Cooperative Functions of ZnT1, Metallothionein and ZnT4 in the Cytoplasm Are Required for Full Activation of TNAP in the Early Secretory Pathway

The activation process of secretory or membrane-bound zinc enzymes is thought to be a highly coordinated process involving zinc transport, trafficking, transfer and coordination. We have previously shown that secretory and membrane-bound zinc enzymes are activated in the early secretory pathway (ESP) via zinc-loading by the zinc transporter 5 (ZnT5)-ZnT6 hetero-complex and ZnT7 homo-complex (zinc transport complexes). However, how other proteins conducting zinc metabolism affect the activation of these enzymes remains unknown. Here, we investigated this issue by disruption and re-expression of genes known to be involved in cytoplasmic zinc metabolism, using a zinc enzyme, tissue non-specific alkaline phosphatase (TNAP), as a reporter. We found that TNAP activity was significantly reduced in cells deficient in ZnT1, Metallothionein (MT) and ZnT4 genes (ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells), in spite of increased cytosolic zinc levels. The reduced TNAP activity in ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells was not restored when cytosolic zinc levels were normalized to levels comparable with those of wild-type cells, but was reversely restored by extreme zinc supplementation via zinc-loading by the zinc transport complexes. Moreover, the reduced TNAP activity was adequately restored by re-expression of mammalian counterparts of ZnT1, MT and ZnT4, but not by zinc transport-incompetent mutants of ZnT1 and ZnT4. In ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells, the secretory pathway normally operates. These findings suggest that cooperative zinc handling of ZnT1, MT and ZnT4 in the cytoplasm is required for full activation of TNAP in the ESP, and present clear evidence that the activation process of zinc enzymes is elaborately controlled.     

A histidine supplement and regulation of the zinc status in Swiss random mice

The effects of histidine on the zinc status are controversial. In mice, we studied the effects of a moderate histidine supplement on the regulation of the zinc status using subcutaneously administered⁶⁵Zn. In animals fed a zinc-adequate diet, histidine supplement did not cause changes in the zinc status (zinc concentrations,⁶⁵Zn tissue distribution, and tissue specific activities). Neither effects on the regulation of the zinc status (⁶⁵Zn retention, excretion and biological half-life) could be demonstrated. However, the combination of a low zinc diet and moderate histidine supplementation caused changes in the regulation of the zinc status (lower⁶⁵Zn retention, associated with increased fecal excretion and a shorter biological half-life), aggravating the dietary deficiency (lower bone zinc, a shift in the⁶⁵Zn tissue distribution). Reviewing the literature, it seems that only a molar histidine/zinc ration of 2,000 or higher will cause zinc deficiency.     

Uptake and accumulation of zinc in juvenile rainbow trout,Salmo gairdneri

Synopsis The toxicity of zinc to rainbow trout was determined and the 72 h median lethal concentration was found to be 2.00 mg l^–1 in freshwater, hardness 7.50 mg l^–1 as calcium. An insignificant increase in zinc concentration of internal tissues occurred in fish exposed to 1.52 mg l^–1 in freshwater for 72 h. However, there was a significant uptake of zinc by gills and the body surface. Fish exposed to 10 mg l^–1 zinc for 72 h in two-thirds sea water showed significant zinc uptake by liver, rectum and muscle, when compared to control fish. Drinking rate decreased from 1.43 to 0.26 ml kg^–1 h^–1 when zinc sulphate was added to freshwater. Trout adapted to two-thirds sea water showed no decrease in drinking, about 7 ml kg^–1 h^–1 when zinc was added to the water.     

Comparison of chromosome damage induced by three zinc compounds using human leukocyte culture

The degree of chromosome damage induced by three compounds of zinc (zinc chloride, zinc sulfate, zinc acetate) was compared in human leucocytes in vitro. Three concentrations of each salt, 3.0×10⁻⁵ M, 3.0×10⁻⁴ M, and 1.5×10⁻³ M, were added to leukocyte cultures. The cells were harvested after 48 and 72 h and chromosome spreads were prepared following a colchicine-hypotonic-fixation-air drying-Giemsa staining schedule. The end point screened was chromosome aberrations. All three salts were lethal at the highest concentration. The degree of chromosome damage was directly proportional to the concentrations used for zinc sulfate and zinc acetate but not for zinc chloride.     

Metabolism of zinc-binding ligands in rat small intestine

Studies were conducted to determine the effects of zinc deficiency and excess zinc intake on the relative⁶⁵Zn-binding activities of metallothionein (MT) and low-molecular-weight zinc-binding ligand (LMW-ZBL) in vitro and in vivo. Zinc-binding ligands of small intestine from four groups, each of five rats (normal, zinc-deficient, excess zinc injected, and excess zinc given orally), were separated by column chromatography on Sephadex G-75. The ratio of⁶⁵Zn binding activities of MT to LMW-ZBL (MT/LMW-ZBL) in zinc-deficient rats was decreased both in vitro and in vivo compared to the control. When excess zinc was administered orally,⁶⁵Zn-binding activity of MT was low in vitro and substantially increased in vivo. However, when excess zinc was injected intraperitoneally,⁶⁵Zn-binding activity of MT in vitro greatly increased, but⁶⁵Zn-binding activities of both MT and LMW-ZBL were significantly reduced in vivo as compared to the control. Based onA ₂₈₀ readings of isolated MT and densities of protein bands in disc gel electrophoresis, the⁶⁵Zn-binding activity of MT in vitro appeared to be proportional to the MT content. Hence, these data indicate that oral administration of excess zinc decreases MT whereas intraperitoneal injection of excess zinc stimulates its synthesis. Zinc deficiency has little to no effect on the intestinal MT metabolism. These results suggest that MT may be important in zinc secretion but not involved in zinc absorption; while LMW-ZBL participates both in zinc absorption and secretion.     

Metabolism of parenterally administered zinc and cadmium in livers of newborn rats

The interaction of injected zinc and cadmium with metallothionein was investigated in newborn rats. Tissues of 5-day-old rats were removed 24 h after a single injection (Sc) of saline or zinc (20 mg/kg, body wt.) or cadmium (1 mg/kg, body wt.) with 2.5 μCi of ⁶⁵Zn or ¹⁰⁹Cd or 5 μCi of [³⁵S]cysteine. Injection of zinc resulted in a 75% increase in the hepatic zinc concentration with a concomitant elevation of metallothionein (P < 0.001), zinc in metallothionein increased by 45% (P < 0.05); [³⁵S]cysteine incorporation indicated the induced synthesis of metallothionein. Injection of cadmium did not alter either metallothionein or zinc levels in liver, but cadmium in cytosol was preferentially bound to metallothionein. Neither treatment altered hepatic copper metabolism and copper in metallothionein, nor renal zinc and metallothionein levels. These data indicate that zinc injection can elevate hepatic zinc levels and induce metallothionein synthesis in newborn rats despite high basal levels; cadmium injection does not induce metallothionein synthesis, though cadmium is avidly sequestered by pre-existing metallothionein. The differences in the induction of metallothionein by these divalent cations can be explained by the differences in their binding affinities for thiol groups in intracellular metallothionein.     

Determination of relative bioavailability of zinc in a petit suisse cheese using weight gain and bone zinc content in rats as markers

The aim of the study was to determine the relative bioavailability of zinc gluconate stabilized with glycine in a Petit Suisse cheese from an infant dessert. Weight gain and bone zinc content were the nutritional responses evaluated for the diets of different zinc content: 2 ppm (basal) and 5, 10, and 30 ppm from zinc gluconate stabilized with glycine and zinc sulfate. Nonlinear regression analysis of the fitted curves for weight gain determined a relative zinc bioavailability of 100% for the Y _max ratio and 96% for Y _max/t _1/2 ratio for zinc gluconate stabilized with glycine (R ²=0.7996 for zinc sulfate and 0.8665 for zinc gluconate stabilized with glycine). The slope ratio analysis from linear regression of femur zinc determined a relative zinc bioavailability of 93% for zinc gluconate stabilized with glycine (R ²=0.8693 for zinc sulfate and 0.8307 for zinc gluconate stabilized with glycine). Zinc gluconate stabilized with glycine has similar bioavailability as zinc sulfate in a Petit Suisse cheese nutritional matrix, with the advantage that the stabilized compound does not modify the sensorial characteristics of the fortified cheese.     

Relationship between1H and13C NMR chemical shifts and the secondary and tertiary structure of a zinc finger peptide

Summary Essentially complete assignments have been obtained for the¹H and protonated¹³C NMR spectra of the zinc finger peptide Xfin-31 in the presence and absence of zinc. The patterns observed for the¹H and¹³C chemical shifts of the peptide in the presence of zinc, relative to the shifts in the absence of zinc, reflect the zinc-mediated folding of the unstructured peptide into a compact globular structure with distinct elements of secondary structure. Chemical shifts calculated from the 3D solution structure of the peptide in the presence of zinc and the observed shifts agree to within ca. 0.2 and 0.6 ppm for the backbone C^aH and NH protons, respectively. In addition, homologous zinc finger proteins exhibit similar correlations between their¹H chemical shifts and secondary structure.     

Zinc uptake by blood cells of rats in zinc deficiency and inflammation

In zinc deficiency, the function of leukocytes is impaired. However, the results of studies on the zinc concentration of blood cells in zinc deficiency are conflicting, probably in part because of technical and analytical problems. The aim of this study was to investigate, under standard conditions, the uptake of⁶⁵Zn-labeled zinc by blood cells, taken from zinc-deficient rats and from rats in which an inflammation is induced. In both conditions, the serum zinc concentration is reduced. In clinical practice, this makes it difficult to determine whether the decrease in serum zinc is the result of a real or an apparent zinc deficiency. In stress, like an inflammatory disease, the decrease of zinc reflects an apparent zinc deficiency because of redistribution of serum zinc into the liver and because of decrease in serum albumin concentration. Over 70% of the serum zinc is bound to albumin. Blood cells from zinc-deficient and control rats were isolated using a discontinuous Percoll gradient and incubated under nearly physiological conditions in a⁶⁵Zn-containing medium. A significant increase in the in vitro uptake of⁶⁵Zn-labeled zinc by the blood cells of zinc-deficient rats was seen: erythrocytes 1.3, mononuclear cells 2.0, and polymorphonuclear cells 2.6 times the control values. During inflammation, no change in⁶⁵Zn-labeled zinc uptake by erythrocytes and mononuclear cells was demonstrated after 2 d, although the serum zinc and albumin concentrations were decreased, but a small but significant increase in zinc uptake by polymorphonuclear cells was observed. This study of⁶⁵Zn uptake in vitro under standard conditions may prove of value for distinguishing in patients real zinc deficiency from apparent zinc deficiency owing to, e.g., stress, although additional experiments should be performed. A part of this study has been presented at the Meeting of The American Gastroenterological Association on May 12–18, 1990, San Antonio, TX, and has been published in abstract inGastroenterology 98 suppl., A423.     

A Binding Shift Assay for the Zinc-Bound and Zinc-Free HIV-1 Nucleocapsid Protein by Capillary Electrophoresis

Affinity capillary electrophoresis was used to detect a shift in mobility when a zinc ion binds to the highly basic nucleocapsid protein (NCp7) of HIV-1. NCp7 contains two Cys-X₂- Cys-X₄-His-X₄-Cys zinc fingers. With constant concentrations of NCp7 as a receptor and various concentrations of zinc as a ligand in the sample buffer and the electrophoresis buffer, we observed changes in electrophoretic mobilities of NCp7 protein when complexes were formed with zinc. Scatchard analysis of the mobility indicates the presence of at least two types of binding sites for zinc. At pH 6.0, one site is shown to bind zinc strongly with a binding constantK_b= 3.25 × 10⁵M⁻¹and the second site has aK_b= 1.8 × 10⁵M⁻¹. The binding of zinc to the first zinc finger decreased the affinity of zinc for the second zinc finger approximately twofold. The Hill coefficient for this negative cooperativity is 0.9. A series of NCp7 mutants were also examined in the assay to determine their ability to bind zinc. This assay affords a quick method to observe a zinc ion binding to NCp7 and to calculate binding constants.     

The effect of zinc on the biosynthesis of tryptophan,andol auxins and gibberellins in barley

The action of zinc on the growth of barley and the biosynthesis of indol compounds and gibberellin-like substances was investigated in a number of concentrations of zinc from doses stimulating growth to toxic doses. The seeds were soaked before sowing in solutions of zinc sulphate (5.10^?5 to 5.10^?1% Zn), and the plants cultivated for 7 days in water. Lower concentrations of zinc increased both plant growth and the biosynthesis of tryptophan and auxins. At the optimum concentration of 5.10^?3% Zn this increase in tryptophan amounted to 241% of the variant without zinc; in substances with an R_F corresponding to indolyacetic acid, the increase determined by the biological test, was 207% as against the variant without zinc. Higher concentrations of zinc inhibited growth, the tryptophan content was decreased to below that of the control without zinc and the auxin content also fell to below the control values. Zinc also influenced the content of gibberellin-like substances in the plants. At a concentration of 5.10^?3% Zn the increase in the growth activity in the gibberellic acid area of the chromatogram was 294% of the variant without zinc. At toxic concentrations of zinc, the content of gibberellin-like substances fell to below that of the controls. The finding that zinc acts simultaneously on the biosynthesis of auxins and gibberellins is also evidence for the common action of growth substances of various chemical types on plant growth.     

Effects of histidine on tissue zinc distribution in rats

Histidine has been reported to affect body zinc status by increasing urinary zinc excretion. The effects of experimental histidinemia on distribution of⁶⁵Zn in anesthetized rats were studied. Infusion ofl-histidine at a rate sufficient to raise plasma concentrations to approximately 2mm for 6h starting 48 h after a single intraperitoneal⁶⁵Zn injection did not alter⁶⁵Zn activities in a variety of tissues when compared with anesthetized uninfused animals. However, plasma⁶⁵Zn and erythrocyte⁶⁵Zn were decreased, and liver⁶⁵Zn was increased. If⁶⁵Zn was injected intravenously during histidine infusion, net accumulation of zinc by some tissues was increased, but uptake by others was reduced relative to uninfused animals. In all cases, however, uptake expressed relative to plasma⁶⁵Zn levels was increased when allowance was made for the more rapid fall in plasma⁶⁵Zn during histidine infusion. Similar infusions ofd-histidine produced quantitatively similar effects. Since enzymatic mechanisms and amino acid carriers would be expected to show stereoselectivity, such processes are unlikely to be involved in the zinc distribution changes described. The possibility of zinc transport by a hitherto unidentified carrier is discussed. These experiments confirm that histidinemia can affect zinc status, but any associated changes in urinary zinc excretion do not seem adequate to account for the tissue changes found.     

Regulation of the adaptation to zinc deficiency in plants

The molecular mechanisms by which plants sense their micronutrient status, and adapt to their environment in order to ensure a sufficient micronutrient supply, are poorly understood. Zinc is an essential micronutrient for all living organisms. when facing a shortage in zinc supply, plants adapt by enhancing the zinc uptake capacity. The molecular regulators controlling this adaptation were recently identified. in this mini-review, we highlight recent progress in understanding the adaptation to zinc deficiency in plants and discuss the future challenges to fully unravel its molecular basis.Key words: adaptation, zinc deficiency, biofortification, molecular regulators, plant nutritionIn an increasingly populated world, agricultural production is an essential element of social development. Agriculture is the primary source of all nutrients required for human life, and nutrient sufficiency is the basis for good health and welfare of the human population.¹ Soils with zinc deficiency are widespread in the world, affecting large areas of cultivated soils in India, Turkey, China, Brazil and Australia,^2,3 making zinc the most common crop micronutrient deficiency.⁴ In addition, risk of inadequate zinc diet and zinc malnutrition are estimated to affect one-third of the global human population, i.e., around two billion people.⁵ Most affected are people living in developing countries, where diets are rich in cereal-based foods. Cereal grains are rich in phytate, which is a potent anti-nutrient, limiting micronutrient bioavailability.⁶ Zinc deficiency in crop production can be easily ameliorated through zinc fertilization, making agronomic biofortification an important strategy,³ however in the poorer regions, the required infrastructure to provide a reliable supply of zinc fertilizers of sufficient quality, is often not available. In those situations, biofortified crops, in which the zinc status of crops is genetically improved by selective breeding or via biotechnology, offer a rural-based intervention that will more likely reach the population.⁷ Different traits can be targeted to developing such improved crops, such as plant zinc deficiency tolerance, zinc use efficiency and the accumulation of zinc in edible parts. However, insufficient knowledge on the molecular mechanisms and the regulation of the zinc homeostasis network in plants is a serious bottleneck when pursuing zinc biofortification.     

In Vitro effects of zinc on markers of bone formation

Zinc deficiency is associated with a reduced rate of bone formation that can be corrected by supplementation of the deficient diet with adequate amounts of zinc. This study was conducted to examine the effects of zinc on bone cell parameters associated with bone formation. Tibiae were removed from 19-d-old chicken embryos and incubated for 48 h in Dulbecco’s modified Eagle’s medium supplemented with antibiotics, bovine serum albumin, and HEPES. The addition of zinc (25–200 Μg/dL) to tibial cultures resulted in a concentrationdependent increase in alkaline phosphatase activity, an increase in the incorporation of proline into bone protein and an increase in the posttranslational oxidation of proline to peptidyl hydroxyproline. These effects of zinc were all diminished by the addition of 2,6-pyridine dicarboxylic acid, a chelator of zinc. The addition of either cycloheximide (10^-5M), dactinomycin (10^-8M), or hydroxyurea (10^-3M) to tibial cultures also attenuated the effects of zinc. The effect of zinc on bone cell DNA synthesis was measured by following the incorporation of³H-thymidine into DNA and by fluorometric measurement of cellular DNA content. These methods revealed that the addition of zinc to cultured tibiae resulted in a concentration-dependent increase in tibial DNA content and synthesis rate. The magnitude of the zinc-induced DNA increase was similar to the magnitude of the zinc-induced increases in alkaline phosphatase activity, proline incorporation, and hydroxyproline synthesis. Normalization of these latter responses to tibial DNA content yield data indicating that the effect of zinc on bone formation results from a zinc-induced increase in bone cell proliferation.     

Effect of extraneous zinc on calf intestinal alkaline phosphatase

The effect of extraneous zinc on calf intestinal alkaline phosphatase was studied for quick reversible binding and slow irreversible binding of zinc ions at various concentrations. Under the conditions of slow binding of zinc to CIP increasing Zn²⁺ (less than 1.0 mM, n_M/n_E 1.0 × 10⁶) inhibited enzymatic activity, and further increasing Zn²⁺ resulted in an increase of activity. For quick reversible binding of Zn²⁺, the effect on CIP activity changed at lower concentrations of substrate, indicating a complex cooperativity between Zn²⁺ and pNPP. Both protein intrinsic emission fluorescence and ANS-bound protein fluorescence, as well as circular dichroism spectra have shown that the binding of zinc ions changed the enzyme conformation, which was the reason for the changes in enzyme activity induced by extraneous zinc.     

Effect of zinc on biological half-lives of 65Zn in whole body and liver and on distribution of 65Zn in different organs of rats following nickel toxicity

This study was designed to determine the effect of zinc on the biological half-lives of ⁶⁵Zn in whole body and liver and on distribution of ⁶⁵Zn in different organs of rats following nickel toxicity. Sprague-Dawley (SD) rats received either nickel in the form NiSO₄·6H₂O at a dose of 800 mg/L in drinking water, zinc in the form of ZnSO₄·7H₂O at a dose of 227 mg/L in drinking water, and nickel plus zinc or drinking water alone for a total duration of 8 wk. All of the rats were injected with a tracer dose of 0.37 MBq ⁶⁵Zn at the end of the treatment period. The effects of different treatments were studied on biological half-lives of ⁶⁵Zn in whole body and liver and on the distribution of ⁶⁵Zn in different organs of rats. In the present study, we have noted that nickel treatment to normal rats caused a significant decrease in the slow component (Tb₂) in liver, which improved following zinc supplementation. Nickel administration to normal-diet-fed animals caused significant lowering in the percentage uptake of ⁶⁵Zn values in the brain, liver, and intestine. However, the administration of zinc to nickel-treated rats improved the status of ⁶⁵Zn in different organs. The Tb₂ in the liver and the percentage uptake of ⁶⁵Zn values elevated following zinc supplementation to nickel-treated rats.     

Transformation and movement of zinc in an alkali soil and their influence on the yield and uptake of zinc by rice and wheat crops

Summary In the summer of 1980, a field experiment was started to evaluate the direct and residual effect of applied zinc (as zinc sulphate) on the yield and chemical composition of rice and wheat grown as crops in sequence, on an alkali soil. The treatments comprised six rates of zinc 0, 2.25, 4.5, 9.0, 18.0 and 27.0 kg ha⁻¹ applied either only once to the first crop, or repeated to each successive crop in a split plot design with 4 replications. Gypsum at 14 t ha⁻¹, was applied uniformly to all plots. The results show that with respect to increase of yield and available zinc content of soil, an application of 2.25 kg ha⁻¹ zinc frequently to each crop was better than a single high dose. A major portion of the applied zinc accumulated in the 0 to 10 cm soil layer; the movement of zinc to lower layers was negligible. Zinc applications increased the concentration of exchangeable < complexed < amorphous sesquixoides-bound zinc > crystalline sesquioxide-bound zinc fractions. Amorphous sesquixoides bound the major portion of the applied zinc compared to other fractions. Exchangeable and amorphous sesquioxide-bound zinc fractions contributed significantly more to zinc uptake by rice, than the other fractions. DTPA extracted zinc more readily from exchangeable and complexed fractions than from sesquioxides. Application of zinc increased the DTPA extractable zinc and hence zinc uptake by plants.     

Zinc biosorption from aqueous solution by a halotolerant cyanobacterium Aphanothece halophytica

We have investigated the characteristics of zinc biosorption by Aphanothece halophytica. Zinc could be rapidly taken up from aqueous solution by the cells with an equilibrium being reached within 15 min of incubation with 100 mg L⁻¹ ZnCl₂. The adsorbed zinc was desorbed by treatment with 10 mM EDTA. The presence of glucose, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and N,N′-dicyclohexylcarbodiimide (DCCD) did not affect the uptake of zinc. The specific uptake of zinc increased at low cell concentration and decreased when cell concentration exceeded 0.2 g L⁻¹. The binding of zinc followed Langmuir isotherm kinetics with a maximum zinc binding capacity of 133 mg g⁻¹ and an apparent zinc binding constant of 28 mg L⁻¹. The presence of an equimolar concentration of Mn²⁺, Mg²⁺, Co²⁺, K⁺, or Na⁺ had no effect on zinc biosorption, whereas Ca²⁺, Hg²⁺, and Pb²⁺ showed an inhibitory effect. The biosorption of zinc was low at a pH range from 4 to 6, but increased progressively at pH 6.5 and 7. Received: 12 December 2001 / Accepted: 11 January 2002     

Effect of excess dietary histidine on rate of turnover of65Zn in brain of rat

The effect of the chronic administration of histidine on the brain zinc level was examined in growing, male Wistar rats. Using a purified diet, the minimum zinc requirement for normal growth and normal plasma and tissue zinc levels was found to be around 10 ppm. Given this zinc content; the diet was supplemented with 5% and 8% histidine, respectively, or with 10% glycine (as control). Brain zinc was analyzed by measuring the rate of turnover of⁶⁵Zn from 2–4 weeks after a single injection of the tracer. Feeding the diet supplemented with 5% histidine caused a small decrease in the plasma zinc concentration and a slight increase in the rate of turnover of⁶⁵Zn in the cerebrum and the cerebellum as compared to the control group. The animals fed the diet supplemented with 8% histidine became severely zinc deficient (as evidenced by a 50% reduction in the plasma zinc content), however, the rate of turnover of⁶⁵Zn in all brain regions examined was significantly decreased as compared to the control group. The results indicate that histidine has no specific complexing action on the brain zinc.