Zur Metallspezifität der Hexokinase

Summary The activity of hexokinase has been determined in the presence of different metal ions. Besides Mg²⁺, the ions Co²⁺, Ni²⁺, Mn²⁺, Zn²⁺, and Cd²⁺ show remarkable activation. The differences are explained by superposition of an activating and an inhibiting function. The specifity problem is discussed.     

Recombinant synthesis of mouse Zn3-β and Zn4-α metallothionein 1 domains and characterization of their cadmium(II) binding capacity

Genetic engineering, coupled with spectro scopic analyses, has enabled the metal binding proper ties of the α and β subunits of mouse metallothionein 1 (MT) to be characterized. A heterologous expression system in E.coli has led to high yields of their pure zinc-complexed forms. The cadmium(II) binding properties of recombinant Zn₄-αMT and Zn₃-βMT have been studied by electronic absorption and circular dichroism. The former binds Cd(II) identically to α fragments obtained from mammalian organs, showing that the recombinant polypeptide behaves like the na tive protein. Titration of Zn₃-βMT with CdCl₂ results in the formation of Cd₃-βMT. The addition of excess Cd(II) leads to Cd₄-βMT which, with the extra loading of Cd(II), unravels to give rise isodichroically to Cd₉-βMT. The effect of cadmium-displaced Zn(II) ions and excess Cd(II) above the full metal occupancy of three has been studied using Chelex-100. The Cd₃-βMT species is stable in the presence of this strong metal-chelating agent. Received 20 May 1997; received after revision 7 July 1997; accepted 9 July 1997     

The effects of various agents in vitro on homocarnosine-carnosine synthetase from rat brain

Summary The present paper reports the first evidence that homocarnosine-carnosine synthetase from rat brain requires free sulfhydryl groups for activity. The activity of the synthetase can be stabilized by dithioerythritol and inhibited strongly by Cu²⁺, Cd²⁺, Hg²⁺, Zn²⁺, and to a lesser extent by Ca²⁺, Ni²⁺, Li, chlorpromazine, -methyl DOPA and norepinephrine at the concentrations tested.Acknowledgment. This work was supported by United States Public Health Service Grant No. NS-06137.     

Zinc ions block the second but not the first phasic response to repetitive application of carbachol and histamine in guinea-pig taenia caeci

In the presence of Zn²⁺ (0.3 mM), carbachol (10^–6 M) or histamine (10^–5 M) induced the phasic response in guinea-pig taenia caeci while the tonic response was markedly inhibited. However, when the muscles were kept in Zn²⁺-containing medium following the first stimulation with either carbachol or histamine, neither application of carbachol nor of histamine elicited another phasic contraction. Caffeine (25 mM) did not induce contraction in the presence of Zn²⁺. After the washing out of caffeine in the presence of Zn²⁺, however, the muscle did then develop the phasic response on the application of carbachol or histamine. In conclusion, Zn²⁺ did not affect the carbachol or histamine-induced Ca²⁺ release from the storage sites. However, when Zn²⁺ was continuously present, Ca²⁺ was not supplied to the storage sites. Furthermore, carbachol and histamine mobilized a common cellular Ca²⁺ store, but they activated Ca²⁺ release channels different from the ones activated by caffeine in the Ca²⁺ storage sites.     

Blockade by zinc ions of K+-induced contraction and calcium in guinea pigTaenia coli

Preincubation with 0.3 mM Zn²⁺ markedly inhibited both the tonic response and Ca²⁺ binding at low affinity sites induced by K⁺ (60 mM), with smaller effects on the phasic response and the high affinity Ca²⁺ sites, inTaenia coli. However, when the muscle was kept in Zn²⁺-containing medium following the first stimulation with the K⁺, the phasic response and the high affinity Ca²⁺ sites were more severely inhibited during the second stimulation with K⁺. This probably indicates that Zn²⁺ reduced the tonic tension response to K⁺ mainly by inhibiting Ca²⁺ influx at the cell membranes ofTaenia coli. However, when Zn²⁺ is continuously present, Ca²⁺ is not supplied at the storage sites and is not available for the phasic response to a second stimulation with K⁺.     

Zinc antagonizes the effect of botulinum type A toxin at the mouse neuromuscular junction

Summary Zn²⁺ (10–100 M) elevated the frequency of miniature end-plate potentials (MEPPs) in the mouse diaphragm. The effect did not depend on external Ca²⁺. Botulinum type A toxin (BTX_A, 50 ng/ml) abolished MEPPs almost completely within 30 min. Zn²⁺ (100 M) restored MEPPs and increased their frequency after they had been abolished by BTX_A in Ca²⁺-free solutions. The antagonistic effect of Zn²⁺ in the Ca²⁺-free solution was reduced by exposing the diaphragm to the toxin in the Ca²⁺-free solutions containing high K⁺. Thus, the action of BTX_A is probably enhanced by depolarization of the motor nerve terminals.     

Zinc increases the longevity of unfertilized sea urchin eggs

Summary We have found that Zn²⁺ prevented lysis of unfertilized sea urchin eggs, and the eggs retained the ability to form fertilization membranes and to divide. SDS polyacrylamide gel electrophoresis showed that proteolysis of several proteins accompanied egg lysis, but Zn²⁺ inhibited this proteolysis. Therefore, Zn²⁺ blocks protease activity directly or indirectly and thereby prolongs the longevity of unfertilized sea urchin eggs.     

GPR39: a Zn<Superscript>2+</Superscript>-activated G protein-coupled receptor that regulates pancreatic,gastrointestinal and neuronal functions

GPR39 is a vertebrate G protein-coupled receptor related to the ghrelin/neurotensin receptor subfamily. The receptor is expressed in a range of tissues including the pancreas, gut/gastrointestinal tract, liver, kidney and in some regions of the brain. GPR39 was initially thought to be the cognitive receptor for the peptide hormone, obestatin. However, subsequent in vitro studies have failed to demonstrate binding of this peptide to the receptor. Zn²⁺ has been shown to be a potent stimulator of GPR39 activity via the Gα_q, Gα_12/13 and Gα_s pathways. The potency and specificity of Zn²⁺ in activating GPR39 suggest it to be a physiologically important agonist. GPR39 is now emerging as an important transducer of autocrine and paracrine Zn²⁺ signals, impacting upon cellular processes such as insulin secretion, gastric emptying, neurotransmission and epithelial repair. This review focuses on the molecular, structural and biological properties of GPR39 and its various physiological functions.     

Calmodulin mediates melatonin cytoskeletal effects

In this article, we review the data concerning melatonin interactions with calmodulin. The kinetics of melatonin-calmodulin binding suggest that the hormone modulates cell activity through intracellular binding to the protein at physiological concentration ranges. Melatonin interaction with calmodulin may allow the hormone to modulate rhythmically many cellular functions. Melatonin's effect on tubulin polymerization, and cytoskeletal changes in MDCK and N1E-115 cells cultured with melatonin, suggest that at low concentrations (10^–9 M) cytoskeletal effects are mediated by its antagonism to Ca²⁺-calmodulin. At higher concentrations (10^–5 M), non-specific binding of melatonin to tubulin occurs thus overcoming the specific melatonin antagonism to Ca²⁺-calmodulin. Since the structures of melatonin and calmodulin are phylogenetically well preserved, calmodulin-melatonin interaction probably represents a major mechanism for regulation and synchronization of cell physiology.     

Zinc ions block the intracellular calcium release induced by caffeine in guinea-pig taenia caeci

Zn²⁺ in low concentrations (0.005–0.1 mM) inhibited the transient contractions in response to caffeine (25 mM) in a dose-dependent manner in smooth muscle of intact guinea-pig taenia caeci. At Zn²⁺ concentrations higher than 0.1 mM, caffeine did not elicit any response. After saponin-treatment of the fibres, which leaves the Ca²⁺ storage sites intact, caffeine contraction was completely inhibited by Zn²⁺ at a relatively low concentration (0.03 mM). However, in Triton-X-100-treated fibres, in which the Ca²⁺ release sites are destroyed, the contraction could be induced in the presence of Zn²⁺ by an increase in Ca²⁺. In conclusion, Zn²⁺ can block the intracellular Ca²⁺ release from caffeine-sensitive release sites in taenia caeci.     

Osmotic adaptation of moderately halophilic methanogenic Archaeobacteria,and detection of cytosolicN,N-dimethylglycine

Methanohalophilus mahii SLP andMethanohalophilus halophilus Z-7982, two closely-related, moderately halophilic, methylotrophic methanogens, were tested for their adaptation to saline conditions. They grew in a wider range of salinities than previously reported, in a defined medium with as little as 0.1 M NaCl, and with a high as 4.0 M NaCl forM. halophilus and 4.5 M NaCl forM. mahii. Fastest growth occurred with 1.5 M NaCl forM. mahii and 1.0 M NaCl forM. halophilus. M. mahii also grew in media in which NaCl was replaced by sucrose or KCl as osmolytes up to the osmolal equivalent of 2 and 2.5 M NaCl (these media contained other sodium salts totaling about 0.1 M Na⁺). In media with either sucrose of KCl replacing NaCl,M. mahii grew fastest at osmolalities approximately equiosmolal to 1 M NaCl.M. mahii not only grew well at a wide range of osmosities, it also tolerated rapid shifts in osmolality. Cells subjected to a rapid 10-fold hypertonic shift resumed growth without a prolonged lag. When cells were subjected to a rapid 10-fold hypotonic shift, 90% of cells lysed, but the remaineder continued to swell with little further lysis during the next 45 min. Surviving cells resumed growth.Methanohalophilus strains grown in defined medium had low cytosolic Na⁺ concentrations; K⁺ concentrations were as high as 0.35 M. Organic osmotica in the cytosol include glycine betaine and larger amounts of N,N-dimethylglycine.     

Kinetic and structural evidence of the alkenal/one reductase specificity of human ζ-crystallin

Human ζ-crystallin is a Zn²⁺-lacking medium-chain dehydrogenase/reductase (MDR) included in the quinone oxidoreductase (QOR) family because of its activity with quinones. In the present work a novel enzymatic activity was characterized: the double bond α,β-hydrogenation of medium-chain 2-alkenals and 3-alkenones. The enzyme is especially active with lipid peroxidation products such as 4-hydroxyhexenal, and a role in their detoxification is discussed. This specificity is novel in the QOR family, and it is similar to that described in the distantly related alkenal/one reductase family. Moreover, we report the X-ray structure of ζ-crystallin, which represents the first structure solved for a tetrameric Zn²⁺-lacking MDR, and which allowed the identification of the active-site lining residues. Docking simulations suggest a role for Tyr53 and Tyr59 in catalysis. The kinetics of Tyr53Phe and Tyr59Phe mutants support the implication of Tyr53 in binding/catalysis of alkenal/one substrates, while Tyr59 is involved in the recognition of 4-OH-alkenals.     

Zur Wirkung von Zn++ auf die Nukleinsäurebiosynthese von Aszites-Tumorzellen

Summary Ascites tumour cells have been employed to study the reactivity of Zn⁺⁺ on nucleic acid biosynthesis. 10^–4 M Zn⁺⁺ caused a selective inhibition of DNA synthesis of intact cells. The rate of RNA- and protein-biosynthesis, however, remained unchanged. The activity of DNA polymerase as well as DNA dependent RNA polymerase was strongly affected by Zn⁺⁺ in vitro.     

Zur biologischen Bedeutung von Fe3+ und Cu2+ als Komplexbildner

Summary The possible role of the metabolism of heavy metal ions in the process of ageing is discussed. It is suggested that, during this process, Cu²⁺ and Fe³⁺ as strong complexing ions, inhibit the activity of other metal enzymes by replacing their metal ion-activator. The relative stability of Cu²⁺- and Fe³⁺-complexes with various chelating compounds related to biological systems has been determined.     

Complex modulation of Cav3.1 T-type calcium channel by nickel

Nickel is considered to be a selective blocker of low-voltage-activated T-type calcium channel. Recently, the Ni²⁺-binding site with critical histidine-191 (H191) within the extracellular IS3–IS4 domain of the most Ni²⁺-sensitive Ca_v3.2 T-channel isoform has been identified. All calcium channels are postulated to also have intrapore-binding site limiting maximal current carried by permeating divalent cations (PDC) and determining the blockade by non-permeating ones. However, the contribution of the two sites to the overall Ni²⁺ effect and its dependence on PDC remain uncertain. Here we compared Ni²⁺ action on the wild-type “Ni²⁺-insensitive” Ca_v3.1_w/t channel and Ca_v3.1_Q172H mutant having glutamine (Q) equivalent to H191 of Ca_v3.2 replaced by histidine. Each channel was expressed in Xenopus oocytes, and Ni²⁺ blockade of Ca²⁺, Sr²⁺, or Ba²⁺ currents was assessed by electrophysiology. Inhibition of Ca_v3.1_w/t by Ni²⁺ conformed to two sites binding. Ni²⁺ binding with high-affinity site (IC₅₀ = 0.03–3 μM depending on PDC) produced maximal inhibition of 20–30 % and was voltage-dependent, consistent with its location within the channel’s pore. Most of the inhibition (70–80 %) was produced by Ni²⁺ binding with low-affinity site (IC₅₀ = 240–700 μM). Q172H-mutation mainly affected low-affinity binding (IC₅₀ = 120–160 μM). The IC₅₀ of Ni²⁺ binding with both sites in the Ca_v3.1_w/t and Ca_v3.1_Q172H was differentially modulated by PDC, suggesting a varying degree of competition of Ca²⁺, Sr²⁺, or Ba²⁺ with Ni²⁺. We conclude that differential Ni²⁺-sensitivity of T-channel subtypes is determined only by H-containing external binding sites, which, in the absence of Ni²⁺, may be occupied by PDC, influencing in turn the channel’s permeation.     

Mechanism of inhibition of IgE-dependent histamine release from rat mast cells by xestobergsterol A from the Okinawan marine spongeXestospongia bergquistia

Histamine release from rat peritoneal mast cells induced by anti-IgE was essentially complete within 4–5 min. Xestobergsterol A and B, which are constituents of the Okinawan marine spongeXestospongia bergquistia Fromont, dose-dependently inhibited anti-IgE-induced histamine release from rat mast cells. The IC₅₀ values of xestobergsterol A and B for histamine release in mast cells activated by anti-IgE were 0.07 and 0.11 M, respectively. Anti-IgE stimulated PI-PLC activity in a mast cell membrane preparation. Xestobergsterol A dose-dependently inhibited the generation of IP3 and membrane-bound PI-PLC activity. Moreover, xestobergsterol A inhibited Ca²⁺-mobilization from intracellular Ca²⁺-stores as well as histamine release in mast cells activated by anti-IgE. On the other hand, xestobergsterol B did not inhibit the membrane-bound and cytosolic PI-PLC activity, IP3 generation or the initial rise in [Ca²⁺]_i in mast cells activated by anti-IgE. These results suggest that the mechanism of inhibition by xestobergsterol A of the initial rise in [Ca²⁺]_i, of the generation of IP3, and of histamine release induced by anti-IgE, was through the inhibition of PI-PLC activity.     

Metal binding to myosin and to myosin DTNB-light chain

Summary The effects of various divalent cations, Ca²⁺, Mg²⁺ and Mn²⁺ on the intrinsic fluorescence of heavy meromyosin (HMM) and myosin 5,5-dithio-bis-(2-nitrobenzoate) DTNB-light chain of rabbit striated muscle, are compared. At pH 6.4, the fluorescence change induced by the metal ions is present only in the isolated light chain and disappears in HMM, thus indicating an interaction between the heavy and light chains with respect to the binding of the metal ions. Whereas Mg²⁺ binds more strongly than Ca²⁺ to myosin, this order is reversed in the case of the DTNB-light chain.     

Mn2+ electron spin resonance studies on ATP phosphoribosyltransferase fromE. coli

Summary ESR binding studies of Mn²⁺ with each of the substrates and products suggests that substrate bridge complexes are formed in the reaction. This prediction is confirmed, comparing Mn²⁺+ADP and Mn²⁺+ADP+enzyme spectra.We thank V. M. Fernández for helpful discussions.     

Phtalocyanine in wässriger Lösung. Zum Mechanismus der Metallkomplexbildung

Summary The mechanism of incorporation of different mental ions into phthalocyaninetetrasulfonic acid in aqueous solution has been investigated. It was found that the rate of the reacion with Cu²⁺ depends on the dissociation rate of the N-H-bond of this porphyrin-like chelating agent, whereas the reaction rate with other metal ions, like Zn²⁺, does not.

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II. Mitt. Siehe I. Mitt.:K. Bernauer undS. Fallab, Helv.44, 1287 (1961).

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Herrn Prof. Dr.H. Erlenmeyer sind wir für sein reges Interesse an dieser Arbeit zu bestem Dank verpflichtet.     

The enteric neural receptor for 5-hydroxytryptamine

Summary An enteric neural receptor for serotonin (5-HT) has been characterized. This receptor was assayed, using³H-5-HT as a radiologand, by rapid filtration of isolated enteric membranes and by radioautography. In addition, intracellular recordings were made from ganglion cells of the myenteric plexus. High affinity, saturable, reversible, and specific binding of³H-5-HT was demonstrated both to membranes of the dissected longitudinal muscle with adherent myenteric plexus and the mucosa-submucosa. Radioautographs showed these³H-5-HT binding sites to be in myenteric ganglia and in a broad unresolved band at the mucosal-submucosal interface. Antagonists active at receptors for other neurotransmitters than 5-HT, at either of the two known types of CNS 5-HT receptor, and at 5-HT uptake sites on serotonergic neurons failed to inhibit binding of³H-5-HT. The structural requirements of analogues for binding to the enteric 5-HT receptor matched the known pharmacology of M or neural 5-HT receptors. A novel 5-HT antagonist was found. This compound, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), antagonized the action of 5-HT on type II/AH cells of the myenteric plexus but did not affect the release or actions of acetylcholine (nicotinic or muscarinic) or substance P. 5-HTP-DP was also an equally potent displacer of³H-5-HT from its binding sites on enteric membranes. It is concluded that the sites responsible for specific binding of³H-5-HT are enteric M or neural 5-HT receptors. These receptors differ from those now known to be present in the CNS.