Studies on the transition of the cristal membrane from the orthodox to the aggregated configuration. III. Loss of coupling ability of adrenal cortex mitochondria in the orthodox configuration

When the cristae of adrenal cortex mitochondria are stabilized in the orthodox configuration by the binding of 20–25 mmoles/mg protein of either Ca²⁺ or free fatty acids (oleic acid), both the capacity for carrying out coupled reactions and the capacity for undergoing energized configurational transitions are lost. The coupled reactions studied included ATP synthesis, divalent cation translocation, monovalent cation trnaslocation, and reversed electron transfer. The coupled processes and energized configurational changes are fully operative when the cristae of adrenal cortex mitochondria are in the aggregated configuration. However, two processes that have been shown to depend on conformational changes (the anaerobic-aerobic proton ejection and energized accumulation of inorganic phosphate) still proceed when mitochondria are in the orthodox configuration. When the mitochondria are initially in the orthodox configuration, addition of divalent cations (Mg²⁺ or Mn²⁺) or albumin induces a transition of the cristae to the aggregated configuration and leads to restoration of all the coupled processes. the orthodox to aggregated transition is reversible and the modulation of this reversibility appears to be one of the key points of control in the mitochondrion and possibly of cellular functions.On leave of absence from the Department of Pathology, Nagoya University School of Medicine, Nagoya, Japan.     

The Ca2+-induced membrane transition in mitochondria. III. Transitional Ca2+ release.

The efflux of Ca²⁺ from mitochondria respiring at steady state, and much of uncoupler-induced Ca²⁺ efflux, is shown to be a consequence of the Ca²⁺-induced membrane transition (the Ca²⁺-induced transition is the Ca²⁺-dependent sudden increase in the nonspecific permeability of the mitochondrial inner membrane which occurs spontaneously when mitochondria are incubated under a variety of conditions (D. R. Hunter, R. A. Haworth, and J. H. Southard, 1976, J. Biol. Chem.251, 5069–5077)). Ca²⁺ release from mitochondria respiring at steady state is shown to be transitional by four criteria: (1) Ca²⁺ release is inhibited by Mg²⁺, ADP, and bovine serum albumin (BSA), all inhibitors of the transition; (2) release is selective for Ca²⁺ over Sr²⁺, a selectivity also found for the transition; (3) the time course of Ca²⁺ release is identical to the time course of the change in the mitochondrial population from the aggregated to the orthodox configuration; and (4) from kinetics, Ca²⁺ release from individual mitochondria is shown to occur suddenly, following a lag period during which no release occurs. Ca²⁺ release induced by uncoupler is shown to be mostly by a transitional mechanism, as judged by four criteria: (1) release of Ca²⁺ is ruthenium red-insensitive and is an order of magnitude faster than Sr²⁺ release which is ruthenium red-sensitive; (2) release of Ca²⁺ is strongly inhibited by keeping the mitochondrial NAD⁺ reduced; (3) the kinetics of Ca²⁺ release indicates a transitional release mechanism; and (4) uncoupler addition triggers the aggregated to orthodox configurational transition which, at higher levels of Ca²⁺ uptake, occurs in the whole mitochondrial population at a rate equal to the rate of Ca²⁺ release. Na²⁺-induced Ca²⁺ release was not accompanied by a configurational change; we therefore conclude that it is not mediated by the Ca²⁺-induced transition.     

On the stabilization by fixation of configurational states in beef heart mitochondria

The energized configuration of the cristal membrane of beef heart mitochondria can be maintained only as long as oxygen is available for electron transfer. When the oxygen supply is exhausted, the membrane undergoes a transition to the nonenergized configuration. Since the exhaustion of the available oxygen supply is complete in 5–20 sec, it is impossible to apply the method of sedimenting the mitochondria prior to fixation for studying the energized configurational states of mitochondria. The direct addition of glutaraldehyde followed by osmium tetroxide to the mitochondrial suspension is the most effective way of freezing the configurational state of the cristal membrane. Fixation with glutaraldehyde appears to be complete within 1–2 sec even at 0°. Osmium tetroxide alone can also freeze the energized configuration by fixation but the concentration of the fixative is critical. The problem of capturing the configurational state applies not only to energized transitions (nonenergized to energized) but also to nonenergized transitions (orthodox to aggregated). The freezing by fixation of the cristal membrane in the aggregated configuration is best accomplished by the sequential use of glutaraldehyde and osmium tetroxide. When the levels of glutaraldehyde and osmium tetroxide are respectively too low or too high, the mitochondrion will undergo a transition from the aggregated to the orthodox configuration before fixation is complete. Light-scattering studies provide an independent method for monitoring configurational changes in mitochondria; these light-scattering measurements confirm that the conditions for fixation which lead to stabilization of the energized state as judged by electron microscopy, also show maintenance of configuration as judged by absence of light-scattering changes after the fixatives are introduced. Reagents used in negative staining will induce the geometrical form of the energized configuration of the mitochondrion even under nonenergizing conditions. These reagents are thus unsuitable for use in studies of configurational transitions in mitochondria.     

In Saccharomyces cerevisiae,cations control the fate of the energy derived from oxidative metabolism through the opening and closing of the yeast mitochondrial unselective channel

The yeast mitochondrial unspecific channel (YMUC) sensitivity to inorganic (Ca²⁺ or Mg²⁺) or organic (hexyl or octyl-guanidine) cations was measured. The rate of oxygen consumption in State 3 and State 4, the transmembrane potential (), mitochondrial swelling, and the polyethylene-glycol mediated recontraction were used to follow opening of the YMUC. Addition of 0.4 mM PO₄ did not close the YMUC, although it did enhance the sensitivity to Ca²⁺ (I₅₀ decreased from 50 to 0.3 mM) and Mg²⁺ (I₅₀ decreased from 5 to 0.83 mM Mg²⁺). The Ca²⁺ concentration needed to close the YMUC was higher than the concentrations usually observed in the cell. Nonetheless, Mg²⁺, Ca²⁺, and PO₄ exhibited additive effects. These cations did not inhibit contraction of preswollen mitochondria, suggesting that the YMUC/cation interaction was labile. Octyl-guanidine (OG-I₅₀ 7.5 M) was the only cation which inhibited mitochondrial recontraction, probably as a result of membrane binding stabilization through its hydrophobic tail. The PO₄-dependent, Ca²⁺/Mg²⁺-mediated closure of the YMUC may be a means to control the proportion of oxidative energy producing ATP or being lost as heat.     

Regulation by Magnesium of Potato Tuber Mitochondrial Respiratory Activities

Dehydrogenase activities of potato tuber mitochondria and corresponding phosphorylation rates were measured for the dependence on external and mitochondrial matrix Mg²⁺. Magnesium stimulated state 3 and state 4 respiration, with significantly different concentrations of matrix Mg²⁺ required for optimal activities of the several substrates. Maximal stimulation of respiration with all substrates was obtained at 2-mM external Mg²⁺. However, respiration of malate, citrate, and -ketoglutarate requires at least 4-mM Mg²⁺ inside mitochondria for maximization of dehydrogenase activities. The phosphorylation system, requires a low level of internal Mg²⁺ (0.25 mM) to reach high activity, as judged by succinate-dependent respiration. However, mitochondria respiring on citrate or -ketoglutarate only sustain high levels of phosphorylation with at least 4-mM matrix Mg²⁺. Respiration of succinate is active without external and matrix Mg²⁺, although stimulated by the cation. Respiration of -ketoglutarate was strictly dependent on external Mg²⁺ required for substrate transport into mitochondria, and internal Mg²⁺ is required for dehydrogenase activity. Respiration of citrate and malate also depend on internal Mg²⁺ but, unlike -ketoglutarate, some activity still remains without external Mg²⁺. All the substrates revealed insensitive to external and internal mitochondrial Ca²⁺, except the exogenous NADH dehydrogenase, which requires either external Ca²⁺ or Mg²⁺ for detectable activity. Calcium is more efficient than Mg²⁺, both having cumulative stimulation. Unlike Ca²⁺, Mn²⁺ could substitute for Mg²⁺, before and after addition of A23, showing its ability to regulate phosphorylation and succinate dehydrogenase activities, with almost the same efficiency as Mg²⁺.     

Flufenamic acid as an inducer of mitochondrial permeability transition

To assess the mechanism by which mitochondrial permeability transition (MPT) is induced by the nonpolar carboxylic acids, we investigated the effects of flufenamic acid (3-trifluoromethyl diphenylamine-2-carboxylic acid, FA) on mitochondrial respiration, electrical transmembrane potential difference (), osmotic swelling, Ca²⁺ efflux, NAD(P)H oxidation and reactive oxygen species (ROS) generation. Succinate-energized isolated rat liver mitochondria incubated in the absence or presence of 10 M Ca²⁺, 5 M ruthenium red (RR) or 1 M cyclosporin A (CsA) were used. The dose response-curves for both respiration release and dissipation were nearly linear, presenting an IC₅₀ of approximately 10 M and reaching saturation within 25-50 M, indicating that FA causes mitochondrial uncoupling by a protonophoric mechanism. Within this same concentration range FA showed the ability to induce MPT in energized mitochondria incubated with 10 M Ca²⁺, followed by dissipation and Ca²⁺ efflux, and even in deenergized mitochondria incubated with 0.5 mM Ca²⁺. ADP, Mg²⁺, trifluoperazine (TFP) and N-ethylmaleimide (NEM) reduced the extent of FA-promoted swelling in energized mitochondria by approximately one half, whereas dithiothreitol (DTT) slightly enhanced it. NAD(P)H oxidation and ROS generation (H₂O₂ production) by mitochondria were markedly stimulated by FA; these responses were partly prevented by CsA, suggesting that they may be implicated as both a cause and effect of FA-induced MPT. FA incubated with mitochondria under swelling assay conditions caused a decrease of approximately 40% in the content of protein thiol groups reacting with 5,5-dithiobis(2-nitrobenzoic acid) (DTNB). The present results are consistent with a ROS-intermediated sensitization of MPT by a direct or indirect FA interaction with inner mitochondrial membrane at a site which is in equilibrium with the NAD(P)H pool, namely thiol groups of integral membrane proteins.     

The role of Ca 2+ in control of malic enzyme activity in bovine adrenal cortex mitochondria

Low physiological levels of Ca²⁺, in the presence of Mg²⁺ allow the reduction of extramitochondrial NADP+ via intramitochondrial malic enzyme. The rate of reduction is dependent on the concentration of Ca²⁺ and Mg²⁺. The Ca²⁺ levels producing the appearance of malic enzyme activity also cause an ultrastructural transformation from the aggregated to the orthodox form. The phenomenon requires electron transport and is blocked by agents which interfere with active Ca²⁺ accumulation.     

Effects of palmitoyl CoA and palmitoyl carnitine on the membrane potential and Mg2+ content of rat heart mitochondria

Palmitoyl CoA and palmitoyl carnitine added to rat heart mitochondria in amounts above 20 and 50 nmoles/mg protein, respectively, induced a fall in transmembrane potential and loss of endogenous Mg²⁺. The dissipation of membrane potential by low concentrations of palmitoyl CoA in the presence of C²⁺, but not that of high concentrations of palmitoyl CoA alone, was prevented by either ruthenium red, Cyclosporin A or Mg²⁺, but reversed only by Mg²⁺. The fall of membrane potential induced by palmitoyl carnitine was not prevented by any of these factors. It is suggested that the action of both palmitoyl CoA and palmitoyl carnitine at high concentrations is due to a non specific disruption of membrane architecture, while that of low concentrations of palmitoyl CoA in the presence of Ca²⁺ is associated specifically with energy dissipation due to Ca²⁺ cycling.Abbreviations LCACoA Long-Chain Acyl CoAs - LCAcar Long-Chain Acyl carnitines - Pcar Palmitoyl carnitine - PCoA Palmitoyl CoA - Transmembrane Potential     

Regulation of calcium currents and secretion by magnesium in crustacean peptidergic neurons

The effect of varying the external Mg²⁺ concentration on Ca²⁺ currents through voltage-operated Ca²⁺ channels has been examined with the patch-clamp technique in acutely isolated neuronal somata from the X-organ-sinus gland (XOSG) of the crab,Cardisoma carnifex. Neurons from this neurosecretory system were selected for morphology associated with crustacean hyperglycemic hormone (CHH) content. In parallel, the effects of Mg²⁺ concentration on K⁺-evoked secretion of CHH from isolated, intact XOSGs have been assayed by ELISA. At physiological Ca²⁺ levels the high-voltage-activated Ca²⁺ currents were attenuated with increasing Mg²⁺ concentration, with 50% inhibition at 75 mM. Mg²⁺ block was voltage-dependent, relief from block occurring with increasing depolarization. Thus, in 24 mM Mg²⁺ inhibition of the Ca²⁺ current was 55% at –10 mV and 30% at +20 mV. Secretion of CHH varied almost linearly with the log of Mg²⁺ concentration; in 2.4 mM Mg²⁺ it was double that in 24 mM Mg²⁺ and almost completely inhibited in 100 mM. Thus, Mg²⁺ produces a parallel inhibition of Ca²⁺ currents and CHH secretion and may play a role as a physiological modulator of neuronal activity and secretion in the XOSG of these crabs.     

Uncoupling of Ca2+ transport from ATP hydrolysis activity of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase

Summary In reconstituted rabbit skeletal muscle (Ca²⁺ + Mg²⁺)-ATPase proteoliposomes, Ca²⁺-uptake is decreased by more than 90% with T₂ cleavage (Arg-198). However, no difference in the ATP dependence of hydrolysis activity is seen between SR and trypsin-treated SR. A large decrease in E-P formation and hydrolysis activity of the enzyme appear only at T₃ cleavage, which represents the cleavage of A₁ fragment to A_1a + A_1b forms. The disappearance of hydrolysis activity due to digestion is prior to the disappearance of E-P formation. No significant difference is found in the passive Ca²⁺ efflux between control SR and tryptically digested SR in the absence of Mg⁺ ruthenium red or in the presence of ATP. However, the passive Ca²⁺ efflux rate for tryptically digested SR is much larger than control SR in the presence of Mg²⁺ + ruthenium red. These results show that the Ca²⁺ channel cannot be closed after trypsin digestion of SR membranes by the presence of the Ca²⁺ channel inhibitors, Mg²⁺ and ruthenium red. In the reconstituted ATPase proteoliposomes, the Ca²⁺ efflux rates are the same regardless of digestion (T₂); also, efflux is not affected by the presence or absence of Mg²⁺ + ruthenium red. These results indicate that T₂ cleavage causes uncoupling of the Ca²⁺-pump from ATP hydrolytic activity.A theoretical model is developed in order to fit the extent of tryptic digestion of the A fragment of the (Ca²⁺ + Mg²⁺)-ATPase polypeptide with the loss of Ca²⁺-transport. Fits of the theoretical equations to the data are consistent with that Ca²⁺-transport system appears to require a dimer of the polypeptide (Ca²⁺ + Mg²⁺)-ATPase.     

Conformational basis of energy transduction in membrane systems VIII. Configurational changes of mitochondria

A method has been devised for the study of configurational changes in mitochondriain situ during the transition from nonenergized to energized conditions. The method depends upon the following component features: (a) subdivision of the tissue into finely diced sections; (b) the use of a modified Krebs-Ringer phosphate solution as the suspending medium; (c) aerobic conditions as the tactic for imposing the energized state; (d) anaerobic conditions or the presence of uncoupler under aerobic conditions as the tactic for imposing the nonenergized state; and (e) rapid fixation of the diced sections by addition of a mixture of formaldehyde and glutaraldehyde at a controlled temperature. Regardless of the tissue of source (heart, liver, skeletal muscle, retina, kidney) or the species (beef, rat, canary), all mitochondria show unambiguous configurational changes during the transition from nonenergized to energized conditions. The present study has revealed various optional features of the configurational states. Thus, there are two nonenergized configurations of the crista—orthodox and aggregated. The osmotic pressure of the suspending medium determines which nonenergized configuration will be observed. There are at least two variant forms of the energized-twisted configuration—tubular and zigzag. Again the osmotic pressure of the medium is an important factor in determining the form of the crista in the energized-twisted configuration. Mitochondria, such as those of heart muscle with relatively little matrix protein, show the clearest and most regular configurational changes, whereas mitochondria, such as those of liver with an abundance of matrix protein, show a more complex and less regular pattern of configurational change. From this comparative study of mitochondriain situ, it can be concluded that no exceptions have been found to the generalization that changes in configurational state of the cristae accompany changes in the energy state; this exact correlation provides additional support for the hypothesis of the conformational basis of energy transduction in the mitochondrion.Postdoctoral Trainee of the University of Wisconsin.Established Investigator of the American Heart Association.This work was supported in part by U.S. Public Health Service Program Project Grant GM-12847 and by a training grant GM-88 from the National Institute of Medical Sciences.     

Closure of VDAC causes oxidative stress and accelerates the Ca-induced mitochondrial permeability transition in rat liver mitochondria

The electron transport chain of mitochondria is a major source of reactive oxygen species (ROS), which play a critical role in augmenting the Ca²⁺-induced mitochondrial permeability transition (MPT). Mitochondrial release of superoxide anions (O₂⁻) from the intermembrane space (IMS) to the cytosol is mediated by voltage dependent anion channels (VDAC) in the outer membrane. Here, we examined whether closure of VDAC increases intramitochondrial oxidative stress by blocking efflux of O₂⁻ from the IMS and sensitizing to the Ca²⁺-induced MPT. Treatment of isolated rat liver mitochondria with 5 μM G3139, an 18-mer phosphorothioate blocker of VDAC, accelerated onset of the MPT by 6.8 ± 1.4 min within a range of 100-250 μM Ca²⁺. G3139-mediated acceleration of the MPT was reversed by 20 μM butylated hydroxytoluene, a water soluble antioxidant. Pre-treatment of mitochondria with G3139 also increased accumulation of O₂⁻ in mitochondria, as monitored by dihydroethidium fluorescence, and permeabilization of the mitochondrial outer membrane with digitonin reversed the effect of G3139 on O₂⁻ accumulation. Mathematical modeling of generation and turnover of O₂⁻ within the IMS indicated that closure of VDAC produces a 1.55-fold increase in the steady-state level of mitochondrial O₂⁻. In conclusion, closure of VDAC appears to impede the efflux of superoxide anions from the IMS, resulting in an increased steady-state level of O₂⁻, which causes an internal oxidative stress and sensitizes mitochondria toward the Ca²⁺-induced MPT.     

Relationship between Ca2+-transport and ATP hydrolytic activities in guinea-pig pancreatic acinar plasma membranes

Partially purified plasma membrane fractions were prepared from guinea-pig pancreatic acini. These membrane preparations were found to contain an ATP-dependent Ca²⁺-transporter as well as a heterogenous ATP-hydrolytic activity. The Ca²⁺-transporter showed high affinity for Ca²⁺ (K_Ca ²⁺ = 0.04 ± 0.01 M), an apparent requirement for Mg²⁺ and high substrate specificity. The major component of ATPase activity could be stimulated by either Ca²⁺ or Mg²⁺ but showed a low affinity for these cations. At low concentrations, Mg²⁺ appeared to inhibit the Ca²⁺-dependent ATPase activity expressed by these membranes. However, in the presence of high Mg²⁺ concentration (0.5–1 mM), a high affinity Ca²⁺-dependent ATPase activity was observed (K_Ca ²⁺ = 0.08 ± 0.02 M). The hydrolytic activity showed little specificity towards ATP. Neither the Ca²⁺-transport nor high affinity Ca²⁺-ATPase activity were stimulated by calmodulin. The results demonstrate, in addition to a low affinity Ca²⁺ (or Mg⁺)-ATPase activity, the presence of both a high affinity Ca²⁺-pump and high affinity Ca²⁺-dependent ATPase. However, the high affinity Ca²⁺-ATPase activity does not appear to be the biochemical expression of the Ca²⁺-pump.Abbreviations Ca²⁺-ATPase calcium-activated, magnesium-dependent adenosine triphosphatase - CaM calmodulin - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate - EDTA ethylene-diaminetetraacetate - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetate - NADPH reduced form of nicotinamide adenine dinucleotide phosphate     

Ca2+ transport by chondrocyte mitochondria of the epiphyseal growth plate

Summary In a study of the Ca²⁺ kinetics of mitochondria of chick epiphyseal chondrocytes, the rate of Ca²⁺ uptake was linear up to a medium Ca²⁺ concentration of 30 m. The half maximal transport rate occurred at 34 m Ca²⁺. The Ca²⁺ uptake rate, expressed as a function of time, was 35 nmoles/mg protein/min; the presence of Mg²⁺ had little effect on Ca²⁺ accumulation. While these kinetic parameters did not differ significantly from mitochondria of cells of nonmineralizing tissues, the respiratory characteristics of the chondrocyte organelles exhibited functional differences. Thus, up to 350 nmoles Ca²⁺/mg protein, chondrocyte mitochondria performed coupled oxidative phosphorylation. Calcium uptake was energy supported, while Ca²⁺ binding was low. Addition of respiratory inhibitors and uncouplers to these mitochondria resulted in a rapid loss of more than 80% of the total Ca²⁺. The Ca/Pi ratio of the extrudate was very similar to the ratio of these ions in cartilage septum fluid. In the most mineralized zones of the epiphyseal plate, there was little change in the state 4 respiratory rate, but nonspecific Ca²⁺ binding was elevated and a high percentage of the total Ca²⁺ was in a nonextrudable form. The results indicate that in cells preparing for mineralization, much of the total mitochondrial Ca²⁺ is in a form that can be transported to the calcification front. In cells close to the calcification front, nonextrudable Ca²⁺ may form calcium phosphate granules described by other investigators.     

Some effects of Ca2+, Mg2+, and Mn2+ on the ultrastructure,light-scattering properties,and malic enzyme activity of adrenal cortex mitochondria

The ultrastructure and 90 ° light-scattering capacity of adrenal cortex mitochondria have been examined under conditions which lead to an activation of malic enzyme activity in these mitochondria. After isolation, the mitochondria display an aggregate ultrastructure which does not resemble the vesicular (orthodox) form normally seen in vivo. Under conditions of malic enzyme activation (presence of malate, NADP⁺, Mg²⁺ and 1 mm Ca²⁺), the ultrastructure reverts to a vesicular form as seen in vivo. Of these required components, only Ca²⁺ affects the ultrastructure. The ultrastructural transformation from the aggregate to the orthodox form is always accompanied by a decrease in the 90 ° light-scattering capacity. When produced by Ca²⁺, transformation requires energy-dependent Ca²⁺ uptake if an oxidizable substrate is present. In the absence of substrate, the transformation occurs as an apparent energy-independent effect. Mn²⁺ can substitute for Ca²⁺ only in the presence of substrate. In de-energized mitochondria, Mn²⁺ prevents the effects of Ca²⁺. The activation of malic enzyme is always preceded by a decrease in light scattering and transformation to the orthodox ultrastructure; however, the presence of the orthodox form is not a sufficient condition since subsequent chelation of free Ca²⁺ fails to reverse either the decrease in light scattering or ultrastructural transformation but does reverse the enzyme activation. In addition, levels of Mn²⁺ which effectively depress light-scattering capacity and produce the orthodox form, fail to activate malic enzyme significantly. The data are discussed as they relate to Ca²⁺-induced damage in mitochondria.     

Purification and characterization of phenylacetate-coenzyme A ligase from a denitrifying Pseudomonas sp., an enzyme involved in the anaerobic degradation of phenylacetate

The enzyme catalysing the first step in the anaerobic degradation pathway of phenylacetate was purified from a denitrifying Pseudomonas strain KB 740. It catalyses the reaction phenylacetate+CoA+ATP phenylacetyl-CoA+AMP+PP_i and requires Mg²⁺. Phenylacetate-CoA ligase (AMP forming) was found in cells grown anaerobically with phenylacetate and nitrate. Maximal specific enzyme activity was 0.048 mol min^-1 x mg^-1 protein in the mid-exponential growth phase. After 640-fold purification with 18% yield, a specific activity of 24.4 mol min^-1 mg^-1 protein was achieved. The enzyme is a single polypeptide with Mr of 52 ±2 kDa. The purified enzyme shows high specificity towards the aromatic inducer substrate phenylacetate and uses ATP preferentially; Mn²⁺ can substitute for Mg²⁺. The apparent K _m values for phenylacetate, CoA, and ATP are 60, 150, and 290 M, respectively. The soluble enzyme has an optimum pH of 8.5, is insensitive to oxygen, but is rather labile and requires the presence of glycerol and/or phenylacetate for stabilization. The N-terminal amino acid sequence showed no homology to other reported CoA-ligases. The expression of the enzye was studied by immunodetection. It is present in cells grown anaerobically with phenylacetate, but not with mandelate, phenylglyoxylate, benzoate; small amounts were detected in cells grown aerobically with phenylacetate.     

Ca2+ transport in mitochondrial and microsomal fractions from higher plants

Mitochondria from etiolated corn possess a much greater Ca²⁺ uptake capacity per mg protein than microsomes from the same source. Differences in energy requirements, sensitivity to specific inhibitors, and sedimentation properties enabled us to study both Ca²⁺ uptake mechanisms without mutual contamination. The microsomal Ca²⁺ uptake does not vary much among different plants as compared to the mitochondrial Ca²⁺ uptake; this is also true for different organs of the same plant. Mitochondrial Ca²⁺ uptake is more dependent on the age of the seedlings than microsomal uptake, because of changes in active Ca²⁺ uptake activity rather than of changes in efflux. Intactness and the oxidative and phosphorylative properties of the mitochondria remained unchanged during this time period. Na⁺ and Mg²⁺ do not induce Ca²⁺ release from mitochondria.Abbreviations ATP adenosine triphosphate - ADP adenosine diphosphate - NADH₂ -nicotinamide adenin dinucleotide, reduced form - Mops 3-(N-morpholino)propane-sulfonic acid - Tris tris-(hydroxymethyl)-aminomethane - Hepes hydroxyethylpiperazine-N-2-ethanesulfonic acid - BSA bovine serum albumin - EDTA (ethylene-dinitrilo)-tetraacetic acid - EGTA ethylene glycol-bis(-aminoethylether)-N,N-tetraacetic acid - CCCP carbonyl cyanide m-chlorophenylhydrazone - DTE 1,4-dithiothreitol     

Effects of antibiotic ionophore, A23187, on oxidative phosphorylation and calcium transport of liver mitochondria

A23187, a new antibiotic with ionophore properties, uncoupled oxidative phosphorylation in mitochondria which oxidized either malate plus glutamate or succinate. Ca²⁺, but not Mg²⁺, enhanced the uncoupling effect. Fluorescence of ANS¹ was increased by A23187 suggesting the mitochondrial membranes were de-energized. This de-energization was presumably by activation of the energy-dependent uptake of Ca²⁺. The steady-state measurements of murexide-divalent cation complexes showed that A23187 caused mitochondria to release the accumulated Ca²⁺ to the medium. This reduced the transmembrane Ca²⁺ gradient even though normal active Ca²⁺ uptake could take place. A23187 inhibited activity of ATPase induced by 2,4-dinitrophenol, valinomycin, and Ca²⁺. The addition of Mg²⁺ could prevent this inhibition presumably by maintaining the endogenous Mg²⁺ concentration. The above metabolic events could be explained by the fact that molecules of A23187 function in the mitochondrial inner membrane as mobile carriers for divalent cations.     

Expression and regulation of insulin-like growth factor binding protein-1 in the rat uterus throughout estrous cycle

The role of Ca²⁺-stimulated adenosine 5-triphosphatase (Ca²⁺-ATPase) in Ca²⁺ sequestering of rat liver nuclei was investigated. Ca²⁺-ATPase activity was calculated by subtracting Mg²⁺-ATPase activity from (Ca²⁺–Mg²⁺)-ATPase activity. Ca²⁺ uptake and release were determined with a Ca²⁺ electrode. Nuclear Ca²⁺-ATPase activity increased linearly in the range of 10–40 M Ca²⁺ addition. With those concentrations, Ca²⁺ was completely taken up by the nuclei dependently on ATP (2 mM). Nuclear Ca²⁺-ATPase activity was decreased significantly by the presence of arachidonic acid (25 and 50 M), nicotinamide-adenine dinucleotide (NAD⁺; 2 mM) and zinc sulfate (2.5 and 5.0 M). These reagents caused a significant decrease in the nuclear Ca²⁺ uptake and a corresponding elevation in Ca²⁺ release from the nuclei. Moreover, calmodulin (10 g/ml) increased significantly nuclear Ca²⁺-ATPase activity, and this increase was not seen in the presence of trifluoperazine (10 M), an antogonist of calmodulin. The present findings suggest that Ca²⁺-ATPase plays a role in Ca²⁺ sequestering by rat liver nuclei, and that calmodulin is an activator. Moreover, the inhibition of Ca²⁺-ATPase may evoke Ca²⁺ release from the Ca²⁺-loaded nuclei.     

Electrical tolerance (breakdown) of theChara corallina plasmalemma: I. Necessity of Ca2+

Summary The relationship between the external Ca²⁺ concentrations [Ca²⁺]₀ and the electrical tolerance (breakdown) in theChara plasmalemma was investigated. When the membrane potential was negative beyond –350–400 mV (breakdown potential, BP), a marked inward current was observed, which corresponds to the so-called punch-through (H.G.L. Coster,Biophys. J. 5:669–686, 1965). The electrical tolerance of theChara plasmalemma depended highly on [Ca²⁺]₀. Increasing [Ca²⁺]₀ caused a more negative and decreasing it caused a more positive shift of BP. BP was at about –700 mV in 200 M La³⁺ solution. [Mg²⁺]₀ depressed the membrane electrical tolerance which was supposed to be due to competition with Ca²⁺ at the Ca²⁺ binding site of the membrane. Such a depressive effect of Mg²⁺ was almost masked when the [Ca²⁺]₀/[Mg²⁺]₀ ratio was roughly beyond 2.