Characterization of a dCTP transport activity reconstituted from human mitochondria

A protein fraction of mitochondria from human acute lymphocytic leukemia ce lls, which could be reconstituted into proteoliposomes to have dCTP transpo rt activity, has been partially purified by hydroxyapatite and blue Sepharo se chromatography. The dCTP transport activity in proteoliposomes was time- dependent and could be activated by Ca2+ and to a lesser extent by Mg2+. No ne of the other divalent cations tested could activate the transport activi ty. The K-m value of dCTP in the presence of Ca2+ was shown to be 3 mu M. d CDP but not dCMP or dCyd could inhibit the transport activity. Other deoxyn ucleoside triphosphates could also inhibit the uptake of dCTP with the pote ncy dGTP = dATP > TTP. Although ATP could competitively inhibit dCTP uptake with a K-i value of 8 mu M, the reconstituted dCTP uptake activity was not sensitive to the ATP/ADP carrier inhibitor atractyloside or the sulfhydryl reagent N-ethylmaleimide. This suggests that the dCTP transport system stu died is not the same as the ATP/ADP carrier. In conclusion, these studies d escribe the first functionally reconstituted mitochondrial carrier that dis plays an efficient transport activity for dCTP.