Glutamate transport in pig heart mitochondria. Binding and structural properties of high-glutamate affinity proteolipid: Reconstitution studies

Previously, a proteolipid that can bind glutamate with high affinity has been isolated from pig heart mitochondrial membranes. A final affinity chromatography on γ-methylglutamate-albumin coreticulated on glass fiber was necessary. This procedure includes long dialysis steps which tend to denature the high-glutamate affinity proteolipid.Here is described a new method of isolation which avoids long dialysis steps and yields greater amounts of the high-glutamate affinity proteolipid.The binding of glutamate or aspartate on high-glutamate affinity proteolipid has been studied by gel filtration, by equilibrium dialysis or by a new procedure of rapid centrifugation based on the insolubility of high-glutamate affinity proteolipid in water. The latter method permits the detection of low and high affinity sites for glutamate with a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ 60 mM and 55 μM, respectively. Among a series of analogues, aspartate appeared to be the best competitor: $\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 30 μ}\text{M}$ and two $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ values, 0.37 mM (at high glutamate concentration) and 3.8 μM (at low glutamate concentration). High-glutamate affinity proteolipid binds 0.4 nmol of glutamate but only 0.1 nmol of aspartate per mg protein. The sites for glutamate and aspartate appear to be different but interdependent.In the presence of high-glutamate affinity proteolipid, externally added glutamate stimulated the efflux of aspartate from preloaded liposomes.High-glutamate affinity proteolipid contains cardiolipin, phosphatidyl choline and phosphatidyl ethanolamine the distribution of which is different from that of the inner membrane.The effects of various phospholipases, trypsin, and thiol reagents were studied on the binding of glutamate. High-glutamate affinity proteolipid binds 9 nmol $\text{N-}\text{ethylmaleimide}$ per mg protein but only 6.1 nmol in the presence of glutamate. The dissociation of high-glutamate affinity proteolipid caused by thiol reagents yielded a soluble protein fraction with higher affinity for glutamate.Electrophoresis and an immunological approach allowed the detection and titration of the glutamate dehydrogenase and aspartate aminotransferase present in high-glutamate affinity proteolipid in inhibited forms, the latter being 26-fold more concentrated than the former.