The distribution of inorganic phosphate and malate between intra- and extramitochondrial spaces. Relationship with the transmembrane pH difference.

N L Greenbaum,D F Wilson

doi:10.1016/s0021-9258(20)71180-4

Abstract

The steady-state distribution of inorganic phosphate and malate between the intra- and extramitochondrial spaces was measured in suspensions of nonrespiring and respiring rat liver mitochondria in which the transmembrane pH difference was incrementally varied. In respiration-inhibited mitochondria, the slope of log [Pi]in/[Pi]out (ordinate) versus delta pH approached 2 by either chemical or isotopic determination of [Pi], and the slope of log [malate]in/[malate]out versus delta pH was 2.0 with an extrapolated log [Pi]in/[Pi]out value of 0.3 at delta pH = 0. We conclude that the distribution of Pi and malate for nonrespiring mitochondria were quantitatively consistent with those predicted by exchange of Pi- for OH- (or cotransport with H+) and of malate 2- for Pi2-. In respiring mitochondria using glutamate + malate as substrate, there was very little pH dependence of Pi or malate accumulation (the slopes were less than 0.5) unless n-butylmalonate (inhibitor of Pi-dicarboxylate exchange) was added before the glutamate and malate, in which case the distribution patterns at delta pH less than 0.4 were similar to those in nonrespiring mitochondria. In either case, however, after reaching a maximal value of 1.1, log [Pi]in/[Pi]out did not further increase with increasing delta pH. Thus, in normally metabolizing mitochondria, the distributions of Pi and malate are not directly correlated with the difference in pH across the membrane.

Highlights

The steady-statedistribution of inorganic phosphatemersalyl inhibits both transporptrocesses),but only the phosand malate between the intra- and extramitochondriaplhate-dicarboxylate exchange is inhibited by substrate anaspaces was measured in suspensions of nonrespiring logs such as n-butylmalonate and phenylsuccinate
As substrate, therewas very little pH dependenceof Pi A correlation between the transmembrane pH difference or malate accumulationnless and Pi distribution has been noted previously in respirationn-butylmalonate(inhibitorofPi-dicarboxylate ex- inhibited mitochondria [4,5,6,7, 11, 16], as well as in vesicles change) was added before the glutamate and malate, into which the Pi transport protein had been reconstituted in which case the distribution patterns at ApH c 0.4 [17]
While ATP is stoichiometrically exchanged for ADP by the adenine nucleotide translocase, the majority of Pi uptake occurs via a separate transport protein of approximately 34 and respiring mitochondria

Summary

RELATIONSHIPWITHTHE TRANSMEMBRANEpHDIFFERENCE*

From the Departmentof Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania19104. While ATP is stoichiometrically exchanged for ADP by the adenine nucleotide translocase, the majority of Pi uptake occurs via a separate transport protein of approximately 34 and respiring mitochondria From these studies, we have been able to conclude that while in the absence of other transport and metabolic processes, phosphate and dicarboxylate distributions follow a predictable (approximately second power) dependence on the pH difference, this behavior is not observed under more physiologicalconditions. = 6.8-6.9) for the extramitochondrial medium (determined directly by titrating the Pi in the assay medium with acid and base) These latter values are consistent with those of Roberts et al [35].Derivation of the predicted equilibrium distributions of Pi and malate for the exchange mechanisms depicted in Fig. 1 can be found in the “Appendix.” The final relationships are as follows. Aliquots from each resulting supernatant fraction were separately quenched by addition to equal volumes of cold 4%

RESULTS

Pi MalaPtreopionate

Pi and Malate Distribution inMitochondria

DISCUSSION

The total chemically measurable phosphate is the sum of