Zinc-binding Ligand Research Articles

A Vancomycin Derivative with a Pyrophosphate-Binding Group: A Strategy to Combat Vancomycin-Resistant Bacteria.

Vancomycin, the drug of last resort for Gram-positive bacterial infections, has also been rendered ineffective by the emergence of resistance in such bacteria. To combat the threat of vancomycin-resistant bacteria (VRB), we report the development of a dipicolyl-vancomycin conjugate (Dipi-van), which leads to enhanced inhibition of cell-wall biosynthesis in VRB and displays in vitro activity that is more than two orders of magnitude higher than that of vancomycin. Conjugation of the dipicolyl moiety, which is a zinc-binding ligand, endowed the parent drug with the ability to bind to pyrophosphate groups of cell-wall lipids while maintaining the inherent binding affinity for pentapeptide termini of cell-wall precursors. Furthermore, no detectable resistance was observed after several serial passages, and the compound reduced the bacterial burden by a factor of 5 logs at 12 mg kg(-1) in a murine model of VRB kidney infection. The findings presented in this report stress the potential of our strategy to combat VRB infections.

Ratiometric and Intensity-Based Zinc Sensors Built on Rhodol and Rhodamine Platforms

A xanthene-forming condensation reaction yields rhodol and rhodamine dyes carrying a zinc-binding ligand that includes the aniline-type nitrogen donor of the fluorophores. Upon zinc coordination in neutral aqueous solution, rhodol RF3 behaves as a ratiometric sensor, and rhodamine RA1 acts as a turn-off intensity-based indicator. Both fluorescent compounds bind the divalent zinc cation with micromolar affinity.

Synthesis and structure–activity relationship of a novel, non-hydroxamate series of TNF-α converting enzyme inhibitors

A novel series of TNF-α converting enzyme (TACE) inhibitors which are non-hydroxamate have been discovered. These compounds use a triazolethione moiety as the zinc binding ligand and exhibit IC50 values from 1.5 to 100nM in a porcine TACE assay. They also have excellent selectivities over other MMPs.

Zinc ligands in an astacin family metalloprotease meprin A.

A conserved tyrosine residue in the 'astacin family' of metalloproteases is one of five ligands proposed to coordinate zinc at the active site. Site-directed mutagenesis of the conserved Tyr (Y226) of recombinant mouse meprin alpha was used to test the hypothesis that this residue is essential for zinc binding and enzymatic activity. In addition, another proposed zinc binding ligand, H167, in the conserved (HEXXH) zinc binding motif of the meprin alpha protease domain was replaced by an alanine residue. Both mutants were expressed and secreted with the same subunit mass as wild type (90 kDa). The Y226F mutant retained the capacity to oligomerize to higher covalently and noncovalently-linked oligomers as the wild type, whereas H167A was predominantly a monomer. The kcat/Km for Y226F against a fluorgenic bradykinin substrate analog was approximately 15% of the wild type, while the H167A mutant had no detectable activity. Both Y226F and H167A were more susceptible to extensive degradation by trypsin compared with the wild-type protein. The zinc content in the wild-type and Y226F mutant proteins were similar, one molecule of zinc per subunit. The results indicate that Y226 is not essential for zinc binding, but Y226 and H167 are essential for full enzymatic activity and stability of the metalloproteinase.

Role of the conserved histidine and aspartic acid residues in activity and stabilization of human gelatinase B: An example of matrix metalloproteinases

Gelatinase B (MMP-9), a member of the matrix metalloproteinase family, is a zinc- and calcium-dependent endopeptidase that is known to play a role in tumor cell invasion and in destruction of cartilage in arthritis. It contains a conserved sequence. 400His-(X)3-His-(X)28-Asp-Asp-(X)2-436Gly, the function of which is under investigation. The conserved Asp-432 and Asp-433 residues were individually replaced with Gly; these substitutions reduced the gelatinolytic activity of the enzyme to 23% and 0%, respectively. Replacing Asp-433 with Glu, however, decreased the gelatinolytic activity of the enzyme by 93% and proteolytic activity of the enzyme for the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 substrate by 79%. The wild-type and D432G and D433E, mutant enzymes had similar Km values for the synthetic substrate and similar Ki values for the competitive inhibitor, GM6001. The kcat/Km values for D432G and D433E mutant enzymes, however, were reduced by a factor of approximately 4 and their KaCa values were increased by four- and sixfold, respectively. The significance of His-400 in the activity of the enzyme was assessed by replacing this residue with Ala and Phe. Both H400A and H400F mutants were inactive toward gelatin substrate. These data demonstrate that Asp-432, Asp-433, and His-400 residues are important for the activity of gelatinase B. His-400 may act as a zinc-binding ligand similar to the His-197 in interstitial collagenase (MMP-7) and Asp-432 and Asp-433 residues are probably involved in stabilization of the active site of the enzyme. The His-400 and Asp-433 residues are conserved in all members of the MMP family. Therefore, our results are relevant to this group as a whole.

Chromatographic analysis of low-molecular-mass copper-binding ligands from the crab species Syclla serrata and Portunis pelagicus

Two copper-binding proteins and a zinc-binding ligand were isolated from the hepatopancreas of the crab Portunus pelagicus. The copper-binding proteins behave similarly to those from the crabs Carcinus maenas and Scylla serrata, and were shown to be metallothioneins. Reversed-phase high-performance liquid chromatographic (HPLC) analysis confirmed the relative purity of both proteins with only cross-contamination between the two different forms of metallothioneins, and offers a good method to separate the two forms of metallothioneins. The vast difference in the retention times (and hence the hydrophobicity) in reversed-phase HPLC indicates that the two proteins could be conformationally very different.

Chromatographic Analysis of Metallothionein Proteins from the Crab Species Scylla Serrata and Portunus Pelagicus

Abstract Different chromatographic techniques have been used successfully to isolate and purify two copperbinding proteins and a zinc-binding ligand from the hepatopancreas of the crab Portunus pelagicus. Comparison studies were made using the crab Scylla serrata which had been studied previously. Based on the identical purification protocol and the very similar retention times on reversed phase HPLC, the copper-binding proteins are found to be similar to those from the crab Scylla serrata and are shown to be metallothioneins. Reversed phase HPLC analysis confirms that there are two distinct forms as purified by standard molecular sieve and ion exchange chromatography.

Abnormal zinc content in human milk. Risk for development of nutritional zinc deficiency in infants.

An abnormally low concentration of zinc in mother's milk has been associated with clinical nutritional zinc deficiency in premature and term newborns. Defective mammary gland secretion of zinc was suggested as the causative factor. We investigated whether low milk zinc concentration might be reflected in an abnormality of the distribution of zinc between casein, whey, and fat components of the milk, in a reduction in the levels of milk citrate, a zinc-binding ligand, or in abnormal levels of copper and iron. Milk from mothers of four low-birth-weight infants was identified as being deficient in zinc content for the lactational stage. One infant had clinical signs of nutritional zinc deficiency. For two of the three infants with subclinical deficiencies in whom balance study data were available, the apparent zinc balance was negative (-5.7 and -6.0 mumol/kg per day). These milks were examined for content and distribution of zinc, copper, and iron, and citrate concentration. Although total zinc concentration (4.5 to 17.2 mumol/L) was below the 95% prediction interval (15.2 to 59.7 mumol/L) for the lactational stage, the distribution of zinc was normal between milk components. Levels of citrate (1.46 to 5.13 mmol/L) were also within normal limits, as were levels of copper (0.27 to 0.85 mumol/L and iron (0.41 to 1.13 mumol/L). We concluded that there was no indirect evidence of a secretory abnormality in milk zinc deficiency, but speculated that there might instead be an abnormality of zinc uptake by the mammary gland from the plasma.

The effect of dithiothreitol on the subcellular distribution of zinc in the cytosol of mussel kidney ( Mytilus Edulis): Isolation of metallothionein and a unique low molecular weight zinc-binding ligand

1. A reducing agent such as dithiothreitol (DTT) was found to be necessary for the isolation of metallothionein-like zinc-binding proteins (MLP) from the cytosol of the kidney of the mussel, Mylilus edulis using Sephadex G-75 gel filtration chromatography. 2. When DTT-free buffer was used, little or no zinc was found to elute in the region usually associated with metallothionein-like proteins (MLP) in mussels. However, when a small amount of DTT was added to the buffer (0.7 mM), a substantial amount of zinc was found to be eluted in the MLP zone. When the DTT concentration was increased (4 mM), serious artefacts were seen. 3. Both in the presence and absence of DTT, most cytosolic kidney zinc from uncontaminated mussels was bound to a unique non-metallothionein low mol. wt ligand with a mol. wt around 1000. This very low mol. wt zinc-binding ligand was responsible for nearly all of the increase in cytosolic zinc when mussels were exposed to 400 ppb zinc.

The addition of picolinic acid to low protein diets — a word of caution

The effect of supplementing a protein deficient diet with picolinic acid on parotid salivary proteins was examined. Female rats were fed one of three synthetic diets containing 18% protein, 8% protein, or 8% protein plus 0.03% picolinic acid. After 38 days, although food intake did not differ, the body weights of rats consuming 18% protein were significantly greater than those maintained on the 8% protein diets with or without picolinic acid. Zinc content of serum and liver was significantly lower in the low protein groups and these values were unaffected by the addition of picolinic acid. Although zinc concentration and salivary flow rate did not differ, total protein concentration as determined by the Lowry and by absorption at 215 nm was highest in the group receiving diets of 8% protein supplemented with picolinic acid. Furthermore, the bands corresponding to basic proline-rich proteins were consistently increased in the rats maintained on the low protein diet supplemented with picolinic acid compared to rats fed the 18% protein diet. These results demonstrate that dietary picolinic acid supplementation alters parotid gland function in a manner which appears to be independent of its proposed effect on dietary zinc absorption. These data suggest that the effect of picolinic acid supplementation is not confined to zinc metabolism and therefore caution is advised in interpretation of dietary studies utilizing this zinc binding ligand.

Characterization of the low mol. wt zinc-binding ligand from rat small intestine by comparison to the organic zinc-binding ligands

1. 1. 65Zn complexes of picolinate (PA), citrate (CA), l-histidine ( l-his), arachidonic acid (AA) or low mol. wt zinc-binding ligand from rat intestine (LMW-ZBL) gave 65Zn eluting peak fraction numbers of 53, 53, 56, 59 and 59 respectively, in a Sephadex G-75 column chromatography. 2. 2. The 65Zn eluting peak fraction numbers with CA, l-his, PA, prostaglandin (PG)E 2, AA, no ligand, arachidonate (AT) or LMW-ZBL were 49, 50, 54, 55, 58, 64, 75 and 76 respectively in a Sephadex G-25 column chromatography. 3. 3. In a Sephadex G-15 column chromatography, the 65Zn eluting peak fraction numbers with CA, PGE 2, AA, l-his, LMW-ZBL or PA were 49, 50, 51, 52, 52 and 55 respectively. 4. 4. The LMW-ZBL in rat small intestine appears to be an AA-like substance.

A unique low molecular weight zinc-binding ligand in the kidney cytosol of the musselMytilus edulis, and its relationship to the inherent variability of zinc accumulation in this organism

Mussels,Mytilus edulis, are known to have a high degree of variability in their whole soft tissue zinc concentrations which cannot be explained by any known ecological or physiological factor. In the present study, 70 individual mussels collected from an uncontaminated site at Bellevue, Newfoundland, in Spring 1987 had kidney zinc concentrations ranging from 148 to 4 907 μg g-1 dry weight while 40 mussel exposed to 25 μg l-1 zinc for 18 d had kidney zinc concentrations ranging from 144 to 14 072 μg g-1. Pooled or individual kidneys were homogenized in 50 mM ammonium bicarbonate buffer (pH 8.0) and ultracentrifuged for 1 h at 105 000 g. On average, about 70% of the total kidney zinc load was found in the 105 000 g pellet. Cytosolic zinc was separated into two peaks on a column of Sephadex G-25. The first peak coincided with the void volume of the column representing molecules with molecular weights of at least 5 000 (exclusion limits of Sephadex G-25). This peak would include any metallothionein present since mussel metallothionein has a molecular weight of 10 000 to 20 000. Little variability was observed in this peak so it was concluded that zinc-thionein did not play a major role in the genesis of the inherent variability. The second peak represented zinc complexed to an unknown substance with an approximate molecular weight of 700 to 1 300. This very low molecular weight zinc showed an extremely high degree of inherent variability and a strong positive correlation with the whole kidney zinc concentration. It is concluded that this very low molecular weight zinc complex plays a major role with regard to kidney zinc variability. Some very low molecular weight zinc was also noted in the digestive gland and gills. It is speculated that this substance may aid in zinc transport as well as in the incorporation of zinc into granules.

Low-molecular-weight zinc-binding ligand: A regulatory modulator for intestinal zinc transport

1. 1. In the last two decades, vast numbers of studies on the zinc nutriture of animals and man have been made. However, the biochemical and physiological events in controlling zinc nutrition are still poorly understood. This report concerns the progress made toward understanding the intestinal zinc absorption and secretion mechanisms. 2. 2. Evidence is accumulating that zinc absorption is a facilitated diffusion while zinc secretion is an active transport. 3. 3. It is known that a low molecular weight zinc-binding ligand (LMW-ZBL) is a key regulator of intestinal zinc absorption, possibly a carrier molecule across the intestinal mucosal cells. Some high molecular weight zinc-binding ligands also appear to be involved in regulating intestinal zinc transport. 4. 4. The identity of the LMW-ZBL is a matter of controversy and its specific role in regulating intestinal zinc transport is not well defined. 5. 5. According to the available literature, no systemic investigations have been made to elucidate the intestinal zinc transport mechanisms, and much more information is needed to fully understand them.

Relative zinc-binding activities of ligands in the cytosol of rat small intestine

1. Relative zinc-binding activities of high-molecular-weight zinc-binding ligand (HMW-ZBL), metallothionein (MT) and low-molecular-weight zinc-binding ligand (LMW-ZBL) in the cytosols of rat small intestines under various experimental conditions were examined. 2. Zinc-binding activities of MT decreased and those of LMW-ZBL increased in the intestinal cytosols from most of the experimental rat groups after incubating at 37°C for 2 hr. 3. The relative zinc-binding activity of MT increased with increasing doses of injected zinc and decreased with orally-administered zinc. 4. Isolated MT did not lose zinc-binding activity during incubation at 37°C for 48 hr, but moved from the MT eluting peak to the LMW-ZBL eluting peak after 1 week.

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 relative(65)Zn-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 of(65)Zn 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,(65)Zn-binding activity of MT was low in vitro and substantially increased in vivo. However, when excess zinc was injected intraperitoneally,(65)Zn-binding activity of MT in vitro greatly increased, but(65)Zn-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, the(65)Zn-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.

Impaired handling of orally administered zinc in pancreatic insufficiency

Five patients with pancreatic insufficiency were evaluated for presence of impaired handling of orally administered zinc. Six hospital employees, eight alcoholic cirrhotics, and four patients with small bowel disease served as healthy or chronically ill controls. Two zinc tolerance tests (ZTT) were performed on each subject, one test using zinc sulfate and the other using zinc dipicolinate, a putative zinc binding ligand. Healthy controls demonstrated normal ZTT curves, with no significant difference between the two forms of zinc. Chronically ill controls had significantly depressed ZTT curves compared to healthy controls with both forms of zinc administered. In contrast, pancreatic insufficiency patients had significantly depressed ZTT curves with zinc sulfate but not with zinc dipicolinate, demonstrating a 40% reduction in the area under the curve with zinc sulfate compared to healthy controls. Our study shows impaired handling of orally administered zinc sulfate but not zinc dipicolinate in patients with pancreatic insufficiency and suggests normal pancreatic function may play a role in zinc metabolism in man.

Factors affecting the bioavailability of zinc

Host factors and dietary factors determine the absorption of zinc by an individual. There is evidence that the entrance of zinc into the body is regulated at the level of the intestinal mucosa by the adequacy of the host's zinc nutriture. Moreover, a variety of gastrointestinal diseases that interfere with the digestion of food, the secretion of zinc-binding ligand, or transmucosal absorption, per se, reduce the efficiency of zinc absorption. Zinc appears to be more available from breast milk than from cow's milk, and, in general, from foods of animal origin than from foods of plant origin. The task of nutrition researchers is to unravel further the mechanisms involved in normal absorption of dietary zinc in human beings and to pursue the identification of foods and to pursue the identification of foods andars to be more available from breast milk than from cow's milk, and, in general, from foods of animal origin than from foods of plant origin. The task of nutrition researchers is to unravel further the mechanisms involved in normal absorption of dietary zinc in human beings and to pursue the identification of foods and chemicals that enhance or inhibit zinc absorption.

Picolinic acid in milk, pancreatic juice, and intestine: inadequate for role in zinc absorption

Picolinic acid (PA) was measured by high pressure liquid chromatography in human milk and other fluids and tissues. Skimmed human milk, intestinal homogenates from human infants and rats, and human and rat pancreatic juice were ultrafiltered and analyzed by high pressure liquid chromatography using an anion-exchange column. Identity of sample components was verified by comparing retention times with those of pure nicotinic acid and PA. The detection limit for PA was 2.5 microM. Human milk contained less than 3.7 microM PA. PA was undetectable in human infant or rat intestine or in human or rat pancreatic juice. The extremely low concentration of PA in milk and its apparent absence in pancreatic juice and intestine provide additional evidence that PA is not the low molecular weight zinc binding ligand of human milk and that it does not have an important physiological role in intestinal zinc absorption.

Picolinic acid as a zinc-binding ligand in human milk: an unconvincing case.

In the paper entitled Characterization and Quantitation of a Zinc-Binding Ligand in Human Milk, Evans and Johnson (4) claim to have identified as picolinic acid a low-molecular-weight ligand binding zinc in human milk. This claim is in conflict with our report identifying this ligand as citrate (13). We now present some experimental data which we hope will clarify the issue. We also question some of the techniques, as well conclusions, described in the paper by Evans and Johnson. To evaluate the possible presence of piwlinic acid in human milk, we subjected to gel filtration samples of (1) human milk, (2) picolinic acid plus zinc, and (3) human milk plus picolinic acid plus zinc. A column (1.6 x 90 cm) was packed with Sephadex G15 (Pharmacia Fine Chemicals, Piscataway, NJ) that had been treated with NaBH, to remove surface charges originally present on the gel matrix (1 1). Under these conditions, recovery of zinc from the column was 95 to 100%; in contrast, when untreated gels are used, recovery of zinc is variable and often lower than 60% (3, 12). The column was equilibrated with 0.1 M ammonium acetate buffer (pH 6.5) at a flow rate of 60 ml/hr. The fractions were analyzed for zinc by flame atomic absorption spectrophotometry (Perkin-Elmer 370) and for aromatic absorption at 280 nm (Varian, Cary 2 19). As shown in Figure IA, zinc in human milk eluted in two peaks. The first peak consisted of high-molecular-weight zinc-binding protein@), and the second was a low-molecular-weight peak coeluting with citrate. This was demonstrated by a specific enzymatic assay for citrate (2), confirming our previous report (13). Figure I B shows the elution behavior of picolinic acid plus zinc I300 pM picolinic acid + 0.3 pg/ml of zinc in the form of Zn (NOR)2]. It is clear that picolinic acid coelutes with zinc, but at a position considerably different from that of the low-molecularweight zinc complex of human milk seen in Figure IA. No such zinc peak was observed in the elution pattern of human milk. It can also be noted that this peak is positioned after the total volume of the column. This is in accordance with the elution behavior of aromatic compounds on Sephadex gels (8); such compounds are retarded because of hydrophobic interaction and thus elute after the total volume of the column. When picolinic acid plus zinc (in the same concentrations as above) was added to human milk and this combination was subjected to gel filtration, three zinc-binding peaks were observed (Figure 1C). The first two peaks were the same as those shown in Figure 1A for human milk; the third peak appeared in the same elution position as that shown for picolinic acid plus zinc in Figure 1B. Therefore, it is evident that if there were a zinc picolinate complex in human milk, this complex would elute after the total volume of the column. Our results demonstrate that zinc picolinate cannot be a significant naturally occurring zinc complex in human milk. Furthermore, examination of Figure 1 in Evans and Johnson's paper shows that their low-molecular-weight zinc complex eluted before the total volume of the wlumn. This result is, in fact, in accordance with the elution pattern we observed for zinc citrate.

Characterization and Quantitation of a Zinc-Binding Ligand in Human Milk

Summary: A low-molecular-weight zinc-binding ligand from human milk has been isolated and characterized. The ligand was isolated by chromatography on Dowex 50, Dowex 1, and Sephadex G-15 equilibrated with 0.153 mM Zn(NO3)2. Mass spectroscopy, thin-layer chromatography, and infrared spectroscopy proved that the zinc-binding ligand isolated by this method from human milk is pyridine-2-carboxylic acid, commonly known as picolinic acid. The concentration of picolinic acid in human milk was 308 μM, the concentration in one brand of processed cow's milk was 20 μM, but picolinic acid was undetectable in a second brand of cow's milk and in four different infant formulas. Weanling rats fed supplemental picolinic acid absorbed significantly more dietary zinc and gained significantly more weight than rats fed an unsupplemented diet. The results suggest that the high bioavailability of zinc in human milk results from the presence of picolinic acid, a bidentate chelating ligand which facilitates zinc absorption from the intestine. Speculation: The high bioavailability of zinc in human milk results from the presence of picolinic acid, a bidentate chelating ligand which facilitates zinc absorption from the intestine.