Zn-binding Ligands Research Articles

Zn isotope fractionation during uptake into marine phytoplankton: Implications for oceanic zinc isotopes

The extreme scarcity of zinc (Zn) in the euphotic zone, coupled to deep enrichments, is consistent with biological uptake at the surface and regeneration at depth. In the context of a nutrient-type depth profile so clearly shaped by uptake into phytoplankton, the growing dataset for Zn isotopes presents a challenge. These data either show very minor isotope effects associated with extreme depletion, or enrichment of the light isotopes in the upper ocean. In contrast, culturing of eukaryotes in the laboratory suggests that light Zn isotopes are preferentially taken up into diatoms and coccoliths, implying that Zn depletion at the surface should be associated with extremely heavy residual dissolved signals.Here we present the first Zn isotope measurements for cultured marine cyanobacteria and compare these data to those for eukaryotic diatoms grown under identical conditions. Of the four cyanobacteria cultured, belonging to the genera Synechococcus and Prochlorococcus, three preferentially take up light Zn into the cell, with a variability that is not fundamentally different between pro- and eukaryotic phytoplankton. We also observe only very subtle differences between Zn/P and Fe/P uptake ratios for these three cyanobacteria groups relative to diatoms grown under the same conditions. A fourth strain exhibits preferential uptake of heavy Zn isotopes, and very high Zn/P ratios. Overall, we speculate that the observed variability among cyanobacteria may be related to the molecular structure of their photosynthetic light harvesting apparatus, adapted to significantly different light niches.These new and published culture data support the hypothesis that cellular δ66Zn in culture might largely be controlled by the organic ligands that bind Zn in the medium. Given that the Zn-binding ligands in the ocean have thermodynamic stability constants that are orders of magnitude smaller than the EDTA used in culture media, the surprisingly subtle Zn isotope variability in some parts of the surface ocean may be reconciled with culture data by the lesser, near zero, preference of these weaker complexes for heavy Zn isotopes.“This article is part of a special issue entitled: “Cycles of trace elements and isotopes in the ocean - GEOTRACES and beyond” - edited by Tim M. Conway, Tristan Horner, Yves Plancherel, and Aridane G. González.”

Application of CE-ICP-MS and CE-ESI-MS/MS for identification of Zn-binding ligands in Goji berries extracts

The identification of groups of ligands binding metals is a crucial issue for the better understanding of their bioaccessibility. In the current study, we have intended an approach for identification of Zn-binding ligands based on using capillary electrophoresis combined with inductively coupled plasma mass spectrometry (CE-ICP-MS) and tandem electrospray ionization mass spectrometry (CE-ESI-MS/MS). The approach, which featured the use of the coupling of capillary electrophoresis with inductively coupled plasma mass spectrometry allows to separate and observe zinc ions present in complexes with respect to their size and charge and to identify nine compounds with zinc isotopic profile. CE-ICP-MS provides us with information about presence of zinc species and elemental information about zinc distribution. CE-ESI-MS/MS provide us with information about the most favorable Zn binding ligands: amino acids, flavonols, stilbenoids, fenolic acids and carotenoids.The presented work is the continuation of previous studies based on using LC-ESI-MS/MS, though, now we presented a new solutions with the possibility of changing detectors without changing the separation techniques, what is important without re-optimizing the method. The new presented method allows to identify the zinc-binding ligands in shorter time.

A proteomics approach to investigate the process of Zn hyperaccumulation in Noccaea caerulescens (J & C. Presl) F.K. Meyer

Zinc (Zn) is an essential trace element in all living organisms, but is toxic in excess. Several plant species are able to accumulate Zn at extraordinarily high concentrations in the leaf epidermis without showing any toxicity symptoms. However, the molecular mechanisms of this phenomenon are still poorly understood. A state-of-the-art quantitative 2D liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS) proteomics approach was used to investigate the abundance of proteins involved in Zn hyperaccumulation in leaf epidermal and mesophyll tissues of Noccaea caerulescens. Furthermore, the Zn speciation in planta was analyzed by a size-exclusion chromatography/inductively coupled plasma mass spectrometer (SEC-ICP-MS) method, in order to identify the Zn-binding ligands and mechanisms responsible for Zn hyperaccumulation. Epidermal cells have an increased capability to cope with the oxidative stress that results from excess Zn, as indicated by a higher abundance of glutathione S-transferase proteins. A Zn importer of the ZIP family was more abundant in the epidermal tissue than in the mesophyll tissue, but the vacuolar Zn transporter MTP1 was equally distributed. Almost all of the Zn located in the mesophyll was stored as Zn-nicotianamine complexes. In contrast, a much lower proportion of the Zn was found as Zn-nicotianamine complexes in the epidermis. However, these cells have higher concentrations of malate and citrate, and these organic acids are probably responsible for complexation of most epidermal Zn. Here we provide evidence for a cell type-specific adaptation to excess Zn conditions and an increased ability to transport Zn into the epidermal vacuoles.

Determination of Zn-citrate in human milk by CIM monolithic chromatography with atomic and mass spectrometry detection

In human milk zinc (Zn) is bound to proteins and low molecular mass (LMM) ligands. Numerous investigations demonstrated that Zn bioavailability in human milk is for infant much higher than in cow's milk. It was presumed that in the LMM human milk fraction highly bioavailable Zn-citrate prevails. However, literature data are controversial regarding the amount of Zn-citrate in human milk since analytical procedures reported were not quantitative. So, complex investigation was carried out to develop analytical method for quantitative determination of this biologically important molecule. Studies were performed within the pH range 5–7 by the use of synthetic solutions of Zn-citrate prepared in HEPES, MOPS and MES buffers. Zn-citrate was separated on weak anion-exchange convective interaction media (CIM) diethylaminoethyl (DEAE) monolithic chromatographic column using NH4NO3 as an eluent. Separated Zn species were determined by flame atomic absorption spectrometry (FAAS) or inductively coupled plasma mass spectrometry (ICP-MS). Quantitative separation of Zn-citrate complexes ([Zn(Cit)]− and [Zn(Cit)2]4−; column recoveries 94–102%) and good repeatability and reproducibility of results with relative standard deviation (RSD±3.0%) were obtained. In fractions under the chromatographic peaks Zn-binding ligand was identified by electrospray ionization tandem mass spectrometry (ESI-MS-MS). Limits of detection (LOD) for determination of Zn-citrate species by CIM DEAE-FAAS and CIM DEAE-ICP-MS were 0.01μg ZnmL−1 and 0.0005μg ZnmL−1, respectively. Both techniques were sensitive enough for quantification of Zn-citrate in human milk. Results demonstrated that about 23% of total Zn was present in the LMM milk fraction and that LMM-Zn corresponded to Zn-citrate. The developed speciation method represents a reliable analytical tool for investigation of the percentage and the amount of Zn-citrate in human milk.

Biological factors regulating the chemical speciation of Cu, Zn, and Mn under different nutrient regimes in a marine mesocosm experiment

Previous mesocosm experiments and field studies have indicated a central role of major nutrients (e.g., N, P, and Si) in regulating phytoplankton abundance and species composition and influencing the rate of uptake of bioactive metals by phytoplankton. We monitored the changes in bioactive metals (Cu, Zn, and Mn) concentration and speciation that occurred over the course of a bloom of the coccolithophore Emiliana huxleyi under three different nutrient treatments. In each treatment, the electrochemically labile fraction of Cu decreased from 1.1 to 0.3 nmol L−1 during the formation of the E. huxleyi bloom. This decrease was likely due to the active release of organic ligands by phytoplankton, which shut off as soon as the level of labile Cu reached 0.3 nmol L−1 ([Cu2+] = 0.02 nmol L−1). Organic Zn‐binding ligands showed elevated concentrations in bag 1 (the N‐ and P‐replete bag) only. The release of these ligands coincided with a 15‐fold increase in the concentration of dead E. huxleyi cells in bag 1 but only a threefold increase in the P‐ or N‐depleted bags. This suggests that the Zn‐binding ligands may have originated from dead or decaying E. huxleyi cells. In both the P‐ and N‐limited bags, labile Mn concentrations showed a succession of two peaks and troughs over the 2‐week period; these were synchronized—but inversely correlated—with bacterial abundance. Labile Mn showed weaker fluctuations and no clear synchronization with bacterial abundance in the N‐ and P‐replete bag. We hypothesize that the passive release of organic Zn‐binding ligands under the nutrient‐replete conditions may have helped restrict the detrimental effect of Zn2+ on Mn uptake by phytoplankton cells, thus diverting part of the dissolved Mn away from sequestration or oxidation by bacteria.

Accumulation and Elimination of Cadmium and Zinc in the Asian Periwinkle Littorina brevicula

To elucidate the differences between the detoxification mechanisms of essential metal (Zn) and non-essential metal (Cd) in Littorina brevicula that is highly resistant to a wide range of heavy metal concentrations, Asian periwinkles were exposed to Cd (400 µg/L), Zn (3000 µg/L) and a mixture of both metals. We examined metal accumulation, elimination and subcellular distribution for binding to proteins. The metal concentration in L. brevicula increased gradually with exposure time (up to 70 days), following which accumulated levels reached saturation point. The accumulated Zn content was increased in the presence of Cd, while Cd uptake was decreased when Zn was present. During the depuration period (42 days), Cd was not removed from periwinkles, while Zn was eliminated in a rate of 2.19 µg Zn g−1day−1. This elimination rate was particularly high on exposure to the metal mixture (3.80 µg Zn g−1day−1). Subcellular distribution studies on Cd and Zn revealed that most Cd (80%) was bound to cytosolic ligand, while more than 75% Zn was distributed in the membrane (insoluble) fraction. An additional difference in sequestering of metal in the cytosol was noted between Cd and Zn; most Cd in the cytosol was bound to metallothionein-like cadmium binding protein, MBP-1 (9.8 kDa), while the profile for Zn distribution revealed the presence of four Zn-binding ligand peaks, specifically, HMW (60 kDa), MBP-1 (9.8 kDa), MBP-2 (5 kDa) and LMW (<1 kDa). Our data confirm that metallothionein-like cadmium binding protein, MBP-1, has the same affinity for Zn, while MBP-2 displays comparatively higher affinity for Zn than Cd.

Chemical speciation of copper and zinc in surface waters of the western Black Sea

The chemical speciation of Cu and Zn was investigated by voltammetric titration methods in the surface waters (∼10 m) of the western Black Sea during an Istanbul–Sevastopol cruise conducted in November 1998. Supporting parameters (temperature (T), salinity (S), pH, alkalinity (Alk), suspended particulate matter (SPM) and dissolved and particulate 234Th) were obtained in order to distinguish hydrographic features against involvement of the metals in biogeochemical processes. In the Turkish continental slope region, the cruise track intersected a narrow vein of colder water originating on the western shelf. The core of this cold water vein was characterised by a relatively low salinity, higher specific alkalinity and higher metal (especially Cu) and metal-binding ligand concentrations. A very large portion of Cu (93–99.8%) and Zn (82–97%) was organically complexed. The degree of complexation was highest in shelf waters and lowest in the central gyre. Titration data for Cu were modelled by two classes of organic binding ligands characterised by (CL1=3–12 nM, log K1′=13.1–13.9) and (CL2=20–70 nM, log K2′=9.4–11.2). These ligands occurred mainly in the ‘dissolved’ phase, as defined by 0.4-μm filtration. The stronger Cu-binding ligand seemed to be produced in situ in response to Cu concentration, whereas the weaker Cu-binding ligand appeared to be derived from terrestrial sources and/or reducing shelf sediments. Titration results for Zn were generally represented by one class of ligands (CL1=8–23 nM, log K1′=9.4–10.2), which were almost uniformly distributed between the ‘dissolved’ (78±8%) and the particulate phase (22±8%). The concentration of these strong Zn-binding ligands showed a very good correlation with SPM (r2=0.64), which improved when the dissolved ligands alone were considered (r2=0.78). It is hypothesised that these ligands were produced in situ by the bacterial breakdown of particulate organic matter.

Potential for the speciation of Zn using fast protein liquid chromatography (FPLC) and convective interaction media (CIM) fast monolithic chromatography with FAAS and electrospray (ES)-MS-MS detection.

Analytical procedures were developed for the speciation of Zn using fast protein liquid chromatography (FPLC), flame atomic absorption spectrometry (FAAS) and convective interaction media (CIM) fast monolithic chromatography with FAAS and electrospray (ES)-MS-MS detection. The investigation was performed on synthetic solutions (2 microg cm-3 Zn) of hydrated Zn2+ species and Zn complexes with citrate, oxalate and EDTA (ligand-to-Zn molar ratio 100:1) over a pH range from 5.4 to 7.4. It was found that Zn interacts with various buffers and the careful adjustment of the pH with diluted solutions of KOH is, therefore, required. FPLC separations were carried out on a Mono Q HR 5/5 strong anion-exchange column, applying an aqueous 1 mol dm(-3) NH4NO3 linear gradient elution over 15 min, at a flow rate of 1.0 cm3 min(-1). The separated Zn species were determined in 1.0 cm3 eluate fractions "off line" by FAAS. Speciation of Zn was also performed on a weak anion-exchange CIM DEAE fast monolithic disc by applying an aqueous 0.4 mol dm(-3) NH4NO3 linear gradient elution over 7.5 min, at a flow rate of 2.0 cm3 min(-1) and determination of the separated Zn species in 1.0 cm3 eluate fractions "off line" by FAAS. Zn-binding ligands in separated fractions were also characterized by electrospray (ES)-MS-MS analysis. The CIM DEAE disc was found to be more efficient in the separation of negatively charged Zn complexes than the Mono Q FPLC column. On the CIM DEAE disc Zn-citrate was separated from both Zn-oxalate and from Zn-EDTA. All these species were also separated from hydrated Zn2+, which was eluted with the solvent front. This method has an advantage over commonly used analytical techniques for the speciation of Zn which are only able to distinguish between labile and strong Zn complexes. Good repeatability of the measurements (RSD 2-4%), tested for six parallel determinations (2 microg cm(-3) Zn) of Zn-EDTA, Zn-citrate and Zn-oxalate was found at a pH of 6.4 on a CIM DAEA disc. The limit of detection (3s) for the separated Zn species was 10 ng cm(-3). The proposed analytical procedure was applied to the speciation of Zn in aqueous soil extracts and industrial waste water from a lead and zinc mining area.

The role of the pancreas in intestinal zinc secretion in metallothionein-null mice.

The distribution and excretion of endogenous Zn, including the role of the pancreas, were examined in fasted MT+/+ and MT-/- mice. At 3 and 6 h after receiving 65Zn tracer by subcutaneous injection, 65Zn levels were compared in tissues of MT+/+ and MT-/- mice. 65Zn levels were significantly higher in the liver and pancreas of the MT+/+ mice, whereas in the MT-/- mice, 65Zn levels were significantly higher in muscle, skin, and most of the gastrointestinal tract other than the stomach and upper small intestine. In MT-/- mice, 3% of the injected 65Zn was recovered in the luminal contents of the small intestine over 3-6 h, compared with <1.5% in the MT+/+ mice. A loading dose of Zn (150 microg, s.c.) sufficient to raise the plasma Zn concentration by fourfold to fivefold in both MT+/+ and MT-/- mice resulted in similar increases in pancreatic Zn levels in each genotype, although more Zn appeared in the lower small intestine of MT-/- mice. Pancreatectomy decreased the level of 65Zn in the small intestine of MT-/- but not MT+/+ mice. Longer-term studies over 4 days demonstrated few differences in tissue 65Zn between MT+/+ and MT-/- mice, with the exception of the pancreas, where 65Zn retention after fasting in MT-/- mice was half that of MT+/+ mice. MT-/- mice also had significantly lower Zn concentrations in the pancreas. Fecal excretion of 65Zn in MT-/- mice was greater than that of MT+/+ mice in the first 24 h (24.7 vs. 18.2% of injected dose; p < 0.05). Besides metallothionein (MT), there were no significant differences in the molecular weight distribution of Zn binding ligands in the lumen of the small intestine between MT+/+ and MT-/- mice. Mice lacking MT I and II lose more endogenous Zn into the gut because of a relative failure of the pancreas to retain Zn. However, increased Zn secretion via the small intestinal mucosa may also contribute to intestinal Zn loss in MT-/- mice.

Identification of an α2-Macroglobulin Receptor in Human Mammary Epithelial Cells

Several cases of zinc (Zn) deficiency in human infants caused by abnormally low concentrations of Zn in breast milk were recently reported, the underlying mechanism of which is not known. Alpha2-macroglobulin (α2-M), a major Zn-binding ligand in serum, presents a potential vehicle for mammary Zn uptake. This study was conducted to determine if an α2-M receptor is present in human mammary epithelial cells, where it may be involved in the endocytosis of α2-M into the mammary gland. Normal human mammary epithelial cells were grown to confluency in serum-free medium. For all binding and uptake studies, α2-M, preactivated with methylamine and labeled with 125I, was added to cells for varied lengths of time to determine saturation over time and at varied concentrations to determine saturation over increasing concentration of ligand. Nonspecific and competitive binding were measured by addition of a 100-fold molar excess of unlabeled α2-M and serum albumin or lactoferrin, respectively. Binding at 4°C was specific for α2-M and approached saturation kinetics at 56 nmol/L. Scatchard plot analysis of the binding data demonstrated more than one binding site: a high affinity, saturable binding site and a low affinity, nonsaturable binding site. Uptake of α2-M at 37°C was rapid and continuous over increasing concentrations of α2-M, and internalized α2-M was rapidly degraded. Results from this study present evidence for receptor-mediated uptake of α2-M in human mammary epithelial cells, which in turn, provides a potential mechanism for Zn acquisition by the cell.

Chemical speciation of dissolved Cu, Zn, Cd, Pb in Narragansett Bay, Rhode Island

The chemical speciation of `dissolved' Cu, Zn, Cd, and Pb was determined by differential pulse anodic stripping voltammetry (DPASV) at three sampling sites in the high salinity region (24–30 psu) of the Narragansett Bay estuary. Speciation results show that organic chelates of these four trace metals are the dominant forms in conventionally filtered ( 99% for Cu in these estuarine waters. Dissolved Zn ([ZnT]) varied from 72 nM at the Upper Bay site to 16 nM at the Lower Bay site, the concentration of the Zn-binding ligands ([LT]) varied from 48 to 11 nM with a relatively uniform conditional stability constant (KZnL,Zn′cond=109.0 M−1). At the Upper and Lower Bay sites, where [ZnT]>[LT], there was less organic complexation (50 to 60%) of [ZnT] as compared to the Mid-Bay (bloom) station, where [ZnT]<[LT] and the degree of organic complexation was considerably higher (97%). The concentration of Cd-binding ligands was nearly constant throughout the bay (ca. 4 nM with mean KCdL,Cd′cond=108.9 M−1) and present at much higher levels than the dissolved Cd ([CdT]=0.80–0.29 nM, decreasing down bay). Titration results for Pb were modeled by two classes of Pb-binding ligands. Dissolved Pb ranged from 0.32 to 0.13 nM and the strong class of Pb-binding ligands (L1) was in slight excess of these values (ca. 0.8 nM) with a mean KcondPbL1,Pb′=1010.0 M−1. A weaker class of Pb-binding ligands (L2) existed at 4.0–8.2 nM with a mean KcondPbL2,Pb′=108.8 M−1. Titration data for Cu were interpreted as being influenced by three classes of Cu-binding ligands (L1, L2, L3), whose sum was always in excess of the dissolved [CuT] (12.7–27.9 nM). Our DPASV estimates of [L1] were 16–38 nM with a KcondCuL1,Cu′≥1012 M−1. There were higher concentrations of intermediate and weaker classes of Cu-binding ligands: [L2]=40 to 15 nM with a mean KcondCuL2,Cu′=108.8 M−1 and [L3]=100 to 54 nM with a mean KcondCuL3,Cu′=107.7 M−1. The two stronger classes of Cu-binding ligands (L1 and L2) resulted in CuL1 and CuL2 as the dominant dissolved Cu species. Free ion concentrations were calculated for all four metals at the three sampling sites: [Cu2+]≤10−12 M, [Cd2+]≤2×10−12 M, [Pb2+]∼10−12 M, while [Zn2+] ranged from 0.3–13×10−9 M. For Cu, Pb, and Cd, these ambient values are below the concentrations of toxic responses reported from laboratory studies of marine microorganisms. At the Upper Bay site, any toxic effect of the elevated free zinc ion concentration is likely to be alleviated by the antagonistic effect of high dissolved Mn.

Zinc uptake and transcellular movement by CACO-2 cells: Studies with media containing fetal bovine serum

A series of experiments using CACO-2 cells, have been conducted to describe the cellular events occurring during Zn uptake and transcellular movement in the presence of media containing fetal bovine serum (FBS). CACO-2 cells were grown for 21 days on either T-25 cell culture flasks or semipermeable membrane inserts maintained in six-well culture plates. Experiments were then conducted using normal growth medium containing FBS, to which 65Zn was added. The rate of Zn transport in an apical to basolateral direction was much greater than the rate in the opposite direction. To study whether uptake and movement exhibited saturation kinetics, different concentrations of Zn (1–200 μ m ) were added with 65Zn. Uptake at the basolateral membrane was saturable; apical to basolateral movement, basolateral to apical movement, and uptake at the apical membrane were not. 65Zn moved apically to basolaterally at a small but constant rate regardless of the basolateral concentration of Zn. The binding ligands for Zn in the cytosol differed depending on the prior Zn status of the cell and the time postlabeling. Endogenous 65Zn was released to the apical and basolateral sides at different rates. These data confirm a previous report that uptake and transcellular movement of Zn is different at the apical and basolateral membrane, and they further show that the presence of Zn-binding ligands alters these processes.

Stereochemistry-activity relationships of ACE inhibitors. Conformational studies by1H and13C NMR of perindopril and selected stereoisomers

Perindopril, the tert-butylamine salt of 1-{(2S)-2-[(1S)-(1-carbethoxybutyl)amino]-1-oxopropyl}-(2S,3aS,7aS)-perhydroindole-2-carboxylic acid, is an inhibitor of angiotensin-converting enzyme (ACE) and a new drug for the treatment of hypertension. The interaction between the inhibitor and the enzyme was investigated by studying the active diacid metabolite of perindopril, its stereoisomers and a desmethyl analogue. The pharmacological study allowed the measurement of the in vitro activities of the different compounds. The 1H and 13C NMR studies have shown that the cis-trans equilibrium about the amide bond is strongly dependent on the configuration of the chiral centres and on the pH of the solution. The pKa of the different acid-base species were measured. The results show that perhydroindole derivatives are potent inhibitors of ACE as long as they fulfil the following basic requirements: (1) an S configuration of the carbon bearing the terminal carboxy group; (2) an S methyl substituent in the alanine residue; however, the inhibitor potency is not modified on the replacement of the alanine residue of the perindopril by a glycine residue; and (3) less stringently, an S configuration of the C-1 butyl carbon. Under these conditions the Zn binding ligand (chain carboxylate group) is devoid of steric hindrance in the trans conformers. No direct relationship appeared between the relative amount of the trans form and the activity. The cyclic skeleton of the perhydroindole derivatives provided a strong hydrophobic interaction with the active enzymatic site, whatever the configurations at C-3a and C-7a. Lipophilic interactions involving the different parts of the inhibitor are not independent of each other.

65Zinc Metabolism Is Altered during Diabetic Pregnancy in Rats

To assess further diabetes-induced alterations in gestational Zn metabolism we examined the uptake/retention and distribution of 65Zn during pregnancy. Control and streptozotocin-induced diabetic rats, with and without insulin treatment, were gavaged with 65Zn on gestational d 12 or 18. Six hours post-intubation, untreated diabetic dams at d 12 of gestation had a lower percentage of retained 65Zn in bone, plasma, erythrocytes, pancreas and spleen than did controls. Retention of 65Zn in amniotic fluid and sacs and in embryos was similar among groups. Untreated diabetic dams at d 18 of gestation had a higher percentage of retained 65Zn in liver, intestine and urine, and most notably a lower percentage in amniotic fluid, placenta, fetus and fetal liver. Lower percentages of 65Zn were found in bone, muscle, plasma, erythrocytes, lung, spleen, heart, pancreas and uterus of diabetic dams compared with controls. HPLC fractionation of samples from maternal livers showed a higher percentage of Zn, Cu and 65Zn associated with the metallothionein peak in samples from untreated diabetic dams compared with other groups, whereas the percentages of Zn, Cu and 65Zn per fraction in fetal liver were similar. The marked decrease in retained 65Zn in products of conception at d 18 suggests either decreased placental transport or altered maternal and/or fetal Zn-binding ligands. Insulin treatment significantly reversed the streptozotocin diabetes-induced derangements in maternal and fetal Zn metabolism.

Significance of extracellular zinc‐binding ligands in the uptake of zinc by human fibroblasts

Extracellular zinc (Zn)-binding ligands were investigated as vehicles for uptake of Zn by human fibroblasts. The uptake of alpha 2-macroglobulin, a major serum Zn-binding protein proposed to have a function in Zn transport, was less than 1/200 that of the Zn uptake rate. The fibroblast growth medium, BME with 10% FBS, contains several Zn-binding ligands. These were separated into components of MW greater than 30,000 and components of MW less than 30,000 using an Amicon microconcentrator. Cells accumulated Zn from both fractions; however, there was more uptake from the filtrate (MW less than 30,000), containing ligands with low affinity for Zn, hence with greater free Zn concentration. Zn uptake from a number of ligands with a range of affinities for Zn was examined and found to be inversely proportional to the Ka value for the ligands and therefore proportional to the free Zn concentration. When histidine and desferrioxamine, two structurally different Zn-binding ligands were compared, analysis of the concentration curves of calculated free Zn against Zn uptake gave similar Vmax and Km values (+/- S.E.M.) of 373 +/- 6 pmol/micrograms DNA/h and 0.08 +/- 0.004 microM for histidine, and 349 +/- 10 pmol/micrograms DNA/h and 0.06 +/- 0.008 microM for DFO, suggesting that the same transport mechanism was operating in both systems. We conclude that no specific ligands are essential for transport of Zn into fibroblasts, but that "free" Zn is acquired by the cell.

The effects of dietary ligands on zinc uptake at the porcine intestinal brush-border membrane

Intestinal brush-border-membrane vesicles were prepared from the porcine small bowel by magnesium precipitation and differential centrifugation, and were functionally intact. The influence of dietary ligands on 65Zn uptake was determined using a 65Zn concentration of 5 microM, an incubation time of 1 min and a reaction temperature of 27 degrees, with a rapid filtration technique. At this low Zn concentration the addition of an excess of folate, histidine or glucose had no effect on Zn uptake. Addition of picolinate, citrate and phytate to the incubation medium significantly reduced Zn uptake at all concentrations of ligand examined. Any inhibitory effects of folic acid in vivo may thus be due to a mucosal rather than lumen interaction. Those ligands inhibiting absorption may have done so through the formation of Zn-ligand complexes, which are either insoluble, or which reduce the binding of Zn to its mucosal receptor. This in vitro model of Zn absorption is useful for comparing the effects of potential Zn-binding ligands in the diet.

Complete amino acid sequence of neutral protease from Bacillus subtilis var. amylosacchariticus.

The neutral protease of Bacillus subtilis var. amylosacchariticus was cleaved chemically or digested with proteolytic enzymes, and the resultant peptides were separated and purified by high performance liquid chromatography. The sequence analyses of these peptides by the manual Edman procedure established the complete amino acid sequence of the enzyme. The neutral protease consisted of 300 amino acid residues with Ala and Leu as its amino- and carboxyl-termini, respectively, and the molecular weight was calculated to be 32,633. The sequence was found to be identical to that of B. subtilis 1A72 neutral protease, which was deduced from nucleotide sequencing. Comparison of the sequence with those of other Bacillus proteases revealed that the putative active site amino acid residues, Zn-binding ligands, and two Ca-binding sites were well conserved among them, as compared with those of thermolysin.

A low molecular weight Zn-binding ligand in the shore crab Carcinus maenas

1. Hepatopancreas extracts of Carcinus maenas typically contain a Zn-binding complex of low mol. wt ( ca 300). 2. The complex is highly polar, lacks nitrogen and aromatic groups and is non-proteinaceous. The solid phase is transparent-white, deliquescent and easily charred with a melting point of 178–186°C. 3 It is proposed that the Zn-binding ligand consists of a dicarboxylic acid with complex aliphatic backbone, or of several related carboxylic acid species.

Interactions of cis-platinum with cellular zinc and copper in rat liver and kidney tissues

In the present study the effects of cis-diamminedichloroplatinum II (cis-DDP) on tissue and subcellular concentrations of endogenous Zn and Cu was investigated in liver and kidneys of rats injected i.p. with 5 mg/kg of cis-DDP. The binding of Pt to cytosolic ligands and its effect on Zn and Cu binding was also investigated. cis-DDP significantly reduced cellular Zn (20%) and Cu (23%) levels particularly in the kidneys. Intracellularly, these decreases were largely reflected in the corresponding cytosolic fractions, with 64% decrease in Zn and 56% in Cu concentrations. At 24 h following cis-DDP injection, in both liver and kidneys, about 50% of cytosolic Pt was present as low molecular weight (LMW) species and the remainder as protein-bound. A small proportion of cellular Pt was bound to a metallothionein-like protein in the liver as well as the kidneys. The binding of renal Zn to cytosolic proteins and LMW Zn-binding ligands was significantly decreased by cis-DDP. In the liver cytosol, there was a marked redistribution of Zn and Cu from LMW Zn/Cu-binding ligand to the MT-like proteins.

Chemical factors affecting the intestinal absorption of zinc in vitro and in vivo.

Everted sacs of rat duodenum and ileum were used to study the effect of anions and organic ligands on the absorption of zinc. The uptake per unit weight of tissue was greater in duodenum than ileum, and it was influenced by the Zn concentration and pH of the incubation medium. The Zn uptake from inorganic salts in simple buffered medium varied in the order zinc sulphate greater than zinc chloride greater than zinc phosphate. Zinc acetate was more effective and zinc citrate less effective than ZnCl2. Addition of aspartic acid or histidine to ZnCl2 increased the uptake but galactose or lactose decreased it. 2-Picolinic acid greatly increased the Zn uptake but 4-picolinic acid reduced it. When incubated with intestinal sacs after incorporation into a synthetic rat diet, only ZnSO4 and 2-picolinic acid increased Zn uptake compared with ZnCl2, but zinc citrate and 4-picolinic acid still tended to decrease it. Metabolic balance studies showed no significant differences in the faecal excretion, total excretion or retention of Zn between rats receiving diets containing different forms of Zn. ZnSO4, zinc citrate and particularly 2-picolinic acid increased the urinary excretion of Zn. The significance of these results is discussed in relation to the suitability of methods for investigating Zn absorption and the importance of Zn-binding ligands.