The effect of catecholamines on transport (Na,K) adenosine triphosphatase.

The effects of norepinephrine and dopamine were investigated on NaK-ATPase (ATP phosphohydrolase EC 3.6.1.3) activity from a beef brain microsomal preparation. These catecholamines can produce a 1.5-fold activation in the NaK-ATPase activity. The catecholamines exert their effect by reversal of divalent metal inhibition of NaK-ATPase activity. The physiologically important divalent metals, Cu²⁺, Zn²⁺, Ca²⁺, and Fe²⁺ are well-known inhibitors of NaK-ATPase. Norepinephrine and dopamine could reverse only the inhibitory effects of Fe²⁺. A model system was devised wherein the effects of several catecholamines and related compounds were tested for their ability to reverse the Fe²⁺ inhibition of NaK-ATPase activity. Effective agents must have an ethylamine side chain and two hydroxyl groups on the phenyl ring to completely reverse the inhibitory effects of Fe²⁺ on NaK-ATPase activity. The order of efficacy is isoproterenol > epinephrine = norepinephrine > phenylephrine. Propranolol can block the effect of isoproterenol. No effect of cyclic AMP either in the presence or absence of theophylline was observed.     

Studies with tryptophan metabolites in vitro. effect of zinc,manganese, copper and cobalt ions on kynurenine hydrolase and kynurenine aminotransferase in normal mouse liver

The possible mechanisms by which increasing concentrations of Zn²⁺, Mn²⁺, Cu²⁺, or Co²⁺ may affect the vitamin B₆-dependent enzymes kynurenine hydrolase and kynurenine aminotransferase. were studied in normal mouse liver homogenates. It was found that Zn²⁺ inhibited kynurenine hydrolase. whereas Mn²⁺ activated this enzyme, but both Zn²⁺ and Mn²⁺ activated kynurenine aminotransferase. Co²⁺ and Cu²⁺ inhibited both enzymes. This inhibition is attributed to the blocking and inactivation of the -SH groups of these enzymes and may be due to the adequate sequence of the -SH groups in both enzymes for Cu²⁺ or Co²⁺ action. This is in contrast to the inadequate sequence of these groups in kynurenine aminotransferase for Zn²⁺ action. The decreasing order by which these metal ions inhibit (a) kynurenine hydrolase is Cu²⁺ > Co²⁺ > Zn²⁺, and (b) kynurenine aminotransferase is Cu²⁺ > Co²⁺. The decreasing order of the per cent activation of the aminotransferase enzyme is Mn²⁺ > Zn²⁺. These decreasing orders fall into a more reasonable order approximating the order of complex stability of these metal ions.     

EFFECTS OF ENDURANCE TRAINING ON SUPEROXIDE DISMUTASE ACTIVITY,CONTENT AND mRNA EXPRESSION IN RAT MUSCLE#

1. The purpose of the present study was to investigate the changes in superoxide dismutase (SOD) isoenzyme (Mn²⁺-SOD and Cu²⁺, Zn²⁺-SOD) activities, contents and mRNA expressions in rat skeletal muscle during endurance training and a single bout of exercise. 2. Thirty-eight male Wistar rats were divided into untrained (U) and trained (T) groups. The T group rats were treadmill-trained for 9 weeks. The activity, content and mRNA expression of Mn²⁺-SOD and Cu²⁺, Zn²⁺-SOD were determined in the soleus muscle of each rat. 3. Mn²⁺-SOD activity and content in the T group were significantly higher than in the U group, both at rest (22 and 21%, respectively) and after exercise (24 and 46%, respectively), while a single bout of exercise affected neither the activity nor content of Mn²⁺-SOD in either group. 4. The content of Cu²⁺, Zn²⁺-SOD in both groups was not different at rest and after exercise, although its activity at rest was significantly higher in the T group than in the U group (by 29%). 5. After exercise, the expression of Mn²⁺-SOD mRNA was markedly attenuated only in the U group (49%); the expression of Cu²⁺, Zn²⁺-SOD mRNA was not influenced by exercise. 6. Our results suggest that adequate endurance training increases both the activity and content of Mn²⁺-SOD and that untrained rats are rather susceptible to oxidative stress during physical exercise. It thus appears that Mn²⁺-SOD provides a reliable index of physical training. 7. The results obtained in the present study also suggest that muscle has the capacity of responding to training in such a manner as to reduce the potential harm arising from the accumulation of oxygen free radicals resulting from enhanced metabolic activity.     

The influence of diethylenetriaminepentaacetate on the synthesis of DNA, RNA and proteins in the regenerating rat liver

Administration of toxic doses of Ca-DTPA (diethylenetriaminepentaacetate) after partial hepatectomy inhibits the synthesis of DNA, RNA and proteins in the regenerating rat liver. Zn-DTPA proved to be ineffective. Impairment by Ca-DTPA of DNA synthesis can be completely restored by subsequent joint administration of Zn²⁺ and Mn²⁺. The dependence of the inhibitory action of Ca-DTPA on dosage and on the time of its administration is consistent with the assumption that the inhibition of DNA synthesis is not the primary reaction but the consequence of an impaired synthesis of proteins which is tentatively ascribed to a disturbed conformation of RNA due to removal of Zn²⁺ and Mn²⁺. Parallel studies on cell-free systems revealed a direct inhibitory action of Ca- and Zn-DTPA on the syntheses. The different activity pattern in vitro and in vivo is explained by the assumption that in the former case the inhibitory action is partly due to the formation of ternary complexes; a mechanism which is not operative in vivo, because the distribution of the chelates is confined to the extracellular space.     

Correlation of the physicochemical properties of metal ions with their activation and inhibition of human erythrocytic δ-aminolevulinic acid dehydratase (ALAD) in vitro

Using normal adult human whole blood hemolysates this study determined, in a dose-response fashion, the in vitro effects of Na⁺, Mg²⁺, Al³⁺, Mn²⁺, Cu²⁺, Ag⁺, Zn²⁺, Cd²⁺, Hg²⁺, Ga³⁺, In³⁺, Sn²⁺, Sn⁴⁺, and Pb²⁺ on normal erythrocytic ALAD. The effects of these 14 metal ions on erythrocytic ALAD 50% inhibited by Pb²⁺ were also determined as was the ability of a maximum stimulatory concentration of Zn²⁺ to prevent or reverse the effects of these metal ions on erythrocytic ALAD. The effects of these metal ions were then classified in terms of their oxidation state, characteristic coordination number, coordination geometry, and hard and soft Lewis acid characteristics in order to determine the physical and chemical properties associated with the ability of a metal ion to activate or inhibit erythrocytic ALAD and whether these properties are unique to a single metal ion. Preincubation studies established Zn²⁺ to be the only metal ion to both activate erythrocytic ALAD and to prevent or reverse the Pb²⁺-induced inhibition of erythrocytic ALAD in vitro even after prolonged contact with the enzyme. Therefore future investigations of the use of nontoxic salts of Zn²⁺ as a prophylactic agent or therapeutic adjunct in the prevention or treatment of lead poisoning with its possibly toxic accumulation of ALA are proposed.     

THERMOPHILIC AMINOPEPTIDASES FROM BAC. STEAROTHERMOPHILUS.

Neutron activation analysis of AP I isolated in the presence of 10^-3 M Co²⁺ indicated 20.4 g atoms cobalt and 2.5 g atoms zinc bound per molecule (API _{isol. co, Zn}) The apoenzyme contains 0.90 g atoms cobalt, and after recombination of the apoenzyme in the presence of 10^-3 M Co²⁺ or Zn²⁺, 17.8 g atoms cobalt (AP I_rec.Co) or 0.74 g atoms cobalt and 16.0 g atoms zinc (AP I_rec.Zn), respectively, are bound per molecule. The AP Irec.Zn has only 5% of the activity of the AP I_isol.Co,Zn but is more thermostable in metal free (Co²⁺ or Zn²⁺) buffer. The zinc atoms are more strongly bound in the zinc enzyme.     

Species and agonist dependent zinc modulation of endogenous and recombinant ATP-gated P2X7 receptors

Zinc (Zn²⁺) and copper (Cu²⁺) are key signalling molecules in the immune system and regulate the activity of many ion channels. Both Zn²⁺ and Cu²⁺ potently inhibit rat P2X₇ receptors via a binding site identified by mutagenesis. Here we show that extracellular Cu²⁺ also potently inhibits mouse P2X₇ receptors. By contrast, the receptor expression system and agonist strongly influence the action of extracellular Zn²⁺ at mouse P2X₇ receptors. Consistent with previous reports, Zn²⁺ inhibits recombinant rat P2X₇ receptors. However, recombinant mouse P2X₇ receptors are potentiated by Zn²⁺ when activated by ATP⁴⁻ but inhibited when stimulated with the ATP analogue BzATP⁴⁻. Endogenous murine macrophage P2X₇ receptors are not modulated by Zn²⁺ when stimulated by ATP⁴⁻ however Zn²⁺ inhibits BzATP⁴⁻ mediated responses. In summary, these findings provide a fundamental insight into the differential actions of Zn²⁺ and Cu²⁺ between different P2X₇ receptor species.     

Zinc regulates the dopamine transporter in a membrane potential and chloride dependent manner

The dopamine transporter (DAT), a membrane protein specifically expressed by dopaminergic neurons and mediating the action of psychostimulants and dopaminergic neurotoxins, is regulated by Zn²⁺ which directly interacts with the protein. Herein, we report a host-cell-specific direction of the Zn²⁺ effect on wild type DAT. Whereas low μmolar Zn²⁺ decreased dopamine uptake by DAT expressing HEK293 cells, it stimulated uptake by DAT expressing SK-N-MC cells. Inhibition or stimulation was lost in a DAT construct without the binding site for Zn²⁺. Also reverse transport was differentially affected by Zn²⁺, dependent on whether the DAT was expressed in HEK293 or SK-N-MC cells. Pre-treatment of DAT expressing cells with phorbol-12-myristate-13-acetate, an activator of protein kinase C, attenuated the inhibitory effect of Zn²⁺ on uptake in HEK293 cells and increased the stimulatory effect in SK-N-MC cells. Patch-clamp experiments under non-voltage-clamped conditions revealed a significantly higher membrane potential of HEK293 than SK-N-MC cells and a reduced membrane potential after phorbol ester treatment. Lowering chloride in the uptake buffer switched the stimulatory effect of Zn²⁺ in SK-N-MC cells to an inhibitory, whereas high potassium depolarization of HEK293 cells switched the inhibitory effect of Zn²⁺ to a stimulatory one. This study represents the first evidence that DAT regulation by Zn²⁺ is profoundly modulated by the membrane potential and chloride.     

Adenosine regulation of canine cardiac adenylate cyclase

Adenosine has a biphasic, [Mg²⁺]-dependent effect on the catalytic activity of dog heart adenylate cyclase. In the presence of 0.5 mM Mg²⁺, adenosine stimulated cyclic AMP formation, but when the cyclase was activated with 4 mM Mg²⁺ plus 0.5 mM Mn²⁺, adenosine inhibited catalytic activity in a dose-dependent fashion. Adenine, 3'-deoxyadenosine and selected purine-modified adenosine analogs stimulated the enzyme, whereas 2'-deoxyadenosine, 5'-deoxyadenosine and adenine-α-l-lyxofuranoside mimicked the inhibitory effect of adenosine on the Mg²⁺ plus Mn²⁺ stimulated enzyme. These results are consistent with the ‘two receptor’ model of Londos and Wolff [C. Londos and J. Wolff, Proc. natn. Acad. Sci. U.S.A.74, 5482 (1978)], but they raise the possibility of subtle organ and species differences in the chemical determinants of adenosine binding. Adenosine in both intracellular and extracellular compartments may modulate adenylate cyclase activity in the beating heart, in addition to its putative role in the regulation of coronary vascular resistance.     

Solid phase synthesis of the retro viral nucleocapsid protein NCp10 of Moloney Murine Leukaemia virus and related “zinc-fingers” in free SH forms

The core of retroviruses contains a highly conserved, low molecular weight, basic protein that binds nucleic acids and is essential for genomic RNA packaging. The 56 amino acid protein, NCp10, of Moloney Murine Leukaemia virus (MoMuLV) has the CysX₂CysX⁴HisX⁴Cys zinc finger-like motif shared by all retrovirus nucleocapsid proteins. The native protein and five modified peptides containing the zinc binding domain were synthesized by solid phase in order to investigate the structural and biochemical role of Zn²⁺ chelation in MoMuLV NCp10 activity. The purity of the synthetic molecules was verified by HPLC and their sequences were confirmed by amino acid analysis and sequencing in the case of NCp10. Thiol dosage agreed with the theoretical value of free cysteine for all these molecules. Fluorescence measurements performed on synthetic NCp10 and zinc finger fragments showed that the tryptophan quantum yield was Zn²⁺ -dependent allowing a 1:1 stoichiometry for the complex to be determined. The apparent affinity constant of NCp10 for the metal was estimated to be superior to 10⁶ M^-1. The synthetic protein, in the presence of Zn²⁺ ions, possesses all the biological properties of NCp10 isolated from virions. It catalyzes both the MoMuLV RNA dimerization and the annealing of the replication primer tRNA^Pro onto MoMuLV RNA.