FUNCTIONAL-PROPERTIES OF THE RECONSTITUTED PHOSPHATE CARRIER FROM BOVINE HEART-MITOCHONDRIA - EVIDENCE FOR ASYMMETRIC ORIENTATION AND CHARACTERIZATION OF 3 DIFFERENT TRANSPORT MODES

The phosphate carrier from bovine heart mitochondria was reconstituted into liposomes by the removal of detergent using hydrophobic ion-exch ange columns. Reversible blocking of the carrier function during chrom atographic steps was possible by the application of the inhibitor p-(c hloromercuri)benzenesulfonate at low temperature. Thus, both forward a nd backward exchange experiments for kinetic characterization of P(i)/ P(i)-antiport as well as the P(i)/H+-symport could be performed. The m aximum rate of P(i)/P(i)-antiport was 90 mumol min-1(mg protein)-1. On ly one single half-saturation constant (K(m)) for phosphate was observ ed at each side of the membrane under antiport conditions, 1.8 mM at t he external and 9.4 mM at the internal side. By comparing the K(m) val ues at both sides of the membrane with the values found in intact mito chondria, a right-side-out orientation of the reconstituted phosphate carrier was concluded. Furthermore, the influence of various sulfhydry l reagents on the carrier was investigated. After modification with Hg Cl2, the phosphate carrier reveals a third (nonphysiological) unidirec tional transport mode. This was characterized by a significantly reduc ed substrate specificity. In view of similar observations with several other mitochondrial carriers, these results again indicate that the p hosphate carrier is a member of the postulated functional family of mi tochondrial carrier proteins.