Lithium induced changes in intracellular free magnesium concentration in isolated rat ventricular myocytes

We have examined the effect of exposing isolated rat ventricular myocytes to lithium while measuring cytosolic free magnesium ([Mg²⁺]_i) and calcium ([Ca²⁺]_i) levels with the fluorescent, ion sensitive probes mag-fura-2 and fura-2. There was a significant rise in [Mg²⁺]_i after a 5 min exposure to a solution in which 50% of the sodium had been replaced by Li⁺, but not when the sodium had been replaced by bis-dimethylammonium (BDA). However, there were significant increases in [Ca²⁺]_i when either Na⁺ substitute was used. The possibility that Li⁺, which enters the cells, interferes with the signal from mag-fura-2 was eliminated as Li⁺ concentrations up to 10 mM had no effect on the dye's fluorescence signal. A possible explanation for these findings is that Li⁺ displaces Mg²⁺ from intracellular binding sites. Having considered the binding constants for Mg²⁺ and Li⁺ to ATP, we conclude that Li⁺ can displace Mg²⁺ from Mg-ATP, thus causing a rise in [Mg²⁺]_i. This work has implications for other studies where Li⁺ is used as a Na⁺ substitute.     

Properties of (Mg2+ + Ca2+)-ATPase of erythrocyte membranes prepared by different procedures: Influence of Mg2+, Ca2+, ATP,and protein activator

Erythrocyte membranes prepared by three different procedures showed (Mg²⁺ + Ca²⁺)-ATPase activities differing in specific activity and in affinity for Ca²⁺. The (Mg²⁺ + Ca²⁺)-ATPase activity of the three preparations was stimulated to different extents by a Ca²⁺-dependent protein activator isolated from hemolystes. The Ca²⁺ affinity of the two most active preparations was decreased as the ATP concentration in the assay medium was increased. Lowering the ATP concentration from 2 mM to 2–200 μM or lowering the Mg:ATP ratio to less than one shifted the (Mg²⁺ + Ca²⁺)-ATPase activity in stepwise hemolysis membranes from mixed “high” and “low” affinity to a single high Ca²⁺ affinity. Membranes from which soluble proteins were extracted by EDTA (0.1 mM) in low ionic strengh, or membranes prepared by the EDTA (1–10 mM) procedure, did not undergo the shift in the Ca²⁺ affinity with changes in ATP and MgCl₂ concentrations. The EDTA-wash membranes were only weakly activated by the protein activator. It is suggested that the differences in properties of the (Mg²⁺ + Ca²⁺)-ATPase prepared by these three procedures reflect differences determined in part by the degree of association of the membrane with a soluble protein activator and changes in the state of the enzyme to a less activatable form.     

Heart sarcolemmal Ca2+ transport in endotoxin shock: I. Impairment of ATP-dependent Ca2+ transport

Effects of endotoxin administration on the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma were investigated. The results show that the sidedness of the sarcolemmal vesicles was not affected but the ATP-dependent Ca²⁺ transport in cardiac sarcolemma was decreased by 22 to 46% (p < 0.05) at 4 h following endotoxin administration. The kinetic analysis indicates that the V_max for ATP and for Ca²⁺ were decreased by 50% (p < 0.01) and 32% (p < 0.01), respectively, while the Km values for ATP and Ca²⁺ were not significantly affected after endotoxin administration. Magnesium (1–5 mM) stimulated while vanadate (0.25–3.0 M) inhibited the ATP-dependent Ca²⁺ transport, but the Mg²⁺-stimulated and the vanadate-inhibitable activities remained significantly lower in the endotoxin-treated animals. These data demonstrate that endotoxin administration impairs the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma and that the impairment is associated with a mechanism not affecting the affinity towards ATP and Ca²⁺. Additional experiments show that the Ca²⁺ sensitivity of the Ca²⁺-ATPase activity was indifferent between the control and endotoxic groups suggesting that endotoxic injury impairs Ca²⁺ pumping without affecting Ca²⁺-ATPase activity. Since sarcolemmal ATP-dependent Ca²⁺ transport plays an important role in the regulation of cytosolic Ca²⁺ homeostasis, an impairment in the sarcolemmal ATP-dependent Ca²⁺ transport induced by endotoxin administration may have a pathophysiological significance in contributing to the development of myocardial dysfunction in endotoxin shock.     

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.     

Characterization of Mg2+- and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg²⁺-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca²⁺ + Mg²⁺-ATPase) of relatively low activity. Mg²⁺-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca²⁺ + Mg²⁺)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg²⁺-ATPase activity revealed apparent K_m values of 115 μm for ATP, 333 μm for magnesium, and 200 μm for calcium. Maximum Mg²⁺-ATPase activity was obtained with no added calcium and 1 mm magnesium. Potassium was found to inhibit Mg²⁺-ATPase activity at concentrations greater than 100 mm. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca²⁺ + Mg²⁺)-ATPase was 13.7 nmol ³²P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol ³²P/mg/min. Characterization of (Ca²⁺ + Mg²⁺)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent K_m^s for magnesium and calcium were 125 and 0.6–1.2 μm, respectively. (Ca²⁺ + Mg²⁺)-ATPase activity was stimulated by potassium with an apparent K_m of 10 mm and maximum activity reached at 100 mm potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca²⁺ + Mg²⁺)-ATPase are similar to those of the (Ca²⁺ + Mg²⁺)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca²⁺ + Mg²⁺)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.     

Characteristics of Ca2+-stimulated ATPase in rat heart sarcolemma in the presence of dithiothreitol and alamethicin

We have studied the activities of Ca²⁺-stimulated ATPase in rat heart sarcolemma upon modulating the redox state of membrane thiol groups with dithiothreitol (DTT). The suitability of alamethicin to unmask the latent activity of this enzyme was also investigated. The Ca²⁺-stimulated ATPase in sarcolemma exhibited two activation sites — one with low affinity (Km = 0.70 ± 0.2 mM; Vmax = 10.0 ± 2.2 mol Pi/mg/h) and the other with high affinity (Km = 0.16 ± 0.7 mM; Vmax = 4.6 ± 0.8 mol Pi/mg/h) for Mg²⁺ATP. Alamethicin at a ratio of 1:1 with the sarcolemmal protein caused a 3-fold activation of Ca²⁺-stimulated ATPase without affecting its sensitivity to Ca²⁺ or Mg²⁺ATP. Treatment of sarcolemma with deoxycholate or sodium dodecyl sulfate resulted in a total loss of the enzyme activity; high concentrations of alamethicin also showed a detergent-like action on the sarcolemmal vesicles. DTT at 5–10 mM concentrations caused a 4–5 fold activation of Ca²⁺-stimulated ATPase in sarcolemma and this effect was observed to be dependent on the concentration of Mg²⁺ATP. DTT increased the affinity of the enzyme to Mg²⁺ATP at the high affinity site and enhanced the Vmax at the low affinity site in addition to increasing the sensitivity of Ca²⁺-stimulated ATPase to Ca²⁺. DTT protected the Ca²⁺-stimulated ATPase against deterioration by detergents and restored the enzyme activity after treatment with N-ethylmaleimide. The mechanism of action of DTT on Ca²⁺-stimulated ATPase may involve the reduction of essential thiols at the active site of the enzyme or its interaction with specific DTT-dependent inhibitor protein. No changes in the sensitivity of sarcolemmal Ca²⁺-stimulated ATPase to orthovanadate was evident in the absence or presence of DTT and alamethicin. The results suggest the use of both DTT and alamethicin for the determination of Ca²⁺-stimulated ATPase activity in sarcolemmal preparations.     

Yeast mitochondrial calcium uptake: Regulation by polyamines and magnesium ions

Spermine, spermidine, and magnesium ions modulate the kinetic parameters of the Ca²⁺ transport system ofEndomyces magnusii mitochondria. Mg²⁺ at concentrations up to 5 mM partially inhibits Ca²⁺ transport with a half-maximal inhibiting concentration of 0.5 mM. In the presence of 2 mM MgCl₂, theS _0.5 value of the Ca²⁺ transport system increases from 220 to 490 µM, which indicates decreased affinity for the system. Spermine and spermidine exert an activating effect, having half-maximal concentrations of 12 and 50 µM, respectively. In the case of spermine, theS _0.5 value falls to 50–65 µM, which implies an increase in the transport system affinity for Ca²⁺. Both Mg²⁺ and spermine cause a decrease of the Hill coefficient, giving evidence for a smaller degree of cooperativity. Spermine and spermidine enable yeast mitochondria to remove Ca²⁺ from the media completely. In contrast, Mg²⁺ lowers the mitochondrial buffer capacity. When both Mg²⁺ and spermine are present in the medium, their effects on theS _0.5 value and the free extramitochondrial Ca²⁺ concentration are additive. The ability of spermine and Mg²⁺ to regulate yeast mitochondrial Ca²⁺ transport is discussed.     

Opiates inhibit calmodulin activation of a high-affinity Ca2+-stimulated Mg2+-dependent ATPase in synaptic membranes

A high affinity Ca²⁺/Mg²⁺ ATPase has been identified and localized in synaptic membrane subfractions. This enzyme is stimulated by low concentrations of Ca²⁺ (1 M) believed to approximate the range of Ca²⁺ in the synaptosomal cytosol (0.1 to 5.0 M). The opiate agonist levorphanol, in a concentration-dependent fashion, inhibited Ca²⁺-stimulated ATP hydrolysis in lysed synaptic membranes. This inhibition was reversed by naloxone, while dextrorphan, the inactive opiate isomer, was without effect. Inhibition by levorphanol was most pronounced in a subfraction of synaptic membranes (SPM-1). The inhibition of Ca²⁺-stimulated ATP hydrolysis was characterized by a reduction inV _max for Ca²⁺. Levorphanol pretreatment reduced the Hill coefficient (H_N) of 1.5 to 0.7, suggesting cooperative interaction between the opiate receptor and the enzyme protein. Levorphanol, but not dextrorphan, also inhibited (28%) ATP-dependent Ca²⁺ uptake by synaptic membranes. Opiate ligand stereoisomers were tested for their effects on calmodulin stimulating of high affinity Ca²⁺/Mg²⁺ ATPase in synaptic membranes. Levorphanol (10 M), but not the inactive stereoisomer (+)dextrorphan, significantly inhibited (35%) the calmodulin-activated Ca²⁺-dependent ATP hydrolysis activity in a preparation of lysed synaptic membranes. Both Ca²⁺-dependent and calmodulin-dependent stimulation of the enzyme in the presence of optimal concentrations of the other co-substrate were inhibited by levorphanol (35–40%) but not dextrorphan. Inhibition of ATP hydrolysis was characterized by a reduction inV _max for both Ca²⁺ and calmodulin stimulation of the enzyme. Calmodulin stimulation of enzyme activity was most pronounced in SPM-1, the membrane fraction which also exhibits the maximal opiate inhibition (40%) of the Ca²⁺-ATPase. The results demonstrate that opiate receptor activation inhibits a high affinity Ca²⁺/Mg²⁺ ATPase in synaptic plasma membranes in a stereospecific fashion. The inhibition of the enzyme may occur by a mechanism involving both Ca²⁺ and calmodulin. Inhibition of calmodulin activation may contribute to the mechanism by which opiate ligands disrupt synaptosomal Ca²⁺ buffering mechanisms. Changes in the cytosolic distribution of synaptosomal Ca²⁺ following inhibition of Ca²⁺/Mg²⁺ ATPase may underlie some of the pharmacological effects of opiate drugs.     

Effects of Ca2+, Mg2+, and depolarizing agents,on the32Pi-labeling and degradation of phosphatidylinositols in rat brain synaptosomes

In isolated synaptosomes from rat brain, 100 M antimycin A and 10 M oxamic acid inhibit the³²Pi-labeling of phosphatidylinositol-4,5-bisphosphate (PIP₂) and phosphatidylinositol-4-phosphate (PIP) by 90% and 95–99% respectively. 10 mM sodium fluoride inhibits the labeling by 50–60% and 10 mM A23187 inhibits the labeling by 63–70%. Phospholipase A₂ inhibits the labeling of PIP₂ and PIP by 93–94% and stimulates their degradation by 84–92%. Depolarization of synaptosomes with 75 mM K⁺ or 100 M veratrine decreases the labeling of PIP₂ and PIP by 66–74%. The decreased labeling results in large part from the Ca²⁺-dependent degradation of³²P-labeled PIP₂ and PIP as shown by pulse-chase experiments in which PIP₂ and PIP were prelabeled with³²Pi. Depolarization of synaptosomes results in the stimulation of⁴⁵Ca²⁺ uptake with the concomitant hydrolysis of PIP and PIP₂. Addition of 1 mM Ca²⁺ accounts for 25% of the enhanced degradation whereas depolarization with 75 mM K⁺ accounts for 75% of the enhanced degradation of PIP₂ and PIP. Depolarization with 100 mM veratrine results in a 223% increase in inositol trisphosphate as evidenced by stimulation of⁴⁵Ca²⁺ uptake. EGTA (10mM) and Mg²⁺ (5–10 mM) inhibit the degradation of PIP and PIP₂ and counteract the action of 1 mM Ca²⁺. Our data demonstrate that⁴⁵Ca²⁺, Mg²⁺, and membrane depolarization play an important role in the turnover of membrane phosphatidylinositols.Abbreviations ATP adenosine triphosphate - Pi inorganic orthophosphate - PIP phosphatidylinositol-4-phosphate - PIP₂ phosphatidylinositol-4,5,-bisphosphate - IP₃ inositol-1,4,5-trisphosphate     

The anticalmodulin effect of aflatoxin B1 on purified erythrocyte Ca2+-AtPase

The genotoxic carcinogen aflatoxin B₁ (AFB₁) inhibited the calmodulin-stimulated membrane-bound (Ca²⁺Mg²⁺)-ATPase. Using the purified enzyme, 12 nmoles per ml of AFB₁ caused maximum inhibition of 28% and 50%, of the acidic phospholipid-stimulated and calmodulin-activated Ca²⁺-ATPase activity respectively. Treatment of red cell ghosts with increasing concentrations of Triton X-100, a non-ionic detergent caused a progressive loss of both the basal and calmodulin-stimulated Ca²⁺-ATPase activity. The activity of the phospholipid-free, detergent-solubilized enzyme was almost fully restored by phosphatidyl serine (PS) and its sensitivity to calmodulin was restored in the presence of phosphatidyl choline (PC). Analysis of the results obtained using varying concentrations of ATP shows that AFB₁ did not affect the Km and Vmax of the unstimulated enzyme whereas these parameters were reduced by about 75% and 50%, respectively, in the presence of calmodulin. Using the product of limited proteolysis by trypsin i.e. the 90 kDa fragment which still retains its calmodulin binding-domain and the 76 kDa fragment which has lost this domain, kinetic studies on the enzyme activity revealed that AFB₁ inhibited the calmodulin-activated 90 kDa fragment by about 50% while the 76 kDa was not affected at all by the toxin and calmodulin. The toxin had no significant affect on the basal activity of the 90 kDa limited proteolysis fragment of the enzyme. These observations suggest that AFB₁ inhibits the activated Ca²⁺-ATPase by binding to an important site in the calmodulin-binding domain of the enzyme. It seems likely that the toxin binds to tryptophan in the calmodulin-binding domain, thus causing a reduction in the rate at which this domain can interact with Ca²⁺-calmodulin or acidic phospholipids. The implication of these observations is that Ca²⁺-extrusion and other calmodulin-activated enzymes and processes may be slowed down during prolonged exposure to AFB₁ because of its anticalmodulin effect.Abbreviations ATP adenosine 5-triphosphate - EGTA ethylenglycolbis (-aminoethylether) N,N-tetraacetic acid - Hepes 4-(2 hydroxyethyl)-1-piperazine ethanesulphonic acid - AFB₁ aflatoxin B₁ - PMSF phenylmethylsulfonylfluoride - TLCK N--p-tosyl-L-lysine chloromethyl ketone - PC phosphatidycholine - PS phosphatidylserine - PI phosphatidyl inositol - DPG diphosphatidyl glycerol - SDS sodium dodecyl sulphate - Tris-HCl Tris (hydroxymethyl)aminomethane hydrochloride     

Biochemical characterization of a calcium ion stimulated-ATPase from goat spermatozoa

The goat spermatozoa membranes isolated after treatment with octa (ethylene glycol) mono n-dodecyl ether (C₁₂E₈) followed by discontinuous sucrose density gradient centrifugation have been found to contain an ATPase that is stimulated by externally added Ca²⁺ only. The membrane fraction has also found to contain Mg²⁺-dependent Ca²⁺-ATPase activity, however the former activity is about 2 fold higher than the latter. The molecular weight of the enzyme is found to be about 97,000 on SDS-polyacrylamide gel. The optimum concentration of Ca²⁺ required for maximum activity is 3 mM for both Mg²⁺-dependent and Mg²⁺-independent Ca²⁺-ATPase. Histidine and imidazole buffers are found to be the most suitable for dependent and independent enzyme activities respectively. ATP with an optimum concentration of 4 mM is observed to be the best substrate than any other nucleotides. The inhibitors like trifluoperazine and vanadate and group specific probes e.g. DTNB and TNBS inhibit these two enzymes but at different rates. Ca²⁺-uptake study shows that the uptake in the presence of Ca²⁺ and ATP is higher than in the presence of Mg²⁺, Ca²⁺ and ATP. The findings lead us to believe that the Mg²⁺-independent Ca²⁺-ATPase has some role in Ca²⁺ transport like Mg²⁺-dependent enzyme.Abbreviations Tris Tris (hydroxymethyl) amino ethane - Hepes-N 2-hydroxy ethyl piperizine-N¹-2-ethane sulfonic acid - Pipes-Piperizine-N N¹-bis(2-ethane sulfonic acid) - EGTA Ethylene Glycol-bis (-amino ethyl ether) - N, N, N¹, N¹ Tetraacetic Acid, sodium salt - TFP Trifluoperazine - DTNB 5,5¹ Dithiobis (2 nitrobenzoic acid) - TNBS 2, 4, 6-Trinitrobenzene Sulfonate - C₁₂E₈ Octa (ethylene glycol) mono n-dodecyl ether - PMSF Phenylmethyl Sulfonyl Fluoride - PAGE Polyacrylamide Gel Electrophoresis - PME -Mercapto Ethanol     

The possibility that Ca2+-ATPase from the plasma membrane-rich fraction of bovine parotid gland is ecto-Ca2+-dependent nucleoside triphosphatase

• 1.1. Parotid plasma membrane nonpump low-affinity Ca²⁺-ATPase, which possesses high-affinity (Ca²⁺ + Mg²⁺ )-ATPase activity, was characterized.
• 2.2. Purified Ca²⁺-ATPase hydrolyzed the nucleoside triphosphates, GTP, ITP, CTP, UTP, TTP (67–93% of ATP) and nucleoside diphosphates, ADP. GDP, IDP, CDP, TDP (12–40% of ATP) but not AMP and p-NPP.
• 3.3. The maximum activities of Ca²⁺- and (Ca²⁺ +Mg²⁺ )-ATPases were obtained in the presence of 1 mM and 0.13 μ M Ca²⁺, respectively.
• 4.4. The K_m values for Ca²⁺ in Ca²⁺- and (Ca²⁺+ Mg²⁺ )-ATPases were 0.2 mM and 22 nM. respectively.
• 5.5. The activities of both Ca²⁺- and (Ca²⁺ + Mg²⁺ )-ATPases were found in the right-side-out-vesicles obtained from the plasma membrane-rich fraction.
• 6.6. These features suggest that Ca²⁺-ATPase is an ecto-Ca²⁺-dependent nucleoside triphosphatase.

     

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     

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.     

Involvement of Ca2+-stimulated adenosine 5′-triphosphatase in the Ca2+ releasing mechanism of rat liver nuclei

The regulatory role of Ca²⁺-stimulated adenosine 5-triphosphatase (Ca²⁺-ATPase) in Ca²⁺ transport system of rat liver nuclei was investigated. Ca²⁺ uptake and release were determined with a Ca²⁺ electrode. Ca²⁺-ATPase activity was calculated by subtracting Mg²⁺-ATPase activity from (Ca²⁺–Mg²⁺)-ATPase activity. The release of Ca²⁺ from the Ca²⁺-loaded nuclei was evoked progressively after Ca²⁺ uptake with 1.0 mM ATP addition, while it was only slightly in the case of 2.0 mM ATP addition, indicating that the consumption of ATP causes a leak of Ca²⁺ from the Ca²⁺-loaded nuclei. The presence of N-ethylmaleimide (NEM; 0.1 mM) caused an inhibition of nuclear Ca²⁺ uptake and induced a promotion of Ca²⁺ release from the Ca²⁺-loaded nuclei. NEM (0.1 and 0.2 mM) markedly inhibited nuclear Ca²⁺-ATPase activity. This inhibition was completely blocked by the presence of dithiothreitol (DTT; 0.1 and 0.5 mM). Also, DTT inhibited the effect of NEM (0.1 mM) on nuclear Ca²⁺ uptake and release. Meanwhile, verapamil and diltiazem (10 M), a blocker of Ca²⁺ channels, did not prevent the NAD⁺ (1.0 and 2.0 mM), zinc sulfate (1.0 and 2.5 M) and arachidonic acid (10 M)-induced increase in nuclear Ca²⁺ release, suggesting that Ca²⁺ channels do not involve on Ca²⁺ release from the nuclei. These results indicates that an inhibition of nuclear Ca²⁺-ATPase activity causes the decrease in nuclear Ca²⁺ uptake and the release of Ca²⁺ from the Ca²⁺-loaded nuclei. The present finding suggests that Ca²⁺-ATPase plays a critical role in the regulatory mechanism of Ca²⁺ uptake and release in rat liver nuclei.     

The calmodulin-activated form of the Ca2+-pumping ATPase of the cardiac sarcolemmal membrane produces Ca2+ gradients with a thermodynamic efficiency of 100%

The thermodynamic efficiency of the calmodulin-activated form of the Ca²⁺-pumping ATPase of the bovine cardiac sarcolemma (SL) was evaluated in sealed vesicles under reversible conditions. The free internal Ca²⁺ concentration ([Ca²⁺]_i) established in the SL vesicle lumen by action of the ATPase was determined as a function of the [ATP]/([ADP][P_i]) ratio for the following experimental conditions: 250mM sucrose, 100mM KCI, 0.1mM Mg²⁺, 25mM HEPES, 25mM Tris, pH 7.40, at 37°C, [Ca²⁺]_o=50nM (1mM Ca/EGTA buffer), 0.75mM Mg-ATP, 0.1mM P_i, variable [ADP]. Under these conditions, with the pump working near itsK _m of 64nM, the [Ca²⁺]_i achieved was 18mM, decreasing with increasing [ADP] for [ADP] 0.84mM. A plot of the square of the [Ca²⁺]_i/[Ca²⁺]_o ratio against [ATP]/([ADP][P_i]) gave a straight line with a slope of 1.5×10⁷M. This was in agreement, within the experimental error, with the equilibrium constant for ATP hydrolysis under these conditions (1.09×10⁷M). These results demonstrate (1) tight coupling between Ca²⁺ transport and ATP hydrolysis with a stoichiometry of 2 Ca²⁺ moved per ATP split and (2) a low degree of passive leakage. Analysis at low [ADP] (<0.83mM) showed the unexpected result that ADP increases the rate of theforward reaction of the pump. The maximal effect on the initial rate is a 96±5% increase, with an EC₅₀ of approximately 0.4mM (ADP). Similar but lesser stimulation was observed with CDP. The implications of the above results for the energetics of the pump and for its physiological function in the beating heart are discussed.     

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.     

Phospholipid requirement of Ca2+-stimulated,Mg2+-dependent ATP hydrolysis in rat brain synaptic membranes

The phospholipid requirement for Ca²⁺-stimulated, Mg²⁺-dependent ATP hydrolysis (Ca²⁺/Mg²⁺-ATPase) and Mg²⁺-stimulated ATP hydrolysis (Mg²⁺-ATPase) in rat brain synaptosomal membranes was studied employing partial delipidation of the membranes with phospholipase A₂ (Hog pancreas), phospholipase C (Bacillus cereus) and phospholipase D (cabbage). Treatment with phospholipase A₂ caused an increase in the activities of both Ca²⁺/Mg²⁺-ATPase and Mg²⁺-ATPase whereas with phospholipase C treatment both the enzyme activities were inhibited. Phospholipase D treatment had no effect on Ca²⁺/Mg²⁺-ATPase but Mg²⁺-ATPase activity was inhibited. Inhibition of Mg²⁺-ATPase activity after phospholipase C treatment was relieved with the addition of phosphatidylinositol-4,5-bisphosphate (PIP₂) and to a lesser extent with phosphatidylinositol-4-phosphate (PIP) and phosphatidylcholine (PC). Phosphatidylserine (PS), phosphatidic acid (PA), PIP and PIP₂ brought about the reactivation of Ca²⁺/Mg²⁺-ATPase. Phosphatidylinositol (PI) and PA inhibited Mg²⁺-ATPase activity.K _ms for Ca²⁺ (0.47 M) and Mg²⁺ (60 M) of the enzyme were found to be unaffected after treatment with the phospholipases.     

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.