Lumenal calcium modulates unitary conductance and gating of a plant vacuolar calcium release channel

The patch clamp technique has been used to investigate ion permeation and Ca²⁺-dependent gating of a voltage-sensitive Ca²⁺ release channel in the vacuolar membrane of sugar beet tap roots. Reversal potential measurements in bi-ionic conditions revealed a sequence for permeability ratios of Ca²⁺ Sr²⁺ Ba²⁺ > Mg²⁺ K⁺ which is inversely related to the size of the unitary conductances K⁺ Mg²⁺ Ba²⁺ > Sr²⁺ Ca²⁺, suggesting that ion movement is not independent. In the presence of Ca²⁺, the unitary K⁺ current is reduced in a concentration- and voltage-dependent manner by Ca²⁺ binding at a high affinity site (K _0.5 = 0.29 mm at 0 mV) which is located 9% along the electric field of the membrane from the vacuolar side. Comparison of reversal potentials obtained under strictly bi-ionic conditions with those obtained in the presence of mixtures of the two ions indicates that the channel forms a multi-ion pore. Lumenal Ca²⁺ also has an effect on voltage-dependent channel gating. Stepwise increases of vacuolar Ca²⁺ from micromolar to millimolar concentrations resulted in a dramatic increase in channel openings over the physiological voltage range via a shift in threshold for channel activation to less negative membrane potentials. The steepness of the concentration dependence of channel activation by Ca²⁺ at –41 mV predicts that two Ca²⁺ ions need to bind to open the gate. The implications of the results for ion permeation and channel gating are discussed.We thank Ian Jennings for writing and implementing some of the software used in this study and Anna J. Bate for technical assistance. The work was supported by grants from the Biotechnology and Biological Sciences Research Council to E.J. (PDF/14) and DS (PG87/529).     

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     

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.     

α-adrenergic receptor regulation of Ca2+/Mg2+-ATPase in brain synaptic membranes

The effects of ₁ and ₂ receptor ligands on Ca²⁺/Mg²⁺-ATPase have been studied using synaptosomal plasma membranes isolated from rat brain cortex. Both phenylephrine and clonidine inhibited Ca²⁺/Mg²⁺-ATPase, in a concentration-dependent fashion. IC₅₀ values for half-maximal inhibition for phenylephrine and clonidine were 29 M and 18 M, respectively. The inhibitory effect of phenylephrine was reversed by the alpha antagonist prazosin while yohimbine and rauwolscine reversed the inhibition of enzyme activity by clonidine. The two antagonist subtypes were effective only against the respective agonist subtypes, demonstrating distinct subtype preferences. Analysis of the kinetics of enzyme inhibition indicate both agonists to be noncompetitive. Some evidence suggests that yohimbine may exhibit mixed agonist/antagonist properties which depend on [Ca²⁺]. The present study provides biochemical evidence to support auto receptor adrenergic receptor regulation of neurotransmitter release.     

Partial purification and characterization of a soluble protein phosphatase fromLeishmania donovani promastigotes

A soluble protein phosphatase from the promastigote form of the parasitic protozoanLeishmania donovani was partially purified using Sephadex G-100, DEAE-cellulose and again Sephadex G-100 columns. The partially purified enzyme showed a native molecular weight of about 42, 000 in both Sephadex G-100 and sucrose density gradient centrifugation. The sedimentation constant, stokes radius and frictional ratio were found to be 3.43S, 2.8 nm and 1.20 respectively. The enzyme preferentially utilized phosphohistone as the best exogenous substrate. Mg²⁺ ions were essential for enzyme activity; among other metal ions Mn²⁺ can replace Mg²⁺ to a certain extent whereas Ca²⁺, Co²⁺ and Zn²⁺ could not substitute for Mg²⁺. The pH optimum of the enzyme was 6.5–7.5 and the temperature optimum 37°C. The apparent Km for phosphohistone was 7.14 M. ATP, ADP, inorganic phosphate and pyrophosphate had inhibitory effect on the enzyme activity whereas no inhibition was observed with sodium tartrate and okadaic acid. These results suggest thatL. donovani promastigotes possess a protein phosphatase which has similar characteristics with the mammalian protein phosphatase 2C.Abbreviations PMSF phenylmethylsulfonyl fluoride - DTT dithiothreitol - TCA trichloroacetic acid - BSA bovine serum albumin - EDTA ethylenediamine tetraacetic acid - ATP adenosine triphosphate - ADP adenosine diphosphate - AMP adenosine monophosphate - EGTA Ethyleneglycol-bis-(-aminoethyl ether) N,N,N,N-tetraacetic acid     

Biophysical characterization of rat cardiac Ca2+/Mg2+ ecto-ATPase (Myoglein)

Sarcolemmal Ca²⁺/Mg²⁺ ecto-ATPase (Myoglein; MW 180 kD) is a membrane bound enzyme which requires a millimolar concentration of either Ca²⁺ or Mg²⁺ for maximal hydrolysis of ATP. The isoelectric point (pI) of the cardiac ecto-ATPase was 5.7. The purified Ca²⁺/Mg²⁺ ecto-ATPase from the rat heart sarcolemmal appeared as a single band with MW 90 kD in the SDS-PAGE. In order to understand the nature of this enzyme, the 90 kD band in the SDS-PAGE was electroeluted; the analysis of the eluate showed 2 prominent bands with MW 90 and 85 kD. The presence of 2 bands was further confirmed by gradient gel (10-20%) electrophoresis in 0.375 M Tris-HCl buffer, pH 8.8. Analysis of the purified Ca²⁺/Mg²⁺ ecto-ATPase as well as the electroeluted protein in a non-equilibrium linear two dimensional electrophoresis (Ampholyte pI 3.0-10.0) also showed two distinct bands. Mass spectroscopic analysis of the enzyme using different matrix combinations revealed the presence of multi-components indicating microheterogeneity in the protein structure. Treatment of the ecto-ATPase with DL-dithiothreitol did not alter the pattern of mass spectroscopic analysis and this indicated that the microheterogeneity may be due to some posttranslational modifications. It is concluded that rat cardiac Ca²⁺/Mg²⁺ ecto-ATPase is an acidic protein having two subunits. Furthermore, the enzyme shows microheterogeneity in its molecular structure.     

Regulatory effect of regucalcin on (Ca2+−Mg2+)-ATPase in rat liver plasma membranes: comparison with the activation by Mn2+ and Co2+

The effect of various metals and regucalcin, a calcium-binding protein isolated from rat liver cytosol, on (Ca²⁺–Mg²⁺)-ATPase activity in the plasma membranes of rat liver was investigated. Of various metals (Zn²⁺, Cu²⁺, Ni²⁺, Mn²⁺, Co²⁺ and Al³⁺; 100 M as a final concentration), Mn²⁺ and Co²⁺ increased markedly (Ca²⁺–Mg²⁺)-ATPase activity, while other metals had no effect. When Ca²⁺ was not added into enzyme reaction mixture, Mn²⁺ and Co²⁺ (25–100 M) did not significantly increase the enzyme activity, indicating that heavy metals act on Ca²⁺-stimulated phosphorylation of the enzyme. Meanwhile, regucalcin (0.25–1.0 M) caused a remarkable elevation of (Ca²⁺–Mg²⁺)-ATPase activity. This increase was not inhibited by the presence of 100 M vanadate, although the effects of Mn²⁺ and Co²⁺ (100 M) were inhibited by vanadate. Also, the inhibition of the Mn²⁺ and Co²⁺ effects by vanadate was not seen in the presence of regucalcin. Moreover, regucalcin (0.5 M) increased significantly the enzyme activity in the absence of Ca²⁺. This effect of regulcalcin was not altered by increasing concentrations of Ca²⁺ added, indicating that the regucalcin effect does not depend on Ca²⁺. The present results suggest that regucalcin activates directly (Ca²⁺–Mg²⁺)-ATPase in liver plasma membranes, and that the activation is not involved in the Ca²⁺-dependent phosphorylation of the enzyme.     

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.     

Characterization of Melittin Effects in Synaptosomes

The effects of melittin at increasing concentrations on: [³H]GABA release from mouse brain synaptosomes; on the radioactivity released from [³H]arachidonic acid labeled synaptosomal membranes; on synaptosomes ultrastructure and on the leakage of the cytoplasmic marker, lactate-dehydrogenase (LDH) was investigated. Melittin 0.3, 1, 3, 7, and 10 M progressively increases [³H]GABA release, but the efficacy of melittin is decreased when the amount of tissue exposed to a constant concentration of the toxin increases. The release of [³H]GABA induced by melittin below 3 M is Ca²⁺ dependent, but not that induced by the higher concentrations. The Ca²⁺ dependent fraction of the [³H]GABA released by 0.3 M melittin is selectively inhibited by 10 M quinacrine and 1 M nordihydroguaiaretic acid (NDGA) and facilitated by 3 M indomethacin, whereas the Ca²⁺ independent fraction of the [³H]GABA released by melittin is not. In the presence of Ca²⁺, melittin 0.3, 1 and 10 M progressively increases [³H]arachidonic acid release over control release, but the effectiveness of melittin is also decreased as the amount of tissue increases. No apparent changes in synaptosomes ultrastructure are observed in 0.3 M treated synaptosomes, but a noticeable disorganization is produced in 10 M melittin-treated synaptosomes, independently on the presence of external Ca²⁺. LDH activity only increases over control activity in the supernatant solutions of 10 M melittin treated synaptosomes, also in a Ca²⁺ independent manner. Our interpretation of these results is that the Ca²⁺-dependent, pharmacologic sensitive component of melittin-induced release of [³H]GABA, unmasked when 0.3 M melittin was used, involves the activation of a Ca²⁺-dependent type of membrane PLA₂. The Ca²⁺-independent release of [³H]GABA is in contrast, highly probable to be due to the membrane perturbation produced by complex melittin/lipid interactions.     

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     

Calcium-binding and its effect on circular dichroism of plant calmodulin

The Ca²⁺-binding properties of calmodulin purified from zucchini (Cucurbita pepo L.) has been determined. A value of 3.3 mol Ca²⁺ per mol of zucchini calmodulin was measured at pH 7.5 by equilibrium chromatography. The far-and near-UV circular-dichroic spectra of the Ca²⁺-and Mg²⁺-saturated as well as from the metal-free forms of zucchini calmodulin reveal that upon Ca²⁺-binding the -helix content increases. A comparison with the spectra of vertebrate calmodulin indicates that both calmodulin have a similar secondary structure, similar Ca²⁺-induced conformational changes and the same number of Ca²⁺-binding sites.Abbreviations CAPP 10-(3-aminopropyl)-2-chloro-phenothiazine - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - EDTA ethylenediaminetetraacetic acid Dedicated to Prof. Dr. Karl Decker on the occasion of his 60th birthday     

Electrogenic calcium transport in plasma membrane of rat pancreatic acinar cells

Summary ATP-dependent⁴⁵Ca²⁺ uptake was investigated in purified plasma membranes from rat pancreatic acinar cells. Plasma membranes were purified by four subsequent precipitations with MgCl₂ and characterized by marker enzyme distribution. When compared to the total homogenate, typical marker enzymes for the plasma membrane, (Na⁺,K⁺)-ATPase, basal adenylate cyclase and CCK-OP-stimulated adenylate cyclase were enriched by 43-fold, 44-fold, and 45-fold, respectively. The marker for the rough endoplasmic reticulum was decreased by fourfold compared to the total homogenate. Comparing plasma membranes with rough endoplasmic reticulum, Ca²⁺ uptake was maximal with 10 and 2 mol/liter free Ca²⁺, and half-maximal with 0.9 and 0.5 mol/liter free Ca²⁺. It was maximal at 3 and 0.2 mmol/liter free Mg²⁺ concentration, at an ATP concentration of 5 and 1 mmol/liter, respectively, and at pH 7 for both preparations. When Mg²⁺ was replaced by Mn²⁺ or Zn²⁺ ATP-dependent Ca²⁺ uptake was 63 and 11%, respectively, in plasma membranes; in rough endoplasmic reticulum only Mn²⁺ could replace Mg²⁺ for Ca²⁺ uptake by 20%. Other divalent cations such as Ba²⁺ and Sr²⁺ could not replace Mg²⁺ in Ca²⁺ uptake. Ca²⁺ uptake into plasma membranes was not enhanced by oxalate in contrast to Ca²⁺ uptake in rough endoplasmic reticulum which was stimulated by 7.3-fold. Both plasma membranes and rough endoplasmic reticulum showed cation and anion dependencies of Ca²⁺ uptake. The sequence was K⁺>Rb⁺>Na⁺>Li⁺>choline⁺ in plasma membranes and Rb⁺K⁺Na⁺>Li⁺>choline⁺ for rough endoplasmic reticulum. The anion sequence was Cl^–Br^–I^–>SCN^–>NO ₃ ^– >isethionate^– >cyclamate^–>gluconate^–>SO ₄ ^2– glutarate^– and Cl^–>Br>gluconate>SO ₄ ^2– >NO ₃ ^– >I^–>cyclamate^–SCN^–, respectively. Ca²⁺ uptake into plasma membranes appeared to be electrogenic since it was stimulated by an inside-negative K⁺ and SCN diffusion potential and inhibited by an inside-positive diffusion potential. Ca²⁺ uptake into rough endoplasmic reticulum was not affected by diffusion potentials. We assume that the Ca²⁺ transport mechanism in plasma membranes as characterized in this study represents the extrusion system for Ca²⁺ from the cell that might be involved in the regulation of the cytosolic Ca²⁺ level.     

Activating effect of regucalcin on (Ca2+-Mg2+)-ATPase in rat liver plasma membranes: relation to sulfhydryl group

The activating mechanism of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on (Ca²⁺–Mg²⁺)-ATPase in the plasma membranes of rat liver was investigated. (Ca²⁺–Mg²⁺)-ATPase activity was markedly increased by a sulfhydryl (SH) group protecting reagent dithiothreitol (DTT; 2.5 and 5 mM as a final concentration), while the enzyme activity was significantly decreased by a SH group modifying reagent N-ethylmaleimide (NEM; 0.5–5 mM). The effect of DTT (5 mM) to increase the enzyme activity was clearly blocked by NEM (5 mM). Regucalcin (0.25–1.0 M) significantly increased (Ca²⁺-Mg²⁺)-ATPase activity. This increase was completely blocked by NEM (5 mM). Meanwhile, digitonin (0.04%), which can solubilize the membranous lipids, significantly decreased (Ca²⁺–Mg²⁺)-ATPase activity. Digitonin did not have an effect on the DTT (5 mM)-increased enzyme activity. However, the effect of regucalcin (0.25 M) increasing (Ca²⁺–Mg²⁺)-ATPase activity was entirely blocked by the presence of digitonin. The present results suggest that regucalcin activates (Ca²⁺–Mg²⁺)-ATPase by the binding to liver plasma membrane lipids, and that the activation is involved in the SH groups which are an active site of the enzyme.     

Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes

Although low Na⁺ is known to increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiac muscle, the exact mechanisms of low Na⁺-induced increases in [Ca²⁺]_i are not completely defined. To gain information in this regard, we examined the effects of low Na⁺ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca²⁺]_i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na⁺-induced increase in [Ca²⁺]_i was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na⁺ did not alter the ATP-induced increase in [Ca²⁺]_i in the cardiomyocytes. This increase in [Ca²⁺]_i due to low Na⁺ and elevated KCl was dependent on the extracellular concentration of Ca²⁺ (0.25–2.0 mM). The L-type Ca²⁺-channel blockers, verapamil and diltiazem, at low concentrations (1 M) depressed the low Na⁺, KCl-induced increase in [Ca²⁺]_i without significantly affecting the response to low Na⁺ alone. The low Na⁺, high KCl-induced increase in [Ca²⁺]_i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 M), and diltiazem (5 and 10 M) as well as with amiloride (5–20 M), nickel (1.25–5.0 mM), cyclopiazonic acid (25 and 50 M) and thapsigargin (10 and 20 M). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 M). These data suggest that in addition to the sarcolemmal Na⁺–Ca²⁺ exchanger, both sarcolemmal Na⁺–K⁺ATPase, as well as the sarcoplasmic reticulum Ca²⁺-pump play prominent roles in the low Na⁺-induced increase in [Ca²⁺]_i. (Mol Cell Biochem 263: 151–162, 2004)     

Subcellular localization of calcium in sporangiophores of Phycomyces blackesleeanus

The in situ localization of Ca²⁺ in stage I sporangiophores of the fungus Phycomyces blakesleeanus was achieved with the potassium pyroantimonate technique. Precipitates of calcium-antimonate were present in mitochondria, vacuoles, endoplasmic reticulum and adjacent cytoplasm, Golgi-like bodies, and nuclei but not cell walls. Material treated with the calcium chelator EGTA lacked these precipitates. The preferential localization of Ca²⁺ in mitochondria, endoplasmic reticulum and vacuoles suggests that these organelles modulate the level of this cation in sporangiophores of P. blakesleeanus.Abbreviations EGTA ethyleneglycol-bis-(-aminoethyl ether) N,N, tetraacetic acid     

Alterations in the Function of Endoplasmic Reticulum and Neuronal Signalling

For many years, the endoplasmic reticulum (ER) was considered to be involved in rapid signalling events due to its ability to serve as a dynamic calcium store capable of accumulating large amounts of Ca²⁺ ions and of releasing them in response to physiological stimulation. Recent data significantly increased the importance of the ER as a signalling organelle, by demonstrating that the ER is associated with specific pathways regulating long-lasting adaptive processes and controlling cell survival. The ER lumen is enriched by enzymatic systems involved in protein synthesis and correcting post-translational folding of these proteins. The processes of post-translational protein processing are controlled by a class of specific enzymes known as chaperones, which in turn are regulated by the free Ca²⁺ concentration within the ER lumen ([Ca²⁺]_L). At the same time, a high [Ca²⁺]_L determines the ability of the ER to generate cytosolic Ca²⁺ signals. Thus, the ER is able to produce signals interacting within different temporal domains. Fast ER signals result from Ca²⁺ release via specific Ca²⁺-release channels and from rapid movements of Ca²⁺ ions within the ER lumen (calcium tunneling). Long-lasting signals involve Ca²⁺-dependent regulation of chaperones with subsequent changes in protein processing and synthesis. Any malfunctions in the ER Ca²⁺ homeostasis result in accumulation of unfolded proteins, which in turn activates several signalling systems aimed at appropriate compensatory responses or (in the case of severe ER dysregulation) in cellular pathology and death (ER stress responses). Thus, the Ca²⁺ ion emerges as a messenger molecule, which integrates various signals within the ER: fluctuations of the [Ca²⁺]_L induced by signals originating at the level of the plasmalemma (i.e., Ca²⁺ entry or activation of the metabotropic receptors) regulate in turn protein synthesis and processing via generating secondary signalling events between the ER and the nucleus.     

The effect of some glycolytic intermediates and long-chain acyl-CoA esters on rat skeletal muscle mitochondrial α-glycerophosphate dehydrogenase

Summary -Glycerophosphate dehydrogenase (sn-glycerol-3-phosphate: acceptor oxidoreductase, EC 1.1.99.5) activity in mitochondria isolated from rat skeletal muscle has been studied. The pH optimum of the enzyme activity was about 7.4 and the apparent K_m value for DL--glycerophosphate was approxinately 1.6mm. The activity of this enzyme was found to be inhibited by DL-glyceraldehyde-3-phosphate, phosphoenolpyruvate and 3-phosphoglycerate in a competitive manner: the apparent K_i values at pH 7.4 being 0.3mm, 1.5mm and 4.0mm respectively. The enzyme was found to be more sensitive to phosphoenolpyruvate at pH 7.0 than 7.6.The activity of -glycerophosphate dehydrogenase in rat skeletal muscle was also inhibited by palmitoyl-CoA and stearoyl-CoA in a competitive manner. The K_i values being about 9.0 m for both metabolites. This inhibition was partly reversed by Ca²⁺ and Mg²⁺ ions. Palmitoylcarnitine also exerted inhibitory effect on -glycerophosphate dehydrogenase activity but palmitate, carnitine and CoA added alone was without effect. It is proposed that the activity of -glycerophosphate dehydrogenase in rat skeletal muscle mitochondria may be controlled by changes of the cytosolic levels of some glycolytic intermediates and long-chain acyl-CoA esters. These results are discussed with respect to the regulation of -glycerophosphate shuttle activity in skeletal muscle.     

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.     

Effect of magnesium on calcium influx activated by glutamate and its agonists in cultured cerebellar granule cells

The effects of Mg²⁺ on the glutamate-, kainate-, N-methyl-d-aspartate- and quisqualate-induced influx of⁴⁵Ca²⁺ were studied in cultured cerebellar granule cells. The N-methyl-d-aspartate- and quisqualate-evoked influx was totally and the kainate- and glutamate-evoked influx partially blocked in 1.3 mM extracellular Mg²⁺. The increase in influx induced by kainate, quisqualate and glutamate was maximal at 0.1 mM Mg²⁺, whereas N-methyl-d-aspartate was most effective in totally Mg²⁺-free media.d-2-Amino-5-phosphonovalerate blocked partially and phencyclidine completely the enhancement of Ca²⁺ influx by 1 mM quisqualate in 0.1-mM Mg²⁺ medium. The effect of 10 M quisqualate was also significantly inhibited by antagonists specific for different glutamate receptor subtypes, including N-methyl-d-aspartate, (RS)-amino-3-hydroxy-5-methyl-4-isozazolepropionate and metabotropic recptors. This evidences a heterogeneous action of quisqualate, mediated by different glutamate receptor subtypes in 0.1 mM Mg²⁺ medium. The efficacy of quisqualate in inducing influx of Ca⁺ and the selectivity of antagonists for different receptors are also modified by extracellular Mg²⁺.     

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.