Role of substrates in Sr2+-induced oscillations of ionic fluxes in rat liver mitochondria

(1) The reason for substrate specificity of Sr²⁺-induced oscillating cation fluxes in isolated rat liver mitochondria was investigated. (2) With succinate as substrate, rotenone prevented oscillation. In this case the mitochondria were only partially able to take up added Sr²⁺ and did not take up any of the released K⁺. Addition of substances decreasing the mitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio (oxaloacetate or acetoacetate) restored the ability for reuptake of K⁺ and for complete uptake of Sr²⁺ and, therefore, oscillation. (3) Inhibition of substrate-level phosphorylation by arsenite or uncoupling of substrate-level phosphorylation by arsenate in the presence of oligomycin also suppressed the reuptake of cations. This effect of inhibition of substrate-level phosphorylation on oscillation could be circumvented by addition of ATP in the presence of oligomycin. (4) Prevention of the intramitochondrial regeneration of 2-oxoglutarate from acetyl-CoA and oxaloacetate by fluorocitrate or from endogenous glutamate by aminoxyacetate shortened the time during which oscillation with succinate as substrate could be observed. (5) From the key role of substrate level phosphorylation it is concluded that for the reuptake of K⁺ and Sr²⁺ during oscillation, sufficient GTP generation by the succinyl thiokinase (EC 6.2.1.4) reaction is essential. Therefore substrate level phosphorylation seems to be a necessary energy source additional to the respiratory chain. Since the latter process drives the active cation movements, the former may be required for the restoration of a sufficiently low proton conductance of the mitochondrial inner membrane. Oscillation in the absence of exogenous ATP therefore demands 2-oxoglutarate as substrate or the intramitochondrial generation of 2-oxoglutarate for the maintenance of a sufficient GTP production for a longer time.     

Inhibition by Sr2+ of specific mitochondrial Ca2+-efflux pathways

The effect of Sr²⁺ on the set point for external Ca²⁺ was studied in rat heart and liver mitochondria with the aid of a Ca²⁺-sensitive electrode. In respiring mitochondria the set point is determined by the rates of Ca²⁺ influx on the Ca²⁺ uniporter and efflux by various mechanisms. We studied the Ca²⁺-Na⁺ exchange pathway in heart mitochondria and the Δψ-modulated efflux pathway in liver mitochondria. Prior accumulation of Sr²⁺ was found to shift the set points towards lower external Ca²⁺ both in heart mitochondria under conditions of Ca²⁺-Na⁺ exchange and in liver mitochondria under conditions that should promote opening of the Δψ-modulated pathway. The effect on the set point was found to be due to inhibition of Ca²⁺ efflux by Sr²⁺ taken up by the mitochondria, while Sr²⁺ efflux was too slow to be measurable.     

Cation/proton antiport systems in Escherichia coli.

Three distinct systems which function as proton/cation antiports have been identified in $\text{E.}\text{coli}$ by the ability of the ions to dissipate the ΔpH component of the protonmotive force in everted vesicles. System I exchanges H⁺ for K⁺, Rb⁺ or Na⁺; System II has Na⁺ and Li⁺ as substrates; and System III catalyzes proton exchange for Ca²⁺, Mn²⁺ or Sr²⁺.     

Involvement of periodic deacylation-acylation cycles of mitochondrial phospholipids during Sr2+-induced oscillatory ion transport in rat liver mitochondria

Lysophosphatidylcholine and lysophosphatidylethanolamine levels were determined during Sr²⁺-induced oscillating ion fluxes in mitochondria prelabelled in vivo with ³²P_i. Periodic fluctuations of both lyso compounds were established with an increase at the stage of simultaneously monitored K⁺ influx and a decrease at K⁺ efflux. The periodic activations and inactivations of phospholipase were found to be associated with periodic changes in the incorporation rates of labelled polyunsaturated fatty acids with an apparent phase difference of 180°. Periodic deacylation-acylation cycles of phospholipids accompanying the periodic cycles of reversible ion accumulation and release are suggested to be involved in the trigger mechanism generating the permeability changes during oscillatory ion transport.     

Divalent cation stimulation of substrate oxidation by corn mitochondria

The oxidation of reduced nicotinamide adenine dinucleotide, malate-pyruvate, and succinate by corn mitochondria in buffered 0.2 m KCl was determined as a function of divalent cations. Ni²⁺, Mg²⁺, Co²⁺, Ca²⁺, Mn²⁺, Sr²⁺, and Ba²⁺ stimulated reduced nicotinamide adenine dinucleotide oxidation in the absence of inorganic phosphate, with Ca²⁺ and Sr²⁺ having the greatest effect. Malate-pyruvate and succinate oxidation was stimulated by Ca²⁺, Ba²⁺, and Sr²⁺, but only in the presence of inorganic phosphate. Ca²⁺, Sr²⁺, and Ba²⁺ produced a simulated state 4 to state 3 transition with all three substrates, but only with malate-pyruvate and succinate was there a return to state 4. The order of divalent cation effectiveness suggests that the rate of water substitution from the cation inner coordination hydration sphere may be a rate-limiting step in certain mitochondrial reactions involving electron transport and phosphorylation.     

Rapid calcium exchange for protons and potassium in cell walls of Chara

Net fluxes of Ca²⁺, H⁺ and K⁺ were measured from intact Chara australis cells and from isolated cell walls, using ion-selective microelectrodes. In both systems, a stimulation in Ca²⁺ efflux (up to 100 nmol m^?2 s^?1, from an influx of ～40 nmol m^?2 s^?1) was detected as the H⁺ or K⁺ concentration was progressively increased in the bathing solution (pH 7.0 to 4.6 or K⁺ 0.2 to 10mol m^?3, respectively). A Ca²⁺ influx of similar size occurred following the reverse changes. These fluxes decayed exponentially with a time constant of about 10 min. The threshold pH for Ca²⁺ efflux (pH 5.2) is similar to a reported pH threshold for acid-induced wall extensibility in a closely related characean species. Application of NH₄⁺ to intact cells caused prolonged H⁺ efflux and also transient Ca²⁺ efflux. We attribute all these net Ca²⁺ fluxes to exchange in the wall with H⁺ or K⁺. A theoretical treatment of the cell wall ion exchanges, using the ‘weak acid Donnan Manning’ (WADM) model, is given and it agrees well with the data. The role of Ca²⁺ in the cell wall and the effect of Ca²⁺ exchanges on the measured fluxes of other ions, including bathing medium acidification by H⁺ efflux, are discussed.     

Osmotic Sensitivity of Ca2+ and H+ Transporters in Corn Roots: Effect on Fluxes and Their Oscillations in the Elongation Region

Seedling roots of corn were treated with different concentrations of mannitol-containing solution for 1 to 1.5 hr, and net fluxes of Ca²⁺ and H⁺ were measured in the elongation region. H⁺ fluxes were much more sensitive to osmotic pressure than were Ca²⁺ fluxes. Oscillations of 7-min period in H⁺ flux, normally observed in the control, were almost fully suppressed at high osmotic concentrations. Net H⁺ flux was shifted from average efflux of 25 ± 3 nmol m⁻² sec⁻¹ to average influx of 10 ± 5 nmol m⁻² sec⁻¹ after the incubation in 100 mm mannitol. The larger the osmotic concentration, the larger was the H⁺ influx. This flux caused the unbuffered solution of pH 4.85 to change to pH 5.3 after mannitol application. It appears that the osmoticum suppresses oscillatory H⁺ extrusion at the plasma membrane. Discrete Fourier Transforms of the H⁺ flux data showed that, apart from suppression of the 7-min oscillations in H⁺ flux, mannitol also promoted the appearance of faster 2-min oscillations. Ca²⁺ influx slightly increased after mannitol treatment. In addition the 7-min oscillatory component of Ca²⁺ flux remained apparent thereby showing independence of H⁺ flux. Received: 25 April 1997/Revised: 11 August 1997     

Tobacco pollen tubes as cellular models for ion dynamics: improved spatial and temporal resolution of extracellular flux and free cytosolic concentration of calcium and protons using pHluorin and YC3.1 CaMeleon

The presence of both calcium (Ca²⁺) and proton (H⁺) apical gradients is necessary for polarized cell elongation to occur in pollen tubes. So far, most of these studies have been carried out in lily pollen tubes, using chemical probes. Yet, lily is a refractory model for molecular genetics, with no easy protocol available for the construction of stable transgenic lines. Tobacco, however, is well suited for both transformation and cell biology, with sexual organs that are accessible, easy to handle and visualize. Pollen tubes are in an ideal size range for sub-cellular imaging analyses using modern microscopy techniques. Ion homeostasis in tobacco pollen tubes has not been precisely characterized so far. Here, we characterize the H⁺ and Ca²⁺ spatial and temporal patterns in tobacco pollen tubes by the use of two fluorescent genetic probes, pHluorin and the YC3.1 yellow CaMeleon, and direct measurement of extracellular flux by ion-sensitive vibrating probes. A distinct 0.4 pH unit acidic gradient was found to stretch from the tip up to 40 μm into the tube shank. This gradient intensity displayed 1–4 min period oscillations and is reduced in the non-growing phase of an oscillatory cycle. Furthermore, sub-membrane and sub-apical alkaline domains were detected. Extracellular H⁺ fluxes oscillated between 10 and 40 pmol cm⁻² s⁻¹. Fourier and continuous wavelet analyses showed tubes with one or two major oscillatory components in both extra and intracellular H⁺ oscillations. Cytosolic Ca²⁺ was imaged by confocal microscopy, showing a V-shaped 40 μm gradient extending from the tip, from 0.2 to 1.0 μM, which oscillates with a 1–4 min period, but with only one major oscillatory component. Extracellular Ca²⁺ fluxes oscillate in most pollen tubes, between 2 and 50 pmol cm⁻² min⁻¹ and, like in H⁺, with one or two major oscillatory peaks. A combination of confocal and widefield microscopy showed that H⁺ and Ca²⁺ displayed different patterns and shapes inside the cell, sometimes suggesting a structurally complementary role for these 2 second messengers in the growth process. These data suggest that fluxes at the apex of the pollen tube are directly responsible for establishment and maintenance of the gradient. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fluxes of Ca2+, Sr2+ and Mg2+ in synaptosomes.

Synaptosomes isolated from sheep brain cortex accumulate Ca²⁺, Sr²⁺ and Mg²⁺ when incubated in isosmotic sucrose media containing 5 mM of either of these cations. The maximal levels of cations retained per mg of protein are 100 nmol of Ca²⁺, 85 nmol of Mg²⁺ and 80 nmol of Sr²⁺. The loss of Ca²⁺ or Sr²⁺ from the preloaded synaptosomes is increased by monovalent cations in the following order: Na⁺> K⁺ > Li⁺> choline, whereas for the loss of Mg²⁺ this order is different: K⁺ > Na⁺ > Li ～ choline. The efflux of Ca²⁺ or Sr²⁺ induced by monovalent cations decreases as the temperature is lowered and it is nearly abolished at 0°C, whereas the efflux of Mg²⁺ is much less influenced by temperature. The results suggest that the mechanism of exchange of Ca²⁺ for Na⁺ in synaptosomes operates similarly for Sr²⁺, but not for Mg²⁺.     

Electroneutral H+-K+ exchange in liver mitochondria Regulation by membrane potential

The paper analyzes the factors affecting the H⁺-K⁺ exchange catalyzed by rat liver mitochondria depleted of endogenous Mg²⁺ by treatment with the ionophore A23187. The exchange has been monitored as the rate of K⁺ efflux following addition of A23187 in low-K⁺ media. (1) The H⁺-K⁺ exchange is abolished by uncouplers and respiratory inhibitors. The inhibition is not related to the depression of ΔpH, whereas a dependence is found on the magnitude of the transmembrane electrical potential, Δψ. Maximal rate of K⁺ efflux is observed at 180–190 mV, whereas K⁺ efflux is inhibited below 140–150 mV. (2) Activation of H⁺-K⁺ exchange leads to depression of ΔpH but not of Δψ. Respiration is only slightly stimulated by the onset of H⁺-K⁺ exchange in the absence of valinomycin. These findings indicate that the exchange is electroneutral, and that the Δψ control presumably involves conformational changes of the carrier. (3) Incubation in hypotonic media at pH 7.4 or in isotonic media at alkaline pH results in a marked activation of the rate of H⁺-K⁺ exchange, while leaving unaffected the level of Mg²⁺ depletion. This type of activation results in partial ‘uncoupling’ from the Δψ control, suggesting that membrane stretching and alkaline pH induce conformational changes on the exchange carrier equivalent to those induced by high Δψ. (4) The available evidence suggests that the activity of the H⁺-K⁺ exchanger is modulated by the electrical field across the inner mitochondrial membrane.     

Ca2+/H+ exchange in the plasma membrane of Arabidopsis thaliana leaves

A large number of plant Ca²⁺/H⁺ exchangers have been identified in endomembranes, but far fewer have been studied for Ca²⁺/H⁺ exchange in plasma membrane so far. To investigate the Ca²⁺/H⁺ exchange in plasma membrane here, inside-out plasma membrane vesicles were isolated from Arabidopsis thaliana leaves using aqueous two-phase partitioning method. Ca²⁺/H⁺ exchange in plasma membrane vesicles was measured by Ca²⁺-dependent dissipation of a pre-established pH gradient. The results showed that transport mediated by the Ca²⁺/H⁺ exchange was optimal at pH 7.0, and displayed transport specificity for Ca²⁺ with saturation kinetics at K _m = 47 μM. Sulfate and vanadate inhibited pH gradient across vesicles and decreased the Ca²⁺-dependent transport of H⁺ out of vesicles significantly. When the electrical potential across plasma membrane was dissipated with valinomycin and potassium, the rate of Ca²⁺/H⁺ exchange increased comparing to control without valinomycin effect, suggesting that the Ca²⁺/H⁺ exchange generated a membrane potential (interior negative), i.e. that the stoichiometric ratio for the exchange is greater than 2H⁺:Ca²⁺. Eosin Y, a Ca²⁺-ATPase inhibitor, drastically inhibited Ca²⁺/H⁺ exchange in plasma membrane as it does for the purified Ca²⁺-ATPase in proteoliposomes, indicating that measured Ca²⁺/H⁺ exchange activity is mainly due to a plasma membrane Ca²⁺ pump. These suggest that calcium (Ca²⁺) is transported out of Arabidopsis cells mainly through a Ca²⁺-ATPase-mediated Ca²⁺/H⁺ exchange system that is driven by the proton-motive force from the plasma membrane H⁺-ATPase.     

K+/H+ antiport in mitochondria

Mitochondria contain a latent K⁺/H⁺ antiporter that is activated by Mg²⁺-depletion and shows optimal activity in alkaline, hypotonic suspending media. This K⁺/H⁺ antiport activity appears responsible for a respiration-dependent extrusion of endogenous K⁺, for passive swelling in K⁺ acetate and other media, for a passive exchange of matrix⁴²K⁺ against external K⁺, Na⁺, or Li⁺, and for the respiration-dependent ion extrusion and osmotic contraction of mitochondria swollen passively in K⁺ nitrate. K⁺/H⁺ antiport is inhibited by quinine and by dicyclohexylcarbodiimide when this reagent is reacted with Mg²⁺-depleted mitochondria. There is good suggestive evidence that the K⁺/H⁺ antiport may serve as the endogenous K⁺-extruding device of the mitochondrion. There is also considerable experimental support for the concept that the K⁺/H⁺ antiport is regulated to prevent futile influx-efflux cycling of K⁺. However, it is not yet clear whether such regulation depends on matrix free Mg²⁺, on membrane conformational changes, or other as yet unknown factors.     

H+ Fluxes at Plasmalemma Level: In Vivo Evidence for a Significant Contribution of the Ca2+-ATPase and for the Involvement of its Activity in the Abscisic Acid-Induced Changes in Egeria Densa Leaves

Abstract: The features of Ca²⁺ fluxes, the importance of the Ca²⁺ pump‐mediated H⁺/Ca²⁺ exchanges at plasmalemma level, and the possible involvement of Ca²⁺‐ATPase activity in ABA‐induced changes of H⁺ fluxes were studied in Egeria densa leaves. The results presented show that, while in basal conditions no net Ca²⁺ flux was evident, a conspicuous Ca²⁺ influx (about 1.1 ìmol g^?1 FW h^?1) occurred. The concomitant efflux of Ca²⁺ was markedly reduced by treatment with 5 íM eosin Y (EY), a specific inhibitor of the Ca²⁺‐ATPase, that completely blocked the transport of Ca²⁺ after the first 20 ‐ 30 min. The decrease in Ca²⁺ efflux induced by EY was associated with a significant increase in net H⁺ extrusion (?ÄH⁺) and a small but significant cytoplasmic alkalinization. The shift of external [Ca²⁺] from 0.3 to 0.2 mM (reducing Ca²⁺ uptake by about 30 %) and the hindrance of Ca²⁺ influx by La³⁺ were accompanied by progressively higher ?ÄH⁺ increases, in agreement with a gradual decrease in the activity of a mechanism counteracting the Ca²⁺ influx by an nH⁺/Ca²⁺ exchange. The ABA‐induced decreases in ?ÄH⁺ and pH_cyt were accompanied by a significant increase in Ca²⁺ efflux, all these effects being almost completely suppressed by EY, in line with the view that the ABA effects on H⁺ fluxes are due to activation of the plasmalemma Ca²⁺‐ATPase. These results substantially stress the high sensitivity and efficacy of the plasmalemma Ca²⁺ pump in removing from the cytoplasm the Ca²⁺ taken up, and the importance of the contribution of Ca²⁺ pump‐mediated H⁺/Ca²⁺ fluxes in bringing about global changes of H⁺ fluxes at plasmalemma level.     

Swelling and contraction of potato mitochondria

Mitochondria isolated from potato tubers fail to undergo passive osmotic swelling when suspended in isotonic Na⁺ acetate or phosphate, in NaCl following addition of tripropyltin, or in Na⁺ nitrate following addition of an uncoupler. Swelling under each of these conditions in mitochondria from other sources has been attributed to the inward movement of Na⁺ on an endogenous Na⁺/H⁺ exchanger. Such a monovalent cation/H⁺ exchanger has also been implicated in respiration-dependent cation extrusion and contraction of swollen mitochondria. Potato mitochondria swollen in chloride and nitrate salts extrude ions and contract when respiration is initiated. The contraction reaction is slower and less efficient than that in beef heart mitochondria, but like the latter, is sensitive to uncouplers and stimulated by nigericin, butacaine, and Mg²⁺. These comparative studies suggest that a cation⁺/H⁺ exchanger is present in potato tuber mitochondria, but that it functions exclusively as a cation-extruding mechanism. They further suggest that cation⁺/H⁺ exchange activity is not identical in mitochondria from different sources and that these exchange components may have a directionality and regulatory features which differ with the metabolic needs of the source tissue.     

BIOCHEMICAL AND ULTRASTRUCTURAL ASPECTS OF CA2+ TRANSPORT BY MITOCHONDRIA OF THE HEPATOPANCREAS OF THE BLUE CRAB CALLINECTES SAPIDUS

Mitochondria isolated from the hepatopancreas of the blue crab Callinectes sapidus show up to 12-fold stimulation of respiration on addition of Ca²⁺, which is accompanied by Ca²⁺ accumulation (Ca²⁺:site = 1.9) and H⁺ ejection (H⁺:Ca²⁺ = 0.85). Sr²⁺ and Mn²⁺ are also accumulated; Mg²⁺ is not. A strongly hypertonic medium (383 mosM), Mg²⁺, and phosphate are required for maximal Ca²⁺ uptake. Ca²⁺ uptake takes precedence over oxidative phosphorylation of ADP for respiratory energy. Once Ca²⁺ is accumulated by the crab mitochondria, it is stable and only very slowly released, even by uncoupling agents. ATP hydrolysis also supports Ca²⁺ uptake. Respiration-inhibited crab hepatopancreas mitochondria show both high-affinity and low-affinity Ca²⁺-binding sites, which are inactive in the presence of uncoupling agents. Crab hepatopancreas mitochondria have an enormous capacity for accumulation of Ca²⁺, up to 5,500 ng-atoms Ca²⁺ per mg protein, with an equivalent amount of phosphate. Freshly isolated mitochondria contain very large amounts of Ca²⁺, Mg²⁺, phosphate, K⁺, and Na⁺; their high Ca²⁺ content is a reflection of the vary large amount of extra-mitochondrial Ca²⁺ in the whole tissue. Electron microscopy of crab mitochondria loaded with Ca²⁺ and phosphate showed large electron-dense deposits, presumably of precipitated calcium phosphate. They consisted of bundles of needle-like crystals, whereas Ca²⁺-loaded rat liver mitochondria show only amorphous deposits of calcium phosphate under similar conditions. The very pronounced capacity of crab hepatopancreas mitochondria for transport of Ca²⁺ appears to be adapted to a role in the storage and release of Ca²⁺ during the molting cycle of this crustacean.     

Early Sr2+-induced effects on membrane potential,proton pumping- and ATP hydrolysis-activities of plasma membrane vesicles from maize root cells

When released in plant environment, strontium (Sr²⁺) can be absorbed predominantly by the plant roots. As the plasma membrane of root cells is amongst the first barriers encountered by Sr²⁺ during its soil/plant transfer and the main entry point of Sr²⁺ into the roots, the main objective of this work aimed to enlighten on some of the Sr²⁺-induced effects at this level in Zea mays L. cv. “Liberal”.Thus this study focused on the Sr²⁺-induced changes on membrane potential of cortical root cells and on proton fluxes in maize roots, in order to determine whether the activity of some of the ion transport systems present in the plasma membrane of maize root cell could be among the first targets of Sr²⁺. We focused in particular on the plasma membrane H⁺-ATPase, known to be one of the major transport systems found in the plasmalemma where it generates a proton motive force (contributing to membrane potential maintaining, and providing energy for ion transport through membrane).The data presented here showed that Sr²⁺ triggered an early and transient membrane depolarisation whose magnitude and duration were dependent on the Sr²⁺-concentration. The time course pattern of a second longer lasting depolarisation could be examined in perspective with the Sr²⁺-induced decrease of the spontaneous proton extrusion observed in root tissues, suggesting a relationship between Sr²⁺-effects on membrane potential and H⁺ excretion. Furthermore, the inhibitory effect exerted by Sr²⁺ on the fusicoccin (FC)-enhanced proton extrusion strongly suggested an inhibition of the plasma membrane H⁺-ATPase. This hypothesis was supported by the inhibition induced by Sr²⁺ on proton pumping- and ATP hydrolysis-activities measured in plasma membrane vesicles (PMV) prepared from maize roots.Taken together the data reported here evidence that, with however a lower efficiency, Sr²⁺ behaved in a quite similar way to Ca²⁺ when inhibiting the H⁺-ATPase activity, and suggest that Sr²⁺ could partially mimic Ca²⁺ onto regulation of the H⁺-ATPase activity.     

Involvement of palmitate/Ca2 +(Sr2 +)-induced pore in the cycling of ions across the mitochondrial membrane

The palmitate/Ca^2 +-induced (Pal/Ca^2 +) pore, which is formed due to the unique feature of long-chain saturated fatty acids to bind Ca^2 + with high affinity, has been shown to play an important role in the physiology of mitochondria. The present study demonstrates that the efflux of Ca^2 + from rat liver mitochondria induced by ruthenium red, an inhibitor of the energy-dependent Ca^2 + influx, seems to be partly due to the opening of Pal/Ca^2 + pores. Exogenous Pal stimulates the efflux. Measurements of pH showed that the Ca^2 +-induced alkalization of the mitochondrial matrix increased in the presence of Pal. The influx of Ca^2 + (Sr^2 +) also induced an outflow of K⁺ followed by the reuptake of the ion by mitochondria. The outflow was not affected by a K⁺/H⁺ exchange blocker, and the reuptake was prevented by an ATP-dependent K⁺ channel inhibitor. It was also shown that the addition of Sr^2 + to mitochondria under hypotonic conditions was accompanied by reversible cyclic changes in the membrane potential, the concentrations of Sr^2 + and K⁺ and the respiratory rate. The cyclic changes were effectively suppressed by the inhibitors of Ca^2 +-dependent phospholipase A₂, and a new Sr^2 + cycle could only be initiated after the previous cycle was finished, indicating a refractory period in the mitochondrial sensitivity to Sr^2 +. All of the Ca^2 +- and Sr^2 +-induced effects were observed in the presence of cyclosporin A. This paper discusses a possible role of Pal/Ca^2 + pores in the maintenance of cell ion homeostasis.     

Partial characterization of placental 3ß-hydroxysteroid dehydrogenase (EC 1.1.1.145), Δ4–5isomerase (EC 5.3.3.1) in human term placental mitochondria

A partial characterization of human term placental 3ß-HSDH in mitochondria is reported. Apparent K_M of pregnenolone: 70 nM. A dose-dependent stimulation of 3ß-HSDH by NAD⁺ or NADP⁺ was observed in the range from 10⁻⁶ to 10⁻³ M (K_M value of NAD⁺: 20 μM). At equimolar concentrations NAD⁺ is more than 10-fold as effective a cofactor of the 3ß-HSDH than NADP⁺. pH optimum: 9.5 (glycine-NaOH buffer). Temperature optimum 40–45°C. A rapid loss of 3ß-HSDH activity was found after preincubation of the enzyme at 37°C after 30 min: less than 50% of initial enzyme activity is present. No inhibition was obtained by Mg²⁺, Ca²⁺ Sr²⁺ and Ba²⁺ (1–100 mM). A strong inhibition was achieved with 1 mM Zn²⁺, Cd²⁺, Cu²⁺ and 10 mM and 100 mM Fe²⁺, Mn²⁺, Co²⁺ and Ni²⁺.     

Induction of Na+ / K+ exchange in swollen heart mitochondria

Heart mitochondria swollen passively in nitrate salts contract in a respiration-dependent reaction which can be attributed to an endogenous cation/H⁺ exchange component (or components). The rate of contraction increases with increased extent of passive swelling in both Na⁺ and K⁺ salts. Since nearly constant internal cation concentrations are maintained during osmotic swelling, this result suggests that both Na⁺/H⁺ and K⁺/H⁺ exchange is enhanced by increased matrix volume. Endogenous Mg²⁺ is also lost with increased matrix volume, and this observation, in conjunction with other evidence available in the literature, suggests that monovalent cation/H⁺ exchanges may be regulated by divalent cations. Passive exchange of Na⁺/K⁺,⁴²K⁺/K⁺, and²⁴Na⁺/Na⁺ can be readily demonstrated in mitochondria swollen in nitrate. All these exchanges are low or not detectable in unswollen control mitochondria, and it appears that they are manifestations of the activated cation/H⁺ component (or components) functioning in the absence of pH.     

Properties of Ca2+ uptake and release by Golgi membrane vesicles from rat intestine

A previous study of energy-independent in vitro Ca²⁺ uptake by rat intestinal epithelial membrane vesicles demonstrated that uptake by Golgi membrane vesicles was greater than that by microvillus or lateral-basal membrane vesicles, was markedly decreased in vitamin D-deficient rats, and responded specifically to 1,25-(OH)₂D₃ repletion (R. A. Freedman, M. M. Weiser, and K. J. Isselbacher, 1977, Proc. Nat. Acad. Sci. USA74, 3612–3616; J. A. MacLaughlin, M. M. Weiser, and R. A. Freedman, 1980, Gastroenterology78, 325–332). In the present study, properties of Ca²⁺ uptake and release by intestinal Golgi membrane vesicles have been investigated. The initial rate of uptake was found to be saturable, suggesting carrier-mediated uptake. Uptake was markedly inhibited by Mg²⁺ and Sr²⁺, but not by Na⁺ or K⁺. Lowering the external [H⁺] or raising the internal [H⁺] resulted in enhancement of the initial rate of uptake; the intial rate was found to correlate with the internal-to-external [H⁺] gradient. The initial rate of uptake could be enhanced by preloading the vesicles with MgCl₂ or SrCl₂ but not CaCl₂, NaCl, or KCl. Vesicles preloaded with K₂SO₄ failed to show enhanced uptake in the presence of valinomycin, suggesting that enhancement in uptake by vesicles preloaded with MgCl₂ was not due to transmembrane potentials. The internal volume of the Golgi membrane vesicles was determined and found to be 9 μl/mg protein; this volume could accomodate less than 1% of the Ca²⁺ uptake maintained at equilibrium. Therefore, the remainder of the Ca²⁺ taken up was presumably bound to the Golgi membranes. A dissociation constant of 3.8 × 10^?6m was found for this binding. The bound Ca²⁺ could be rapidly released by external Mg²⁺ or Sr²⁺, but not Ca²⁺, Na⁺, or K⁺. Release of bound Ca²⁺ could also be induced by raising the [H⁺] of the external medium. Failure of external Ca²⁺ to release bound Ca²⁺ suggested that the release induced by external Mg²⁺, Sr²⁺, or H⁺ was not due to competitive displacement of Ca²⁺ from its binding sites. These results indicated that Ca²⁺ uptake by intestinal Golgi membrane vesicles consists of carrier-mediated transport followed by binding of Ca²⁺ to the vesicle. The effects of H⁺, Mg²⁺, and Sr²⁺ on Ca²⁺ uptake and release suggest the existence of cation countertransport in the Golgi membrane vesicles.