Berton C. Pressman

The direction of polarity of the mitochondrial trans-membrane potential

Abstract The concentration of various anions in the sucrose impermeable space of ratliver mitochondria relative to the applied concentration can be used to calculate an equilibrium potential if one supposes that the anion distribution is passive. Values so calculated for anions of different net charge are about 30 mV, with the interior positive. The relative merits of the hypotheses that an internal positivity is generated by active cation movement or Mitchell s chemiosmotic generation of an internal negativity coupled with hydroxyl-for-anion exchanges are examined.

Two-phase partition studies of alkali cation complexation by ionophores

SummaryThe partition of alkali cations and anions between an aqueous and an immiscible organic phase has been studied in the absence and presence of neutral and carboxylic ionophores of the valinomycin and nigericin types, respectively. Cation extraction into the organic phase was augmented considerably by the ionophores, and a cation specificity of K+≧Rb+>Cs+≫Na+ was found for all the neutral ionophores tested. Evidence is given that the actual values of ion specificity are a function of the solvent polarity, especially for valinomycin where an inversion of the K+/Rb+ specificity was observed. The ionophores examined have the following rank order of effectiveness for K+ extraction into a standard organic phase consisting of 70% toluene-30%n-butanol: valinomycin>18-crown-6≫trinactin>enniatin B≈dinactin>monactin>nonactin. The ion affinity and selectivity data thus obtained have been compared with data previously reported.In a toluene-butanol solvent, extraction of cations in the absence of ionophores occurs as ion pairs. On the other hand, the neutral ionophores extract the cations by the mechanism of complexation, with the lipophilic anions coextracted as free gegenionic species at lower ionophore complex concentrations. When the concentration of extracted cations exceeds 1×10−4m, ion pairing between the ionophore complex and the anion occurs, and this tendency increases with increasing concentration and decreasing polarity of the organic phase. Anion pairing with the complexed cations is much less than for the free cations and this effect appears to be due to the larger distance of closest approach of the anion for the complexed cation.

Rates of exchange of mitochondrial Mg2+ determined from 28Mg flux measurements

Abstract The rate of exchange of the endogenous Mg 2+ of isolated rat liver mitochondria was measured by means of the radioisotope 28 Mg. Two different centrifugation techniques yielding similar results, were utilized for separating the mitochondria from suspenpension media for analysis. Mitochondrial Mg 2+ exchanges very slowly with externally added Mg 2+ . The influx of labeled Mg 2+ observed in the presence of substrate is blocked by treatment with the respiratory inhibitor, rotenone, or the uncoupler, p -trifluoromethoxy(carbonylcyanide) phenylhydrazone. The observed rates of Mg 2+ influx are similar in order of magnitude to rates of K + influx measured under comparable conditions. Parathyroid hormone stimulates Mg 2+ influx, but only when metabolic energy is available to support net Mg 2+ uptake.

Competition between magnesium and guanidine for mitochondrial binding sites.

Abstract A number of recent, independent investigations have shown that the binding of Mg++ ( Brierley et al. , 1962 ), Ca++ ( Vasington and Murphy, 1962 ) and Mn++ ( Chappell et al. , 1962 ) to mitochondria share in common a characteristic concomitant binding of phosphate. It has been suggested by Brierley et al. (1962) that the known affinities of these metal ions for phosphate may be involved in the binding and indeed that actual intramitochondrial precipitation of insoluble phosphates is an integral part of the process. We wish to call attention to the similarity of these binding phenomena to that obtainable with guanidine and alkylguanidines ( Pressman, 1963 ). The present communication will extend the similarity by showing that the binding of guanidine shows a common pattern with that of Mg++ in response to various inhibitors, and furthermore, that guanidine and Mg++ are mutually competetive for mitochondrial binding sites.

Turnover numbers for ionophore-catalyzed cation transport across the mitochondrial membrane

SummaryThe turnover numbers of the ionophores valinomycin, the macrolide actins, enniatin B and dicyclohexyl 18-crown-6 for translocation of cations through the mitochondrial membrane have been compared quantitatively. The rank order of decreasing maximum turnover number for K+ transport calculated on the basis of the ionophore concentration within the membrane is trinactin>dinactin∼monactin∼ valinomycin>nonactin>18-crown-6>enniatin B. The strength of binding of the ionophores to the mitochondria has the following rank order: valinomycin>macrolide actins>enniatin B>18-crown-6.A rough proportionality was observed between the transport rate of K+, Rb+, Cs+ or Na+ with a given ionophore and the heterogeneous complexation constant of the corresponding ionophore-cation pair in two-phase extraction experiments. However, the proportionality constants between transport and the heterogeneous complexation constant differ between the ionophores. These comparisons indicate that valinomycin and enniatin B transport cations about 10 times slower than would be expected from their two-phase complexation behavior, using the complexation and transport reactions of the macrolide actins as a basis for comparison. Transport with 18-crown-6 was about 1,000 times slower than predicted. These observations are discussed in terms of partitioning of the ionophores between various regions of the mitochondrial membrane.The data are discussed in terms of a carrier model involving hydrophilic complexes on the membrane surface in addition to hydrophobic complexes which cross the membrane.

Electromeric analysis of cytochromes in mitochondria

Abstract Potentiometric techniques have been used to examine the interactions of certain redox dyes with mitochondria. Most of the dyes tested proved unsuitable in low concentrations for analysis by potentiometry, and, at high concentrations, they either had untoward effects on the mitochondria or served themselves as electron transfer reagents. However, tetramethyl p -phenylenediamine proved an exception in that it gave a rapid response at the platinum electrode at 10 μ m concentration and equilibrated rapidly with a component in the electron transfer system. Since this reagent was used in catalytic concentrations, it did not serve as a donor or sink of electrons, but rather as an indicator of the redox potential of a component of the mitochondria during respiration. Analysis of potential changes on adding ADP, inhibitors, or uncouplers, and comparison with spectroscopic data, enabled a correlation of the potential of TMPD with the response of cytochrome c . Polarographic analysis facilitated the examination of redox reagents which did not react rapidly in potentiometric analysis. The specificity of the reagent toward particular components of the mitochondrial chain depended on the reagent used. Vitamin K 5 proved to have a preferential specificity toward cytochrome b as shown by its polarographic response to the addition of reagents that alter the electron transfer characteristics of the mitochondria.

Correlation between changes in metabolite concentrations and rate of ion transport following glucose addition to Escherichia coli B

Abstract Washed, starved preparations of Escherichia coli B metabolize glucose with concomitant uptake of K + and O 2 and extrusion of H + . Abrupt changes in the rates of these processes are correlated with apparent sequential shifting in time of the control sites of aerobic glycolysis from the glyceraldehyde-3-phosphate dehydrogenase-3-phosphoglyceric phosphokinase couple to phosphofructokinase to hexikinase. It is suggested that the principal inhibitors of phosphofructokinase activity are citrate and ATP. The rate of K + uptake is neither a function of the rate of glucose dissimilation nor of the concentration of ATP in these short-term experiments, and the energy available for the active transport of ions appears to vary as the intracellular chemical environment changes.

[72] The use of K+ concentration gradients for the synthesis of ATP by mitochondria

Publisher Summary The release of endogenous mitochondrial K + caused by the ionophore valinomycin can be coupled to net adenosine triphosphate (ATP) synthesis. Thus, the free energy of a K + concentration gradient can drive an endergonic process, such as phosphorylation; preloading mitochondria with K+ augments the yield of ATP considerably. The use of ionophores to generate a cation diffusion potential to drive various endergonic processes has been applied to a broad spectrum of energy transductions. Exergonic valinomycin-induced K + transport has been shown to drive (1) ATP synthesis by chloroplasts, (2) ATP synthesis by submitochondrial particles, (3) reversed mitochondrial electron transport, (4) Ca 2+ transport by mitochondria, (5) energy-linked spectral changes in bacterial carotenoids, and (6) energy-dependent fluorescence shifts of various membrane probes. These and related phenomena are explained in terms of membrane potential generation coupled to H + transport. The reproducible demonstration of ATP synthesis via mitochondrial K + gradients and includes a description of related processes. Factors important for investigating the relationships between ion gradients and various energy transformations are discussed in the chapter.

Subcellular distribution in surviving rat heart slices of Persantin-2,6-14C and its fixation to isolated subcellular particles☆

Abstract In experiments with surviving rat heart slices it has been shown that Persantin and 2,6- 14 C-Persantin permeate the myocardial cell. Under our conditions, 50% or more of the drug in the medium was passed to the intracellular space and distributed among the intracellular compartments. The labeled drug was found in all the particles and in the cytoplasm. Fixation studies in vitro with sarcosomes or with the microsomal fraction, in amounts comparable to those found in the myocardial cell, showed that about 30% of labeled Persantin in the medium was fixed to the sarcosomes; 60% of this appeared to be tightly bound, whereas the remainder could be washed out with isotonic sucrose. The microsomal fraction in vitro fixed about 6% of labeled Persantin in the medium. These differences largely reflect the fact that there is about ten times more nitrogen in the cell attributable to sarcosomes than to the microsomal fraction. From paper Chromatographic studies of the labeled lipid-soluble fraction combined from all intracellular compartments, it can be stated that most of the 14 C-Persantin is not chemically altered under the conditions employed for these studies.