Effects Of Several Divalent Cations Research Articles

Divalent Cations Modulate TMEM16A Calcium-Activated Chloride Channels by a Common Mechanism

The gating of Ca²⁺-activated Cl⁻ channels is controlled by a complex interplay among [Ca²⁺](i), membrane potential and permeant anions. Besides Ca²⁺, Ba²⁺ also can activate both TMEM16A and TMEM16B. This study reports the effects of several divalent cations as regulators of TMEM16A channels stably expressed in HEK293T cells. Among the divalent cations that activate TMEM16A, Ca²⁺ is most effective, followed by Sr²⁺ and Ni²⁺, which have similar affinity, while Mg²⁺ is ineffective. Zn²⁺ does not activate TMEM16A but inhibits the Ca²⁺-activated chloride currents. Maximally effective concentrations of Sr²⁺ and Ni²⁺ occluded activation of the TMEM16A current by Ca²⁺, which suggests that Ca²⁺, Sr²⁺ and Ni²⁺ all regulate the channel by the same mechanism.

Manganese is highly effective in protecting cells from cadmium intoxication

Cadmium poisoning results in cell death. Although several intracellular pathways have been identified in this response, transport systems responsible for cadmium entry into cells remain poorly understood and controversial. Here, we analyzed the effects of several divalent cations on cadmium toxicity in different cell types. We found that zinc, previously reported as a protective agent against cadmium poisoning, is actually much less efficient than manganese. We show that manganese dramatically reduces cadmium intake, and that this is associated with the inhibition of our recently reported sustained activation of ERK, characteristic of cadmium intoxication. Finally, we show that this inhibition of cadmium entry and ERK-sustained activation perfectly correlates with a high cellular resistance to cadmium exposure. Our results, together with previously published data, support the idea that the yet to be characterized manganese transporter system(s) may be responsible for cadmium entry into cells.

Zn2+-Mediated Structure Formation and Compaction of the “Natively Unfolded” Human Prothymosin α

Human recombinant prothymosin α (ProTα) is known to have coil-like conformation at neutral pH; i.e., it belongs to the class of “natively unfolded” proteins. By means of circular dichroism, SAXS, and ANS fluorescence, we have investigated the effect of several divalent cations on the structure of this protein. Results of these studies are consistent with the conclusion that ProTα conformation is unaffected by large excess of Ca2+, Mg2+, Mn2+, Cu2+, and Ni2+. However, Zn2+ induces compaction and considerable rearrangement of the protein structure. This means that ProTα can specifically interact with Zn2+ (KD ∼ 10−3 M), and such interactions induce folding of the natively unfolded protein into a compact partially folded (premolten globule-like) conformation. It is possible that these structural changes may be important for the function of this protein.

Skeletal T−O−T Vibrations as a Tool for Characterization of Divalent Cation Complexation in Ferrierite

The effect of several divalent cations (Mn2+, Mg2+, Ni2+, and Co2+) exchanged in Na-ferrierite and ammonia adsorption/desorption was studied by FTIR spectroscopy in the range 4000−400 cm-1. The complete cation exchange eliminated zeolite bridging acidic hydroxyls and provided for monitoring of the stretching and bending modes of ammonia interacting with cations and the accompanying parallel changes of the zeolite lattice vibrations induced by bare cations and cation−ligand complexes at various ammonia coverage. Dehydration of ferrierites with ion-exchanged divalent cations gives formation of new IR bands in the transmission window between antisymmetric (around 1070 cm-1) and symmetric (around 780 cm-1) T−O−T stretching lattice modes. These new bands disappear upon full saturation by ammonia and reappear under complete ammonia desorption. They are assigned to antisymmetric stretching T−O−T mode of zeolitic lattice shifted to a lower frequency due to the reversible local deformation of the flexible zeolitic...

Studies on Uroporphyrinogen Biosynthesis in Pig Liver

Porphobilinogen-deaminase (PBG-D) and PBG-D-isomerase complex (PBG-D-I) from pig liver were isolated and partially purified. Uroporphyrinogen I and III formation was found to be linear with time and protein concentration. Optimal pH was about 7.4 and 7.6-7.8 for PBG-D and PBG-D-I complex, respectively. Some properties of the isolated enzymes were studied. Molecular mass determination gave a value of 40,000 Da for PBG-D and 50,000 Da for the complex. Both enzymes exhibited classical Michaelis-Menten kinetics. Km and Vmax parameters were estimated. The effect of several divalent cations, ammonia and thiol reagents was also investigated. The differential action of some of these chemicals on PBG-D and PBG-D-I system would suggest that PBG-D and isomerase may not be only physically adjacent but actually associated.

Assay and properties of N-acetylglucosamine-6-phosphate deacetylase from rat liver

A single-vial assay has been developed for N-acetylglucosamine-6-phosphate deacetylase, in which [ 3H]acetate released from 3H-acetyl-labeled substrate is measured in a biphasic liquid scintillation counting system after acidification of the reaction mixture. The deacetylase was partially purified from rat liver, and some of its properties were determined. Chromatography on a calibrated Sepharose CL-6B column indicated a molecular weight of 345,000. The K m for the substrate at pH 8.0 was 0.3 m m. Glucosamine 6-phosphate and glucose 6-phosphate inhibited the enzyme, whereas N-acetylgalactosamine, N-acetylglucosamine, N-acetylglucosamine 1-phosphate, and glucosamine 1-phosphate were without effect. The effects of several divalent cations were also examined. Under the conditions tested, Ca 2+, Mg 2+, and Ba 2+ had essentially no effect, whereas Mn 2+, Ni 2+, and Cu 2+ were inhibitory and Co 2+ stimulated activity at low concentrations but inhibited above 5 m m. An increase in the ionic strength of the reaction mixture to 0.3 m decreased the activity by 40%.

Optimal conditions for prolidase assay by proline colorimetric determination: application to iminodipeptiduria

Prolidase assay was reinvestigated by determining proline, using Chinard's method. Although several authors had previously tested this colorimetric reaction, accurate details regarding enzyme activity were not available. The need for greater sensitivity led to the introduction of several modifications: dialysis was eliminated and the substrate concentration and incubation time were changed. In addition, the reaction mixture was preincubated with Mn 2+ for 24 h in order to triple prolidase activity. Color development followed at 90°C, because of partial glycylproline hydrolysis at higher temperatures. The effect of several divalent cations on prolidase activity were tested with and without Mn 2+. This modified assay was applied to erythrocytes, plasma and skin fibroblasts from a female patient with iminodipeptiduria.

The effects of several divalent cations on the activation or inhibition of RNA polymerases II

Eukaryotic RNA polymerases II or B have been generally shown to be more active with Mn 2+ rather than Mg 2+ as the required divalent metal ion. However, very little was known about the activation or inhibition of RNA polymerases by metal ions other than Mg 2+ and Mn 2+. In this study the effects of several divalent cations were observed on in vitro transcription by highly purified RNA polymerases II from the mushroom Agaricus bisporus and calf thymus. Cobalt (II), Fe 2+, Mg 2+, or Mn 2+ may function as the divalent metal activator for RNA synthesis catalyzed by RNA polymerase II from calf thymus with the maximal activity observed using Fe 2+ as the activating metal ion. Of the 13 divalent cations used in this study, only Co 2+ or Mn 2+ can serve as the required divalent metal for RNA polymerase II from A. bisporus. Barium (II) had an apparent synergistic effect on the activation of RNA polymerase II from A. bisporus and calf thymus, and Sr 2+ exhibited a strong positive synergism on the activity of the mushroom enzyme. RNA polymerases II from both the mushroom and calf thymus were significantly inhibited by Be 2+, Cd 2+, Hg 2+, Ni 2+, and Zn 2+. A. bisporus RNA polymerase II was strongly inhibited by Cu 2+ and Fe 2+. The particularly potent metal inhibitor, Zn 2+, was shown to manifest competitive inhibition for both enzymes with respect to the substrates as determined with Mn-uridine triphosphate. Zinc (II) was also shown to manifest mixed inhibition with respect to the DNA template cofactor for both the calf thymus and mushroom RNA polymerase II.

Teh pH-dependence of the inhibitory effects of several divalent cations on the ovine intestine adenosine deaminase activity

1. 1. In the reaction performed in an alkaline region, the inhibitory effects of Ba 2+, Ca 2+, Mg 2+, Mn 2+ and Sr 2+ on the bovine intestine adenosine deaminase were observed. 2. 2. Mn 2+ completely inhibited its activity at pH 8.0, and Ca 2+ and Mg 2+ at pH 9.0. The inhibition of the latter two metals was reversible, and low hydrogen ion concentration was required when binding. The addition of EDTA reduced the inhibitory effects but a difference was found between Ca 2+ and Mg 2+ in the recovery process.

Metal-induced infidelity during DNA synthesis.

The effect of several divalent cations on the accuracy of DNA replication in vitro has been examined. Only Be2+ altered the accuracy of DNA synthesis using purified DNA polymerase (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase; EC 2.7.7.7) from avian myeloblastosis virus. The Be2+-induced base substitutions occurred with all templates and with all nucleotides tested. Analysis of the product by equilibrium density centrifugation and processive hydrolysis with snake venom phosphodiesterase suggested that the noncomplementary nucleotides were present in phosphodiester linkage. Nearest neighbor studies indicated that many of the Be2+-induced errors were present as single base substitutions. The enhancement of error frequency could be duplicated by the pretreatment of the enzyme, but not the template, with Be2+. Glycerol gradient centrifugation dissociated the Be2+-DNA polymerase complex and restored the initial error frequency of the polymerase. Thus, the weak binding of a metal cation to a DNA polymerase could alter the accuracy with which that polymerase copied DNA. Beryllium is a known carcinogen. The potential use of this system as a screening technique to detect chemical mutagens and carcinogens is considered.

Adenyl Cyclase: KINETIC PROPERTIES AND NATURE OF FLUORIDE AND HORMONE STIMULATION

Abstract The effects of several divalent cations, ATP, F-, and epinephrine, on adenyl cyclase from cardiac tissue were examined. Mn++ was more effective than Mg++ in activating the enzyme and the apparent Ka for Mn++ (0.7 mm) was less than that for Mg++ (2 to 3 mm). Co++ produced weaker stimulation than Mg++. ATP concentrations in excess of divalent metal ion inhibited the cardiac enzyme. F- increased the velocity of the reaction at all Mg++ concentrations, even those above saturation. It was concluded that the stimulatory effect of F- was due to an increase in Vmax, there being no significant effect on the Km for substrate (MgATP) or on the apparent Ka for Mg++. Epinephrine, like F-, stimulated the cardiac enzyme at all Mg++ concentrations and did not prominently affect the apparent Ka for Mg++ or the Km for the MgATP substrate. The actions of F- and epinephrine on the enzyme in washed particles of brain and of F- and glucagon on particulate preparations of liver were also examined. In each case the prominent effect of F- and hormone was to increase reaction velocity. It is suggested that a common feature of hormonal (and F-) stimulation of adenyl cyclase may be an increase in Vmax of the enzyme, reflecting increased reactivity of the catalytic site with substrate.

The effect of divalent cations on cell adhesion.

The effect of several divalent cations on cell adhesion was estimated in monolayer cell cultures. The exclusion of the divalent cations (Ca and Mg) from Hanks' balanced salt solution resulted in rapid cell detachment from the glass substratum. Detached cells would reattach when these divalent cations again were introduced into the medium.Other divalent cations were found to have effects similar to those of calcium or magnesium on cell detachment and reattachment, with the exception of strontium which showed no interference with cell contact abilities.

Serologic and enzymatic investigations of Trichinella spiralis II. Characterization of larval lactic dehydrogenase

A comparison has been made between lactic dehydrogenase (LDH) of extracts of Trinchinella spiralis larvae and pooled sera from normal rabbits and animals 8 weeks postinfection. Polyacrylamide gel electrophoresis demonstrated differences between the LDH isozymes of the parasite and those separated in the sera from normal and infected rabbits. The pH optimum of the parasite LDH was observed at 9.0, while optimal enzymatic activity of the sera enzymes was demonstrated at pH 10.5. It was shown that the larval LDH was considerably more heat stable than the serum enzyme of either the normal or trichina-infected rabbits. Differences in the inhibitory effects of several divalent cations were demonstrated between the enzyme of Trichinella extract and that from sera from normal and infected rabbits. Serological studies did not show cross-reactions between the rabbit and larval LDH by antibodies made by the rabbit. An isozyme found in the larvae was demonstrated in the adult worms. These studies indicate basic differences between physicochemical properties of the LDH of the parasite and the rabbit sera.