Paolo Dell'Antone

Evidence for an ATP-driven “proton pump” in rat liver lysosomes by basic dyes uptake

Abstract When rat liver lysosomes are suspended in a medium containing acridine orange at neutral pH, accumulation of the dye may be observed within the vesicles. The uptake appears driven by a pH gradient between the external medium and the interior of the lysosomes since it is inhibited by NH 4 + , nigericin and other electroneutral proton-cation exchangers. FCCP is ineffective in inhibiting the uptake. In the presence of Mg ++ and anions such as Cl − , ATP promoted a further and more extensive but slower oligomycin and ouabain-insensitive dye uptake, which was also inhibited by FCCP. Very similar results were obtained with neutral red and atebrin. When the rate of the ATP-induced acridine uptake in preparations of different purification grade was compared, it was observed that the uptake rate increased in parallel with lysosomal enzymatic activity. These results suggest that an electrogenic ATP-driven-Mg ++ dependent “proton pump” is operating in the lysosomal membrane, as previously proposed.

ATP-dependent and ionophore-induced proton translocation in pea stem microsomal vesicles

Abstract The presence of an electrogenic pump in pea stem microsomal vesicles has already been demonstrated, but no evidence on the nature of the electrogenic ion has been presented (Rasi-Caldogno, F., De Michelis, M.I. and Pugliarello, M.C. (1981) Biochim. Biophys. Acta 642, 37–45). In this work we tested the usefulness of the ΔpH probe Acridine orange to monitor both ATP-dependent and ionophore-induced H+ fluxes in pea stem microsomal vesicles. The H+/K+ exchanger nigericin causes a marked uptake of protons into the vesicles that can be followed, with similar results, both as Acridine orange absorbance changes and pH changes of the external medium. ATP induces an uptake of Acridine orange into the vesicles which is reversed by FCCP and abolished by the presence of Triton X-100 in the incubation medium, thus indicating an inward, ATP-driven, H+ translocation. The ATP-dependent acridine orange uptake is Mg2+-requiring and KCl-stimulated. Such activity is inhibited by two specific ATPase inhibitors, dicyclohexylcarbodiimide and diethylstilbestrol, while it is unaffected by oligomycin and Na3VO4. These results show that Acridine orange is a useful probe to measure pH gradients in our membrane system and are consistent with the hypothesis that an ATPase of plasmalemma may act as a proton pump.

H+/site ratio and steady state distribution of divalent cations in mitochondria.

Mitochondria as well as bacteria are known to operate as proton pumps, i.e. the metabolic force is used to transport primarily protons across the membrane. The primary transport of protons is then coupled through various mechanisms to the movement of other species, cations, anions and water. The mechanism of proton transport has been assumed to occur via either respiratory loops [ 1 ] or membrane Bohr effect [2,3] or H’ carrier or channel [4] . Since the membrane Bohr effect and the proton channel may accomodate a stoicheometry of 4 H’/ site while the respiratory loops are compatible with a ratio only of 2 H/site, much interest has been concentrated over the exact determination of the H/site ratio. While Mitchell and Moyle [5 ] reported a ratio of 2 H/site, recently Lehninger and associates [6,7] reported that when the movement of endogenous phosphate is restricted a ratio of 3 or 4 H/site could be measured. This observation supports the earlier conclusions of Azzone and associates [8-lo], who not only measured a ratio of 4 K’/site but also indicated the opportunity of measuring the stoicheometry on cation rather than on H’ charges [ 1 l] . This is due to the masking of the H’ fluxes because of overlapping anion fluxes. A role of endogenous phosphate in affecting the H/site ratio is confirmed also by the appearance of a spin exchange signal in the ESR spectrum during Mr? uptake [ 121; the signal is attributed to (Mn)@O& precipitate. While the view of 4 H/site is gaining increasing consensus, Moyle and Mitchell [ 131 have brought a new argument in favour of 2 H/site, based on the

Aluminum inhibits the lysosomal proton pump from rat liver

Lysosomes are cytoplasmatic organelles, delimitated by a single lipoprotein membrane, that contain several enzymes mostly belonging to the hydrolases in that they function mainly for intracellular digestion. Lysosomal internal pH is characteristically acidic and it is maintained around pH 4.5 by a proton pump, an ATPase, that uses energy from ATP hydrolysis to translocate H+ ions into lysosomes. In the presence of Al3+ the proton pump activity is markedly reduced compromising acidic vesicles functionality. Among different species utilized, Al2(SO4)3 and AlF3 were the most effective. Aluminum effect was not observed when the delta pH was produced artificially by nigericin.

Nucleophilic sites in energized mitochondrial membranes

pH indicators have been used in studies with mitochondria and submitochondrial particles with increasing frequency in recent years [ 1 3 ] . The absorbancy changes observed have been so far interpreted mostly as due to acid-base transitions [ 1 3 ] . The acid-base transition however has also been suggested to occur because of migration of the indicator between the inner and the outer mitochondrial aqueous phase [3]. The hypothesis that some of the responses of the indicator might reflect conformational changes of the membrane has not yet been confirmed [4]. Fluorescent probes have also been used to follow the metabolic transitions of mitochondrial membranes [5]. The fluorescence changes during energization have been interpreted as due either to conformational changes of the membrane [5,6] or to migration of the probes between phases of different hydrophobicity [7]. In the present paper we report our studies with another pH indicator, neutral red. It will be shown that at acidic pH the indicator responses to energization of submitochondrial particles cannot be interpreted as acid-base transitions but rather as due to an attractive interaction between dye molecules leading to phenomena of the stacking type.

Mitochondrial bioenergetics as affected by cationic detergents

This paper examines the accumulation and toxicity mechanism of a cationic detergent, cetyltrimethylammonium (bromide) (CTAB), in energized rat liver mitochondria. The results suggest that: (1) the CTAB ion is accumulated in the mitochondrial matrix by a membrane potential-driven uptake mechanism; (2) accumulation may lead to a toxic effect, since it gives rise to collapse of the mitochondrial membrane potential (ΔΦ) which is correlated with ATP synthesis and which regulates Ca++ uptake; (3) collapse of ΔΦ may be due to enhanced permeability of the mitochondrial membrane to the ions (detergent effect); and (4) ΔΦ collapse and Ca++ and K+ release were also observed in another cationic detergent, NTAB, but not in the presence of anionic detergents.

ESR determination of Mn++ uptake and binding in mitochondria.

Active transport in energy transducing membranes involves the utilization of metabolic energy to move ions against electrochemical gradients. In the case of the divalent cations which are actively transported by mitochondria, namely Ca++, Mn++ and Sr++ [l] , the problem arises as to the determination of the matrix concentration of osmotically active cations. Massari et al. [2] have calculated the ion uptake from absorbancy changes. Gunther and Puskin [3-51 have used the ESR signal of Mn++ to distinguish between free and bound cations and to calculate the matrix concentration of free Mn++. According to Gunther and Puskin [3], during active uptake the Mn++ spectrum can be split into two components E and S belonging to membrane bound and matrix free Mn++, respectively. The binding of divalent cations to the membrane has also been followed qualitatively through the enhancement of the fluorescent chelate of tetracycline with divalent cations [6]. In the present paper the changes of e.s.r. signal of Mn++ during active uptake have been correlated with absorbance and.rhatrix volume changes. On the basis of the combined volume and e.s.r. data a procedure is described for the determination of free Mn++ concentration in the mitochondrial matrix.

Anticholinesterasic drugs: tacrine but not physostigmine, accumulates in acidic compartments of the cells

Tacrine (THA) and physostigmine (PHS) have been used in Alzheimers disease therapy for their anticholinesterasic activity. Here we show that THA is taken up in rat lysosomes in an energy-dependent manner, and that it is also accumulated in acidic compartments of rat thymocytes and neuroblastoma cells. A concentration of THA less than 1 mM dissipated the pH gradient (delta pH) in all the above mentioned in vitro systems. On the contrary higher concentrations of PHS (1-2 mM) were ineffective. The accumulation of THA in acidic organelles of the cell may be relevant for the pharmacological action of THA in Alzheimers treatment.

Binding changes and apparent pKa shifts of bromthymol blue as tools for mitochondrial reactions

Abstract The passive interaction of bromthymol blue and other anionic and neutral dyes with mitochondria and particles is accompanied by an increase of the apparent p K a , is enhanced at higher ionic strengths, is slightly inhibited by neutral dyes, is high only for the acidic, un-ionized form, and is independent of the presence of the sulfonic side chain. The pH dependence for the binding of the dyes follows the ionization of the chromophoric group: the pK of binding, defined as the pH for 50% binding, is identical with the apparent p K a of the dye. The interaction of bromthymol blue results in shifts of the apparent p K a , toward the alkaline, upon energization of particles or of mitochondria in the absence of cation uptake, and toward the acid upon energization of mitochondria in the presence of Ca 2+ uptake. In the case of the energized particles there is a decrease in the dissociation constant of the membrane for the dye. The increase of apparent p K a is accounted for by the increased binding of the acidic dye and conversion of the free alkaline into acidic form. Bromthymol blue is seen as localized at the external interface with the apolar part in a hydrophobic environment and the hydrophilic part in water. The absorbance changes of bromthymol blue with mitochondria and particles are ascribed to increases of the activity coefficients of one or the other forms of the dye, and bromthymol blue may be taken as a probe of hydrophobic and electrostatic interactions. The hypothesis that the uptake of organic anions in energized particles be due to modifications of the met membrane charge or potential is criticized. It is suggested that the conformational changes accompanying energization involve local increases of membrane hydrophobicity. The higher affinity of the anions is due to stronger electrostatic interactions to electrophilic sites in less polar environments.

Metachromatic effects and binding of organic cations to energized submitochondrial particles

Abstract Energization of submitochondrial particles results in a marked increase of binding, measured as number of sites and binding constants, of the cationic dyes Acridine Orange and Neutral Red. The binding of the dyes is accompanied by spectral changes which are identified as metachromatic effects. The findings are interpreted in terms of interaction with electron-negative sites and stacking of the dye molecules.