartate efflux occurs by protonation of a group on the carrier (pK = 7.9) at the external side of the inner mitochondrial membrane, followed by deprotonation at the matrix surface. It is postulated that glutamate binds to the protonated form of the carrier and aspartate to the deprotonated form. Because of the alkaline pK, aspartate efflux is inhibited with increased matrix [H+] due to limitation of the availability of deprotonated carrier for aspartate binding. For the reverse exchange, aspartate uptake is inhibited by increasing external [H+]. Thus the rate of aspartate uptake by mitochondria is apparently impeded both by a proton motive force (Δp) unfavorable to entry of ions with net negative charge as well as by the small proportion of deprotonated carrier at the outer surface of the membrane. This conclusion is further illustrated by inhibition of the aspartate-aspartate exchange with increased [H+] and by addition of an energy source. The glutamate-glutamate exchange, however, showed a slight stimulation by increased [H+] and was unaffected by the energy state. The model initially proposed for the carrier, in which a neutral glutamate-carrier complex exchanges for a negatively charged aspartate-carrier complex, is tested further. Glutamate uptake was noncompetitively inhibited by external aspartate, which indicates that aspartate and glutamate bind to separate forms of the carrier. Intramitochrondrial glutamate at a concentration of 18 mm, however, had no effect on aspartate efflux. Arrhenius plots for the glutamate-aspartate and aspartate-glutamate exchanges were linear over the range of temperatures tested (1-35 °C and 5-25 °C, respectively) and provided an average value of 14.3 kcal/mol for the energy of activation. In addition, there appear to be two pools, exchangeable and nonexchangeable, of matrix aspartate available to the translocator, since extramitochondrial radiolabeled aspartate can equilibrate only with unlabeled matrix aspartate at low aspartate loading (1-2 nmol of aspartate/mg of protein). The physiological significance of the data is discussed.

Original languageEnglish (US)
Pages (from-to)448-462
Number of pages15
JournalArchives of Biochemistry and Biophysics
Volume173
Issue number2
DOIs
StatePublished - Apr 1976
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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