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At least 523 records · Page 29Linked to original sources
PubMed1993

Localization of the tightly bound divalent-cation-dependent and nucleotide-dependent conformation changes in G-actin using limited proteolytic digestion.

Using proteolytic susceptibility as a probe, we have identified four regions of the actin polypeptide chain where structural rearrangements, dependent on the nature of the tightly bound metal ion and/or nucleotide, take place. Replacement of the tightly bound Ca2+ by Mg2+ in ATP-actin strongly affected the regions around Arg26 and Lys68, as judged from nearly complete inhibition of tryptic cleavages of the polypeptide chain at these residues. It also significantly diminished the rates of splitting by trypsin of the peptide bonds involving carbonyl groups of Arg372 and of Lys373 in the C-terminal segment. Conversion of ATP-actin to ADP-actin (with Mg2+ as the tightly bound cation) abolished the protective effect of Mg2+ on specific tryptic cleavage and, in contrast, largely inhibited proteolysis at specific sites for subtilisin and for a novel protease from Escherichia coli A2 strain within a surface loop of residues 39-51. We also examined the effect of proteolytic cleavage or chemical modification at certain sites on the kinetics of proteolysis at other sites of the molecule. These experiments demonstrated structural relationships between loop 39-51 and regions involving Lys61 and Lys68. It is suggested that the conformational transitions reflected in the observed changes in proteolytic susceptibility may underlie the known influence of the nature of the tightly bound cation and nucleotide on the kinetics of actin polymerization and stability of the polymer.

Actins↗
PubMed2000

Inactivation of P2X2 purinoceptors by divalent cations.

1. P2X2 channels are activated by extracellular ATP. Despite being commonly described as non-desensitizing, P2X2 receptors do desensitize or inactivate. In the unspliced, 472 amino acid isoform of the P2X2 receptor, inactivation required membrane disruption and the presence of extracellular Ca2+. 2. The ability to inactivate whole-cell currents developed slowly after breaking in. In contrast, currents from excised patches exhibited rapid (approximately 100 ms) inactivation with a dependence on extracellular Ca2+, ATP and voltage. 3. The inactivation rate increased with the fourth power of [Ca2+] suggesting that the functional channel may be a tetramer. Ca2+ had both a higher affinity and a larger Hill coefficient for inactivation than Mg2+, Ba2+ or Mn2+. Trivalent cations at concentrations up to the solubility product of ATP had no effect. The change in apparent co-operativity with ionic species suggests the presence of experimentally unresolved ligand-insensitive kinetic steps. 4. Based on the weak voltage dependence of inactivation and the lack of effect of intracellular Ca2+ buffers, the Ca2+-binding sites are probably located near the extracellular surface of the membrane. 5. The recovery from inactivation was slow, with a time constant of approximately 7 min. 6. Ca2+-sensitive inactivation only appeared when the membrane was disrupted in some manner. Treatment with actin and microtubule reagents did not induce inactivation, suggesting that an intact cytoskeleton is not necessary. 7. Inactivation rates observed in different patch configurations suggest that the induction of Ca2+-dependent inactivation was due to the loss of a diffusible cofactor located in the membrane or the cytoplasm.

Actins↗
PubMed1986

Antibacterial activity in human amniotic fluid: dependence on divalent cations.

Antibacterial activity, tested against Bacillus subtilis, was present in all 34 samples of human amniotic fluid collected between 37 and 41 weeks gestation. High-molecular-weight (beta-lysin) antibacterial activity was reduced after treatment with ethylene diamine tetra-acetic acid (EDTA), but not after treatment with 1, 10-phenanthroline or diphenylthiocarbazone. The activity was restored following incubation with magnesium, calcium, or zinc. Low-molecular-weight antibacterial activity (less than 1000 daltons) in acid-alcohol extracts was reduced after treatment with dithizone, but not after treatment with EDTA or 1, 10 phenanthroline. Activity was restored after treatment with manganese, cobalt or zinc. High-molecular-weight (beta-lysin) activity was therefore biochemically similar to serum beta-lysin but biochemically different from the low-molecular-weight antibacterial component of amniotic fluid.

Adult↗
PubMed1983

Phosphoinositide metabolism in the retina: localization to horizontal cells and regulation by light and divalent cations.

Isolated retinas from Xenopus laevis incorporated greater amounts of [3H]inositol and 32Pi into phosphoinositides when incubated in light than did control retinas incubated in the dark. Inositol was primarily incorporated into phosphatidylinositol (83-86%), while phosphate labeled the polyphosphoinositides (72-79%). The incorporation of radioactive glycerol, serine, choline, or ethanolamine into retinal lipids was unaffected by light. Following incubation with [3H]inositol, the cell type involved in the light response was identified by light and electron microscope autoradiography to be the horizontal cell. These results are consistent with a classic phosphatidylinositol effect in the retina. An interesting feature of this response is that the stimulus (light) is received in the photoreceptor cell and the effect is manifest in the horizontal cell.

Animals↗
PubMed1984

Activation of adenylyl cyclase by preincubation of rat cerebral-cortical membranes under phosphorylating conditions: role of ATP, GTP, and divalent cations.

The effects of preincubation under phosphorylating conditions on adenylyl cyclase activity were studied in preparations containing synaptic membranes from rat cerebral cortex. Preincubation of the membranes with 2 mM ATP and 10 mM MgCl2 resulted in a 50% increase of adenylyl cyclase activity which withstood sedimentation and washing. This activation was maximal after 5 min of preincubation, was reversed after longer preincubations, and paralleled the time course of endogenous phosphorylation-dephosphorylation of proteins observed under these conditions. The activation showed a critical requirement for Mg2+ ions and was dependent on ATP concentration. Similar activation was observed after preincubation of cerebral-cortical membranes with adenosine-5'-O-(3-thiophosphate) (ATP gamma S), but this activation was not reversed by prolonged preincubation times. The activation by ATP gamma S was potentiated severalfold by including synaptoplasm in the preincubation. Further experiments indicated that the activity of nucleoside diphosphokinase, which converts ATP gamma S to guanosine-5'-O-(3-thiophosphate) (GTP gamma S), could account for this potentiation. Preincubation of washed membranes for 5 min with 10 microM GTP and 10 mM MgCl2 also produced a 50% activation of adenylyl cyclase which withstood sedimentation and washing and was reversed by longer preincubations. Endogenous phosphorylation of specific protein components in the membranes during the preincubation was examined by including radioactively labeled nucleoside thiophosphates in the preincubation medium. Incorporation of 35S from [35S]ATP gamma S into a protein component with apparent Mr of 54,000 daltons ( 54K ) correlated significantly with the activation of adenylyl cyclase by ATP gamma S. Thiophosphorylation of the 54K protein was potentiated by addition of GDP to reactions carried out with [35S]ATP gamma S. Endogenous activity utilizing [gamma-32P]GTP as a phosphate donor also preferentially phosphorylated the 54K protein band. These results support previous suggestions that protein phosphorylation plays a role in the regulation of adenylyl cyclase activity. Among the numerous membrane-bound phosphoproteins in rat brain, we have identified a specific protein component with an apparent Mr of 54,000 daltons as the most likely candidate for involvement in this mode of regulation. This 54K protein, which is a principal substrate for a GTP-preferring protein kinase activity in brain membranes, can now be at the focus of investigations attempting to demonstrate a direct role for protein phosphorylation in adenylyl cyclase regulation.

Adenosine Triphosphate↗
PubMed1992

Differential effects of Mg2+ and other divalent cations on the binding of tritiated opioid ligands.

The effects of MgCl2 on the binding of tritiated ligands to opioid binding sites in homogenates of guinea-pig brain in HEPES buffer have been studied. The binding of tritiated mu-, delta-, and kappa-opioid agonists was promoted in a concentration-dependent manner over a range of MgCl2 concentrations from 0.1 mM to 10 mM, as was binding of the nonselective antagonists [3H]diprenorphine and [3H]naloxone. At concentrations of MgCl2 above 10 mM reversal of this effect was observed. The effects of MgCl2 on binding parameters differed at each site. The promoting effects of MgCl2 were mimicked by MnCl2, CaCl2, and MgSO4, but CoCl2 and ZnCl2 were inhibitory. Following treatment of guinea-pig brain synaptosomes at pH 11.5 to eliminate G proteins, the binding of the mu-opioid agonist [3H][D-Ala2, MePhe4, Gly-ol5]enkephalin and [3H]naloxone was much reduced but binding of [3H]diprenorphine was unaffected. Under these conditions MgCl2 still promoted binding of [3H]diprenorphine. The results suggest that Mg2+ ions promote binding by an action at the opioid receptor, even in the absence of G protein, and that opioid antagonists may differ in their recognition of opioid receptor binding sites.

Animals↗
PubMed1982

Effects of divalent cations and normorphine on spontaneous excitatory junction potentials in the mouse vas deferens.

1 Excitatory junction potentials (e.j.ps) occurring spontaneously or evoked by nerve stimulation were recorded intracellularly from smooth muscle cells of the mouse isolated vas deferens. 2 The amplitude of the evoked e.j.ps and the amplitude and frequency of spontaneous e.j.ps were measured before and during application of normorphine or solutions which might be expected change the influx of calcium ions into the nerve terminals. 3 Spontaneous e.j.ps could be recorded even in solutins which contained tetrodotoxin (1 microM), no added calcium an EGTA (1mM). A four fold increase in calcium concentration from 1.25 to 5 mM greatly increased the amplitude of the evoked e.j.ps but had no effect on the amplitude or frequency of the spontaneous e.j.ps. 4 Magnesium (12mM) and cobalt (4mM) both greatly reduced the evoked e.j.ps and also reduced the frequency of spontaneous e.j.ps. 5 Normorphine (2 microM) reduced the amplitude of the evoked e.j.p by 70% but had no effect on the amplitude or frequency of spontaneous e.j.ps. 6 It is suggested that normorphine inhibits noradrenaline secretion from nerve varicosities by a mechanism different from that of magnesium and cobalt. One possibility is a block of action potential propagation along varicose fibers.

Action Potentials↗
PubMed1995

Effects of divalent cations and La3+ on contractility and ecto-ATPase activity in the guinea-pig urinary bladder.

1. Several cations (Ba2+, Cd2+, Co2+, Cu2+, Mn2+, Ni2+, Zn2+ and La3+, all as chloride salts, 1-1000 microM) were tested in the guinea-pig urinary bladder for their ability to: (i) modify contractile responses to electrical field stimulation (EFS), ATP, alpha,beta-methylene ATP (alpha,beta-meATP), carbachol (CCh), and KCl; (ii) affect ecto-ATPase activity. 2. Ba2+ (10-1000 microM) concentration-dependently potentiated contractile responses evoked by EFS (4-16 Hz), ATP (100 microM), alpha,beta-meATP (1 microM), CCh (0.5 microM), and KCl (30 mM). Ni2+ at concentrations of 1-100 microM also potentiated contractility of the urinary bladder, but at concentrations tested its effect was not concentration-dependent. Cu2+ at a concentration of 10 microM and Cd2+ at a concentration of 1 microM potentiated responses to all stimuli, except KCl. Ni2+ at a concentration of 1000 microM and Cd2+ at a concentration of 100 microM inhibited contractions evoked by all stimuli, and at a concentration of 1000 microM Cd2+ abolished any contractions. Responses to ATP and alpha,beta-meATP were selectively inhibited by Cu2+, Zn2+ or La3+, each at a concentration of 1 mM. 3. Cu2+, Ni2+, Zn2+ and La3+ (100-1000 microM) concentration-dependently inhibited ecto-ATPase activity in the urinary bladder smooth muscle preparations, while Ba2+ and Mn2+ were without effect, and Cd2+ and Co2+ caused significant inhibition only at a concentration of 1000 microM. 4. There was no correlation between the extent of ecto-ATPase inhibition and the effect on contractile activity of any of the cations. 5. In conclusion, the ability of some divalent cations to inhibit ecto-ATPase activity and to potentiate or inhibit contractile responses in the guinea-pig urinary bladder appear to be independent effects.

Adenosine Triphosphatases↗
PubMed1987

Mechanism of inhibition of bovine pineal gland hydroxyindole-O-methyltransferase (EC 2.1.1.4) by divalent cations.

The effect of inhibitory cations on hydroxyindole-O-methyltransferase (HIOMT) was investigated in order to elucidate the mechanisms involved. Copper inhibition of HIOMT can be classified as partial, classic noncompetitive inhibition with respect to S-adenosyl methionine and as partial, mixed noncompetitive inhibition with respect to N-acetylserotonin. Binding of copper to the enzyme impaired but did not prevent catalysis, and the difference in inhibitory pattern observed with both substrates may be due to the obligatory order of substrate binding to HIOMT. Calcium and magnesium were uncompetitive inhibitors of HIOMT with respect to both substrates and binding of these cations to the enzyme prevents catalysis. The cations presumably bind to HIOMT in the vicinity of the catalytic site and in some way prevent or impair the catalytic process, possibly by interfering with substrate binding to the enzyme, interfering with methyl transfer or product release after catalysis, or a combination of these effects. The different inhibitory patterns observed may result from differences in molecular size or outer electron shell valence of the cations.

Acetylserotonin O-Methyltransferase↗
PubMed1984

Calcitonin-like immunoreactivity in human milk, longitudinal alterations and divalent cations.

The concentration of immunoreactive calcitonin (iCT) in human milk was 10-40 times the serum level. Thus in milk samples collected from 29 mothers within one week and from 46 mothers three months post partum the levels of iCT were respectively 2.46 +/- 0.99 ng/ml (mean +/- SD) and 0.58 +/- 0.08 ng/ml. Eight of the 29 mothers delivered milk at two occasions, 1-2 and 4-6 days after delivery. There was a significant decline of iCT with about 20% during this period. One mother delivered milk for two months. During the first week after delivery the iCT decreased from 1.4 to 0.5 ng/ml and after that the iCT content remained rather constant. The level of ionized calcium (Ca++) was correlated to iCT the first week post partum (r = 0.72 p less than 0.01) but not in milk samples obtained after three months. The concentration of magnesium was weakly correlated to iCT during the first week post partum (r = 0.37 p less than 0.05). There was no correlation between iCT and the total calcium level nor to the concentration of zinc. The high concentration of iCT in milk compared to serum suggests a local production of iCT within the mammary gland or a specific transport and concentration mechanism.

Calcitonin↗
PubMed1975

Isolation, properties, and structural features of divalent cation ionophores derived from beef heart mitochondria.

The notion of small molecular weight ion carriers in biological systems is herein documented by a description of the isolation and ionophoretic properties of a family of oxyoctadecadienoate congeners derived from beef-heart mitochondria. Although certain members of this family of compounds have been shown to possess unique ionophoretic properties, one should not lose sight of the fact that the compounds that we have described represented only a portion of the total picture. Other chemically unrelated, yet structurally unknown species have been isolated from beef-heart mitochondria, and compounds similar in both chromatographic and spectroscopic properties to the oxyoctadecadienoate family, as well as other unique structures, have been isolated in our laboratory from sarcoplasmic reticulum and chloroplasts. The important points to be derived from these findings are that there is an apparent abundance of natural ionophores and we should no longer concern should address ourselves to the more relevant task of digging them out and ourselves with the question "are there ionophores in biological systems?" but describing their chemical and physical properties. In view of the apparent abundance of natural ionophores, this is an enormous task, especially when one considers that it only represents half of the problem. The isolation and description of the ionophoroprotein or channel-forming complexes share equally in the overall significance and level of understanding attributable to this area of inquiry and it would appear that many fruitful collaborative ventures are, or should be, on the horizon.

Animals↗