INHIBITION OF THE MITOCHONDRIAL CA2+ UNIPORTER BY PURE AND IMPURE RUTHENIUM RED

Commercial ruthenium red is often purified by a single recrystallizati on as described by Luft, J.H. (1971) Anat Rec 171, 347-368, which yiel ds small amounts of material having an apparent molar extinction coeff icient of similar to 67,400 at 533 nm. A simple modification to the pr ocedure dramatically improves the yield, allowing crystallization to b e repeated. Three times recrystallized ruthenium red has an apparent e xtinction coefficient of similar to 85,900, the highest value reported to date. Both crude and highly purified ruthenium red can be shown to inhibit reverse activity of the mitochondrial Ca2+ uniporter (uncoupl ed mitochondria), provided that care is taken to minimize and account for Ca2+ release through the permeability transition pore. Crude ruthe nium red is 7-10 fold more potent than the highly purified material in this regard, on an actual ruthenium red concentration basis. The same relative potency is seen against forward uniport (coupled mitochondri a), however, the I-50 values are 10 fold lower for both the crude and purified preparations. These data demonstrate unambiguously that the e nergy state of mitochondria affects the sensitivity of the Ca2+ unipor ter to ruthenium red preparations, and that both the forward and rever se reactions are subject to complete inhibition. The data suggest, how ever, that the active inhibitor may not be ruthenium red per se, but o ne or more of the other ruthenium complexes which are present in ruthe nium red preparations.