Development of a colorimetric test system for detection of point mutations via ligation of a tandem of short oligonucleotides on methacrylate beads

A new approach for detection of point mutations has been developed. The nonradioactive test system proposed is based on enzymatic ligation of a tandem of three short oligonucleotides B∼pN₈+pN₄+pN′₈ Bio in the presence of a complementary DNA template. The 5′-terminal octanucleotide B∼pN₈ is immobilized on polymer methacrylate beads (B) and the 3′-terminal octanucleotide pN′₈ Bio contains a biotin residue at the 3′-phosphate. Ligation of the tandem produces a 20-mer biotinylated oligonucleotide on a polymer bead, which is then visualized via subsequent treatments with streptavidin-alkaline phosphatase conjugate and chromogenic substrates. Intense staining of the polymer beads is observed when the amount of DNA template (20-mer oligonucleotide) is as low as ∼10⁻¹⁴ mol. It is shown that practically no polymer staining is observed when the complex formed by the tandem and the 20-mer DNA template contains a mismatch either in the tetranucleotide duplex or in the duplex of octanucleotide immobilized on the beads. This suggests a possibility of using the presented approach in test systems for detection of point mutations in PCR-amplified DNA fragments.     

A new approach to detect a particular DNA sequence by UV-immobilization of its hybridization complex with a highly specific probe resulting from ligation of a tandem of short oligonucleotides in solution

A new approach was proposed for detecting amplified DNA fragments by hybridization with a highly selective oligonucleotide probe obtained by ligation of a tandem of three short oligonucleotides (pN8 + pN4 + pN8' Bio) in solution, with subsequent UV-immobilization of the hybridization product on a nylon membrane and its colorimetric detection with the streptavidin-alkaline phosphatase technique. Owing to the high selectivity of ligation, the 20-mer ligation product was detected on a membrane only when it was completely complementary to a template fragment. The results showed that any single-nucleotide substitution in the tetramer-binding site can be localized and identified with the use of all 12 possible tetramers.     

Magnesium reduces nickel inhibition of DNA polymerization

The activities of DNA polymerization and DNA ligation in extract of Chinese hamster ovary cells were both stimulated by MgCl₂. DNA polymerization was stimulated by MgCl₂ above 0.25 mM, whereas, MgCl₂ above 2 mM was required to stimulate DNA ligation. The activity of DNA polymerization maintained a plateau at MgCl₂ 1–12 mM, whereas DNA ligation reached a maximal activity at MgCl₂ 6 mM and decreased thereafter. NiCl₂ 0.1-0.2 mM also had a stimulatory effect on DNA polymerization, but was much less potent than MgCl₂. However, nickel ion (Ni²⁺) had no detectable stimulating effect on the activity of DNA ligation. In the presence of MgCl₂, the activities of DNA polymerization and DNA ligation decreased with increasing concentration of NiCl₂. Ni₂₊ inhibition of DNA polymerization was reduced by increasing the concentration of MgCl2, but increasing the concentration of MgCl₂ did not reduce Ni²⁺ inhibition of DNA ligation. Preincubating cell extract with MgCl₂ decreased the Ni²⁺ inhibition of DNA polymerization but not DNA ligation. These results suggest that Ni²⁺ may compete with magnesium ion (Mg²⁺) to reduce DNA polymerization, but this mechanism seems not applicable to Ni²⁺ inhibition of DNA ligation.     

A comparative study of PCR product detection and quantitation by electrochemiluminescence and fluorescence

Amplification and detection of target DNA sequences are made possible in a polymerase chain reaction (PCR) by using a mixture of biotinylated and ruthenium(II) trisbipyridal (Ru(bpy)₃²⁺)-end-labelled primers. In this way, biotin for capture and Ru(bpy)₃²⁺ for detection are directly incorporated into the PCR product obviating subsequent probe hybridization. PCR of a bacterial DNA template from Alteromonas species strain JD6.5 using a cocktail of biotin- and Ru(bpy)₃²⁺-labelled primers amplified a 1 kilobase region. Serial dilution of PCR product followed by magnetic separation with Streptavidin (SA)-coated magnetic beads and an electrochemiluminescence (ECL) assay using the semi-automated QPCR System 5000 demonstrated sensitive (pg range) DNA detection. ECL assay of probe hybridization to a human immunodeficiency virus (HIV) sequence also produced pg level sensitivity. Quantitative DNA determination by ECL assay correlated well with visual detection of DNA in electrophoretic gels. However, DNA detection by ECL assay was 10 to 100 times more sensitive than conventional ethidium bromide staining. The combination of DNA-based magnetic separation with ECL assay provides a very sensitive and rapid method of quantitating DNA which, owing to its rapid and facile nature, may have many applications in the research, environmental monitoring, industrial and clinical fields.     

The Ca-Transport ATPase of Plant Plasma Membrane Catalyzes a nH/Ca Exchange

Microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings accumulate Ca²⁺ upon addition of MgATP. MgATP-dependent Ca²⁺ uptake co-migrates with the plasma membrane H⁺-ATPase on a sucrose gradient. Ca²⁺ uptake is insensitive to oligomycin, inhibited by vanadate (IC₅₀ 40 micromolar) and erythrosin B (IC₅₀ 0.2 micromolar) and displays a pH optimum between pH 6.6 and 6.9. MgATP-dependent Ca²⁺ uptake is insensitive to protonophores. These results indicate that Ca²⁺ transport in these microsomal vesicles is catalyzed by a Mg²⁺-dependent ATPase localized on the plasma membrane. Ca²⁺ strongly reduces ΔpH generation by the plasma membrane H⁺-ATPase and increases MgATP-dependent membrane potential difference (Δψ) generation. These effects of Ca²⁺ on ΔpH and Δψ generation are drastically reduced by micromolar erythrosin B, indicating that they are primarily a consequence of Ca²⁺ uptake into plasma membrane vesicles. The Ca²⁺-induced increase of Δψ is collapsed by permeant anions, which do not affect Ca²⁺-induced decrease of ΔpH generation by the plasma membrane H⁺-ATPase. The rate of decay of MgATP-dependent ΔpH, upon inhibition of the plasma membrane H⁺-ATPase, is accelerated by MgATP-dependent Ca²⁺ uptake, indicating that the decrease of ΔpH generation induced by Ca²⁺ reflects the efflux of H⁺ coupled to Ca²⁺ uptake into plasma membrane vesicles. It is therefore proposed that Ca²⁺ transport at the plasma membrane is mediated by a Mg²⁺-dependent ATPase which catalyzes a nH⁺/Ca²⁺ exchange.     

Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with k_cat ≈ 8 x 10⁻³ s⁻¹ and K_M < 1 nM at 25°C under conditions where T4 DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower k_cat and a K_M ≈ 300 nM. The rate of ligation increased with addition of Mn²⁺, but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5′-phosphorylated dC or dG residue on the 3′ side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA.     

A New Approach to Revealing Point Mutations in DNA Analyzed by Colorimetric Detection

A new approach to detection of point mutations in an amplified DNA was developed. The approach is based on highly selective ligation (T4 DNA ligase) of a tandem of short oligonucleotides one of which contains the biotin group. The ligation product is formed only when the hybridization complex DNA/tandem is formed and the tandem is perfect. The hybridization complex DNA/(biotinylated ligation product) was separated from the biotinylated component of the tandem by UV‐immobilization of the reaction mixture on a nylon membrane. The immobilized hybridization complex was detected colorimetrically by a streptavidin‐alkaline phosphatase cojugate with a chromogenic substrate.     

Electrochemical synthesis of gold nanostructure modified electrode and its development in electrochemical DNA biosensor

In this article, gold nanostructure modified electrodes were achieved by a simple one-step electrodeposition method. The morphologies of modified electrodes could be easily controlled by changing the pH of HAuCl₄ solution. The novel nanoflower-like particles with the nanoplates as the building blocks could be interestingly obtained at pH 5.0. The gold nanoflower modified electrodes were then used for the fabrication of electrochemical DNA biosensor. The DNA biosensor fabrication process was characterized by cyclic voltammetry and electrochemical impedance spectroscopy with the use of ferricyanide as an electrochemical redox indicator. The DNA immobilization and hybridization on gold nanoflower modified electrode was studied with the use of [Ru(NH₃)₆]³⁺ as a hybridization indicator. The electrochemical DNA biosensor shows a good selectivity and sensitivity toward the detection of target DNA. A detection limit of 1 pM toward target DNA could be obtained.     

Relative Reactivity of Ribosyl 2′-OH vs. 3′-OH in Concentrated Aqueous Solutions of Phosphoimidazolide Activated Nucleotides

Phosphoimidazolide activated ribomononucleotides (*pN, see structure) are useful substrates for the non-enzymatic synthesis of oligonucleotides. In the presence of metal ions, aqueous solutions of *pN yield primarily the two internucleotide-linked (pN^2'pN and pN^3'pN) and the pyrophosphate-linked (N^5'ppN) dimers. Small amounts of cyclic dimers and higher oligomers are also produced. In this study the relative reactivity of 2-OH vs. 3-OH was determined from the ratio of the yields of pN^2'pN vs. pN^3'pN. Experiments were performed at 23 °C in the range 7.2 pH 8.4 with substrates that differ in nucleobase (guanosine (G), cytidine (C), uridine (U), and adenosine (A)) and leaving group (imidazole (Im), 2-methylimidazole (2-MeIm) and 2,4-dimethylimidazole (2,4-diMeIm)). Two metal ions (Mg²⁺ or Mn²⁺) were employed as catalysts. The conditions used here, i.e. a substrate concentration in the range 0.1 M to 1.0 M and metal ion concentration in the range 0.05 M to 0.2 M, favor base-stacking interactions. The ratio pN^2'pN: pN^3'pN = 2-5: 3-5 was found independent of nucleobase and typically varied between 2 to 3 indicating that the 2-OH is about 2 to 3 times more reactive than the 3-OH. *pN with Im, compared to 2-MeIm and 2,4-diMeIm leaving group, produce lower yields of internucleotide linked dimers, and a higher pN^2'pN: pN^3'pN ratio. Trends in the data, observed with all three leaving groups, suggest an increase in pN^2'pN: pN^3'pN ratio with decreasing substrate concentration (up to 5.47 with 0.051 M ImpG). The observations are in accord with earlier studies reporting a relative reactivity 2'-5': 3'-5'= 6 to 9 obtained with Im as the leaving group, in dilute nucleotide solutions and under conditions that disfavor stacking. It is speculated that the concentration induced change in the relative reactivity is the result of self-association via base-stacking that enhances selectively the proximity of the 3-OH of one molecule to the reactive P-N bond of an other molecule. The implication of these conclusions for oligomerization/ligation reactions is discussed.     

Dual mechanism of activation of plant plasma membrane Ca2+-ATPase by acidic phospholipids: Evidence for a phospholipid binding site which overlaps the calmodulin-binding site

The effect of phospholipids on the activity of isoform ACA8 of Arabidopsis thaliana plasma membrane (PM) Ca²⁺-ATPase was evaluated in membranes isolated from Saccharomyces cerevisiae strain K616 expressing wild type or mutated ACA8 cDNA. Acidic phospholipids stimulated the basal Ca²⁺-ATPase activity in the following order of efficiency: phosphatidylinositol 4-monophosphate>phosphatidylserine>phosphatidylcholine?phosphatidylethanolamine?0. Acidic phospholipids increased V_max-Ca²⁺ and lowered the value of K_0.5-Ca²⁺ below the value measured in the presence of calmodulin (CaM). In the presence of CaM acidic phospholipids activated ACA8 by further decreasing its K_0.5-Ca²⁺ value. Phosphatidylinositol 4-monophosphate and, with lower efficiency, phosphatidylserine bound peptides reproducing ACA8 N-terminus (aa 1–116). Single point mutation of three residues (A56, R59 and Y62) within the sequence A56-T63 lowered the apparent affinity of ACA8 for phosphatidylinositol 4-monophosphate by two to three fold, indicating that this region contains a binding site for acidic phospholipids. However, the N-deleted mutant Δ74-ACA8 was also activated by acidic phospholipids, indicating that acidic phospholipids activate ACA8 through a complex mechanism, involving interaction with different sites. The striking similarity between the response to acidic phospholipids of ACA8 and animal plasma membrane Ca²⁺-ATPase provides new evidence that type 2B Ca²⁺-ATPases share common regulatory properties independently of structural differences such as the localization of the terminal regulatory region at the N- or C-terminal end of the protein.     

Differential electrophysiologic effects of global and regional ischemia and reperfusion in perfused rat hearts. Effects of Mg2+ concentration

The effects of regional and global ischemia on cellular electrical activity and on arrhythmias induced by reperfusion were studied at different Mg²⁺ concentrations (Mg²⁺ _o, 0, 1.2, and 4.8 mM) in perfused rat hearts. Surface electrograms and transmembrane potentials were recorded during control, 10 min of ischemia (perfusion arrest or coronary ligation), and reperfusion. Increasing Mg²⁺ _o from 0-4.8 mM decreased heart rate, did not alter action potential morphology, and had a strong antiarrhythmic action on reperfusion following coronary ligation. At low and normal Mg²⁺ _o, the incidence of tachyarrhythmias was between 70 and 80%. Global ischemia led to progressive atrioventricular block and the final ventricular beating rate was similar at all Mg²⁺ _o despite unequal initial values. The severity of arrhythmias was similar to that found after regional ischemia in Mg²⁺ _o = 0, but much lower at normal and high Mg²⁺ _o. The resting depolarization induced by coronary ligation decreased as Mg²⁺ _o was raised, but such a relation was not seen during global ischemia where the depolarization was less marked. The action potential duration did not vary with the ventricular rate between 160 and 380 beats per min but increased considerably when sinus rate was markedly slowed (40 to 80 bpm) by raising Mg²⁺ _o to 9.6 mM. Our data show that a high Mg²⁺ _o exerts a strong protection against reperfusion arrhythmias regardless of the type of ischemia. Modulation of the sinus rhythm by Mg²⁺ may contribute to its protective effect by decreasing K⁺ _o accumulation and Na⁺ _i loading during ischemia.     

P2X<Subscript>7</Subscript> Receptor Stimulation of Membrane Internalization in a Thyrocyte Cell Line

Using fluorescent membrane markers, we have previously shown that extracellular ATP stimulates both exocytosis and membrane internalization in the Fisher rat thyroid cell line FRTL. In this study, we examine the actions of ATP using whole-cell recording conditions that favor stimulation of membrane internalization. ATP stimulation of the P2X₇ receptor activated a reversible, Ca²⁺-permeable, cation conductance that slowly increased in size without changes in ion selectivity. ATP also induced a delayed irreversible decrease in cell capacitance (C_m) that was equivalent to an 8% decrease in membrane surface area. Addition of guanosine 5′-0-2-thiodiphosphate to the pipette solution inhibited the ATP-induced decrease in C_m without affecting channel activation. The effects of ATP on membrane conductance were mimicked by 2′,3′-O-(4-benzoylbenzoyl)-ATP, but not by UTP, adenosine, or 2-methylthio-ATP, and were inhibited by pyridoxal phosphate-6-azophenyl-2′4′-disulfonic acid, adenosine 5′-triphosphate-2′3′-dialdehyde, and Cu²⁺. The capacitance decrease persisted in Na⁺-, Ca²⁺- and Cl⁻-free external saline or with Ca²⁺-free pipette solution. It is concluded that ATP activation of the inotropic P2X₇ receptor stimulates membrane internalization by a mechanism that involves intracellular GTP, but does not require internal Ca²⁺ or influx of Na⁺ or Ca²⁺ through the receptor-gated channel.     

Effect of concanavalin A on the activity of membrane-bound and detergent-solubilized Mg2+-ATPase

Plasma membranes were isolated after binding liver and hepatoma cells to polylysine-coated polyacrylamide beads, and the effect of concanavalin A on the membrane-bound Mg²⁺-ATPase and the Mg²⁺-ATPase solubilized by octaethylene glycol monododecyl ether (C₁₂E₈) was studied. In the experiment of membranebound Mg²⁺-ATPase, plasma membranes were pretreated with Concanavalin A and the activity was assayed. Concanavalin A stimulated the activity of both liver and hepatoma enzymes assayed above 20°C. Concanavalin A abolished the negative temperature dependency characteristic of liver plasma membrane Mg²⁺-ATPase. On the other hand, Concanavalin A prevented the rapid inactivation due to storage at ?20°C, which was characteristic of hepatoma plasma membrane Mg²⁺-ATPase. With solubilized Mg²⁺-ATPase from liver plasma membranes, the negative temperature dependency was not observed. Concanavalin A, which was added to the assay medium, stimulated the activity of the enzyme solubilized in C₁₂E₈ at a high ionic strength. However, Concanavalin A failed to show any effect on the enzyme solubilized in C₁₂E₈ at a low ionic strength. With solubilized Mg²⁺-ATPase from hepatoma plasma membranes, Concanavalin A could not prevent the inactivation of the enzyme during incubation at ?20°C.     

Energy coupling in Mn2+ oxidation by a marine bacterium

ATP synthesis couple to Mn²⁺ oxidation was demonstrated with partially or wholly everted membrane vesicles from marine bacterial strain SSW₂₂. The extent of ATP synthesis in these experiments was greater in earlier experiments. Chemiosmosis is the most probable mechanism for energy coupling because 2,4-dinitrophenol at appropriate concentrations stimulated Mn²⁺ oxidation by intact cells, membrane vesicles or extracts of strains SSW₂₂, S₁₃, and marine pseudomonad 16B. Externally added ADP stimulated Mn²⁺ oxidation by everted membrane vesicles of strain SSW₂₂. This stimulation was oxygen-dependent. It is explained on the basis of a chemiosmotic model for energy coupling in Mn²⁺ oxidation.     

Active transport of Ca in membrane vesicles from pea: Evidence for a H/Ca antiport

Two non mitochondrial systems involved in ATP-dependent Ca²⁺ accumulation have been described and characterized in two membrane fractions from pea internodes purified on a metrizamide-sucrose discontinuous gradient. In the lighter membrane fraction an ATP-dependent Ca²⁺ accumulation system, which shows the characteristics of an ATP-dependent H⁺/Ca²⁺ antiport, predominates. This system is inhibited by FCCP and nigericin and stimulated by 50 mM KCl. It is saturated by 0.8–1.0 mM MgSO₄-ATP, strictly requires ATP and is severely inhibited by an excess of free Mg²⁺ or Mn²⁺. A second system of ATP-dependent Ca²⁺ accumulation, recovered mainly in the heavier membrane fraction, is insensitive to FCCP, is saturated by 8–10 mM MgSO₄-ATP, can utilize also ITP or other nucleoside triphosphates although at lower rate than ATP and is only scarcely affected by an excess of free Mg²⁺ or Mn²⁺. This system is interpreted as corresponding to the (Ca²⁺ + Mg²⁺)-ATPase described by Dieter, P. and Marmé, D. ((1980) Planta 150, 1–8).     

Electrochemical measurement of DNA hybridization using nanosilver as label and horseradish peroxidase as enhancer

A simple and sensitive electrochemical DNA biosensor based on in situ DNA amplification with nanosilver as label and horseradish peroxide (HRP) as enhancer has been designed. The thiolated oligomer single-stranded DNA (ssDNA) was initially directly immobilized on a gold electrode, and quartz crystal microbalance (QCM) gave the specific amount of ssDNA adsorption of 6.3 ± 0.1 ng/cm². With a competitive format, hybridization reaction was carried out via immersing the DNA biosensor into a stirred hybridization solution containing different concentrations of the complementary ssDNA and constant concentration of nanosilver-labeled ssDNA, and then further binding with HRP. The adsorbed HRP amount on the probe surface decreased with the increment of the target ssDNA in the sample. The hybridization events were monitored by using differential pulse voltammetry (DPV) with the adsorbed HRP toward the reduction of H₂O₂. The reduction current from the enzyme-generated product was related to the number of target ssDNA molecules in the sample. A detection of 15 pmol/L for target ssDNA was obtained with the electrochemical DNA biosensor. Additionally, the developed approach can effectively discriminate complementary from non-complementary DNA sequence, suggesting that the similar enzyme-labeled DNA assay method hold great promises for sensitive electrochemical biosensor applications.     

Enantioselective cleavage of supercoiled plasmid DNA catalyzed by chiral macrocyclic lanthanide(III) complexes

The enantiomers of the Sm (III), Eu (III) and Yb (III) complexes [LnL(NO₃)₂](NO₃) of a chiral hexaazamacrocycle were tested as catalysts for the hydrolytic cleavage of supercoiled plasmid DNA. The catalytic activity was remarkably enantioselective; while the [LnL^SSSS(NO₃)₂](NO₃) enantiomers promoted the cleavage of plasmid pBR322 from the supercoiled form (SC) to the nicked form (NC), the [LnL^RRRR(NO₃)₂](NO₃) enantiomers were inactive. Kinetics of plasmid DNA hydrolysis was also investigated by agarose electrophoresis and it indicated typical single-exponential cleavage reaction. The hydrolytic mechanism of DNA cleavage was confirmed by the successful ligation of hydrolysis product by T4 ligase. The NMR study of the solutions of the complexes in various buffers indicated that the complexes exist as monomeric cationic complexes [LnL(H₂O)₃]^3 + in slightly acidic solutions and as dimeric cationic complexes [Ln₂L₂(μ-OH)₂(H₂O)₂]^4 + in slightly basic 8 mM solutions, with the latter form being a possible catalyst for hydrolysis of phosphodiester bonds.     

Plasma Membrane Ca2+-ATPase Isoforms Composition Regulates Cellular pH Homeostasis in Differentiating PC12 Cells in a Manner Dependent on Cytosolic Ca2+ Elevations

Plasma membrane Ca²⁺-ATPase (PMCA) by extruding Ca²⁺ outside the cell, actively participates in the regulation of intracellular Ca²⁺ concentration. Acting as Ca²⁺/H⁺ counter-transporter, PMCA transports large quantities of protons which may affect organellar pH homeostasis. PMCA exists in four isoforms (PMCA1-4) but only PMCA2 and PMCA3, due to their unique localization and features, perform more specialized function. Using differentiated PC12 cells we assessed the role of PMCA2 and PMCA3 in the regulation of intracellular pH in steady-state conditions and during Ca²⁺ overload evoked by 59 mM KCl. We observed that manipulation in PMCA expression elevated pH_mito and pH_cyto but only in PMCA2-downregulated cells higher mitochondrial pH gradient (ΔpH) was found in steady-state conditions. Our data also demonstrated that PMCA2 or PMCA3 knock-down delayed Ca²⁺ clearance and partially attenuated cellular acidification during KCl-stimulated Ca²⁺ influx. Because SERCA and NCX modulated cellular pH response in neglectable manner, and all conditions used to inhibit PMCA prevented KCl-induced pH drop, we considered PMCA2 and PMCA3 as mainly responsible for transport of protons to intracellular milieu. In steady-state conditions, higher TMRE uptake in PMCA2-knockdown line was driven by plasma membrane potential (Ψp). Nonetheless, mitochondrial membrane potential (Ψm) in this line was dissipated during Ca²⁺ overload. Cyclosporin and bongkrekic acid prevented Ψ_m loss suggesting the involvement of Ca²⁺-driven opening of mitochondrial permeability transition pore as putative underlying mechanism. The findings presented here demonstrate a crucial role of PMCA2 and PMCA3 in regulation of cellular pH and indicate PMCA membrane composition important for preservation of electrochemical gradient.     

Solubilization and reconstitution of ca pump from corn leaf plasma membrane

The Ca²⁺ transport system of corn (Zea mays) leaf plasma membrane is composed of Ca²⁺ pump and Ca²⁺/H⁺ antiporter driven by H⁺ gradient imposed by a H⁺ pump (M Kasai, S Muto [1990] J Membr Biol 114: 133-142). It is necessary for characterization of these Ca²⁺ transporters to establish the procedure for their solubilization, isolation, and reconstitution into liposomes. We attempted to solubilize and reconstitute the Ca²⁺ pump in the present study. A nonionic detergent octaethyleneglycol monododecyl ether (C₁₂E₈) was the most effective detergent for a series of extraction and functional reconstitution of the Ca²⁺ pump among seven detergents examined. This was judged from activities of ATP-dependent ⁴⁵Ca²⁺ uptake into liposomes reconstituted with the respective detergent-extract of the plasma membrane by the detergent dilution method. C₁₂E₈-extract of the plasma membrane was subjected to high performance liquid chromatography using a DEAE anion exchange column. Ca²⁺-ATPase was separated from VO₄³⁻-sensitive Mg²⁺-ATPase. These ATPases were separately reconstituted into liposomes, and their ATP-dependent Ca²⁺ uptake was measured. The liposomes reconstituted with the Ca²⁺-ATPase, but not with the VO₄³⁻-sensitive Mg²⁺-ATPase, showed ATP-dependent Ca²⁺ uptake. Nigericin-induced pH gradient (acid inside) caused only a little Ca²⁺ uptake into liposomes reconstituted with the Ca²⁺-ATPase, suggesting that the Ca²⁺/H⁺ antiporter was not present in the preparation. These results indicate that the Ca²⁺-ATPase actually functions as Ca²⁺ pump in the corn leaf plasma membrane.     

Pharmacological dose of melatonin reduces cytosolic calcium load in response to cholecystokinin in mouse pancreatic acinar cells

Intracellular Ca²⁺ overload has been considered a common pathological precursor of pancreatic injury. In this study, the effects of melatonin on Ca²⁺ mobilization induced by cholecystokinin octapeptide (CCK-8) in freshly isolated mouse pancreatic acinar cells have been examined. Changes in intracellular free Ca²⁺ concentration were followed by single cell fluorimetry. For this purpose, cells were loaded with the Ca²⁺-sensitive fluorescent dye fura-2-acetoxymethyl ester. In order to evaluate the contribution of Ca²⁺ transport at the plasma membrane, at the endoplasmic reticulum (ER) or at the mitochondria, cells were incubated with CCK-8 alone or in combination with LaCl₃, thapsigargin (Tps), or FCCP to, respectively, uncouple Ca²⁺ transport at these localizations. The experiments were performed in the absence or in the presence of melatonin in combination with the stimuli mentioned. Our results show that the total Ca²⁺ mobilization evoked by CCK-8 was attenuated by a 30 % in the presence of 100 µM melatonin compared with the responses induced by CCK-8 alone. Upon inhibition of Ca²⁺ transport into the ER by Tps, Ca²⁺ mobilization was also reduced in the presence of melatonin. In the presence of LaCl₃ plus melatonin, the total Ca²⁺ mobilization induced by CCK-8 was significantly decreased, compared with the response obtained without melatonin but in the presence of LaCl₃. No major differences were found when the cells were incubated with CCK-8 or Tps alone or in combination with LaCl₃ plus melatonin and FCCP, compared with the responses obtained in the absence of FCCP. The initial Ca²⁺ release from intracellular stores evoked by CCK-8 or Tps was not significantly reduced in the presence of melatonin. The effect of melatonin could be explained on the basis of a stimulated Ca²⁺ transport out of the cell through the plasma membrane and by a stimulation of Ca²⁺ reuptake into the ER. Accumulation of Ca²⁺ into mitochondria might not be a major mechanism stimulated by melatonin. We conclude that melatonin alleviates intracellular Ca²⁺ accumulation, a situation potentially leading to cell damage in the exocrine pancreas.