Citrate transport in corn mitochondria

Citrate uptake by corn mitochondria (Zea mays L. B73 × Mol9) was investigated by osmotic swelling and [¹⁴C]citrate accumulation. Uptake driven by passive influx, ammonium gradients, and respiration was followed. There was no requirement for phosphate and/or malate to secure citrate uptake, although under some conditions these additives were promotive. Inhibition of the phosphate and dicarboxylate carriers did not eliminate citrate uptake. Citrate_in/malate_out exchange occurs, but at a rate too slow to account for observed citrate uptake, and depletion of endogenous malate only reduced citrate uptake by 38%. It was concluded that citrate can be rapidly accumulated by a mechanism other than by exchange for dicarboxylates. The effect of uncoupler on respiration-driven [¹⁴C]citrate accumulation, and studies of passive swelling using ionophores and uncouplers indicated that the major avenue of citrate uptake is by H⁺/citrate co-transport with a pH optimum near 4.5. The in vivo role of this mechanism is not yet understood.     

The effects of valinomycin on respiration and volume changes in plant mitochondria

The effects of valinomycin on the respiration and volume changeshave been studied with isolated mitochondria from bean hypocotyl(Phaseolus vulgaris L.) and cauliflower bud (Brassica oleraceaL.). In the presence of 10 mM K salts of chloride, acetate,phosphate, and sulfate respiration is stimulated by valinomycinconcomitant with osmotic swelling. When swelling declines respirationwith organic acid substrates also declines. In the presenceof the K salts of acetate and PO₄ but not Cl^– the terminationof respiration leads to contraction. The contraction in K-PO₄is inhibited by addition to the external medium of between 65to 100 mM K-PO₄. The results are interpreted to suggest thatvalinomycin in the presence of KCl facilitated the movementof K down an electrical gradient, with the Cl anion followingand osmotic swelling resulting. However, in a medium containingacetate or PO₄ the anions are actively transported against anelectrical gradient at the expense of metabolic energy. Valinomycinfacilitates the influx of K⁺ with the actively transported anionand swelling follows. When respiration terminates the activelytransported anions move passively back down their electrochemicalgradient and osmotic contraction follows. ¹ Present address: Department of Biology, Fort Lewis College,Durango, Colorado 81301, U.S.A. (Received July 21, 1972; )     

Permeability Properties of the Inner Membrane of Mung Bean Mitochondria and Changes during Energization

The permeability properties of the inner membrane of mung bean mitochondria were studied by osmotic swelling techniques. Rapid mitochondrial swelling was observed in isotonic ammonium phosphate, which indicated that an active phosphate/hydroxyl antiporter was present. The phosphate carrier was specifically inhibited by sulfhydryl reagents. Mitochondria did not swell in isotonic ammonium salts of malate, succinate, or fumarate, either in the presence or absence of 10 millimolar phosphate. Additionally, no swelling was observed in ammonium citrate upon addition of malate plus phosphate. Consequently, no evidence was obtained with the osmotic swelling technique for a coupled exchange of phosphate for dicarboxylic acids across the membrane.     

Ion transport induced by polycations and its relationship to loose coupling of corn mitochondria

Treatment of corn mitochondria (Zea mays L., WF9 (Tms) × M14) with polycations (protamine, pancreatic ribonuclease, or polylysine) releases acceptorless respiration if phosphate is present. Concurrently, there is extensive active swelling which is reversed when respiration is uncoupled or stopped. Mersalyl, the phosphate transport inhibitor, blocks both the release of respiration and the active swelling. Diversion of energy into phosphate transport lowers respiratory control and ADP: O ratios. This response is termed “loose coupling” in distinction to “uncoupling” in which energy is made unavailable for either transport or ATP formation. Corn mitochondria as used here are endogenously loose coupled to some extent, and show state 4 respiration linked to active transport.     

Amino acid and divalent ion permeability of the pores formed by the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac in insect midgut brush border membrane vesicles

The pores formed by Bacillus thuringiensis insecticidal toxins have been shown to allow the diffusion of a variety of monovalent cations and anions and neutral solutes. To further characterize their ion selectivity, membrane permeability induced by Cry1Aa and Cry1Ac to amino acids (Asp, Glu, Ser, Leu, His, Lys and Arg) and to divalent cations (Mg²⁺, Ca²⁺ and Ba²⁺) and anions (SO₄²⁻ and phosphate) was analyzed at pH 7.5 and 10.5 with midgut brush border membrane vesicles isolated from Manduca sexta and an osmotic swelling assay. Shifting pH from 7.5 to 10.5 increases the proportion of the more negatively charged species of amino acids and phosphate ions. All amino acids diffused well across the toxin-induced pores, but, except for aspartate and glutamate, amino acid permeability was lower at the higher pH. In the presence of either toxin, membrane permeability was higher for the chloride salts of divalent cations than for the potassium salts of divalent anions. These results clearly indicate that the pores are cation-selective.     

Ion transport by heart mitochondria. XXII. Spontaneous, energy-linked accumulation of acetate and phosphate salts of monovalent cations

The accumulation of monovalent cations by isolated beef heart mitochondria has been studied by evaluating the efficiency of energy-dependent osmotic swelling. Extensive osmotic swelling occurs spontaneously when isolated heart mitochondria are suspended in 0.1 m acetate or phosphate salts. The swelling and ion uptake depend on either respiration or the presence of exogenous ATP, and the initial rate of swelling is proportional to the initial rate of respiration or ATP hydrolysis, respectively. The efficiency of the reaction varies somewhat from preparation to preparation but approaches a limit of about 2 cations accumulated per pair of electrons traversing a phosphorylation site. All monovalent cations tested support the reaction, but the most efficient energy-dependent swelling occurs with K⁺. Weak acid anions are required for the ion accumulation and swelling and the reaction appears to depend on the amount of free acid available in the suspension. Permeant strong acid anions, such as NO₃⁻, fail to support the swelling reaction in the presence of energy. Valinomycin increases both the amount and the efficiency of ion uptake under these conditions. Mg²⁺ decreases both of these values whereas p-chloromercuriphenyl sulfonate increases both. These responses are discussed in terms of current models of mitochondrial ion transport.     

Transport protein synthesis in non-growing yeast cells

Addition of a metabolizable substrate (glucose, ethanol and, to a degree, trehalose) to non-growing baker's yeast cells causes a boost of protein synthesis, reaching maximum rate 20 min after addition of glucose and 40–50 min after ethanol or trehalose addition. The synthesis involves that of transport proteins for various solutes which appear in the following sequence: H⁺, l-proline, sulfate, l-leucine, phosphate, α-methyl-d-glucoside, 2-aminoisobutyrate. With the exception of the phosphate transport system, the K_t of the synthesized systems is the same as before stimulation. Glucose is usually the best stimulant, but ethanol matches it in the case of sulfate and exceeds it in the case of proline. This may be connected with ethanol's stimulating the synthesis of transport proteins both in mitochondria and in the cytosol while glucose acts on cytosolic synthesis alone. The stimulation is often repressed by ammonium ions (leucine, proline, sulfate, H⁺), by antimycin (proline, trehalose, sulfate, H⁺), by iodoacetamide (all systems tested), and by anaerobic preincubation (leucine, proline, trehalose, sulfate). It is practically absent in a respiration-deficient petite mutant, only little depressed in the op₁ mutant lacking ADP/ATP exchange in mitochondria, but totally suppressed (with the exception of transport of phosphate) in a low-phosphorus strain. The addition of glucose causes a drop in intracellular inorganic monophosphate by 30%, diphosphate by 45%, ATP by 70%, in total amino acids by nearly 50%, in transmembrane potential (absolute value) by about 50%, an increase of high-molecular-weight polyphosphate by 65%, of total cAMP by more than 100%, in the endogenous respiration rate by more than 100%, and a change of intracellular pH from 6.80 to 7.05. Ethanol caused practically no change in ATP, total amino acids, endogenous respiration, intracellular pH or transmembrane potential; a slight decrease in inorganic monophosphate and diphosphate and a sizeable increase in high-molecular-weight polyphosphate. The synthesis of the various transport proteins thus appears to draw its energy from different sources and with different susceptibility to inhibitors. It is much more stimulated in facultatively aerobic species (Saccharomyces cerevisiae, Endomyces magnusii) than in strictly aerobic ones (Rhodotorula glutinis, Candida parapsilosis) where an inhibition of transport activity is often observed after preincubation with metabolizable substrates.     

Energy-linked Sulfate Uptake by Corn Mitochondria via the Phosphate Transporter

Corn shoot mitochondria possess an energy-linked transport system for sulfate uptake as demonstrated by osmotic swelling and [³⁵S]SO₄²⁻ accumulation. Maximum uptake is secured in the presence of Mg²⁺ and oligomycin with sucrose for osmotic support. Neither phosphate nor dicarboxylate anions are required. When added simultaneously, millimolar concentrations of phosphate block [³⁵S]SO₄²⁻ uptake after the initial minute. Mersalyl, N-ethylmaleimide, and 2,4-dinitrophenol are strong inhibitors of sulfate uptake; n-butylmalonate is a weak inhibitor. These inhibitors act in the same fashion on phosphate uptake. It is concluded that sulfate uptake in the absence of phosphate is by the phosphate transporter.     

Anion transporters in plant mitochondria

The swelling of potato (Solanum tuberosum L.) mitochondria in isosmotic ammonium salts of phosphate, chloride, malate, succinate, and citrate was investigated by measuring light scattering. Potato mitochondria swell spontaneously in ammonium phosphate, and this swelling can be inhibited in N-ethylmaleimide. They swell in ammonium malate or succinate only after the addition of inorganic phosphate and in ammonium citrate only after the addition of both phosphate and a dicarboxylic acid. Pentylmalonate inhibits swelling in ammonium citrate solutions by competing for dicarboxylate entry. The results indicate that potato mitochondria possess a phosphate-hydroxyl carrier, a dicarboxylate carrier, and a tricarboxylate carrier.     

Citrate and succinate uptake by potato mitochondria

The uptake of [¹⁴C]citrate and [¹⁴C]succinate was studied in potato mitochondria (Solanum tuberosum var. Russet Burbank) using cellulose pore filtration and was found to occur by the same mechanisms as described for mammalian mitochondria. Potato mitochondria, in the absence of respiration, have a very low capacity for uptake by exchange with endogenous anions, taking up only 2.4 nanomoles citrate and 2.0 nanomoles succinate per milligram protein. Maximum citrate uptake of over 17 nanomoles per milligram protein occurs in the presence of inorganic phosphate, a dicarboxylic acid, and an external energy source (NADH), conditions where net anion accumulation proceeds, mediated by the interlinking of the inorganic phosphate, dicarboxylate, and tricarboxylate carriers. Maximum succinate uptake in the absence of respiratory inhibitors requires only added inorganic phosphate.     

ION SERIES AND THE PHYSICAL PROPERTIES OF PROTEINS. II

1. Our results show clearly that the Hofmeister series is not the correct expression of the relative effect of ions on the swelling of gelatin, and that it is not true that chlorides, bromides, and nitrates have "hydrating," and acetates, tartrates, citrates, and phosphates "dehydrating," effects. If the pH of the gelatin is taken into considertion, it is found that for the same pH the effect on swelling is the same for gelatin chloride, nitrate, trichloracetate, tartrate, succinate, oxalate, citrate, and phosphate, while the swelling is considerably less for gelatin sulfate. This is exactly what we should expect on the basis of the combining ratios of the corresponding acids with gelatin since the weak dibasic and tribasic acids combine with gelatin in molecular proportions while the strong dibasic acid H₂SO₄ combines with gelatin in equivalent proportions. In the case of the weak dibasic acids he anion in combination with gelatin is therefore monovalent and in the case of the strong H₂SO₄ it is bivalent. Hence it is only the valency and not the nature of the ion in combination with gelatin which affects the degree of swelling. 2. This is corroborated in the experiments with alkalies which show that LiOH, NaOH, KOH, and NH₄OH cause the same degree of swelling at the same pH of the gelatin solution and that this swelling is considerably higher than that caused by Ca(OH)₂ and Ba(OH)₂ for the same pH. This agrees with the results of the titration experiments which prove that Ca(OH)₂ and Ba(OH)₂ combine with gelatin in equivalent proportions and that hence the cation in combination with the gelatin salt with these two latter bases is bivalent. 3. The fact that proteins combine with acids and alkalies on the basis of the forces of primary valency is therefore not only in full agreement with the influence of ions on the physical properties of proteins but allows us to predict this influence qualitatively and quantitatively. 4. What has been stated in regard to the influence of ions on the swelling of the different gelatin salts is also true in regard to the influence of ions on the relative solubility of gelatin in alcohol-water mixtures. 5. Conductivity measurements of solutions of gelatin salts do not support the theory that the drop in the curves for swelling, osmotic pressure, or viscosity, which occurs at a pH 3.3 or a little less, is due to a drop in the concentration of ionized protein in the solution; nor do they suggest that the difference between the physical properties of gelatin sulfate and gelatin chloride is due to differences in the degree of ionization of these two salts.     

Glucose Respiration in the Intact Chloroplast of Chlamydomonas reinhardtii

Chloroplastic respiration was monitored by measuring ¹⁴CO₂ from ¹⁴C glucose in the darkened Chlamydomonas reinhardtii F-60 chloroplast. The patterns of ¹⁴CO₂ evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide, glycolate-2-phosphate, and phosphoenolpyruvate were those expected from the oxidative pentose phosphate cycle and glycolysis. The K_m for glucose was 56 micromolar and for MgATP was 200 micromolar. Release of ¹⁴CO₂ was inhibited by phloretin and inorganic phosphate. Comparing the inhibition of CO₂ evolution generated by pH 7.5 with respect to pH 8.2 (optimum) in chloroplasts given C-1, C-2, and C-6 labeled glucose indicated that a suboptimum pH affects the recycling of the pentose phosphate intermediates to a greater extent than CO₂ evolution from C-1 of glucose. Respiratory inhibition by pH 7.5 in the darkened chloroplast was alleviated by NH₄Cl and KCl (stromal alkalating agents), iodoacetamide (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), or phosphoenolpyruvate (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiration in the darkened Chlamydomonas chloroplast is the fructose-1,6-bisphosphatase/phosphofructokinase junction. The respiratory pathways described here can account for the total oxidation of a hexose to CO₂ and for interactions between carbohydrate metabolism and the oxyhydrogen reaction in algal cells adapted to a hydrogen metabolism.     

Endosperm acidification and related metabolic changes in the developing barley grain

The starchy endosperm (SE) of the developing grain (caryopsis) of barley (Hordeum vulgare L.) cv Himalaya, as well as that of other barley cultivars examined, acidifies during maturation. The major decrease in pH begins with the attainment of maximum grain dry weight, onset of dehydration, and completion of chlorophyll loss. Acidification is correlated with the accumulation of malate and lesser amounts of citrate and lactate, produced and probably secreted by the pericarp/testa/aleurone (PTA). It is accompanied by large concurrent rises in phosphoeno/pyruvate carboxylase and alcohol dehydrogenase (ADH) activity in the PTA. The activity of seven other enzymes of oxaloacetate and pyruvate metabolism was found to fall or rise only slightly during acidification. Sequential changes in relative amount of ADH isozymes were found in both PTA and SE. The PTA maintained a high respiration rate and adenylate energy charge (AEC) throughout acidification, whereas the SE showed a low respiration rate and rising AEC. The data are consistent with the occurrence of hypoxia in the SE. It is suggested that the above enzyme changes are required for the development of a malate/ethanol fermentation (i.e. a mixed metabolism) in the aleurone layer during maturation.     

Ion transport by heart mitochondria: XXVII. The relation of mercurial-dependent adenosine triphosphatase activity to ion movements

The properties of a mercurial-dependent adenosine triphosphatase activity have been examined in isolated beef heart mitochondria. The reaction differs from that induced by uncouplers in that it is associated with extensive ion uptake and osmotic swelling, is highly specific for K⁺ over Na⁺, and is enhanced by respiration. Evidence is presented which suggests that the following events can account for the observations: (1) The mercurial blocks the phosphate transporter so that phosphate hydrolyzed from ATP is trapped in the matrix. (2) This interior negative potential causes cations to move inward and swelling results. (3) Permeability to K⁺ but not to Na⁺ is enhanced greatly by the reaction of the mercurial with the membrane. The inward movement of K⁺ closely resembles that produced by valinomycin, in that it is accompanied by proton ejection into the medium and it rapidly establishes a condition in which ion gradients cannot be maintained. This marked increase in permeability may be related to the pH gradient and is manifest as additional passive swelling in the absence of sucrose and passive contraction when sucrose is present. A comparison of the kinetics of swelling and of ATP hydrolysis shows that the elevated rates of ATPase are correlated with this condition of high permeability. When a corresponding condition of high permeability to Na⁺ is established by treatment with gramicidin or EDTA, the mercurial-dependent ATPase is nearly as rapid in Na⁺ as in the K⁺ medium. It appears, therefore, that the K⁺ specificity resides at the level of membrane permeability and is not a feature of the ATPase reaction per se. (4) Respiration appears to affect the ATPase reaction by virtue of its ability to extrude ions from the matrix in the presence of the mercurial. p-Chloromercuriphenyl sulfonate causes a switch from respiration-dependent ion accumulation to respiration-dependent ion extrusion to occur. A model to explain these reactions is presented.     

Remote loading of doxorubicin into liposomes driven by a transmembrane phosphate gradient

This study examines a new method for the remote loading of doxorubicin into liposomes. It was shown that doxorubicin can be loaded to a level of up to 98% into large unilamellar vesicles composed of egg phosphatidylcholine/cholesterol (7/3 mol/mol) with a transmembrane phosphate gradient. The different encapsulation efficiencies which were achieved with ammonium salts (citrate 100%, phosphate 98%, sulfate 95%, acetate 77%) were significantly higher as compared to the loading via sodium salts (citrate 54%, phosphate 52%, sulfate 44%, acetate 16%). Various factors, including pH-value, buffer capacity, solubility of doxorubicin in different salt solutions and base counter-flow, which likely has an influence on drug accumulation in the intraliposomal interior are taken into account. In contrast to other methods, the newly developed remote loading method exhibits a pH-dependant drug release property which may be effective in tumor tissues. At physiological pH-value doxorubicin is retained in the liposomes, whereas drug release is achieved by lowering the pH to 5.5 (approximately 25% release at 25 °C or 30% at 37 °C within two h). The DXR release of liposomes which were loaded via a sulfate gradient showed a maximum of 3% at pH 5.5.     

Mechanisms of citrate transport and exchange in corn mitochondria

Previous work (Birnberg, Jayroe, Hanson 1982 Plant Physiol 70: 511-516) demonstrated that corn mitochondria (Zea mays L.) can accumulate citrate by a malate- and phosphate-independent proton symporter. This uptake and symport of other ions were investigated. Passive swelling experiments indicated that corn mitochondria can accumulate several other anions by proton symport, but only isocitrate is taken up nearly as effectively as citrate. At the optimal pH (4.5), active uptake of carrier-free [¹⁴C]citrate in 50 micromolar mersalyl is inhibited by fourteen anions, but only the I₅₀ (the concentration of inhibitor required to reduce uptake of carrier-free [¹⁴C]citrate by 50%) values of citrate (0.08 millimolar) and d-and l-isocitrate (0.5 millimolar) are less than 4 millimolar. Isocitrate is a competitive inhibitor of citrate uptake and [¹⁴C]isocitrate is accumulated with a K_m similar similar to its I₅₀. Valinomycin reduces net active citrate accumulation at pH 7.5, consistent with the relatively low V_max for citrate uptake. At pH 4.5, mersalyl reduces the rate of citrate uptake without changing the affinity of the carrier for citrate. Thus, the corn mitochondria have a high-affinity, mersalyl-insensitive carrier selective for citrate that also transports isocitrate.     

Inhibition of postoxidative calcium release in corn mitochondria by inorganic phosphate

Respiration drives the accumulation of a small amount of calcium in corn (Zea mays L.) mitochondria, and this calcium is released when respiration ceases. A postenergized addition of phosphate leads to phosphate uptake and enhaced calcium retention. Oligomycin, KCN, 2,4-dinitrophenol, or mersalyl are without effect on the phosphate-induced calcium retention. Addition of phosphate also inhibits the release of endogenous phosphate which normally accompanies the calcium. It is suggested that passive phosphate uptake retards the release of endogenous phosphate which is complexed with the calcium.     

Effect of Various Ions, pH, and Osmotic Pressure on Oxidation of Elemental Sulfur by Thiobacillus thiooxidans

The oxidation of elemental sulfur by Thiobacillus thiooxidans was studied at pH 2.3, 4.5, and 7.0 in the presence of different concentrations of various anions (sulfate, phosphate, chloride, nitrate, and fluoride) and cations (potassium, sodium, lithium, rubidium, and cesium). The results agree with the expected response of this acidophilic bacterium to charge neutralization of colloids by ions, pH-dependent membrane permeability of ions, and osmotic pressure.     

A special role of phosphate in the stability of lactate dehydrogenase against destruction by a polyelectrolyte

It has been shown by X-ray structure analysis that proteins have specific anion-binding sites for sulfate, citrate, and phosphate ions; however, the functional role of these anions is not always clear. Thus, it is unknown which of two phosphate anions, mono- or divalent, determines the stability of cellular proteins under stress conditions. In the present work, the influence of phosphate, sulfate, and chloride on the stability of lactate dehydrogenase (LDH) in the presence of poly(styrenesulfonate) (PSS) has been investigated by the methods of steady-state kinetics and intrinsic protein fluorescence. The study is based on the analysis of differences between the influence of phosphate and sulfate ions on the process at two pH values, 6.2 and 7.0, at which the ratio of the concentrations of mono- and bivalent phosphate forms differs, whereas sulfate remains in the bivalent form. It was shown that the differences between the influence of phosphate and sulfate ions at pH 7.0 were greater; divalent phosphate ions much more effectively stabilized LDH against destruction by a polyelectrolyte compared with sulfate and monovalent phosphate. It was concluded that, of two anion-binding sites of the LDH molecule, the intersubunit center plays the most important role in its stabilization against destruction by polyelectrolyte, and, of two forms of phosphate anions, its bivalent form HPO ₄ ^?2 plays the stabilizing role.     

Amino acid and divalent ion permeability of the pores formed by the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac in insect midgut brush border membrane vesicles

The pores formed by Bacillus thuringiensis insecticidal toxins have been shown to allow the diffusion of a variety of monovalent cations and anions and neutral solutes. To further characterize their ion selectivity, membrane permeability induced by Cry1Aa and Cry1Ac to amino acids (Asp, Glu, Ser, Leu, His, Lys and Arg) and to divalent cations (Mg(2+), Ca(2+) and Ba(2+)) and anions (SO(4)(2-) and phosphate) was analyzed at pH 7.5 and 10.5 with midgut brush border membrane vesicles isolated from Manduca sexta and an osmotic swelling assay. Shifting pH from 7.5 to 10.5 increases the proportion of the more negatively charged species of amino acids and phosphate ions. All amino acids diffused well across the toxin-induced pores, but, except for aspartate and glutamate, amino acid permeability was lower at the higher pH. In the presence of either toxin, membrane permeability was higher for the chloride salts of divalent cations than for the potassium salts of divalent anions. These results clearly indicate that the pores are cation-selective.