Functional Approach to the Catalytic Site of the Sarcoplasmic Reticulum Ca2+-ATPase: Binding and Hydrolysis of ATP in the Absence of Ca2+

Isolated sarcoplasmic reticulum vesicles in the presence of Mg(2+) and absence of Ca(2+) retain significant ATP hydrolytic activity that can be attributed to the Ca(2+)-ATPase protein. At neutral pH and the presence of 5 mM Mg(2+), the dependence of the hydrolysis rate on a linear ATP concentration scale can be fitted by a single hyperbolic function. MgATP hydrolysis is inhibited by either free Mg(2+) or free ATP. The rate of ATP hydrolysis is not perturbed by vanadate, whereas the rate of p-nitrophenyl phosphate hydrolysis is not altered by a nonhydrolyzable ATP analog. ATP binding affinity at neutral pH and in a Ca(2+)-free medium is increased by Mg(2+) but decreased by vanadate when Mg(2+) is present. It is suggested that MgATP hydrolysis in the absence of Ca(2+) requires some optimal adjustment of the enzyme cytoplasmic domains. The Ca(2+)-independent activity is operative at basal levels of cytoplasmic Ca(2+) or when the Ca(2+) binding transition is impeded.     

Light-induced increase in free Mg2+ concentration in spinach chloroplasts: measurement of free Mg2+ by using a fluorescent probe and necessity of stromal alkalinization

Free Mg(2+) in chloroplasts may contribute to the regulation of photosynthetic enzymes, but adequate methodology for the determination of free Mg(2+) concentration ([Mg(2+)]) in chloroplasts has been lacking. We measured internal chloroplast [Mg(2+)] by using a Mg-sensitive fluorescent indicator, mag-fura-2. In intact, dark-kept spinach chloroplasts, internal [Mg(2+)] was estimated to be 0.50 mM, and illumination caused an increase in [Mg(2+)] to 2.0mM in the stroma. The light-induced increase in [Mg(2+)] was inhibited by a blocker of driven electron transport and uncouplers. The K(+)-specific ionophore valinomycin inhibited the [Mg(2+)] increase in the absence of external K(+), and addition of KCl restored the [Mg(2+)] increase. NH(4)Cl, which induces stromal alkalinization, enhanced the [Mg(2+)] increase. A Ca(2+)-channel blocker, ruthenium red, inhibited the [Mg(2+)] increase, but LaCl(3) had no effect. These results indicate that stromal alkalinization is essential for light-induced increase in [Mg(2+)]. This system for measuring internal chloroplast [Mg(2+)] might provide a suitable system for assay of Mg(2+) transport activity of chloroplast membranes.     

Interference of methyl trachyloban-19-oate ester with CF(0) of spinach chloroplast H(+)-ATPase

In isolated spinach chloroplasts, low concentrations (I(50)=14 microM) of methyl trachyloban-19-oate ester inhibited ATP synthesis and coupled electron transport as well as light-activated membrane-bound Mg(2+)-ATPase activity. Basal (-Pi) and uncoupled electron transport and heat-activated Ca(2+)-dependent ATPase activity of isolated coupling factor proteins were unaffected by methyl trachyloban-19-oate. Thylakoids partially stripped of coupled factor by EDTA were unable to accumulate protons in the light. However, increasing concentrations of methyl trachyloban-19-oate ester restored this ability. It is concluded that the methyl trachyloban-19-oate ester effects result from blocking proton transport through the CF(0) channel. Methyl trachyloban-19-oate ester exhibited non-competitive kinetics with DCCD and triphenyltin. These results suggest that the natural products, DCCD and triphenyltin, access inhibition sites in CF(0). The K(i) is 75 microM.     

Effect of nano-TiO2 on photochemical reaction of chloroplasts of spinach

The effects of nano-TiO₂ (rutile) on the photochemical reaction of chloroplasts of spinach were studied. The results showed that when spinach was treated with 0.25% nano-TiO₂, the Hill reaction, such as the reduction rate of FeCy, and the rate of evolution oxygen of chloroplasts was accelerated and noncyclic photophosphorylation (nc-PSP) activity of chloroplasts was higher than cyclic photophosphorylation (c-PSP) activity, the chloroplast coupling was improved and activities of Mg²⁺-ATPase and chloroplast coupling factor I (CF₁)-ATPase on the thylakoid membranes were obviously activated. It suggested that photosynthesis promoted by nano-TiO₂ might be related to activation of photochemical reaction of chloroplasts of spinach.     

Functional implication with the metal-binding properties of KChIP1

To investigate the metal-binding properties of KChIP1, the interaction of KChIP1 and mutated KChIP1 with divalent cations (Mg(2+), Ca(2+), Sr(2+), and Ba(2+)) was explored by 8-anilinonaphthalene-1-sulfonate (ANS) fluorescence. It showed that KChIP1 possessed two types of Ca(2+)-binding sites, high-affinity and low-affinity Ca(2+)-binding sites. However, only low-affinity-binding site for Mg(2+), Sr(2+), and Ba(2+) was observed. The metal-binding properties of KChIP1 are not appreciably affected after removal of the N-terminal portion and EF-hand 1. Deleting the EF-hand 4 of KChIP1 abolishes its high-affinity Ca(2+)-binding site, but retains the intact low-affinity-binding site for metal ions. A decrease in the nonpolarity of ANS-binding site occurs with all mutants. However, the binding of ANS with KChIP1 is no longer observed after removal of EF-hands 3 and 4. Intermolecular interaction assessed by chemical cross-linking suggested that KChIP1 had a propensity to form dimer in the absence of metal ions, and a KChIP1 tetramer was pronouncedly produced in the presence of metal ions. Noticeably, the oligomerization state depends on the integrity of EF-hand 4. Taken together, our data suggest that EF-hand 4 is of structural importance as well as functional importance for fulfilling the physiological function of KChIP1.     

Hormone-stimulated Mg(2+) accumulation into rat hepatocytes: a pathway for rapid Mg(2+) and Ca(2+) redistribution

Many diseases such as cardiac arrhythmia, diabetes, and chronic alcoholism are associated with a marked decrease of plasma and parenchymal Mg(2+), and Mg(2+) administration is routinely used therapeutically. This study uses isolated rat hepatocytes to ascertain if and under which conditions increases in extracellular Mg(2+) result in an increase in intracellular Mg(2+). In the absence of stimulation, changing extracellular Mg(2+) had no effect on total cellular Mg(2+) content. By contrast, carbachol or vasopressin administration promoted an accumulation of Mg(2+) that increased cellular Mg(2+) content by 13.2 and 11.8%, respectively, and stimulated Mg(2+) uptake was unaffected by the absence of extracellular Ca(2+). Mg(2+) efflux resulting from stimulation of alpha- or beta-adrenergic receptors operated with a Mg(2+):Ca(2+) exchange ratio of 1. These data indicate that cellular Mg(2+) uptake can occur rapidly and in large amounts, through a process distinct from Mg(2+) release, but operating only upon specific hormonal stimulation.     

Effect of Cd(2+) and Hg(2+) on the activity of Na(+)/K(+)-ATPase and Mg(2+)-ATPase adsorbed on polystyrene microtiter plates.

In the present study a polystyrene microtiter plate was tested as a support material for synaptic plasma membrane (SPM) immobilization by adsorption. The adsorption was carried out by an 18-h incubation at +4 degrees C of SPM with a polystyrene matrix, at pH 7.4. Evaluation of the efficiency of the applied immobilization method revealed that 10% protein fraction of initially applied SPM was bound to the support and that two SPM enzymes, Na(+)/K(+)-ATPase and Mg(2+)-ATPase, retained 70-80% activity after the adsorption. In addition, adsorption stabilizes Na(+)/K(+)-ATPase and Mg(2+)-ATPase, since the activities are substantial 3 weeks after the adsorption. Parallel kinetic analysis showed that adsorption does not alter significantly the kinetic properties of Na(+)/K(+)-ATPase and Mg(2+)-ATPase and their sensitivity to and mechanism of Cd(2+)- or Hg(2+)-induced inhibition. The only exception is the "high affinity" Mg(2+)-ATPase moiety, whose affinity for ATP and sensitivity toward Cd(2+) were increased by the adsorption. The results show that such system may be used as a practical and comfortable model for the in vitro toxicological investigations.     

Effect of Y-24180 on Ca2+ movement and proliferation in renal tubular cells

In canine renal tubular cells, the effect of Y-24180, a presumed specific platelet activating factor (PAF) receptor antagonist, on intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by using fura-2 as a Ca(2+)-sensitive fluorescent probe. Y-24180 (0.1-10 microM) caused a rapid and sustained [Ca(2+)](i) rise in a concentration-dependent manner. The [Ca(2+)](i) rise was prevented by 30% by removal of extracellular Ca(2+), but was not changed by dihydropyridines, verapamil and diltiazem. Y-24180-induced Ca(2+) influx was confirmed by Mn(2+)-influx induced quench of fura-2 fluorescence. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the increasing effect of 5 microM Y-24180 on [Ca(2+)](i) was abolished; conversely, depletion of Ca(2+) stores with 5 microM Y-24180 abolished thapsigargin-induced [Ca(2+)](i) rise. U73122, an inhibitor of phoispholipase C, inhibited ATP-, but not Y-24180-induced [Ca(2+)](i) rise. Overnight treatment with Y-24180 did not alter cell proliferation rate. Collectively, these results suggest that Y-24180 acts as a potent, but not cytotoxic, Ca(2+) mobilizer in renal tubular cells, by stimulating both extracellular Ca(2+) influx and intracellular Ca(2+) release. Since alterations in Ca(2+) movement may interfere many cellular signaling processes unrelated to modulation of PAF receptors, caution must be applied in using this chemical as a selective PAF receptor antagonist.     

Effects of Ce3+, Cd2+, and Hg2+ on activities and secondary structure of trypsin

The different effects of Ce³⁺, Cd²⁺, and Hg²⁺ on the activities and secondary structure of trypsin were studied. The results showed that trypsin activity was increased substantially by Ce³⁺ in 0.5–5 μmol/L concentration, but the activity was decreased significantly by Cd²⁺ or Hg²⁺ in 0.5–5 μmol/L concentration. The ultraviolet-visible spectrum of trypsin with 4 μmol/L Ce³⁺ treatment was the same as that of the control, but the 232-nm characteristic peak of trypsin with 4 μmol/L Cd²⁺ or Hg²⁺ treatment was blue-shifted and the peak intensity weakened. The circular dichroism (CD) spectrum of trypsin with 4 μmol/L Ce³⁺ treatment was similar to that of the control. The secondary structure of trypsin did not change with Ce³⁺ treatment. However, the CD spectrum of trypsin with 4 μmol/L Cd²⁺ or Hg²⁺ treatment was different from that of the control and Ce³⁺ treatment. The secondary structure of trypsin with Cd²⁺ or Hg²⁺ treatment changed greatly; for example, the α-helix and β-sheet contents were reduced significantly, the β-turn was enhanced greatly, and the random coil contents increased or decreased.     

Lack of nitric oxide synthase depresses ion transporting enzyme function in cardiac muscle

Nitric oxide (NO*) is produced endogenously from NOS isoforms bound to sarcolemmal (SL) and sarcoplasmic reticulum (SR) membranes. To investigate whether locally generated NO* directly affects the activity of enzymes mediating ion active transport, we studied whether knockout of selected NOS isoforms would affect the functions of cardiac SL (Na+ + K+)-ATPase and SR Ca2+-ATPase. Cardiac SL and SR vesicles containing either SL (Na+ + K+)-ATPase or SR Ca2+-ATPase were isolated from mice lacking either nNOS or eNOS, or both, and tested for enzyme activities. Western blot analysis revealed that absence of single or double NOS isoforms did not interrupt the protein expression of SL (Na+ + K+)-ATPase and SR Ca2+-ATPase in cardiac muscle cells. However, lack of NOS isoforms in cardiac muscle significantly altered both (Na+ + K+)-ATPase activity and SR Ca2+-ATPase function. Our experimental results suggest that disrupted endogenous NO* production may change local redox conditions and lead to an unbalanced free radical homeostasis in cardiac muscle cells which, in turn, may affect key enzyme activities and membrane ion active transport systems in the heart.     

Damage to the chloroplast H+-ATPase complex by low concentrations of glutaraldehyde

The energy-dependent processes coupled to electron transport were studied in isolated pea chloroplasts treated with low concentrations (1-5 mM) of glutaraldehyde (GA) in the dark and in the light sufficient to cause energization of the membrane. After GA treatment the chloroplasts exhibited a strong suppression of cyclic and non-cyclic phosphorylation, coupled (+ADP+Pi) electron transport and diminution of the light-activated Mg2+-ATPase activity. The rate of basal electron transport was unaffected. The GA-treated chloroplasts were found to retain the capacity to form the osmotic component of the transmembrane potential. These data and the results of the effect of florizine and ATP on electron transport suggest that the effect of GA on energy transduction processes associated with photophosphorylation may consist in its action on reversible H+-ATPase. In light-adapted samples treated with GA the data characterizing the formation of a high energy state (rate of photophosphorylation, steady-state level of photo-induced quenching of atebrin fluorescence and its dark recovery; photo-induced absorbance changes at 520 nm; rate of the slow phase of delayed fluorescence increment) appear to be changed to a greater extent as compared to the dark-adapted samples. The observed changes may arise from a greater conductivity of thylakoid membranes due to fixation of the H+-ATPase proton channel in the "open" state.     

Ca(2+) homeostasis, Ca(2+) signalling and somatodendritic vasopressin release in adult rat supraoptic nucleus neurones

Multiple mechanisms that maintain Ca(2+) homeostasis and provide for Ca(2+) signalling operate in the somatas and neurohypophysial nerve terminals of supraoptic nucleus (SON) neurones. Here, we examined the Ca(2+) clearance mechanisms of SON neurones from adult rats by monitoring the effects of the selective inhibition of different Ca(2+) homeostatic molecules on cytosolic Ca(2+) ([Ca(2+)](i)) transients in isolated SON neurones. In addition, we measured somatodendritic vasopressin (AVP) release from intact SON tissue in an attempt to correlate it with [Ca(2+)](i) dynamics. When bathing the cells in a Na(+)-free extracellular solution, thapsigargin, cyclopiazonic acid (CPA), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and the inhibitor of plasma membrane Ca(2+)-ATPase (PMCA), La(3+), all significantly slowed down the recovery of depolarisation (50 mM KCl)-induced [Ca(2+)](i) transients. The release of AVP was stimulated by 50 mM KCl, and the decline in the peptide release was slowed by Ca(2+) transport inhibitors. In contrast to previous reports, our results show that in the fully mature adult rats: (i) all four Ca(2+) homeostatic pathways, the Na(+)/Ca(2+) exchanger, the endoplasmic reticulum Ca(2+) pump, the plasmalemmal Ca(2+) pump and mitochondria, are complementary in actively clearing Ca(2+) from SON neurones; (ii) somatodendritic AVP release closely correlates with intracellular [Ca(2+)](i) dynamics; (iii) there is (are) Ca(2+) clearance mechanism(s) distinct from the four outlined above; and (iv) Ca(2+) homeostatic systems in the somatas of SON neurones differ from those expressed in their terminals.     

Conformation and activity of chloroplast coupling factor exposed to low chemical potential of water in cells.

(1) Photophosphorylation, Ca2+-ATPase and Mg2+-ATPase activities of isolated chloroplasts were inhibited 55--65% when the chemical potential of water was decreased by dehydrating leaves to water potentials (psi w) of --25 bars before isolation of the plastids. The inhibition could be reversed in vivo by rehydrating the leaves. (2) These losses in activity were reflected in coupling factor (CF1) isolated from the leaves, since CF1 from leaves with low psi w had less Ca2+-ATPase activity than control CF1 and did not recouple phosphorylation in CF1-deficient chloroplasts. In contrast, CF1 from leaves having high psi w only partially recoupled phosphorylation by CF1-deficient chloroplasts from leaves havig low psi w. This indicated that low psi w affected chloroplast membranes as well as CF1 itself. (3) Coupling factor from leaves having low psi w had the same number of subunits, and the same electrophoretic mobility, and could be obtained with the same yields as CF1 from control leaves. However, direct measurements of fluorescence polarization, ultraviolet absorption, and circular dichroism showed that CF1 from leaves having low psi w differed from control CF1. The CF1 from leaves having low psi w also had decreased ability to bind fluorescent nucleotides (epsilon-ATP and epsilon-ADP). (4) Exposure of isolated CF1 to low psi w in vitro by preincubation in sucrose-containing media inhibited the Ca2+-ATPase activity of the protein in subsequent assays without sucrose. Inclusion of 5 or 10 mM Mg2+ in the preincubation medium markedly inhibited Ca2+-ATPase activity. (5) These results show that CF1 undergoes changes in cells which alter its phosphorylating ability. Since low cell psi w changed the spectroscopic properties but not other protein properties of CF1, the changes were most likely caused by altered confurn, photophosphorylation. The inhibition of ATPase activity in CF1 in vitro at low psi w and high ion concentration mimicked the change in activity seen in vivo.     

Effects of Nanoanatase TiO2 on Photosynthesis of Spinach Chloroplasts Under Different Light Illumination

With a photocatalyzed characteristic, nanoanatase TiO₂ under light could cause an oxidation–reduction reaction. Our studies had proved that nano-TiO₂ could promote photosynthesis and greatly improve spinach growth. However, the mechanism of nano-TiO₂ on promoting conversion from light energy to electron energy and from electron energy to active chemistry energy remains largely unclear. In this study, we report that the electron transfer, oxygen evolution, and photophosphorylation of chloroplast (Chl) from nanoanatase-TiO₂-treated spinach were greatly increased under visible light and ultraviolet light illumination. It was demonstrated that nanoanatase TiO₂ could greatly improve whole chain electron transport, photoreduction activity of photosystem II, O₂-evolving and photophosphorylation activity of spinach Chl not only under visible light, but also energy-enriched electron from nanoanatase TiO₂, which entered Chl under ultraviolet light and was transferred in photosynthetic electron transport chain and made NADP⁺ be reduced into NADPH, and coupled to photophosphorylation and made electron energy be transformed to ATP. Moreover, nanoanatase h⁺, which photogenerated electron holes, captured an electron from water, which accelerated water photolysis and O₂ evolution.     

Maize tonoplast PP(i)-dependent H(+)/Ca(2+) exchange: two K(s) for Ca(2+) and inhibition by thapsigargin

Maize root tonoplasts are able to accumulate Ca(2+) using the energy derived from the H(+) gradient formed during PP(i) hydrolysis. Oxalate increases 6- to 10-fold the amount of Ca(2+) accumulated by tonoplast. Two apparently different K(s) values for Ca(2+) with values of 0.36 and 4.70 microM were detected when oxalate was included in the medium and the free Ca(2+) concentration in the medium was buffered with the use of EGTA. Binding of Ca(2+) to the outer surface of tonoplasts inhibits the outflow of Ca(2+) previously accumulated by the tonoplast, half-maximal inhibition being observed in presence of 1 microM Ca(2+). Thapsigargin, a specific inhibitor of Ca(2+)-ATPase, inhibits the Ca(2+) uptake driven by H(+) gradient but does not inhibit the hydrolysis of PP(i) nor the formation of a H(+) gradient.     

Dibutyryl-Cyclic GMP Stimulation of Ca2+-ATPase Activity in Rat Brain Synaptic Membranes

The effects of dibutyryl cyclic AMP (db-cAMP) and dibutyryl cyclic GMP (db-cGMP) were tested on Ca2+-ATPase, Mg2+-ATPase, and (Ca2+ + Mg2+)-ATPase activities in lysed synaptosomes prepared from whole rat brains (minus cerebellum). At concentrations from 0.1 to 2.0 mM, db-cGMP produced a selective, concentration-dependent increase in Ca2+-ATPase activity. Both db-cGMP and db-cAMP slightly reduced Mg2+-ATPase activity, whereas neither compound had concentration-dependent effects on (Ca2+ + Mg2+)-ATPase activity. These findings suggest that the Mg2+-independent, Ca2+-ATPase activity in rat brain is regulated by a cyclic GMP-dependent process. Further, the data provide evidence that the Ca2+-ATPase activity in lysed synaptosomal membranes represents an enzyme that is distinguishable from both the Mg2+ -and (Ca2+ + Mg2+)-ATPase.     

Effects of Lead on Activities of Photochemical Reaction and Key Enzymes of Carbon Assimilation in Spinach Chloroplast

Photosynthesis is one of the most sensitive processes to lead, but the effects of lead on the transformation of light energy of plants are still not clearly understood. In the present paper, spinach was cultivated in the experimental fields and was sprayed with various concentrations of PbCl2 solution. We investigated the effects of lead on the activities of photochemical reaction and the key enzymes of carbon assimilation in spinach chloroplast. The results showed that Pb2+ treatment could significantly inhibit the Hill reaction activity of spinach chloroplast and photophosphorylation, and it had a more conspicuous effect on cyclic photophosphorylation than non-cyclic photophosphorylation. The activities of ATPase on the thylakoid membrane were severely inhibited under Pb2+-treated condition, and Ca2+ ATPase activity was affected more obviously than Mg2+ ATPase activity. Meanwhile, the activities of the key enzymes of carbon assimilation were also significantly reduced by Pb2+, especially Rubisco activase. The reduction of dry weight of spinach caused by Pb2+ was more significant than that of fresh weight. It implied that Pb2+ could disturb light energy transformation of chloroplast.     

Phospholemman expression is high in the newborn rabbit heart and declines with postnatal maturation

Phospholemman (PLM) is a small sarcolemmal protein that modulates the activities of Na(+)/K(+)-ATPase and the Na(+)/Ca(2+) exchanger (NCX), thus contributing to the maintenance of intracellular Na(+) and Ca(2+) homeostasis. We characterized the expression and subcellular localization of PLM, NCX, and the Na(+)/K(+)-ATPase alpha1-subunit during perinatal development. Western blotting demonstrates that PLM (15kDa), NCX (120kDa), and Na(+)/K(+)-ATPase alpha-1 (approximately 100kDa) proteins are all more than 2-fold higher in ventricular membrane fractions from newborn rabbit hearts (1-4-day old) compared to adult hearts. Our immunocytochemistry data demonstrate that PLM, NCX, and Na(+)/K(+)-ATPase are all expressed at the sarcolemma of newborn ventricular myocytes. Taken together, our data indicate that PLM, NCX, and Na(+)/K(+)-ATPase alpha-1 proteins have similar developmental expression patterns in rabbit ventricular myocardium. Thus, PLM may have an important regulatory role in maintaining cardiac Na(+) and Ca(2+) homeostasis during perinatal maturation.     

Defective Mg2+ regulation of RyR1 as a causal factor in malignant hyperthermia

In skeletal muscle, Mg(2+) exerts a dual inhibitory effect on RyR1, by competing with Ca(2+) at the activation site and binding to a low affinity Ca(2+)/Mg(2+) inhibitory site. Pharmacological activators of RyR1 must overcome the inhibitory action of Mg(2+) before Ca(2+) efflux can occur. In normal muscle, where the free [Mg(2+)](i) is approximately 1mM, even prolonged exposure to millimolar levels of volatile anesthetics does not initiate SR Ca(2+) release. However, when the cytosolic [Mg(2+)] is reduced below the physiological range, low levels of volatile anesthetic within the clinically relevant range (1mM) can initiate SR Ca(2+) release, in the form of a propagating Ca(2+) wave. In human muscle fibers from malignant hyperthermia susceptible patients, such Ca(2+) waves occur when 1mM halothane is applied at physiological [Mg(2+)](i). There is increasing evidence to suggest that defective Mg(2+) regulation of RyR1 confers susceptibility to malignant hyperthermia. At the molecular level, interactions between critical RyR1 subdomains may explain the clustering of RyR1 mutations and associated effects on Mg(2+) regulation.