The effect of calcium on the respiratory responses of corn mitochondria

Tightly coupled respiring corn mitochondria (Zea mays L.) respond to calcium addition with a transitory respiratory increase, proton extrusion, and Ca²⁺ binding. The extent of response is dependent upon the level of endogenous phosphate, and a large sustained respiratory increase can be obtained with addition of phosphate. However, calcium does not act as a permeant cation in that it will not penetrate with acetate. It appears that the transitory respiratory increase must be linked to the uptake of a calcium phosphate complex, but there is no evidence that transport of the complex serves to produce an electrophoretic calcium uniport. It is believed that calcium phosphate transport in corn is a constitutive property, and not produced by membrane damage.     

Oxidation of reduced nicotinamide adenine dinucleotide phosphate by isolated corn mitochondria

Isolated corn (Zea mays L.) mitochondria were found to oxidize reduced nicotinamide adenine dinucleotide phosphate in a KCl reaction medium. This oxidation was dependent on the presence of calcium or phosphate or both. Strontium and manganese substituted for calcium, but magnesium or barium did not. The oxidation of NADPH produced contraction of mitochondria swollen in KCl. Further evidence that the oxidation of NADPH was coupled was observed in respiratory control and adenosine diphosphate-oxygen ratios that were comparable to those reported for reduced nicotinamide adenine dinucleotide. The pathways of electron flow from NADH and NADPH were compared through the addition of electron transport inhibitors. The only difference between the two dinucleotides was that amytal was found to inhibit almost totally the state 3 oxidation of NADPH, but had little effect on the state 3 oxidation of NADH. The hypothetical pathways for electron flow from NADPH are discussed, as are the possible sites of calcium and phosphate stimulation.     

Phosphate-induced Stimulation of Acceptorless Respiration in Corn Mitochondria

By use of the organic mercurial mersalyl to block phosphate transport, it has been shown that only a small fraction of the respiratory increase of corn mitochondria in response to additions of inorganic phosphate is due to energy expended in phosphate accumulation. Most of the respiratory release occurs from accelerated turnover of the coupling mechanism with internal phosphate in an oligomycin-sensitive reaction. Addition of ADP to mersalyl-blocked mitochondria depletes internal phosphate in ATP formation and respiration declines. Arsenate produces the same responses as phosphate but is more effective in respiratory release.     

The effect of calcium on the respiratory responses of mung bean mitochondria.

Purified mung bean hypocotyl mitochondria were examined for their capacity to carry out respiration-dependent accumulation of calcium. The addition of 0.1-1.0 mM calcium to mung bean mitochondria supplemented with succinate gave no stimulation of state 4 respiration even in the presence of inorganic phosphate and the ionophoretic antibiotic A-23187. Even at high calcium concentrations, no transient changes in the respiratory activity occurred and subsequent addition of ADP initiated a further state 3 response. Although the additions of calcium resulted in a rapid H+ ejection, it was insensitive to lanthanum and uncoupling agents. Similarly, additions of calcium failed to initiate any transient changes in the oxidation-reduction states of either pyridine nucleotides or cytochrome b. Direct spectrophotometric recordings of absorbance changes of murexide revealed no respiration-linked calcium transport. It is proposed that although mung bean mitochondria possess a respiration-linked electrochemical potential gradient it would appear that this potential cannot be expressed as calcium transport even at high ion concentrations, probably due to a low calcium membrane permeability.     

The effect of calcium on the respiratory responses of mung bean mitochondria

Purified mung bean hypocotyl mitochondria were examined for their capacity to carry out respiration-dependent accumulation of calcium. The addition of 0.1–1.0 mM calcium to mung bean mitochondria supplemented with succinate gave no stimulation of state 4 respiration even in the presence of inorganic phosphate and the ionophoretic antibiotic A-23187. Even at high calcium concentrations, no transient changes in the respiratory activity occurred and subsequent addition of ADP initiated a further state 3 response. Although the additions of calcium resulted in a rapid H⁺ ejection, it was insensitive to lanthanum and uncoupling agents. Similarly, additions of calcium failed to initiate any transient changes in the oxidation-reduction states of either pyridine nucleotides or cytochrome b. Direct spectrophotometric recordings of absorbance changes of murexide revealed no respiration-linked calcium transport. It is proposed that although mung bean mitochondria possess a respiration-linked electrochemical potential gradient it would appear that this potential cannot be expressed as calcium transport even at high ion concentrations, probably due to a low calcium membrane permeability.     

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.     

Transport mechanism for calcium and phosphate in ram spermatozoa

Calcium uptake into ejaculated ram spermatozoa is highly enhanced by the addition of extracellular phosphate. Under identical conditions, extracellular calcium stimulates the uptake of phosphate by the cells. Both calcium and phosphate uptake are comparably inhibited by the sulfhydryl reagent mersalyl. The I50 was found to be 6.36 and 10.14 nmol mersalyl per mg protein for phosphate and calcium uptake, respectively. Calcium uptake is inhibited by mersalyl whether phosphate is present or not. Extracellular fructose causes a 5-fold increase in calcium uptake. When fructose and phosphate are present in the cell's medium, there is an additive effect, which indicates that two independent systems are involved in calcium transport into the cell. Ruthenium red, which blocks Ca2+ transport into the mitochondria, causes 70% and 95% inhibition of calcium uptake in the absence or in the presence of fructose, respectively. Ruthenium red does not affect phosphate uptake unless calcium was present in the incubation medium. The stimulatory effect of fructose upon calcium uptake can be mimicked by L-lactate and can be inhibited by the glycolytic inhibitor 2-deoxyglucose. Fructose and L-lactate stimulate mitochondrial respiration in a comparable way. Oligomycin, which inhibits mitochondrial ATP synthesis, does not inhibit Ca2+ uptake. This indicates that ATP is not involved in the mechanism by which mitochondrial respiration stimulates Ca2+ uptake. The calcium channel blocker, verapamil, inhibits Ca2+ uptake in the presence or absence of extracellular phosphate. The phosphate-dependent calcium transport mechanism is more sensitive to verapamil than is the phosphate-independent transporter. In summary, the data indicate that the plasma membrane of mammalian spermatozoa contains a calcium/phosphate symporter, a phosphate-independent calcium carrier and a calcium-independent phosphate carrier.     

Calcium-activated phosphate uptake in contracting corn mitochondria

The phosphate inhibition of succinate-powered contraction in corn mitochondria can be reversed with calcium. Associated with this reversal is an accumulation of phosphate and calcium. Both ions are essential for accumulation, although strontium will partially substitute for calcium. Arsenate does not substitute for phosphate except in producing the inhibition of contraction.

The antibiotics oligomycin and aurovertin do not block the phosphate inhibition of contraction or the calcium-activated phosphate uptake associated with the release of the inhibition. Dinitrophenol uncouples the phosphate uptake but permits full contraction.

Calcium promotes inorganic phosphate accumulation in root tissue as well as in mitochondria.

The results are discussed from the viewpoint of theories of calcium reaction with high energy intermediates of oxidative phosphorylation. It is concluded that calcium probably reacts with X~P in corn mitochondria, rather than with X~I as with animal mitochondria.

     

Effect of verapamil and sulfhydryl reagents on calcium transport in bovine spermatozoa

Calcium uptake by intact bovine epididymal spermatozoa is not affected by low concentrations (up to 0.75 mM) of the calcium transport blocker verapamil. Under these conditions, calcium transport into sperm mitochondria is highly inhibited. At higher verapamil concentrations (1.0, 1.5 mM), calcium transport into intact sperm is also inhibited, and this inhibition cannot be relieved by disrupting the plasma membrane with filipin. Calcium uptake into intact sperm is highly inhibited by mersalyl and this inhibitory effect can be completely relieved when the plasma membrane is disrupted by filipin. This effect of mersalyl is not dependent on the presence of phosphate in the incubation medium. Phosphate itself, up to 2 mM, enhances calcium uptake into the cells; this effect decreases at higher concentrations and is depressed 57% at 10 mM phosphate. This inhibitory effect of high phosphate concentration can be blocked by mersalyl. It is suggested that the calcium carrier itself and not a phosphate carrier of the plasma membrane is inhibited by mersalyl. It is possible that there is a symporter for calcium and phosphate in the plasma membrane of bovine spermatozoa.     

Effects of calcium and lanthanum on phosphate efflux from nonmyelinated nerve fibers

Summary Phosphate efflux was measured as the fractional rate of loss of radioactivity from rabbit vagus loaded with radiophosphate. The effects of changes in extracellular calcium and of lanthanum have been investigated. In Locke solution with normal, 0.9mm, calcium and without phosphate, the fractional rate of loss was 1.62×10^–3 min^–1 at 120 min after the beginning of the washing period and fell slowly (9% hr^–1) during washing from 2 to 6 hr. Addition of calcium to the Locke solution produced a transient increase followed by a reversible maintained increase in phosphate efflux. The latter was 40 and 75% above efflux in normal calcium for 20 and 50mm calcium, respectively. Removal of calcium, with or without addition of EGTA, produced only a transient increase in phosphate efflux, with no subsequent maintained change. Addition of low concentrations of lanthanum produced a reversible inhibition of phosphate efflux. Half-maximal inhibition was at 3.5 m lanthanum and appeared to be due to binding of lanthanum to more than one, probably two, sites. Measurements of inhibition by lanthanum at different calcium concentrations did not indicate any competition between calcium and lanthanum. It is suggested that at least a part of phosphate efflux depends on internal calcium and that lanthanum acts by preventing release of phosphate from the phosphate transport mechanism.     

Investigation of the Accompaniment of Calcium During Active Calcium Transport from Human Erythrocyte Ghosts

To determine whether a cell metabolite was involved in active calcium transport, the cell contents of human erythrocytes were subjected to high dilutions and the resultant ghosts were checked for their ability to actively transport calcium. It was found that the diluted erythrocyte ghosts did retain their capacity to actively transport calcium and that the characteristics of this transport process appeared to be unaltered by the high dilutions. Calcium analysis of the cell membrane and cell supernatant indicated that almost all of the calcium was lost from the cell solution rather than the cell membrane as active calcium transport proceeded. Therefore it appeared that calcium was able to cross the cell membrane without the aid of a cell metabolite. Investigations with layered erythrocytes indicated that the active transport of calcium was not assisted by centrifugation. Neither inorganic phosphate, pyrophosphate, nor an adenine nucleotide appeared to accompany calcium across the membrane as indicated by total phosphate and inorganic phosphate analysis and 260-nm readings of the deproteinized supernatant.     

The effects of calcitonin on calcium uptake and respiration in rat-liver mitochondria

The action of calcitonin on both the transport of calcium across the mitochondrial membrane and cellular respiration has been studied in the presence and absence of added phosphate. In the presence of phosphate, both the rate of calcium entry and the amount of calcium accumulated was stimulated by calcitonin, above a threshold concentration, in a saturable manner. In the absence of phosphate, calcitonin enhanced the rate of calcium entry, but had no appreciable effect on the levels of total calcium accumulated. The minimum concentration of calcitonin necessary to produce these effects was in all cases dependent on the external calcium concentration. Mitochondrial respiration was inhibited only at calcitonin levels much higher than those affecting calcium uptake. These results are consistent with the idea that the action of calcitonin is directly related to the mechanism of calcium uptake, and not to the respiratory process.     

The effects of calcitonin on calcium uptake and respiration in rat-liver mitochondria

The action of calcitonin on both the transport of calcium across the mitochondrial membrane and cellular respiration has been studied in the presence and absence of added phosphate. In the presence of phosphate, both the rate of calcium entry and the amount of calcium accumulated was stimulated by calcitonin, above a threshold concentration, in a saturable manner. In the absence of phosphate, calcitonin enhanced the rate of calcium entry, but had no appreciable effect on the levels of total calcium accumulated. The minimum concentration of calcitonin necessary to produce these effects was in all cases dependent on the external calcium concentration. Mitochondrial respiration was inhibited only at calcitonin levels much higher than those affecting calcium uptake. These results are consistent with the idea that the action of calcitonin is directly related to the mechanism of calcium uptake, and not to the respiratory process.     

Roles of phosphorylation and nucleotide binding domains in calcium transport by sarcoplasmic reticulum adenosinetriphosphatase

The roles of the phosphorylation (phosphorylated enzyme intermediate) and nucleotide binding domains in calcium transport were studied by comparing acetyl phosphate and ATP as substrates for the Ca2+-ATPase of sarcoplasmic reticulum vesicles. We found that the maximal level of phosphoenzyme obtained with either substrate is approximately 4 nmol/mg of protein, corresponding to the stoichiometry of catalytic sites in our preparation. The initial burst of phosphoenzyme formation observed in the transient state, following addition of either substrate, is accompanied by internalization of 2 mol of calcium per mole of phosphoenzyme. The internalized calcium is then translocated with a sequential pattern, independent of the substrate used. Following a rate-limiting step, the phosphoenzyme undergoes hydrolytic cleavage and proceeds to the steady-state activity which is soon "back inhibited" by the rise of Ca2+ concentration in the lumen of the vesicles. When the "back inhibition" is released by the addition of oxalate, substrate utilization and calcium transport occur with a ratio of 1:2, independent of the substrate and its concentration. When the nucleotide binding site is derivatized with FITP, the enzyme can still utilize acetyl phosphate (but not ATP) for calcium transport. No secondary activation of acetyl phosphate utilization by the FITC-enzyme was obtained with millimolar nucleotide. These observations demonstrate that the basic coupling mechanism of catalysis and calcium transport involves the phosphorylation and calcium binding domains, and not the nucleotide binding domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of ribonuclease on the binding of calcium by mitochondria

Summary Mitochondria, isolated from potato tuber, pretreated with ribonuclease (RNase) showed an increase in calcium binding at low enzyme concentration. The same dosage-response pattern was obtained whether the enzyme treatment was conducted for 10, 30 or 120 min. However, when the enzyme treatment was carried out at 0° instead of at 30°, no increase in Ca-binding was obtained, suggesting that no interaction occurred between the enzyme and the divalent cation. Oxygen consumption under the same conditions was not affected. Microsomes treated with RNase did not show a change in their Ca-binding capacity, although untreated microsomes showed about the same increase in Ca-binding with increase in temperature as did the mitochondria. When mitochondria prelabeled with ⁴⁵Ca, after extracting their inorganic calcium phosphate, were treated with RNase a liberation of Ca, ribonucleotide, and phosphate was observed. It is suggested that Ca ions form a bridge like bond between ribonucleic acid and either phospholipids or phospholipoproteins, because RNase liberated more phosphate than nucleotides and the extra phosphate cannot be inorganic calcium phosphate, since the calcium phosphate was extracted before addition of RNAse.The investigation reported in this paper (No. 68-3-71) is in connection with a project of the Kentucky Experiment Station and is published with approval of the Director.     

Respiratory activation of 2,4-dinitrophenol-stimulated ATPase activity in plant mitochondria

A comparison has been made of cauliflower mitochondria, which have no 2,4-dinitrophenol-stimulated ATPase (EC 3,6,1,4), with corn mitochondria, which do. Unlike corn mitochondria, cauliflower mitochondria show poor initial respiratory control ratios and phosphate uptake, but these are normalized after the first ADP addition. Sonication or high pH treatment releases a high rate of oligomycin-sensitive ATPase, indicating ATP transport into cauliflower mitochondria is the limiting factor. A brief period of respiration will activate, or “prime,” the 2,4-dinitrophenol-stimulated ATPase of cauliflower mitochondria, and the activity is inhibited by atractyloside, mersalyl, and oligomycin. Influx pumping of phosphate or arsenate extends the time the priming period lasts after respiration ceases to 1–2 min unless the 2,4-dinitrophenol is added before the ATP, in which case the priming is collapsed. Respiratory priming seems to consist of creating a transmembrane potential, possibly in the form of a phosphate gradient, for driving the ATP^4?-ADP^3? transporter.     

2H-NMR, 31P-NMR and DSC characterization of a novel lipid organization in calcium-dioleoylphosphatidate membranes. Implications for the mechanism of the phosphatidate calcium transmembrane shuttle

2H-NMR, 31P-NMR and DSC investigations are presented on the structure and dynamics of the Ca2+-dioleoylphosphatidate complex which is formed upon addition of calcium to dispersions of pure dioleoylphosphatidate or of dioleoylphosphatidate in mixtures with dioleoylphosphatidylcholine (DOPC). It is concluded that the phosphate region in the polar headgroup of dioleoylphosphatidate is immobilized, while the oleate chains remain liquid and have increased disorder. In mixtures of dioleoylphosphatidate and DOPC in the presence of calcium a dioleoylphosphatidate-rich phase is segregated, in which the molecular behaviour of phosphatidate is rather similar to that of the pure Ca2+-dioleoylphosphatidate complex. A hypothetical model is proposed for the structure of this complex and this is correlated with the dioleoylphosphatidate-mediated transmembrane transport of calcium (Smaal, E.B., Mandersloot, J.G., De Kruijff, B. and De Gier, J. (1986) Biochim. Biophys. Acta 860, 99-108). Data indicate that this transmembrane shuttle is an inverted organization of phosphatidate molecules enclosing calcium ions in an anhydrous core.     

Calcium oxalate and calcium phosphate capacities of cardiac sarcoplasmic reticulum

Both oxalate-supported and phosphate-supported calcium uptake by canine cardiac sarcoplasmic reticulum initially increase linearly with time but fall to a steady-state level within 20 min. The departure from linearity could be due to a decrease in influx or to an increase in efflux of calcium. Because Ca2+-ATPase activity is linear, a decrease in the influx of calcium is an unlikely cause of the non-linear calcium uptake curves. A possible cause of an increase in calcium efflux is rupture of the vesicles. This hypothesis was tested by investigating the amount of calcium which could be released upon addition of 5 mM EGTA. The amount of rapidly releasable calcium was zero until a threshold calcium uptake of about 4-6 mumol calcium oxalate or calcium phosphate per mg was reached. After that point the rapidly releasable calcium continued to increase with calcium oxalate to reach more than 23 mumol/mg, but stayed constant at about 0.7 mumol/mg for calcium phosphate. The rapidly releasable calcium was attributed to calcium oxalate or calcium phosphate crystals externalized by vesicle rupture. The differences in the amounts of rapidly releasable calcium were attributed to different kinetics of calcium phosphate and calcium oxalate dissolution. Addition of ryanodine caused a marked increase in the threshold for rapidly releasable calcium oxalate. Transmission electron micrographs showed that vesicles can become filled with calcium oxalate crystals, but the vesicles were heterogeneous with respect to their size and their sensitivity to ryanodine. These observations support the hypothesis that calcium oxalate and calcium phosphate capacities are limited by vesicle rupture and that ryanodine increases the capacity by closing a calcium channel in a subpopulation of vesicles that otherwise would not accumulate calcium.     

Influence of glyoxylic acid on properties of isolated mitochondria]

Glyoxalate is an effector of oxidative phosphorylation in isolated mitochondria : it slows down State 3 but does not affect State 4 respiration. This report presents the findings of our study on the mechanism of action of glyoxalate ; these findings are listed below. The inhibition of Stage 3 respiration by glyoxalate does not set in immediately, can be reversed in part by the addition of an uncoupling agent or a dithiol, is non-competitive against succinate and can be demonstrated with substrates requiring the involvement of other membrane transport systems. Glyoxalate prevents the increased oxygen uptake stimulated by 2,4-DNP or Sr++. Glyoxalate also inhibits phosphate transport and this inhibition can account for most of the effect observed. The inhibition of State 3 respiration is paralleled by a decrease in the mitochondrial accumulation of succinate : this decrease could arise from a direct effect of glyoxalate on dicarboxylic acid transport or could be the result of an inhibiton of the phosphate transport system, which is connected with the former. The decrease in the respiratory rate of uncoupled mitochondria placed in a phosphate free medium demonstrates that the effector acts directly at the substrate transport or/and electron transfer level. Phosphate, by delaying the respiratory inhibiton due to glyoxalate, has a protecting effect on mitochondrial functions. Glyoxalate is thus acting at several mitochondrial sites. It acts presumably by forming hemimercaptals, blocking sulfhydryl groups. Its effects can be accounted for by the unfolding of such (hemicercaptal) groups under the influence of ADP, Pi, uncoupling or others agents which bring about conformational changes in the internal mitochondrial membrane.     

Determination of calcium transport and phosphoprotein phosphatase activity in microsomes from respiratory and vascular smooth muscle.

1. Calcium transport into microsomal vesicles of respiratory (tracheal) smooth muscle was characterized. This calcium transport was ATP dependent and stimulated by the presence of the oxalate ion. The magnitude of transport was similar to that reported for microsomes from other types of smooth muscle. 2. Bovine and rabbit, heavy and light microsomes were isolated from respiratory (tracheal) and vascular (aortic) smooth muscle. Preincubation of these vesicles with cyclic AMP and protein kinase did not alter the transport of calcium into the vesicles. There uas no evidence of phosphate incorporation into microsomal membrane proteins. Similar results were obtained if phosphorylase b kinase replaced the combination of cyclic AMP and protein kinase during the preincubation. 3. The phosphoprotein phosphatase activity of cardiac sarcoplasmic reticulum and smooth muscle microsomes was determined. The activity of this enzyme was found to be several-fold less in the cardiac sarcoplasmic reticulum than in various smooth muscle microsome preparations.