Effect of magnesium on the properties of zinc alkaline phosphatase.

Alkaline phosphatase of Escherichia coli, isolated by procedures which do not alter its intrinsic metal content, contains 4.0 +/- 0.3 g-atoms of tightly bound zinc per mole (Kd less than 1 muM) and 1.3 +/- 0.2 g-atoms of magnesium per mole (Bosron, W.F., Kennedy, F.S., and Vallee, B.L. (1975), Biochemistry 14, 2275-2282). Importantly, the binding of magnesium is dependent both upon pH and zinc content. Hence, the failure to assign the maximal magnesium stoichiometry to enzyme isolated by conventional procedures may be considered a consequence of the conditions chosen for optimal bacterial growth and purification of the enzyme which are not the conditions for optimal binding of magnesium to alkaline phosphatase. Under the conditions employed for the present experimental studies, a maximum of six metal sites are available to bind zinc and magnesium, i.e., four for zinc and two for magnesium. Magnesium alone does not activate the apoenzyme, but it regulates the nature of the zinc-dependent restoration of catalytic activity to apophosphatase, increasing the activity of enzyme containing 2-g-atoms of zinc five-fold and that of enzyme containing 4-g-atoms of zinc 1.4-fold. Moreover, hydrogen-tritium exchange reveals the stabilizing effects of magnesium on the structural properties of phosphatase. However, neither the KM for substrate nor the phosphate binding stoichiometry and Ki are significantly altered by magnesium. Hence, magnesium, which is specificially bound to the enzyme, both stabilizes the dynamic protein structure and regulates the expression of catalytic activity by zinc in alkaline phosphatase.     

Studies on the role of zinc in parturition in the rat.

1. Pregnant rats were fed either low (less than 1 p.p.m.) Zn or control (40 p.p.m. Zn) diets from day 10 of gestation. They were killed at intervals during the last 96 h preceding the normal time for onset of parturition, and differences in plasma progesterone, oestradiol-17 beta and ovarian 20 alpha-hydroxysteroid dehydrogenase were assessed. 2. Gestation was prolonged in Zn-deficient rats. 3. Although the preparturient decline in plasma progesterone began at the same time in all groups, at term, plasma progesterone concentration in Zn-deficient rats remained significantly higher than in normal females. 4. Induction of ovarian 20 alpha-hydroxysteroid dehydrogenase activity was delayed by about 8 h by Zn deficiency. This delay was not observed if prostaglandin F2 alpha was injected previously. 5. The results suggest a Zn-dependent step(s) in uterine synthesis and/or release of prostanoids.     

Interaction of zinc and hemoglobin: binding of zinc and the oxygen affinity.

Stripped human hemoglobin was shown to have a high apparent zinc association constant of 1.3 X 10(7) M-1 with a stoichiometry of one zinc for every two hemes. The saturation of this site produces a dramatic 3.7-fold increase in the oxygen affinity. The effect of zinc on the oxygen affinity is interrelated with the interaction of 2,3-diphosphoglyceric acid (2,3-DPG) and hemoglobin. Thus, a smaller zinc effect is observed in the presence of added 2,3-DPG. Information about the location of the zinc-binding site responsible for the increased oxygen affinity has been obtained by comparing the binding of zinc to various hemoglobins. Blocking the beta93 sulfhydryl group decreases the apparent zinc association constant by an order of magnitude. The substitution of histidine-beta143 in hemoglobin Abruzzo [beta143 (H21) His leads to Arg] and hemoglobin Little Rock [beta143 (H21) His leads to Gln] decreases the apparent zinc association constant by two orders of magnitude. The substitution of histidine-beta143 by other amino acids and the reaction of the beta93 sulfhydryl group are known to produce dramatic increases in the oxygen affinity. The binding of zinc to one or both of these amino acids can, therefore, explain the zinc-induced increase in the oxygen affinity.     

Effect of replacement of "zinc finger" zinc on estrogen receptor DNA interactions.

Exposure of bovine estrogen receptor to the metal chelators EDTA and 1,10-phenanthroline results in a loss of nonspecific DNA binding, presumably because of the removal of "zinc finger" zinc. Nonspecific DNA binding, as measured by a DNA-cellulose binding assay, can be restored by dialysis of the aporeceptor against buffer containing zinc, cadmium, and cobalt but not with buffer containing copper or nickel. More detailed studies were carried out using a bacterially expressed polypeptide encompassing the DNA binding domain of the human estrogen receptor. Apopolypeptide fails to bind DNA specifically, as measured by mobility shift assay using a consensus estrogen response element hexamer containing oligonucleotide, but DNA binding was restored by dialysis of the apopolypeptide against buffer containing zinc, cadmium, and cobalt but not with buffer containing copper or nickel. Dissociation constants of zinc- and cadmium-reconstituted polypeptide for the estrogen response element hexamer (66 and 48 nM, respectively) are virtually indistinguishable from native polypeptide (Kd = 48 nM) whereas cobalt-reconstituted polypeptide has a lower affinity (Kd = 720 nM). However, native, zinc-, cadmium-, and cobalt-reconstituted polypeptides gave identical results in a methylation interference assay. Competition experiments with zinc and copper or nickel suggest that copper and nickel are able to bind to zinc finger residues but do so nonproductively. The relative affinities copper greater than cadmium greater than zinc greater than cobalt greater than nickel for the polypeptide were determined by a zinc blot competition assay. The ability of cadmium and cobalt to substitute for zinc in the zinc fingers demonstrates a structural "flexibility" in the DNA binding domain as each of these metals has slightly different ionic radii. On the other hand, subtle differences in DNA binding affinity and/or specificity could exist, which may not be detectable here. Also, the ability of metals to substitute for zinc in the DNA binding domain suggests that metal substitution in these zinc fingers in vivo may be of relevance to the toxicity and/or carcinogenicity of some of these metals.     

Effects of zinc ion on the conformation of antigenic determinants on insulin.

Comparison of c.d. spectra of Zn-insulin with Zn2+-free insulin demonstrated significant differences. It has been proposed that these differences are due to either changes in the structure of insulin monomers within aggregated insulins or the results of insulin aggregation. The effect of Zn2+ on the immunological activity of insulin indicated that the antigenic determinants of insulin were also altered. The apparent loss of immunological activity of monoiodotyrosylinsulin was demonstrated to be due to the loss of Zn2+ rather than the substitution of iodine. The immunological activity of Zn-insulin and Zn2+-free insulin was compared in both the radioimmune and immune haemolysis-inhibition assays by using an identical population of antibodies and concentrations of inhibitor. Relative to Zn-insulin, Zn2+-free insulin had a markedly attenuated immunological activity in the immune haemolysis-inhibition assay, whereas in the radioimmune assay slightly greater immunological activity was observed with the Zn2+-free insulin. These observations are submitted as evidence that the removal of Zn2+ perturbs the conformation of determinants that react with antibodies operative in the immune haemolysis-inhibition assay (immune haemolysis determinants) and has a minimal effect on the conformation of determinants that react with antibodies operative in the radioimmune assay (radioimmune determinants).     

Oral iron and the bioavailability of zinc.

The oral bioavailability of zinc was studied in nonpregnant adults before and 24 hours after two weeks of oral supplementation with iron and folic acid. Bioavailability was greatly reduced, and the shape of the plasma curves suggested that this was due to impairment of the intestinal absorption of zinc. The findings suggest that the reduced bioavailability of zinc occurs because of interelement competition in the bowel wall. This might induce zinc depletion.     

Changes in zinc ligation promote remodeling of the active site in the zinc hydrolase superfamily.

The zinc hydrolase superfamily is a group of divergently related proteins that are predominantly enzymes with a zinc-based catalytic mechanism. The common structural scaffold of the superfamily consists of an eight-stranded beta-sheet flanked by six alpha-helices. Previous analyses, while acknowledging the likely divergent origins of leucine aminopeptidase, carboxypeptidase A and the co-catalytic enzymes of the metallopeptidase H clan based on their structural scaffolds, have failed to find any homology between the active sites in leucine aminopeptidase and the metallopeptidase H clan enzymes. Here we show that these two groups of co-catalytic enzymes have overlapping dizinc centers where one of the two zinc atoms is conserved in each group. Carboxypeptidase A and leucine aminopeptidase, on the other hand, no longer share any homologous zinc-binding sites. At least three catalytic zinc-binding sites have existed in the structural scaffold over the period of history defined by available structures. Comparison of enzyme-inhibitor complexes show that major remodeling of the substrate-binding site has occurred in association with each change in zinc ligation in the binding site. These changes involve re-registration and re-orientation of the substrate. Some residues important to the catalytic mechanism are not conserved amongst members. We discuss how molecules acting in trans may have facilitated the mutation of catalytically important residues in the active site in this group.     

Proton and zinc effects on HERG currents.

The proton and Zn2+ effects on the human ether-a-go-go related gene (HERG) channels were studied after expression in Xenopus oocytes and stable transfection in the mammalian L929 cell line. Experiments were carried out using the two-electrode voltage clamp at room temperature (oocytes) or the whole-cell patch clamp technique at 35 degrees C (L929 cells). In oocytes, during moderate extracellular acidification (pHo = 6.4), current activation was not shifted on the voltage axis, the time course of current activation was unchanged, but tail current deactivation was dramatically accelerated. At pHo < 6.4, in addition to accelerating deactivation, the time course of activation was slower and the midpoint voltage of current activation was shifted to more positive values. Protons and Zn2+ accelerated the kinetics of deactivation with apparent Kd values about one order of magnitude lower than for tail current inhibition. For protons, the Kd values for the effect on tail current amplitude versus kinetics were, respectively, 1.8 microM (pKa = 5.8) and 0.1 microM (pKa = 7.0). In the presence of Zn2+, the corresponding Kd values were, respectively, 1.2 mM and 169 microM. In L929 cells, acidification to pHo = 6.4 did not shift the midpoint voltage of current activation and had no effect on the time course of current activation. Furthermore, the onset and recovery of inactivation were not affected. However, the acidification significantly accelerated tail current deactivation. We conclude that protons and Zn2+ directly interact with HERG channels and that the interaction results, preferentially, in the regulation of channel deactivation mechanism.     

An effect of zinc deficiency on dental caries.

Rat pups suckled by dams fed a zinc-deficient diet developed higher levels of dental caries following a caries-test challenge than pups suckled by dams fed a zinc-adequate diet. The zinc deficient treatment was administered during the mineralization stage of tooth development before the molars erupted. The data shows that suboptimal zinc nutriture was associated with an increase in dental caries.Fluoride, a trace element, is recognized as a factor in preventing dental caries and its use has had a significant impact on dental health in this country. However, the possible roles of other trace elements have not been closely examined. Zinc is a trace element important to bone mineralization as well as in general nutrition.The effects on dental caries produced by the addition of zinc to rat diets have been reported (1, 2, 3). In those investigations, however, zinc supplements were added to diets that were adequate in zinc. Moreover, zinc supplements were fed after the teeth had appeared in the oral cavity. Because the critical period of tooth maturation occurs before eruption (4), post-eruptive zinc supplements probably should not markedly influence the development and, specifically, the mineralization of the tooth. Therefore, we investigated the influence of pre-eruptive zinc deficiency on caries development in rats.     

The effect of dietary zinc deficiency on the mossy fiber zinc content of the rat hippocampus. A microbeam PIXE study. Particle Induced X-Ray Emission

The effect of dietary zinc deficiency on the mossy fiber zinc content of the rat hippocampus was investigated using PIXE (Particle Induced X-Ray Emission) spectroscopy. Using the proton microbeam (60 X 60 microns), 2 mm line-scans were made on hippocampal sections and the data were expressed as absolute zinc concentrations. Values of 55 and 136 ppm (dry weight) were found for the mean background zinc level and the maximum mossy fiber zinc level, respectively, in animals fed a control diet containing 50 ppm zinc. Treatment of these animals with dithizone caused about 50% reduction in the maximum mossy fiber zinc level. Feeding a zinc-deficient diet for 28 days did not cause a decrease in the mossy fiber zinc level, however, feeding the zinc-deficient diet for 90 days reduced the maximum mossy fiber zinc level by about 30%. The results are discussed in relation to the behavioral abnormalities that have been observed in zinc-deficient animals.     

Kinetic analysis of the effect of zinc ion on the inorganic pyrophosphatase reaction.

A kinetic study is presented in which the effect of Zn(II) on yeast inorganic pyrophosphatase was quantitatively determined. A dual role model for metal ion effect, previously determined for the Mg(II)-pyrophosphatase system (O. A. Moe and L. G. Butler, 1972, J. Biol. Chem.247, 7308–7315), was applied successfully to the analysis of the kinetics for Zn(II)-pyrophosphatase and Zn(II), Mg(II)-pyrophosphatase systems. The model, assigning an activator role to free Zn(II) ion and a substrate role to the Zn(II)-pyrophosphate complex, gave an excellent fit to the data. Inhibition of the Mg(II)-pyrophosphatase system by Zn(II) was analyzed by a model in which competitive binding of the Mg(II)-pyrophosphate and Zn(II)-pyrophosphate complexes occurred at the enzyme active site, with both complexes undergoing reaction at different rates. Relative maximal velocities and enzymeligand dissociation constants for the Zn(II)-pyrophosphate complex were determined for the cases where the metal ion activator role was fulfilled by Zn(II) and Mg(II), respectively. The maximal velocity parameter showed a dependence on the nature of the activator metal ion, demonstrating that the role of the latter is associated both with the process of substrate binding and with the mechanism of catalysis. Values for all kinetic parameters are reported for an ionic strength of 0.2, pH 7.0, and 25.0 °C.