Zinc-binding proteins detected by protein blotting

The Western blotting technique was used for the detection of zinc-binding proteins. Proteins were separated electrophoretically on 15% polyacrylamide-sodium dodecyl sulfate minigels, the gels were soaked in a reduction buffer, and the proteins were transferred to nitrocellulose filters. Zinc-binding proteins were probed with radioactive zinc (65Zn) and were detected by autoradiography. This technique allows the detection of as little as 20 to 100 pmol of zinc metalloproteins.     

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.     

Transferrin function in zinc absorption and transport.

Blood was drawn from the portal vein of rats immediately after the insertion of 65Zn into the duodenal lumen. Analysis of the plasma from the portal blood demonstrated that the major portion of absorbed 65Zn was bound to transferrin. When apotransferrin was incubated with 65Zn-labeled basolateral plasma membranes, 60% of the isotope was removed from the membrane. Zinc-free albumin prepared with histidine removed 25% of the 65Zn from labeled membranes and histidine had no effect. These results demonstrate that transferrin functions in the absorption and transport of zinc.     

The dynamic nature of zinc availability from foods in vivo

This paper presents a review of data from animal experiments demonstrating that the relative availability of zinc from foods is affected by an interaction between the source and amount of zinc consumed. Zinc availability from foods was determined by whole body counting after feeding 65Zn-labeled meals containing varying amounts of zinc. Relative to availability from zinc chloride, zinc availability from foods such as chicken, milk, and peanut butter was greater when determined using 98 rather than 16 micrograms zinc in the meal. In rats fed the higher dose of zinc, there were greater differences in zinc availability among the 15 foods studied, and zinc availability was greater from some foods than from zinc chloride. After an in vitro enzymatic digestion, neither zinc solubility nor the partitioning of zinc between low and high molecular weight substances was useful for predicting zinc availability in vivo. These data indicate that zinc availability from food is not a constant proportion of availability from a zinc salt. In view of the dynamic nature of zinc availability in vivo, the inability to accurately simulate different physiological responses to varying quantities of certain foods may limit the usefulness of in vitro methods.     

Limited Hydrolysis of Ricin D with Alkaline Protease from Bacillus subtilis

Streptomyces naraensis was inoculated into 100 ml of culture broth, containing 50 µCi of ⁶⁵Zn, diluted with ZnCl₂ solution to make 10^-4 m Zn²⁺ ion, at 27°C for 5 days with shaking. ⁶⁵Zn-labeled neutral proteinase from Streptomyces naraensis was prepared by the method described previously. The preparation was homogeneous by disc electrophoresis and contained 1 g-atom of zinc per mole of enzyme in calculation by radioactivity.

It was suggested that the protein-bound zinc of neutral proteinase was not essential for enzymatic activity. Thus, this zinc was an essential component for the higher order structure of the protein, and the removal of zinc treated with EDTA* inactivated the enzyme. The enzymatic activity was maintained in the presence of calcium ion.     

Evidence for zinc binding by two structural proteins of Plodia interpunctella granulosis virus.

Workers in our laboratory previously reported the possibility of cation involvement in the in vitro dissociation of the Plodia interpunctella granulosis virus nucleocapsids (K. A. Tweeten, L. A. Bulla, Jr., and R. A. Consigli, J. Virol. 33:866-876, 1980; M. E. Wilson and R. A. Consigli, Virology 143:516-525, 1985). The current study found zinc associated with both granulosis virus nucleocapsids and granulin by atomic absorption analysis. A blotting assay with 65Zn2+ specifically identified the radioactive cation as binding to two viral structural proteins, granulin and VP12. These findings indicate that zinc may have a critical role in maintaining virus stability.     

Glycoproteomics and glycomics investigation of membrane N-glycosylproteins from human colon carcinoma cells

Aberrant glycosylation of proteins is known to profoundly affect cellular adhesion or motility of tumoral cells. In this study, we used HT-29 human colon epithelial cancer cells as a cellular model of cancer progression, as they can either proliferate or differentiate into enterocyte phenotype. A glycoproteomic approach based on Con A lectin-affinity chromatography, SDS-PAGE and MS analysis, allowed the identification of membrane N-glycoproteins from Triton X-100-solubilized proteins from membrane preparation. Among them, 65% were membrane proteins, and 45% were known to be N-glycosylated, such as alpha chains integrin and dipeptidyl isomerase IV. By lectin-blot analysis, significant changes of alpha-2,3- and alpha-2,6-sialylation of membrane glycoproteins were observed between proliferating and differentiated HT-29 cells. From these results, nano-LC-MS/MS analysis of the tryptic digests of the corresponding bands was performed and led to the identification of several transmembrane glycoproteins, like members of the solute carrier family and adhesion proteins. Finally, we compared N-glycans profiles and monosaccharide composition of proliferating and enterocyte-like HT-29 cells using MALDI-MS and GC-MS analyses of permethylated derivatives. This glycomic approach allowed to underscore significant changes in N-glycans structure, in particular the expression of atypical N-acetylglucosamine (GlcNAc)-ended N-glycans in enterocyte-like HT-29 cells.     

Metals on the move: zinc ions in cellular regulation and in the coordination dynamics of zinc proteins

Homeostatic control maintains essential transition metal ions at characteristic cellular concentrations to support their physiological functions and to avoid adverse effects. Zinc is especially widely used as a catalytic or structural cofactor in about 3000 human zinc proteins. In addition, the homeostatic control of zinc in eukaryotic cells permits functions of zinc(II) ions in regulation and in paracrine and intracrine signaling. Zinc ions are released from proteins through ligand-centered reactions in zinc/thiolate coordination environments, and from stores in cellular organelles, where zinc transporters participate in zinc loading and release. Muffling reactions allow zinc ions to serve as signaling ions (second messengers) in the cytosol that is buffered to picomolar zinc ion concentrations at steady-state. Muffling includes zinc ion binding to metallothioneins, cellular translocations of metallothioneins, delivery of zinc ions to transporter proteins, and zinc ion fluxes through cellular membranes with the result of removing the additional zinc ions from the cytosol and restoring the steady-state. Targets of regulatory zinc ions are proteins with sites for transient zinc binding, such as membrane receptors, enzymes, protein–protein interactions, and sensor proteins that control gene expression. The generation, transmission, targets, and termination of zinc ion signals involve proteins that use coordination dynamics in the inner and outer ligand spheres to control metal ion association and dissociation. These new findings establish critically important functions of zinc ions and zinc metalloproteins in cellular control.     

Zinc uptake by rice,as affected by metabolic inhibitors and competing cations

Summary The effects of zinc application on zinc uptake, distribution and translocation in maize and barley grown in zinc deficient soil with high pH and high calcium content were studied. Zn⁶⁵ content and uptake in roots, sheaths and blades of maize and barley plants increased significantly with increased levels of zinc application. The sheaths contained highest Zn⁶⁵ content followed by roots and blades. The distribution of total zinc, however, differed from that of radioactive zinc. The roots had the highest zinc content, followed by sheaths and blades. The two species differed very little in zinc distribution patterns. The autoradiographs of intact maize and barley plants showed that Zn⁶⁵ was fairly evenly distributed in the main and auxiliary roots, but, there was a relatively higher concentration at the root-stem junction. The Zn⁶⁵ concentration was higher in nodes than in internodes, and in young emerging leaves compared to older leaves. re]19730819 Institute of Soil Science and Isotope Laboratory Agricultural University of Norway     

Aminoacyl transfer from phenylalanyl-tRNA microinjected into Xenopus laevis oocytes.

$\text{Xenopus laevis}$ oocytes were injected with [¹⁴C] phe-tRNA and the fate of the aminoacyl moiety was studied. The radioactive phenylalanine is gradually hydrolized off the tRNA once inside the cell. The rate of deacylation of the tRNA is not affected by inhibition of cellular protein synthesis by puromycin or cycloheximide. Part of the radioactive amino acid that leaves the tRNA (30 to 65%) is transferred directly into the oocyte nascent proteins as evidenced by the fact that its incorporation into proteins is not reduced by coinjection with a large excess of [¹²C] phenylalanine. Aminoacyl transfer from injected phe-tRNA into proteins is inhibited by puromycin and cycloheximide.     

Visualization of biodistribution of Zn complex with antidiabetic activity using semiconductor Compton camera GREI

Various types of zinc (Zn) complexes have been developed as promising antidiabetic agents in recent years. However, the pharmacological action of Zn complex is not elucidated because the biodistribution of the complex in a living organism has not been studied. Nuclear medicine imaging is superior technology for the noninvasive analysis of the temporal distribution of drug candidates in living organisms. Gamma-ray emission imaging (GREI), which was developed by our laboratory as a novel molecular imaging modality, was adopted to visualize various γ-ray–emitting radionuclides that are not detected by conventional imaging techniques such as positron emission tomography and single-photon emission computed tomography. Therefore, we applied GREI to a biodistribution assay of Zn complexes. In the present study, ⁶⁵Zn was produced in the ^natCu(p,n) reaction in an azimuthal varying field cyclotron for the GREI experiment. The distribution was then noninvasively visualized using GREI after the intravenous administration of a ⁶⁵Zn-labeled di(1-oxy-2-pyridinethiolato)zinc [Zn(opt)₂], ZnCl₂, and di(l-histidinato)zinc. The GREI images were validated using conventional invasive assays. This novel study showed that GREI is a powerful tool for the biodistribution analysis of antidiabetic Zn complexes in a living organism. In addition, accumulation of ⁶⁵Zn in the cardiac blood pool was observed for [Zn(opt)₂], which exhibits potent antidiabetic activity. These results suggest that the slow elimination of Zn from the blood is correlated to the antidiabetic activity of [Zn(opt)₂].     

Effect of bile/pancreatic secretions on absorption of radioactive or stable zinc

Biliary/pancreatic (B/P) secretions are a major component of endogenous secretions, and endogenously secreted Zn is a primary means of Zn homeostasis. This study examined whether B/P fluid alters the absorption/reabsorption of Zn and, in doing so, whether this contributes to homeostatic control of Zn. Animal experiments utilized rats fed 10 or 300 μg Zn/kg diet. An open-ended gut perfusion study in which⁶⁵Zn-labeled B/P fluid or⁶⁷Zn-labeled and digested diet found significantly decreased Zn absorption from B/P fluid. Although Zn absorption from both sources was less in animals fed diets higher in Zn, there was no interaction of treatment and diet. Further studies utilizing cultured human colon carcinoma cells (CACO-2) as in vitro models of gut enterocytes found that the presence of B/P fluid significantly decreased Zn retention and/or transport and resulted in a redistribution of cellular Zn after 1200 min of incubation. These studies show that a substance in B/P fluid can decrease the absorption of Zn and also suggest that dietary Zn and Zn associated with B/P secretions are absorbed from distinct pools. However, the lack of an interactive effect with diet, and the amount of time required to see differences in CACO-2 cells, suggest that differences in absorption are not a major contributor to Zn homeostasis. US Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination.     

Metallothionein transfers zinc to mitochondrial aconitase through a direct interaction in mouse hearts

Previous studies have shown that in a cell-free system, metallothionein (MT) releases zinc when the environment becomes oxidized and the released zinc is transferred to a zinc-binding protein if such a protein is present. However, it is unknown whether and how zinc transfers from MT to other proteins in vivo. The present study was undertaken to test the hypothesis that if zinc transfer from MT to other proteins occurs in vivo, the transfer would proceed through a direct interaction between MT and a specific group of proteins. The heart extract obtained from MT-null mice was incubated with 65Zn-MT or 65ZnCl2 and the proteins receiving 65Zn were separated by blue-native PAGE (BN-PAGE) or sodium dodecyl sulfate-PAGE (SDS-PAGE), and detected by autoradiography. A unique 65Zn-binding band was observed from the 65Zn-MT-incubated, but not the 65ZnCl2-incubated preparation. The analysis using matrix assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry revealed that mitochondrial aconitase (m-aconitase) was among the proteins accepting Zn directly from Zn-MT. The m-aconitase, not the cytosolic aconitase (c-aconitase), was co-immunoprecipitated with MT. This study demonstrates that MT transfers zinc to m-aconitase through a direct interaction.     

Zinc retention differs between primary and transformed cells in response to zinc deprivation

Previous studies in our laboratory have demonstrated that reducing the availability of zinc with the extracellular metal chelator DTPA (diethylenetriaminepentaacetate) enhances, rather than inhibits, the thyroid hormone induction of growth hormone mRNA in GH3 rat anterior pituitary tumor cells. To understand the actions of the chelator on cellular zinc status, we observed the effects of DTPA on ⁶⁵Zn uptake and retention. DTPA reduced the uptake of ⁶⁵Zn by GH3 cells from the medium, but when GH3 cells were prelabeled with ⁶⁵Zn, it resulted in greater retention of the isotope. In primary hepatocytes, DTPA both reduced the uptake of ⁶⁵Zn from the medium and increased efflux from prelabeled cells. To investigate this difference, we studied the effects of DTPA on radioactive zinc flux in the H4IIE (rat hepatoma), MCF-7 (human breast cancer) and Hs578Bst (nontransformed human mammary) cell lines and in rat primary anterior pituitary cells. DTPA reduced the uptake of ⁶⁵Zn in all cell lines examined. DTPA increased the retention of ⁶⁵Zn in prelabeled H4IIE, MCF-7 and Hs578Bst cells but reduced it in primary pituitary cells. Time course experiments showed that ⁶⁵Zn efflux is shut down rapidly by DTPA in transformed cells, whereas the chelator causes greater efflux from primary hepatocytes over the first 6 h. Experiments with ¹⁴C-labeled DTPA confirmed that this chelator does not cross cell membranes, showing that it operates entirely within the medium. Expression of ZnT-1, the efflux transporter, was not affected by DTPA in H4IIE cells. Thus, zinc deprivation enhanced zinc retention in established cell lines but increased efflux from primary cells, perhaps reflecting differing requirements for this mineral.     

Poly(ADP-ribose)polymerase: a novel finger protein.

By Energy Dispersive X-ray fluorescence we have determined that calf thymus poly(ADP-ribose) polymerase binds two zinc ions per enzyme molecule. Using 65Zn (II) for detection of zinc binding proteins and polypeptides on western blots, we found that the zinc binding sites are localized in a 29 kd N-terminal fragment which is included in the DNA binding domain. Metal depletion and restoration experiments proved that zinc is essential for the binding of this fragment to DNA as tested by Southwestern assay. These results correlate with the existence of two putative zinc finger motifs present in the N-terminal part of the human enzyme. Poly(ADP-ribose)polymerase fingers could be involved in the recognition of DNA strand breaks and therefore in enzyme activation.     

Molecular aspects of human cellular zinc homeostasis: redox control of zinc potentials and zinc signals

Zinc(II) ions are essential for all forms of life. In humans, they have catalytic and structural functions in an estimated 3,000 zinc proteins. In addition, they interact with proteins transiently when they regulate proteins or when proteins regulate cellular zinc re-distribution. As yet, these types of zinc proteins have been explored poorly. Therefore the number of zinc/protein interactions is potentially larger than that given by the above estimate. Confronted with such a wide range of functions, which affect virtually all aspects of cellular physiology, investigators have begun to elucidate the molecular mechanisms of cellular homeostatic control of zinc, especially the functions of transporter, sensor, and trafficking proteins, such as metallothioneins, in providing the correct amounts of zinc ions for the synthesis of zinc metalloproteins. The sulfur-containing amino acid cysteine in proteins has an important role in the cellular mobility of zinc ions. Sulfur-coordination environments provide sufficiently strong interactions with zinc ions; they can undergo fast ligand-exchange; and they can serve as molecular redox switches for zinc binding and release. For the cellular functions of zinc, the free zinc ion concentrations (zinc potentials, pZn = −log[Zn²⁺]) and the zinc buffering capacity are critically important parameters that need to be defined quantitatively. In the cytoplasm, free zinc ions are kept at picomolar concentrations as a minute fraction of the few hundred micromolar concentrations of total cellular zinc. However, zinc ion concentrations can fluctuate under various conditions. Zinc ions released intracellularly from the zinc/thiolate clusters of metallothioneins or secreted from specialized organelles are potent effectors of proteins and are considered zinc signals. The cellular zinc buffering capacity determines the threshold between physiological and pathophysiological actions of zinc ions. When drugs, toxins, other transition metal ions or reactive compounds compromise zinc buffering, large zinc ion fluctuations can injure cells through effects on redox biology and interactions of zinc ions with proteins that are normally not targeted.

Cellular zinc and redox buffering capacity of metallothionein/thionein in health and disease

Zinc is involved in virtually all aspects of cellular and molecular biology as a catalytic, structural, and regulatory cofactor in over 1000 proteins. Zinc binding to proteins requires an adequate supply of zinc and intact molecular mechanisms for redistributing zinc ions to make them available at the right time and location. Several dozen gene products participate in this process, in which interactions between zinc and sulfur donors determine the mobility of zinc and establish coupling between cellular redox state and zinc availability. Specifically, the redox properties of metallothionein and its apoprotein thionein are critical for buffering zinc ions and for controlling fluctuations in the range of picomolar concentrations of "free" zinc ions in cellular signaling. Metallothionein and other proteins with sulfur coordination environments are sensitive to redox perturbations and can render cells susceptible to injury when oxidative stress compromises the cellular redox and zinc buffering capacity in chronic diseases. The implications of these fundamental principles for zinc metabolism in type 2 diabetes are briefly discussed.     

Isolation of a novel rat testicular metalloprotein binding cadmium and zinc

Various testicular metal-binding proteins having apparent mol wt in the range of 10–30 kD have been demonstrated by gel filtration of¹⁰⁹Cd- or⁶⁵Zn-labeled cytosol, but in no case has a purified metalloprotein been isolated that contains stoichiometric amounts of the metal. The purpose of this work was to purify from rat testes a testes-specific 30 kD Cd-binding protein (Cd-testin) following in vitro addition of¹⁰⁹Cd to testis cytosol. Conventional purification methods similar to those used for purification of metallothionein could not be used because Cd was not retained in stoichiometric amounts by the 30 kD species when these methods were employed. However, using ammonium sulfate fractionation, hydrophobic interaction and gel filtration chromatography, a 30 kD protein containing 2.6 mol of Cd/ mol of protein was isolated. Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the isolated protein contained one major polypeptide with a mol mass of 22 kD and a pI of 4.6 (22 kD/pI 4.6) and two minor polypeptides (16 kD/pI 4.6 and 10±4 kD/pI 6.3) Two-dimensional gel electrophoresis demonstrated that the 22 kD species is a major low mol mass (<60 kD) protein in rat testic cytosol. The 22 kD protein was not detectable in cytosol of rooster testis, a tissue that is insensitive to Cd-induced damage and devoid of the 30 kD Cd-binding protein. Gel filtration and hydrophobic interaction chromatography of¹⁰⁹Cd- and⁶⁵Zn-labeled cytosol demonstrated that¹⁰⁹Cd and⁶⁵Zn cochromatography with the 30 kD protein. The function of this novel 30 kD testicular metal-binding protein is not known, but our work and other studies suggest that its occurrence in testes is linked to the production of a unique 22 kD polypeptide.