Optimal KCl Concentration Research Articles

Effect of Potassium Chloride as a Supporting Electrolyte on the Dispersion Towards the Fabrication of Films by Electrophoretic Deposition of Bi2Sr2CaCu2 O 8 in Ethanol

Bi2Sr2CaCu2O8 (Bi-2212) powder was fabricated using solid state reaction method. The suspensions were prepared containing finely ground Bi-2212 powder with and without potassium chloride (KCl) addition. Settling experiments and measurements of zeta potential, particle size distribution, and electrophoretic mobility were performed to determine the optimal concentration of the supporting electrolyte. Bi-2212 suspensions were used in electrophoretic deposition (EPD), and the films were heat treated. The results showed that the optimal concentration of KCl is 5.21 wt %. The zeta potential at this concentration is highest at about 31.10 mV, the settling velocity is lowest ∼3.84 mm/min, and the particle size is smallest (∼450 nm), which indicate that the dispersion is better due to the reduction of intermolecular attraction. The effects of KCl as a flux were observed in the microstructure and superconducting properties of Bi-2212 film. The smoothest surface morphology, high c-axis orientation, and highest critical temperature (Tc)-onset of 84.73 K were obtained from the film prepared with optimized Bi-2212 suspension.

Hyoscyamine production in hairy roots of three Datura species exposed to high-salt medium

Several species of the genus Datura have been cultivated for their hyoscyamine content. Hyoscyamine production in field-cultivated plants can often be limited by environmental conditions. The culture of hairy roots obtained by inoculation of Datura explants with the A4 strain of Agrobacterium rhizogenes offers promising prospects for the in vitro production of hyoscyamine. The objectives of this study were to select high-quality hairy root lines and then optimize hyoscyamine production by applying a salt stress. Potassium chloride (KCl) and calcium chloride (CaCl2) were added at various elicitation times, and both had significant effects on hyoscyamine biosynthesis. The optimal concentration of KCl was 2 g L−1, combined with a contact time of 10 h for the selected Datura tatula line (LDT) and 24 h for the selected Datura stramonium (LDS) and Datura innoxia (LDI) lines. For CaCl2, the optimum concentration was 2 g L−1 for LDS and LDT, with respective elicitation times of 10 and 24 h. For LDI, the best result was obtained with a CaCl2 concentration of 1 g L−1 and an elicitation time of 24 h. The highest hyoscyamine levels obtained for LDS, LDT, and LDI were, respectively, 2.32-, 1.99-, and 1.85-fold the control levels with KCl elicitation and 2.08-, 2.07-, and 1.85-fold the control levels with CaCl2 elicitation. The line resulting from D. tatula elicited with 2 g L−1 CaCl2 for 24 h produced the most hyoscyamine content (16.978 mg g−1 DW), followed by LDI and then LDS.

Characterization of a family B DNA polymerase from the hyperthermophilic crenarchaeon Ignicoccus hospitalis KIN4/I and its application to PCR.

A family B DNA polymerase gene from the hyperthermophilic crenarchaeon Ignicoccus hospitalis KIN4/I was highly expressed under the control of T7lac promoter of pET-28ARG in Escherichia coli BL21-CodonPlus(DE3)-RIL cells. The produced I. hospitalis (Iho) DNA polymerase was purified by heat treatment followed by HisTrap™ HP column and HiTrap™ SP column chromatographies. The molecular mass of the purified Iho DNA polymerase was 88 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH for Iho DNA polymerase activity was 7.0 and the optimal temperature was 70 °C. Iho DNA polymerase was strongly activated by the presence of magnesium ion at an optimum concentration of 3 mM. The optimal concentration of KCl for Iho DNA polymerase activity was 60 mM. The half-life of the enzyme at 94 °C was about 2 h. The optimal conditions for polymerase chain reaction (PCR) were determined. Iho DNA polymerase possesses 3'→5' exonuclease activity, and the fidelity of the Iho DNA polymerase was similar to that of Pfu and Vent DNA polymerases. However, Iho DNA polymerase provided more enhanced efficiency of PCR amplification than Pfu and Vent DNA polymerases. Iho DNA polymerase could successfully amplify a 2-kb λ DNA target with a 10-s extension time and could amplify a DNA fragment up to 8 kb λ DNA.

Ionic effects on intrinsic gill muscles in the freshwater bivalve, Dreissena polymorpha

The intrinsic gill muscles in Dreissena polymorpha are arranged to affect interfilament distance and ostial dimension. This orientation suggests a role in the general control of gill function including the regulation of water flow. Muscle contraction in the gill results in a decrease in overall surface area and we used this change in dimension as an indirect measure of muscle contraction. The Ringer’s solution bathing the gill was experimentally manipulated to assess the effects of ionic composition on muscle activity elicited by acetylcholine application. Eliminating CaCl 2 in the Ringer’s and adding 1 mM EGTA, or replacing the CaCl 2 with CoCl 2 or MnCl 2 abolished the contractile response. A proper balance between NaCl and KCl was also critical for the maintenance of muscle response. The optimal KCl concentration was between 1 and 2 mM and became most important at higher NaCl concentrations. Acclimation to hyperosmotic conditions was dependent in part on the ouabain-sensitive activity of a Na +/K +ATPase. Overall, these muscles respond to their ionic environment as one might expect from typical molluscan smooth muscles. An alteration of muscle function experienced during changing environmental conditions may limit the distribution of this species.

Isolated human chorionic vascular reactivity: Technical considerations for fresh preparations

1. 1. Sixty chorionic vascular rings from normal term placentas were immersed in an organ bath for isometric tension recording to study (A) the contractile response to 120 mM of potassium chloride (KCI) after adjustment and equilibration to 1–5 g of passive tension; and (B) the concentration-response curve to KCI after adjustment and equilibration to the optimal passive tension. 2. 2. Adjustment to 4 g of passive tension elicited the maximal ( P<0.007) and the latest ( P<0.006) KCl-induced contraction among arterial rings. Venous rings showed the greatest contraction when adjusted to 3 g, but the differences were not significant except when compared to 1 g of passive tension ( P<0.03). 3. 3. The EC 50 for chorionic arteries and veins was 14.2 and 25.7 mM, respectively ( P<0.003). The maximal contraction was already obtained with 40 mM of KCl. 4. 4. Our results suggest that (A) the optimal passive tension for fresh human chorionic arteries is 4 g; (B) chorionic venous reactivity is less influenced by the initial tension; and (C) the optimal concentration of KCl to be used as a contracting agent of these tissues is 40 mM.

Diluent for cool storage of the Japanese eel ( Anguilla japonica) spermatozoa

The appropriate ionic composition for the Japanese eel ( Anguilla japonica) sperm diluent for cool storage was examined. Eel spermatozoa were immotile in isotonic NaCl, KCl, and mannitol solutions, and the motility was initiated when osmolality was increased. Spermatozoa were diluted 50 times with various isotonic solutions and stored for 3 h at 5 °C. Subsequently, this mixture was diluted 21 times with 450 mM NaCl to examine the percentage motility. Spermatozoa diluted with artificial seminal plasma, consisting of 149.3 mM NaCl + 15.2 mM KCl + 1.3 mM CaCl 2 + 1.6 mM MgCl 2 buffered with 5 mM NaHCO 3-NaOH at pH 8.2, or with isotonic NaCl + KCl solution, showed high percentage motility after storage. However, spermatozoa diluted with isotonic NaCl, mannitol, NaCl + CaCl 2, or NaCl + MgCl 2 solutions showed significantly lower motility compared to the spermatozoa stored in the former two solutions. In subsequent experiments, spermatozoa were diluted with isotonic solutions of various potassium concentrations (0, 5, 15, 25, 35, 45, 75, and 164.5 mM) at 50 times and stored at 3 °C for up to 28 days to examine the optimal composition of diluent. Spermatozoa that were diluted with the 5–35 mM KCl solutions showed a decrease of percentage motility at 24 h, but they showed a marked recovery of motility by the seventh day. Increasing the potassium concentration in the isotonic diluent reduced the rate of decline in motility observed at 24 h of storage. Spermatozoa diluted with the 45, 75 or 164.5 mM KCl isotonic solutions did not show the temporal decrease of motility at 24 h. However, the percentage motility indices (percentage motility/initial percentage motility × 100) of spermatozoa in 75 or 164.5 mM KCl solutions at day 21 (26.2% and 0.6%, respectively) were significantly lower than those of 15–45 mM KCl solutions (47.9–64.8%). These results indicate that the potassium ion is an essential constituent of the isotonic diluent for cool storage of the Japanese eel spermatozoa. The optimal KCl concentration for short term storage (up to 7 days) is 45–75 mM, and that for longer storage is 15–45 mM.

Comparison of DNA Polymerases .ALPHA., .DELTA., and .EPSILON. of Mouse Cell Line FM3A and Its Temperature-Sensitive Mutant tsFT20.

DNA polymerases (pol) alpha, delta and epsilon of a mouse cell line FM3A and its temperature-sensitive derivative tsFT20, which is defective in DNA replication at a non-permissive temperature, were purified by chromatographic procedures monitored by a set of relatively specific assays for the respective DNA polymerase activities. The pol epsilon activity was separated into two fractions with similar enzymatic properties except for their optimal KCl concentrations and processivities. The fractions of pol delta and epsilon were not homogeneous, but their identities were confirmed by their sensitivities to DNA polymerase inhibitors, their associated 3'-->5' exonuclease activities, optimal concentrations of salts, dependencies on the proliferating cell nuclear antigen and processivities in polymerization, which also excluded significant contamination with other DNA polymerases. Of the DNA polymerases prepared from tsFT20 cells, only pol alpha showed greatly decreased activity and remarkable sensitivity to the non-permissive temperature, demonstrating that pol delta and epsilon, the other polymerases supposed to be involved in nuclear DNA replication, are unequivocally different entities from pol alpha. The level of pol epsilon activity tsFT20 was also significantly lower than in the parental cells, suggesting cooperation and/or interaction between pol alpha and epsilon, and some relevance of pol epsilon to DNA replication.

Role of metal ions in the assembly and decay of the transcription initiation complex on tRNA gene in yeast extracts

Studies on formation of the transcription initiation complex on the tRNA Tyr gene in yeast extracts with the use of Sarkosyl-challenge assay proved that a single magnesium ion is indispensable for the complex assembly in the absence of nucleotides. Other cations do not substitute for Mg 2+. The optimal KCl concentration for transcription of the gene by the assembled initiation complex was found to be twice as high as that for the assembly process and close to that observed previously for the TFIIIC (or tau) factor dissociation from the promoter. The preformed complex remained stable for several hours at room temperature and its decay was not influenced by ionic strength. The data seem to support the notion that the TFIIIC factor is used only for assembly of the initiation complex and is not necessarily involved in the subsequent steps of transcription,

Biochemical and pharmacological properties of p170 and p180 forms of topoisomerase II

The p170 and p180 forms of topoisomerase II have been compared. The concentration dependence of ATP for catalytic activity of the two forms of the enzyme was identical, and each was equally sensitive to novobiocin. Orthovanadate was found to be a potent inhibitor of catalytic activity of both p170 and p180, with an IC50 value of about 2 microM for each. Under standard reaction conditions, relaxation of supercoiled pBR322 by p180 was highly processive, while p170 performed the same reaction in a distributive manner. The optimal concentration of KCl for catalytic activity of p180 was 20-30 mM higher than that for p170. Comparison of their thermal stability showed that p180 was inactivated at twice the rate of p170. Teniposide and merbarone selectively inhibited catalytic activity of p170, requiring concentrations 3-fold and 8-fold lower, respectively, than those required for equivalent inhibition of p180. Similar selectivity for p170 was seen for teniposide-stimulated DNA cleavage or its inhibition by merbarone. Analysis of sites of DNA cleavage indicated a subset of sites that were either preferred or unique for each of the enzymes. A synthetic oligonucleotide representative of p170 sites selectively inhibited the p170 enzyme. Immunoblotting of p170 and p180 from U937 cells at different stages of proliferation showed that p170 levels declined as the cells reached the plateau phase of growth, while p180 levels were low during rapid proliferation and increased as the growth rate slowed. The data indicate that the p170 and p180 forms of topoisomerase II can be distinguished biochemically, pharmacologically, and by differential cellular regulation.

An improved purification procedure and properties of casein kinase II from brain.

A simple and short purification procedure applicable to casein kinase II has been developed, for fully characterizing the enzyme from calf cerebral cortex cytosol. The procedure consists of four chromatographic steps: DEAE-cellulose, phosphocellulose, phosvitin-Sepharose and ATP-agarose which yields 87% pure casein kinase II. The purified enzyme shows three major bands with apparent molecular masses of 42, 38, and 27 kDa by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and is self-autophosphorylated on its 27 kDa polypeptide. The enzyme shows all the characteristics described for casein kinase II from other sources: it is independent of cyclic nucleotides, calcium/phospholipids, and double-stranded poly(I).poly(C); it can utilize both ATP and GTP as phosphoryl donors and can phosphorylate both casein and phosvitin but not histone. The kinetic studies establish that the Km for ATP is 12.5 microM and 25.1 microM when using phosvitin and casein respectively as phosphoryl acceptors. The Km for phosvitin is 0.91 mg/ml and for casein 1.43 mg/ml, while the Vmax is 315 nmol/min/per mg protein and 479 nmol/min/per mg protein for phosvitin and casein respectively. The activity of the kinase is highly stimulated by KCl or NaCl, and almost completely inhibited by heparin concentrations of 1 microgram/ml (92%). This inhibition is reduced to only 33% in the presence of optimal KCl concentrations (150 mM). Spermine stimulates enzyme activity, whilst hemin produces a slight inhibition.

Characterization of the AIDS-associated retrovirus reverse transcriptase and optimal conditions for its detection in virions

The RNA-dependent DNA polymerase of the AIDS-associated retrovirus (ARV) gives highest activity with the synthetic template, poly(rA)oligo(dT) and prefers Mg 2+ over Mn 2+ as a divalent cation. It can use other template-primer combinations but with poly(rC m)oligo(dG), it prefers Mn 2+ over Mg 2+. Detection of ARV reverse transcriptase in culture fluids is substantially increased with an optimal KCl concentration and a special combination of EGTA and reducing agents. Our results indicate some distinguishing characteristics of the ARV reverse transcriptase and optimal conditions for its detection.

Requirement of different ionic concentrations for optimal translation of α and β globin mRNA in Krebs II ascites cell-free system

The translation of rabbit α globin mRNA in a Krebs II ascites cellfree system was more dependent upon the K + concentration than rabbit β globin mRNA. The optimal KCl concentration was approximately 70 mM for the synthesis of the α chain and between 80 and 90 mM for that of the β chain. With CH 3 CO 2K the optimum concentration for α chain synthesis was also 70 mM but the optimum for the β chain synthesis was not sharp any more and ranged from 70 mM to over 110 mM. In the range of the optimal Mg 2+ concentration for the α and β globin chain synthesis the α β ratio decreased when the Mg 2+ concentration increased. In the presence of DTT and EDTA the optimal KCl concentration for both α and β globin chain synthesis decreased.

Na + ,K + -Dependent ATPase Activity and Effect of K + on In vitro Protein Synthesis and NAD Pyrophosphorylase in Toxoplasma gondii

Although Na+, K+-dependent ATPase activity was barely detectable in extracellular tachyzoites of Toxoplasma gondii, Mg++-dependent ATPase was readily demonstrable. Based on this finding, the effect of K+ on in vitro protein synthesis and NAD pyrophosphorylase of T. gondii was evaluated. Addition of KCl enhanced in vitro protein synthesis. NAD pyrophosphorylase also was activated by KCl. The optimal concentration of KCl for both enzymatic reactions was 150 mM. The supernatant fluid (15,000 g, 20 min) isolated from the homogenate of purified tachyzoites of T. gondii contained more Na+ than K+. Evaluation of leakage of Na+ and K+ from purified tachyzoites suggested that Na+ leaked more than K+. These observations suggest an important role of K+ in the regulation of the metabolism of T. gondii.

Negative homotropic cooperativity in rat muscle AMP deaminase: A kinetic study of the inhibition of the enzyme by ATP

1. 1.|Rat skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) at optimal KCl concentrations shows a biphasic response to increasing levels of the allosteric inhibitor ATP. 2. 2.|Up to 10 μM, ATP appears to convert the enzyme to a form exhibiting sigmoidal kinetics while at higher concentrations its inhibitory effect is manifested by an alteration of AMP binding to AMP deaminase indicative of negative homotropic cooperativity at about 50% saturation. 3. 3.|AMP deaminase is inactivated by incubation with the periodate oxidation product of ATP. The (oxidized ATP)—AMP deaminase complex stabilized by NaBH 4 reduction shows kinetic properties similar to those of the native enzyme in the presence of high ATP concentrations. 4. 4.|A plausible explanation of the observed cooperativety is that ATP induces different conformation states of AMP deaminase subunits, causing the substrate to follow a sequential mechanism of binding to enzyme. 5. 5.|Binding of the radioactive oxidized ATP shows that 3.2 mol of this reagent bind per mol AMP deaminase.

Specific modification of the GTP binding sites of rat 5′-adenylic acid aminohydrolase by periodate-oxidized GTP

1. 1. Rat skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) can be inactivated by incubation with the periodate-oxidized analogue of the enzyme inhibitor GTP. 2. 2. Nucleoside triphosphates and KC1 at high concentrations protect against inactivation, while ADP has no effect. 3. 3. The inactivation can be reversed by the addition of GTP and amino acids and made irreversible by reduction with NaBH 4. This indicates that, in the binding of the oxidized GTP to the enzyme, a Schiff base is formed between the aldehyde groups of the inhibitor and amino groups of the enzyme. 4. 4. The kinetic properties of the reduced (oxidized GTP)-AMP deaminase derivative indicate that the loss of activity results from an increase in K m while no appreciable change in V is observed; consequently, the enzyme shows positive homotropic cooperativity even in the presence of optimal KCl concentration. 5. 5. Since the treated enzyme shows kinetic properties similar to those of the native enzyme in the presence of GTP, and since the loss of sensitivity to GTP is directly proportional to the degree of inactivation, it is concluded that the oxidized GTP specifically modifies the binding sites for GTP. 6. 6. Binding of the radioactive oxidized GTP shows that two binding sites for this reagent exist in the AMP deaminase molecule.

Supramolecular forms of actin from amoebae of Dictyostelium discoideum.

Actin purified from amoebae of Dictyostelium discoideum polymerizes into filaments at 24 degrees upon addition of KCl, as judged by a change in optical density at 232 nm and by electron microscopy. The rate and extent of formation of this supramolecular assembly and the optimal KCl concentrations (0.1 M) for assembly are similar to those of striated muscle actin. The apparent equilibrium constant for the monomer-polymer transition is 1.3 muM for both Dictyostelium and muscle actin. Although assembly of highly purified Dictyostelium actin monomers into individual actin filaments resembles that of muscle actin, Dictyostelium actin but not muscle actin was observed to assemble into two-dimensional nets in 10 mM CaCl2. The Dictyostelium actin also forms filament bundles which are 0.1 mum in diameter and which assemble in the presence of 5 mM MgCl2. These bundles formed from partially purified Dictyostelium actin preparations but not from highly purified preparations, suggesting that their formation may depend on the presence of another component. These actin bundles reconstituted in vitro resemble the actin-containing bundles found in situ by microscopy in many non-muscle cells.