Sub-mitochondrial location of Ruthenium Red-sensitive calcium-ion transport and evidence for its enrichment in a specific population of rat liver mitochondria

Abstract

Seven fractions sedimenting at between 3000 and 120,000 g min were prepared from a rat liver homogenate by differential centrifugation in buffered iso-osmotic sucrose. The following measurements were carried out on each of these fractions: ruthenium red-sensitive Ca2+ transport in the absence and in the presence of Pi as well as in the presence of N-ethylmaleimide to prevent Pi cycling, succinate-supported respiration in the absence and in the presence of ADP, the membrane potential (.DELTA.E) and transmembrane pH gradient (-59 .DELTA.pH) components of the protonmotive force, cytochrome oxidase, uncoupler-stimulated adenosine triphosphatase, .alpha.-glycerophosphate dehydrogenase, Pi content and the effect on the resting rate of respiration of repeated additions of a fixed Ca2+ concentration. Ca2+ transport either in the presence or in the absence of added Pi and in the presence of N-ethylmaleimide exhibits significantly higher rates in the fraction sedimenting at 8000 g min. By contrast, respiration in the presence or in the absence of added ADP and the values for .DELTA.E and -59 .DELTA.pH were similar in those fractions sedimenting between 4000-20,000 g min, indicating that the driving force for Ca2+ transport was similar in each of these fractions. Experiments designed to determine the capacity of the individual fractions for Ca2+, as measured by the effect of repeated additions of Ca2+ on the resting rate of respiration, showed that fraction 2, i.e., that sedimenting at 8000 g min, also exhibited the greatest tolerance towards the uncoupling action of the ion. Of the 3 enzyme activity profiles, only that of .alpha.-glycerophosphate dehydrogenase was similar to that of Ca2+ transport. Since previous workers have assigned this enzyme to loci in the inner peripheral membrane [Werner and Neupert (1972) the ruthenium red-sensitive Ca2+- transport system may also be located in this domain of the inner membrane. The relation of these findings to the mechanisms of mitochondrial Ca2+ transport and the biogenesis of mitochondria is discussed.