Metal ion-dependent binding of gastrin to albumin

Binding of 125I-[Nle15]gastrin to albumin purified from porcine serum, from porcine gastric mucosal cytosol, and from bovine serum has been demonstrated by covalent cross-linking and ultracentrifugation. Binding was enhanced in the presence of Zn2+, Ni2+, Cu2+, Co2+, and Cd2+, but not Ca2+, Mg2+, or Mn2+. The best fit to the binding data for bovine serum albumin was obtained with a model assuming two nonequivalent binding sites. The affinity of both sites for gastrin was increased in the presence of 100 microM Zn2+ or Ni2+ ions. The highest association constant observed was 2.3 X 10(5) M-1 in the presence of 100 microM Zn2+ ions. The similarity of the Zn(2+)-dependence of binding for bovine and porcine serum albumins, despite the replacement of His3 by Tyr, suggested that the N-terminal metal ion-binding site was not involved. Although all gastrin affinities were reduced by 50% in the presence of 150 mM NaCl, the Zn(2+)-dependence of binding was retained. We therefore propose that the ternary complex of gastrin, Zn2+ ions, and albumin may play a physiological role in the serum transport of Zn2+ ions and in the uptake of Zn2+ ions from the lumen of the gastrointestinal tract.     

Interaction of copper nd zinc cations with calcium-binding proteins

Interactions of the calcium binding proteins, parvalbumin from cod muscles, alpha-lactalbumin from cow milk and calmodulin from bovine brain, with Cu2+ and Zn2+ ions have been studied by intrinsic fluorescence and microcalorimetry methods. It was revealed that parvalbumin binds one Cu2+ ion per molecule with association constant from 10(5) to 10(6) M-1. Zn2+ ions seem to compete for the same site which does not coincide with the two Ca2+ and Mg2+ binding sites. alpha-Lactalbumin contains from 2 to 4 Cu2+ and Zn2+ binding sites, the number and affinities of which depend on Ca2+ concentration. Calmodulin has similar Cu2+ and Zn2+ binding sites. The binding of Cu2+ and Zn2+ ions to parvalbumin and alpha-lactalbumin changes the shape and position of their thermal denaturation transitions. The results obtained together with the literature data show that the ability to interact with Cu2+ and Zn2+ ions is a property inherent to many calcium-binding proteins, which may play a physiological role for some of them.     

Zn+2 induces reversible cross-linking of human placental thyroid hormone nuclear receptor with no effect on hormone binding.

Zn+2 is required for specific binding of c-erbA proteins to the hormone response elements of target genes. It is unclear whether Zn+2 is important for the binding of ligand to c-erbA proteins. The present study evaluated the effect of Zn+2 and other divalent cations on the binding of 3,3',5-triiodo-L-thyronine(T3) to the purified human placental c-erbA protein (h-TR beta 1). Zn+2 induced cross-linking of h-TR beta 1 to form aggregates in a dose-dependent manner with an apparent half-maximal concentration of approximately 200 microM at 22 degrees C. Cross-linking was reversible by the addition of 5 microM EDTA or 10 mM dithiothreitol. The cross-linked h-TR beta 1 bound T3. These results indicated Zn+2 had no effect on T3 binding and suggested that the cysteines and histidines involved in cross-linking are not essential for T3 binding.     

Metal ion dependence of the binding of triiodothyronine by cytosol proteins of bullfrog tadpole tissues.

The binding of triiodothyronine by Rana catesbeiana tadpole tail fin, tail muscle, kidney, and liver cytosol was studied using dextran-coated charcoal to separate bound and free hormone. A metal ion dependency was suggested by the fact that EDTA decreased the binding of triiodothyronine 80 to 90% in tail fin and tail muscle cytosol. Inhibition of binding in kidney or liver was less, 40 to 50%. This inhibition could be restored by adding an excess of divalent cations with an order of potency of Mn2+ greater than Ca2+ congruent to Co2+ greater than Sr2+ greater than Ba2+ greater than Mg2+. Other chelators, e.g. o-phenanthroline, 8-hydroxyquinoline, and ethylene glycol bis(beta-aminoethylether)-N,N'-tetraacetate also decreased the binding of triiodothyronine, whereas citrate, oxalate, imidazole, and glycine had no effect. The triiodothyronine binding capacity of tail fin cytosol was reduced by EDTA treatment and dialysis against buffer. Ca2+ in the 1 to 10 mM range and Mn2+ at 1 mM could restore the binding to normal levels. Higher Mn2+ increased binding 70% above normal or to Ca2+-restored levels. The triiodothyronine cytosol binding activity was nondialyzable, heat-labile. pH-dependent, pronase-digestible, but unaffected by incubation with trypsin, RNase, and DNase, suggesting that the cytosol binding sites are acidic proteins. Scatchard analysis of triiodothyronine binding by the cytosol of different tissues, revealed Kassoc of 7.1 x 10(6) M(-1), 11.6 x 10(6) M(-1), 3.6 X 10(6) M(-1), and 68.0 x 10(6) M(-1) for tail fin, tail muscle, kidney, and liver cytosol, respectively. The corresponding maximal binding capacities in picomoles per mg of crude cytosol protein in these four tissues were 10.4, 0.86, 1.3, and 0.04, respectively.     

Activation of alkaline phosphatase with Mg2+ and Zn2+ in rat hepatoma cells. Accumulation of apoenzyme

In Reuber rat hepatoma cells (R-Y121B), alkaline phosphatase activity increased without de novo enzyme synthesis (Sorimachi, K., and Yasumura, Y. (1986) Biochim. Biophys. Acta 885, 272-281). The enzyme was partially purified by butanol extraction from the particulate fractions. The incubation of the extracted alkaline phosphatase with the cytosol fraction induced a large increase in enzyme activity (5-10-fold of control). The dialyzed cytosol was more effective than the undialyzed cytosol during an early period of incubation at 37 degrees C. This difference between the dialyzed and the undialyzed cytosol fractions was due to endogenous Na+. For maximal activation of the enzyme, both Mg2+ above 1 mM and Zn2+ at low concentrations (below 0.01 mM) were needed, although Zn2+ at high concentrations (above 0.1 mM) showed an inhibitory effect. Zn2+ and Mg2+ alone slightly increased alkaline phosphatase activity. This activation of the enzyme was temperature dependent and was not observed at 0 or 4 degrees C. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the increase in alkaline phosphatase activity did not involve the fragmentation of the enzyme and that 65Zn2+ bound to it during enzyme activation with 65Zn2+ and Mg2+. The cytosol fraction not only supplied Zn2+ to the nascent enzyme but also increased the maximal enzyme activity more than did direct addition of metal ions. Ferritin and metallothionein contributed to the activation of alkaline phosphatase with the metal ions. Since the binding of Zn2+ and Mg2+ to the nascent alkaline phosphatase is disturbed in Reuber rat hepatoma cells (R-Y121B), the apoenzyme is accumulated inside the cells. The binding of Zn2+ and Mg2+ to the apoenzyme readily takes place in the cell homogenates accompanied by an increase in catalytic activity without new enzyme synthesis.     

19F-n.m.r. study of trifluoperazine-S100 protein interaction: effects of Ca2+ and Zn2+

19F-n.m.r. spectra were measured to investigate the effects of Ca2+ and Zn2+ on the interaction of trifluoperazine (TFP) with three S100 proteins. It was found that TFP binds to S100a and S100ao proteins irrespective of the presence of Ca2+ and Zn2+, while in the presence of Ca2+ the apparent affinity of TFP to the proteins was greater than that in its absence or in the presence of Zn2+. In contrast, the binding affinity of TRP to S100b protein in the presence and absence of metal ions was lower than to S100a and S100ao proteins. These results suggested that TFP binds to each S100 protein in two ways: one is Ca2(+)- or Zn2(+)-dependent specific manner and another is Ca2(+)- or Zn2(+)-independent non-specific manner.     

Zinc binding stabilizes mitochondrial Tim10 in a reduced and import-competent state kinetically

Tim10 and all the small Tim proteins of the mitochondrial intermembrane space contain a consensus twin CX3C Zn2+-finger motif. While disulphide bond formation between the Cys residues of this motif is essential for complex formation by the small Tim proteins, the specific role of Zn2+-binding during the import and assembly of these proteins is not clear. In this study, we investigated the effects of the biologically relevant thiol-disulphide redox molecule, glutathione, and Zn2+-binding on the oxidative folding of yeast mitochondrial Tim10 using both biochemical and biophysical methods in vitro. We show that, whilst oxidized Tim10 cannot be reduced by reduced glutathione, reduced Tim10 is effectively oxidized at levels of glutathione comparable to those found in the cytosol. The oxidized Tim10 generated in the presence of glutathione is competent for complex formation with its partner protein Tim9, confirming it has a native fold. The standard redox potential of Tim10 at pH 7.4 was determined to be -0.32 V, confirming that Tim10 is a much stronger reductant than glutathione (-0.26 V, at pH 7.4) and could therefore be oxidized rapidly by oxidized glutathione in the cytosol. However, we found that Zn2+-binding can stabilize the reduced Tim10, decreasing the rate of the oxidative folding more than tenfold. In addition, we show that protein disulphide isomerase can catalyse the oxidative folding of Tim10 provided that Zn2+ was removed. We propose that Zn2+-binding is essential to maintain the protein in a reduced and import-competent state in the cytosol, and that zinc has to be removed after the protein is imported into mitochondria to initiate protein oxidative folding and assembly.     

Zinc increases phorbol ester receptors in intact B-cells, neutrophil polymorphs and platelets

In the presence of pyrithione, which was used as a Zn2+ ionophore, Zn2+ (10-100 microM) increased phorbol ester binding by intact B-CLL cells in a dose-dependent fashion. Zn pyrithione increased 2-fold the number of phorbol ester receptors in B-cells (0.74 to 1.4 pmol/10(6) cells), neutrophil polymorphs (0.2 to 0.51 pmol/10(6) cells) and platelets (91 to 209 pmol/10(10) cells). Fractionation of cells after treatment with Zn pyrithione showed that increased binding of PDBu occurred in the particulate fraction of cells and this was accompanied by loss of phorbol ester receptors from the cytosol. These data are compatible with a role for Zn in the subcellular distribution and activation of protein kinase C.     

Liver hypertrophy in winter flounder following exposure to experimentally oiled sediments

1. Male winter flounder were exposed to sediments contaminated with Venezuelan crude oil in 3 laboratory experiments of 4-5 months duration. 2. Oil exposure resulted in significant increases in liver weight. This was particularly evident in fish weighing less than 400 g. 3. The enlarged livers of the oil-exposed flounder had reduced concentrations of DNA, protein, Na+ and Zn2+, and increased concentrations of lipid and phospholipid. 4. The reduced DNA and Na+ concentrations suggested liver hypertrophy rather than hyperplasia. 5. The increased phospholipid concentrations suggested growth of membrane structures such as endoplasmic reticulum.     

Activation of apoalkaline phosphatase by serum albumin with Zn2+ in rat hepatoma cells.

Reuber rat hepatoma cells (R-Y121B) cultured at 0.5% serum accumulated apoalkaline phosphatase in intact cells. When R-Y121B cells were cultured in the presence of bovine serum albumin, alkaline phosphatase activity increased in the cells, and the associated increase in enzyme activity differed amongst bovine serum albumin preparations. The treatment of bovine serum albumin with activated charcoal not only enhanced the effect of serum albumin on alkaline phosphatase activity, but also cancelled the differences due to different preparations of serum albumin. In contrast, no effect from serum albumin was observed in the increase of alkaline phosphatase activity in R-Y121B cell homogenates incubated at 37 degrees C. The activated-charcoal treatment of bovine serum albumin increased the amount of Zn2+ bound to the protein. When R-Y121B cells were cultured with bovine serum albumin, the concentration of Zn2+ in the cytosol fraction slightly increased. However, the effect of serum albumin on Zn2+ concentration in the cytosol fractions was independent of charcoal treatment. It was concluded that serum albumin with Zn2+ induces the activation of apoalkaline phosphatase due to Zn2+ binding.     

Rat Brain S100b Protein: Purification, Characterization, and Ion Binding Properties. A Comparison with Bovine S100b Protein

We purified to homogeneity rat brain S100b protein, which constitutes about 90% of the soluble S100 protein fraction. Purified rat S100b protein comigrates with bovine S100b protein in nondenaturant system electrophoresis but differs in its amino acid composition and in its electrophoretic mobility in urea-sodium dodecyl sulfate-polyacrylamide gel with bovine S100b protein. The properties of the Ca2+ and Zn2+ binding sites on rat S100b protein were investigated by flow dialysis and by fluorometric titration, and the conformation of rat S100b in its metal-free form as well as in the presence of Ca2+ or Zn2+ was studied. The results were compared with those obtained for the bovine S100b protein. In the absence of KCl, rat brain S100b protein is characterized by two high-affinity Ca2+ binding sites with a KD of 2 X 10(-5) M and four lower affinity sites with KD about 10(-4) M. The calcium binding properties of rat S100b protein differ from bovine S100b only by the number of low-affinity calcium binding sites whereas similar Ca2+-induced conformational changes were observed for both proteins. In the presence of 120 mM KCl rat brain S100b protein bound two Zn2+-ions/mol of protein with a KD of 10(-7) M and four other with lower affinity (KD approximately equal to 10(-6) M). The occupancy of the two high-affinity Zn2+ binding sites was responsible for most of the Zn2+-induced conformational changes in the rat S100b protein. No increase in the tyrosine fluorescence quantum yield after Zn2+ binding to rat S100b was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

The relative priority of prekallikrein and factors XI/XIa assembly on cultured endothelial cells

Investigations determined the relative preference of prekallikrein (PK) or factor XI/XIa (FXI/FXIa) binding to endothelial cells (HUVECs). In microtiter plates, biotinylated high molecular weight kininogen (biotin-HK) or biotin-FXI binding to HUVEC monolayers or their matrix proteins, but not fibronectin-coated plastic microtiter plate wells, was specifically blocked by antibodies to each of the receptors of HK, uPAR, gC1qR, or cytokeratin 1. Fluorescein isothiocyanate (FITC)-PK specifically bound to HUVEC suspensions without added Zn2+, whereas FITC-FXI or -FXIa binding to HUVEC suspensions required 10 microM added Zn2+ to support specific binding. Plasma concentrations of FXI did not block FITC-PK binding to HUVECs in the absence or presence of 10 microM Zn2+. In the absence of HK, the level of FITC-FXI or -FXIa binding was half that seen in its presence. At physiologic concentrations, PK (450 nM) abolished FITC-FXI or -FXIa binding to HUVEC suspensions in the absence or presence of HK in the presence of 10 microM Zn2+. Released Zn2+ from 2-8 x 10(8) collagen-activated platelets/ml supported biotin-FXI binding to HUVEC monolayers, but platelet activation was not necessary to support biotin-PK binding to HUVECs. At physiologic concentrations, PK also abolished FXI binding to HUVECs in the presence of activated platelets, but FXI did not influence PK binding. PK in the presence or absence of HK preferentially bound to HUVECs over FXI or FXIa. Elevated Zn2+ concentrations are required for FXI but not PK binding, but the presence of physiologic concentrations of PK and HK also prevented FXI binding. PK preferential binding to endothelial cells contributes to their anticoagulant nature.     

The role of zinc in the stabilization of the dimeric form of bacterial alpha-amylase.

Bacterial alpha-amylase was shown by equilibrium and velocity-sedimentation studies to be a monomer-dimer equilibrium system in 0.10M-NaCl/0.015M-calcium acetate/0.010M-EDTA, pH7.0; an association constant of 2.4 X 10(3)M-1 is obtained. Studies of the binding of Zn2+ to alpha-amylase in 0.10M-NaCl/0.005M-calcium acetate, pH7.0, yielded binding curves that exhibit dependence on the concentration of alpha-amylase (Zn2+-free) used in the equilibrium-dialysis experiments. Results are described very satisfactorily by a reaction scheme in which Zn2+ binds exclusively to the dimer of the above monomer--dimer system with an association constant of 1.0 X 10(6)M-1. The present results refute the earlier scheme for dimer stabilization by Zn2+ in which the metal ion formed a cross-link between two non polymerizing monomer units.     

Interconversion of androgen receptor forms by divalent cations and 8 S androgen receptor-promoting factor. Effects of Zn2+, Cd2+, Ca2+, and Mg2+

The effects of divalent cations (Zn2+, Cd2+, Ca2+, Mg2+) on the cytosol androgen receptor were determined by sedimentation into sucrose gradients. At low ionic strength (25 mM KCl, 50 mM Tris, pH 7.4), Zn2+ (200 microM total, which calculates to 130 nM free Zn2+ in 10 mM mercaptoethanol) causes a shift in the sedimentation coefficient of the rat Dunning prostate tumor (R3327H) cytosol receptor and rat ventral prostate cytosol receptor from 7.5 +/- 0.3 S to 8.6 +/- 0.3 S. Zn2+ stabilizes the 8.6 S receptor form in salt concentrations up to 0.15 M KCl in 50 mM Tris, pH 7.2. In low ionic strength gradients containing Ca2+ (greater than or equal to 200 microM) or Mg2+ (greater than or equal to 1 mM), the receptor sediments as 4.7 +/- 0.3 S. The dissociating effects of Ca2+ and Mg2+ can be fully reversed by sedimentation into gradients containing Zn2+ (200 microM total) or Cd2+ (10 microM total). In the presence of Zn2+ (200 microM total), Ca2+ (10 microM to 3 mM) converts the receptor to an intermediate form with sedimentation coefficient 6.2 +/- 0.2 S, Stokes radius 73 A, and apparent Mr approximately 203,000. The potentiating effect of Zn2+ on formation of the 8.6 S receptor (in the absence of Ca2+) and the 6.2 S receptor (in the presence of Ca2+) requires both the 4.5 S receptor and the 8 S androgen receptor-promoting factor. Sodium molybdate stabilizes the untransformed cytosol receptor but, unlike Zn2+, does not promote reconstitution of the 8.6 S receptor from its partially purified components. These results indicate that divalent cations alter the molecular size of the androgen receptor in vitro and thus may have a role in altering the state of transformation of the receptor.     

A novel regulatory metal binding domain is present in the C terminus of Arabidopsis Zn2+-ATPase HMA2

HMA2 is a Zn2+-ATPase from Arabidopsis thaliana. It contributes to the maintenance of metal homeostasis in cells by driving Zn2+ efflux. Distinct from P1B-type ATPases, plant Zn2+-ATPases have long C-terminal sequences rich in Cys and His. Removal of the 244 amino acid C terminus of HMA2 leads to a 43% reduction in enzyme turnover without significant effect on the Zn2+ K(1/2) for enzyme activation. Characterization of the isolated HMA2 C terminus showed that this fragment binds three Zn2+ with high affinity (Kd = 16 +/- 3 nM). Circular dichroism spectral analysis indicated the presence of 8% alpha-helix, 45% beta-sheet, and 48% random coil in the C-terminal peptide with noticeable structural changes upon metal binding (8% alpha-helix, 39% beta-sheet, and 52% random coil). Zn K-edge XAS of Zn-C-MBD in the presence of one equivalent of Zn2+ shows that the average zinc complex formed is composed of three His and one Cys residues. Upon the addition of two extra Zn2+ ions per C-MBD, these appear coordinated primarily by His residues thus, suggesting that the three Zn2+ binding domains might not be identical. Modification of His residues with diethyl pyrocarbonate completely inhibited Zn2+ binding to the C terminus, pointing out the importance of His residues in Zn2+ coordination. In contrast, alkylation of Cys with iodoacetic acid did not prevent Zn2+ binding to the HMA2 C terminus. Zn K-edge XAS of the Cys-alkylated protein was consistent with (N/O)4 coordination of the zinc site, with three of those ligands fitting for His residues. In summary, plant Zn2+-ATPases contain novel metal binding domains in their cytoplasmic C terminus. Structurally distinct from the well characterized N-terminal metal binding domains present in most P1B-type ATPases, they also appear to regulate enzyme turnover rate.     

Insulin-metal ion interactions: the binding of divalent cations to insulin hexamers and tetramers and the assembly of insulin hexamers

An insulin hexamer containing one B10-bound Co(III) ion and one unoccupied B10 site has been synthesized. The properties of the monosubstituted hexamer show that occupancy of only one B10 site by Co3+ is sufficient to stabilize the hexameric form under the conditions of pH and concentration used in these studies. The experimentally determined, second-order rate constants for the binding of Zn2+ and Co2+ to the unoccupied B10 site are consistent with literature rate constants for the rate of association of these divalent metal ions with similar small molecule ligands. These findings indicate that the rate-limiting steps for Zn2+ and Co2+ binding involve the removal of the first aqua ligand. The rate constant for the binding of Cd2+ is significantly lower than the literature values for small molecule chelators, which suggests that some other protein-related process is rate-limiting for Cd2+ binding to the unoccupied, preformed B10 site. The kinetics of the assembly of insulin in the presence of limiting metal ion provides strong evidence indicating that the B13 site of the tetramer species can bind Zn2+, Cd2+, or Ca2+ prior to hexamer formation and that such binding assists hexamer formation. Both the tetramer and the hexamer B13 sites were found to exhibit similar affinities for Zn2+ and Cd2+ (Kd congruent to 9 microM), whereas the tetramer B13 sites bind Ca2+ much more weakly (Kd congruent to 1 mM for tetramer vs 83 microM for hexamer). The second-order rate constants estimated for the association of Zn2+ and Cd2+ to the tetrameric site indicate that the loss of the first inner-sphere aqua ligand is the rate-limiting step for binding.     

Identification of phosphatidylserine-binding proteins in human white blood cells.

Cytosol and membrane fractions from human neutrophils, monocytes, lymphocytes and platelets were separated by SDS/PAGE, blotted on to nitrocellulose and assayed for selective binding of phosphatidylserine (PS). Two PS-binding proteins with apparent molecular masses of 115 kDa and 100 kDa were identified in the cytosol of neutrophils, monocytes and lymphocytes. Corresponding bands along with other PS-binding proteins were detected in platelets in both cytosol and membrane fractions. These proteins were also found to bind protein kinase C (PKC) provided that PS was present. The 115 kDa and 100 kDa proteins (PS-p115/110) were partially purified from neutrophils and were used for the study of PS and PKC binding. The binding of PS did not require Ca2+ or Mg2+ and was inhibited by phosphatidic acid, by 1-alkyl-2-acetylphosphocholine and, to a lesser extent, by other lipids. The binding of PKC, however, was strictly PS- and Ca2(+)-dependent and seems to occur secondarily to PS binding.