Added Metal Ions Research Articles

Influence of metal ions on the structure of poorly crystallized iron oxide rusts

The poorly crystallized steel rusts have been prepared by hydrolysis of aqueous solutions dissolving Fe(III) and different metal ions such as Ti(IV), Cr(III), Ni(II) and Cu(II) at varied atomic ratios in metal/(Fe+metal). The precipitates formed by the hydrolysis were frozen with liquid N 2 to inhibit crystallization and particle growth. The rusts produced were poorly crystalline and nanosized particles with sizes of 4–10 nm and the rust formed without adding metal ions was assigned to ferrihydrite. The crystallite size decreased from 1.1 to 0.7 nm with increasing the added metal ions. It was shown by extended X-ray fine structure and Mössbauer spectroscopy that the short-range structure of poorly crystalline rusts was disturbed by adding Ti(IV).

Regioselectivity in the intramolecular allyl transfer reaction catalysed by electrogenerated nickel complexes: influence of metal ions

The intramolecular transfer of allyl moieties from substituted allyl aryl ethers to carbonyl groups has been studied by electrosynthesis, in a nickel-catalysed reaction. The influence of metal ions such as Mg 2+, Zn 2+ and Al 3+ has been examined. Regioselectivity towards the branched isomer was better with Zn 2+ than with Mg 2+ ions, but it was higher in the absence of added metal ions.

Trace metal-ion catalysis of oxidation of aqueous hydrogen sulfide by outer-sphere oxidants

Stopped-flow kinetic studies have been conducted on the oxidation of aqueous H 2S in acetate buffers (pH ∼5) at 25 °C. The oxidants studied include [IrCl 6] 2−, [Ru(NH 3) 5isn] 3+, [Ru(NH 3) 5py] 3+, and [Ru(NH 3) 4bpy] 3+. In all cases, the reactions are highly sensitive to catalysis by Cu 2+ at the 2 μM level. In the presence of chelating ligands such as oxalate, EDTA, phenanthroline, and bathophenanthrolinedisulfonate the rates are substantially inhibited relative to reactions with no added metal ions. It is clear that the rates obtained without added chelating ligands or metal ions are due to catalytically active amounts of metal ions unavoidably present as impurities. This sensitivity to adventitious catalysis is likely a general characteristic of outer-sphere oxidations of H 2S in acetate buffers. On the other hand, with Na 9P 2W 15Nb 3O 62 as the oxidant in borate buffers (pH ∼9) the reaction rates are insensitive to the presence of EDTA or 2 μM Cu 2+; this difference may be either a pH effect of a reflection of an inner-sphere mechanism.

Ferric Ions Reduce the Antioxidant Activity of the Phenolic Fraction of Virgin Olive Oil

ABSTRACT: The antioxidant activity of relatively polar extracts from virgin olive oil was investigated in sunflower oil stripped of tocopherols and in tocopherol‐stripped sunflower oil‐in‐water emulsions. The extracts were found to be effective as antioxidants in both media in the absence of added metal ions. However, the antioxidant activity was markedly reduced by the presence of added ferric chloride. In sunflower oil‐in‐water emulsions (pH 5.4) containing ferric chloride, all concentrations of olive oil polyphenols exhibited pro‐oxidant effects. It appears that the reducing action of olive oil polyphenols accelerates oxidation of oil and especially of emulsions containing Fe (III) by reducing ferric ions to ferrous ions, which are effective pro‐oxidants during storage.

Inhibition of glyceraldehyde-3-phosphate dehydrogenase by peptide and protein peroxides generated by singlet oxygen attack.

Reaction of certain peptides and proteins with singlet oxygen (generated by visible light in the presence of rose bengal dye) yields long-lived peptide and protein peroxides. Incubation of these peroxides with glyceraldehyde-3-phosphate dehydrogenase, in the absence of added metal ions, results in loss of enzymatic activity. Comparative studies with a range of peroxides have shown that this inhibition is concentration, peroxide, and time dependent, with H2O2 less efficient than some peptide peroxides. Enzyme inhibition correlates with loss of both the peroxide and enzyme thiol residues, with a stoichiometry of two thiols lost per peroxide consumed. Blocking the thiol residues prevents reaction with the peroxide. This stoichiometry, the lack of metal-ion dependence, and the absence of electron paramagnetic resonance (EPR)-detectable species, is consistent with a molecular (nonradical) reaction between the active-site thiol of the enzyme and the peroxide. A number of low-molecular-mass compounds including thiols and ascorbate, but not Trolox C, can prevent inhibition by removing the initial peroxide, or species derived from it. In contrast, glutathione reductase and lactate dehydrogenase are poorly inhibited by these peroxides in the absence of added Fe2+-EDTA. The presence of this metal-ion complex enhanced the inhibition observed with these enzymes consistent with the occurrence of radical-mediated reactions. Overall, these studies demonstrate that singlet oxygen-mediated damage to an initial target protein can result in selective subsequent damage to other proteins, as evidenced by loss of enzymatic activity, via the formation and subsequent reactions of protein peroxides. These reactions may be important in the development of cellular dysfunction as a result of photo-oxidation.

Alteration of the selectivity of DNA cleavage by a deglycobleomycin analogue containing a trithiazole moiety.

The bleomycin (BLM) group of antitumor antibiotics effects DNA cleavage in a sequence-selective manner. Previous studies have indicated that the metal-binding and bithiazole moieties of BLM are both involved in the binding of BLM to DNA. The metal-binding domain is normally the predominant structural element in determining the sequence selectivity of DNA binding, but it has been shown that replacement of the bithiazole moiety with a strong DNA binder can alter the sequence selectivity of DNA binding and cleavage. To further explore the mechanism by which BLM and DNA interact, a trithiazole-containing deglycoBLM analogue was synthesized and tested for its ability to relax supercoiled DNA and cleave linear duplex DNA in a sequence-selective fashion. Also studied was cleavage of a novel RNA substrate. Solid-phase synthesis of the trithiazole deglycoBLM A(5) analogue was achieved using a TentaGel resin containing a Dde linker and elaborated from five key intermediates. The ability of the resulting BLM analogue to relax supercoiled DNA was largely unaffected by introduction of the additional thiazole moiety. Remarkably, while no new sites of DNA cleavage were observed for this analogue, there was a strong preference for cleavage at two 5'-GT-3' sites when a 5'-(32)P end-labeled DNA duplex was used as a substrate. The alteration of sequence selectivity of cleavage was accompanied by some decrease in the potency of DNA cleavage, albeit without a dramatic diminution. In common with BLM, the trithiazole analogue of deglycoBLM A(5) effected both hydrolytic cleavage of RNA in the absence of added metal ion and oxidative cleavage in the presence of Fe(2+) and O(2). In comparison with BLM A(5), the relative efficiencies of hydrolytic cleavage at individual sites were altered.

Purification and characterization of a putative virulence factor, serine protease, from Vibrio parahaemolyticus

A protease (protease A) was successfully purified from the extracellular proteins of Vibrio parahaemolyticus no. 93, a clinical strain carrying neither tdh nor trh genes, using phenyl-Sepharose CL-4B hydrophobic interaction chromatography. The molecular mass of protease A was 43 kDa using gel filtration, which was in agreement with the results obtained from SDS–PAGE, suggesting that protease A was a monomeric protein. Additionally, the isoelectric point of this protein was 5.0. The optimum temperature and pH of protease A ranged from 40°C to 50°C and pH 8, respectively. Protease A activity was inhibited by serine protease inhibitors, such as phenylmethylsulfonyl fluoride and soybean trypsin inhibitor; moreover, the activity could be blocked by treatment with 20 mM of 1,10-phenanthroline, but could not be restored by adding metal ions. These results indicated that protease A is a serine protease that requires metal. The 12 N-terminal residues of protease A showed a high degree of identity (81%) to the sequence of Vibrio metschnikovii VapT serine protease. The purified protease had significant effects on the growth of Chinese hamster ovary, HeLa, Vero and Caco-2 cells and its cytotoxic activity was not blocked by gangliosides. Protease A lysed erythrocytes well but its hemolytic activity was unstable after heat treatment, indicating that protease A is able to cause hemolysis but is a heat-labile protein. The purified protease caused tissue hemorrhage and death in mice when injected both intraperitoneally and intravenously. In conclusion, this is the first report of a serine protease purified directly from the supernatant of V. parahaemolyticus and identifying it as a potential virulence factor.

Purification and characterization of a putative virulence factor, serine protease, fromVibrio parahaemolyticus

A protease (protease A) was successfully purified from the extracellular proteins of Vibrio parahaemolyticus no. 93, a clinical strain carrying neither tdh nor trh genes, using phenyl-Sepharose CL-4B hydrophobic interaction chromatography. The molecular mass of protease A was 43 kDa using gel filtration, which was in agreement with the results obtained from SDS-PAGE, suggesting that protease A was a monomeric protein. Additionally, the isoelectric point of this protein was 5.0. The optimum temperature and pH of protease A ranged from 40 degrees C to 50 degrees C and pH 8, respectively. Protease A activity was inhibited by serine protease inhibitors, such as phenylmethylsulfonyl fluoride and soybean trypsin inhibitor; moreover, the activity could be blocked by treatment with 20 mM of 1,10-phenanthroline, but could not be restored by adding metal ions. These results indicated that protease A is a serine protease that requires metal. The 12 N-terminal residues of protease A showed a high degree of identity (81%) to the sequence of Vibrio metschnikovii VapT serine protease. The purified protease had significant effects on the growth of Chinese hamster ovary, HeLa, Vero and Caco-2 cells and its cytotoxic activity was not blocked by gangliosides. Protease A lysed erythrocytes well but its hemolytic activity was unstable after heat treatment, indicating that protease A is able to cause hemolysis but is a heat-labile protein. The purified protease caused tissue hemorrhage and death in mice when injected both intraperitoneally and intravenously. In conclusion, this is the first report of a serine protease purified directly from the supernatant of V. parahaemolyticus and identifying it as a potential virulence factor.

Fractal characterization of silica sol prepared by the sol-gel method: From the sol formation to the flocculation process

The fractal characterization of silica particles prepared by the sol-gel method was obtained; from the beginning of the sol-gel synthesis to the aggregation process of these particles by adding metal ions in solution, the fractal dimension was determined. At the beginning of the sol-gel process, unstable structures were formed due, essentially, to the auto-catalytic nature of the sol-gel condensation reactions; these particles are fractal structures with a fractal exponent corresponding to a reaction limited aggregation regime. As the time proceeds, the reactants are consumed approaching the system to equilibrium, stabilizing the size of the silica particles. The silica sol can be flocculated by adding metal ions in solution. The fractal exponent for the aggregation process was determined, obtaining a value corresponding to a diffusion limited aggregation regime.

Fluorescence and FT-IR spectroscopic studies of Suwannee river fulvic acid complexation with aluminum, terbium and calcium

In this study fluorescence emission and IR spectroscopy have been used to investigate the interaction of the class A (oxygen seeking ‘hard acid’) metal Al 3+, with Suwannee River fulvic acid. Addition of Al 3+ ion results in a significant enhancement in fulvic acid fluorescence emission (at λ em=424 nm) and significant red shift of the excitation wavelength (from λ ex=324 nm to λ ex=344 nm) at low pH values (pH∼4.0–5.0). At pH 4.0 (0.1 M ionic strength), where the predominant aluminum ion species is the ‘free’ (aquo) ion, the fulvic acid fluorescence reaches 142% of the value in the absence of added metal ion. Analysis of the pH 4.0 and pH 5.0 fluorescence enhancement data with the nonlinear (single site) model of Ryan and Weber indicated binding constants in the range of 4.67·10 4–2.87·10 6 M −1 and concentrations of ligand sites in the range of 18.6·10 −6–24.0·10 −6 M, both consistent with previous studies performed on both aquatic and soil fulvic acids. Companion fluorescence experiments performed on two other class A metal ions, Ca 2+ and Tb 3+, indicated no significant enhancement or quenching with Ca 2+ and only slight quenching with Tb 3+. Comparison of FT-IR spectra collected on fulvic acid alone and fulvic acid in the presence of the three class A metals (Al 3+, Ca 2+ and Tb 3+) provides strong evidence for the involvement of carboxyl carbonyl functions in the binding of all three metal ions, which is not unexpected. The spectra also reveal, however, a very pronounced difference in the 4000–2000 cm −1 IR spectral region between the Al 3+ spectrum and the Ca 2+ and Tb 3+ spectra. The –OH stretch spectral region in the Al 3+ spectrum has a major component shifted to higher energy (compared to fulvic acid alone or to fulvic acid in the presence of Ca 2+ or Tb 3+). Even more striking is the emergence of a pronounced IR band at 2407 cm −1, which is present only in the Al 3+ spectrum. The results of fluorescence and IR experiments with the model compounds salicylic acid and phthalic acid further confirm that both salicylic acid-like sites and phthalic acid-like sites are likely complexation sites for Al 3+ in fulvic acid and are major contributors to the observed spectroscopic changes associated with Al 3+ ion complexation. From a comparison of both the fluorescence and IR spectral results for all three class A metals, differing most strongly in the value of their ionic index, it seems clear that major sources of the deviation in spectral properties between Al 3+ and Ca 2+/Tb 3+ is the unusually high value of its charge density and relatively low propensity for involvement in covalent bonding interactions (very high ionic index and relatively low covalent index in the Nieboer and Richardson classification of environmental metals), as well as affinity for certain functional groups.

Characterization of the hepatitis C virus NS2/3 processing reaction by using a purified precursor protein.

The NS2-NS3 region of the hepatitis C virus polyprotein encodes a proteolytic activity that is required for processing of the NS2/3 junction. Membrane association of NS2 and the autocatalytic nature of the NS2/3 processing event have so far constituted hurdles to the detailed investigation of this reaction. We now report the first biochemical characterization of the self-processing activity of a purified NS2/3 precursor. Using multiple sequence alignments, we were able to define a minimal domain, devoid of membrane-anchoring sequences, which was still capable of performing the processing reaction. This truncated protein was efficiently expressed and processed in Escherichia coli. The processing reaction could be significantly suppressed by growth in minimal medium in the absence of added zinc ions, leading to the accumulation of an unprocessed precursor protein in inclusion bodies. This protein was purified to homogeneity, refolded, and shown to undergo processing at the authentic NS2/NS3 cleavage site with rates comparable to those observed using an in vitro-translated full-length NS2/3 precursor. Size-exclusion chromatography and a dependence of the processing rate on the concentration of truncated NS2/3 suggested a functional multimerization of the precursor protein. However, we were unable to observe trans cleavage activity between cleavage-site mutants and active-site mutants. Furthermore, the cleavage reaction of the wild-type protein was not inhibited by addition of a mutant that was unable to undergo self-processing. Site-directed mutagenesis data and the independence of the processing rate from the nature of the added metal ion argue in favor of NS2/3 being a cysteine protease having Cys993 and His952 as a catalytic dyad. We conclude that a purified protein can efficiently reproduce processing at the NS2/3 site in the absence of additional cofactors.

Metal-Mediated Self-Assembly of π-Bonded Benzoquinone Complexes into Polymers with Tunable Geometries.

Coordination of Mn(CO)3+ to the π system in hydroquinone facilitates proton loss to afford benzoquinone complexes. Subsequent σ coordination of the benzoquinone oxygen atoms to added metal ions results in neutral one-, two-, or three-dimensional quinoid polymers. The geometrical requirements of the metal ion and the presence of added "spacer" ligands dictate the type of polymer formed.

Purification and characterisation of a serine peptidase from the marine psychrophile strain PA-43

An extracellular serine peptidase, purified from the culture supernatant of the sub-Arctic psychrophilic bacterium strain PA-43, is monomeric, with a relative molecular mass of 76 000, and an unusually low p I of 3.8. The peptidase is active towards N-succinyl AAPF p-nitroanilide and N-succinyl AAPL p-nitroanilide, indicating a chymotrypsin-like substrate specificity. It is inhibited by the serine peptidase inactivator phenylmethylsulfonyl fluoride, but not by EDTA or EGTA, suggesting that added metal ions are not necessary for activity. The enzyme is most active at pH 8.3 and at 55–60°C, although it is unstable at 60°C. It is nevertheless remarkably stable for an enzyme from a psychrophilic microorganism, remaining active after 1 week at 20°C and after five freeze–thaw cycles. Comparison of the N-terminal 40 amino acid residues with other archived sequences revealed highest similarity to the alkaline serine protease ( aprx) from Bacillus subtilis.

Purification and characterisation of a serine peptidase from the marine psychrophile strain PA-43.

An extracellular serine peptidase, purified from the culture supernatant of the sub-Arctic psychrophilic bacterium strain PA-43, is monomeric, with a relative molecular mass of 76000, and an unusually low pI of 3.8. The peptidase is active towards N-succinyl AAPF p-nitroanilide and N-succinyl AAPL p-nitroanilide, indicating a chymotrypsin-like substrate specificity. It is inhibited by the serine peptidase inactivator phenylmethylsulfonyl fluoride, but not by EDTA or EGTA, suggesting that added metal ions are not necessary for activity. The enzyme is most active at pH 8.3 and at 55-60 degrees C, although it is unstable at 60 degrees C. It is nevertheless remarkably stable for an enzyme from a psychrophilic microorganism, remaining active after 1 week at 20 degrees C and after five freeze-thaw cycles. Comparison of the N-terminal 40 amino acid residues with other archived sequences revealed highest similarity to the alkaline serine protease (aprx) from Bacillus subtilis.

Structure and chemistry of grain boundaries in SiO2-doped TZP

The addition of glass phase can control the grain boundary structure and hence the mechanical properties of tetragonal zirconia polycrystals (TZP). To reveal the effect of the glass dopant on the high-temperature deformation behavior of TZP, SiO2-doped TZP, (SiO2—Al2O3)-doped TZP, (SiO2—MgO)-doped TZP and undoped TZP were prepared and their grain boundary structure, chemical composition and chemical bonding state were investigated by high resolution electron microscopy ,HREM), energy dispersive X-ray spectroscopy ,EDS) and electron energy loss spectroscopy (EELS) using a field-emission-type transmission electron microscope (FE-TEM). It was found that no amorphous film was formed along the grain boundaries in any of the specimens examined, but amorphous pockets formed at multiple grain boundary junctions in three kinds of glass-doped specimens. In the glass-doped specimens, the segregation of yttrium, silicon and the added metal ions (Al31 or Mg21) was observed over a width of several nm across the grain boundaries. The addition of pure SiO2 much enhanced the ductility in TZP, although further addition of a small amount of Al2O3 or MgO to SiO2 phase resulted in a marked reduction in the tensile ductility of SiO2-doped TZP. EELS measurements and molecular orbital (MO) calculations using a cluster model revealed that the ductility of TZP was related to the bond overlap population (BOP) at the grain boundaries, which was influenced by the kinds of segregated dopants. That is, the presence of Si41 increases the BOP, strengthening the grain boundary bonding strength and thus preventing cavity formation, but Al31 and Mg21 decrease the BOP, enhancing the grain boundary cavitation and thus reducing the ductility. Furthermore, the dynamic behavior of SiO2 in TZP was observed using a TEM in situ heating technique, and the results supported the fact that that Si segregates along the grain boundaries.

Rheological behavior of sodium alginate solutions with added divalent metal salts and their use as thickeners in cotton printing with reactive dyes

The addition of traces of certain divalent metal ions (Ca2+ and Mg2+) to aqueous solutions of sodium alginate increases their viscosity to a high extent. Further increase in the concentration of the added metal ions causes gelation and precipitation of the metal alginate salt. The effect of adding the divalent metal ions on the viscosity of sodium alginate solutions (before gelation or precipitation stages) was investigated via rheological and viscosity measurements. The optimum concentrations of the divalent metal salts were determined. Calculated traces of divalent metal salts were added to pastes of low-viscosity sodium alginate (5%) to increase its viscosity and subsequently decrease the amount of sodium alginate required for use in screen printing of cotton fabrics with reactive dyes (i.e., decreasing cost). For the sake of comparison, a comparativey was conducted by preparing other series of printing pastes thickened with low-viscosity sodium alginate (8%) and high-viscosity sodium alginate (2.5%). These printing pastes were applied to cotton fabrics by using the same technique. © 2001 John Wiley & Sons, Inc. Adv Polym Techn 20: 58–71, 2001

Differences in Antioxidative Efficiency of Catechins in Various Metal-Induced Lipid Peroxidations in Cultured Hepatocytes.

The effects of tea catechins, including (‐)-epicatechin (EC), (‐)-epigallocatechin (EGC), (‐)-epicatechingallate (ECg) or (‐)-epigallocatechingallate (EGCg) on the lipid peroxidation induced by either ferrous (Fe), copper (Cu) or vanadium (V ) ions were examined in normal and α-linolenic acid-loaded (LNA-loaded) cultured rat hepatocytes. The order of antioxidative activity was almost similar and ranked as follows: EGCg > ECg > EGC > EC against the three metal-induced lipid peroxidations in both groups of cultured hepatocytes. However, each catechin displayed a marked variation in its antioxidative potency depending on the added metal ion species, whereas dibutyl hydroxy toluene (BHT), a typical lipid radical scavenger, exhibited a similar antioxidative potency with all metal ions. The half inhibition concentration (IC50) values of the catechins for Fe ion-induced lipid peroxidation were 2 to 3 times and 5 to 14 times higher than those for V ion and Cu ion, respectively in normal hepatocytes. Only in LNA-loaded hepatocytes, was EC antioxidative at 20 µM Fe concentration but it became prooxidative above 50 µM Fe concentration. Catechins such as (+)-catechin, (+)-epicatechin and (±)-catechin also acted as prooxidants at high Fe concentrations in LNA-loaded hepatocytes. The antioxidative efficiency of EGC, ECg or EGCg decreased with the increase in Fe ion concentrations from 20 µM up to 500 µM in LNA-loaded hepatocytes. These findings suggest that the metal-chelating property of catechins may play a major role in determining antioxidative activity in cultured hepatocytes.

Inhibition of ferric chelate reductase in alfalfa roots by cobalt, nickel, chromium, and copper

This research was initiated to examine the influence of metal cations on ferric chelate reducing capacity of alfalfa grown in hydroponic culture under conditions of iron sufficient or iron limited conditions. In the absence of added metal ions, ferric chelate reductase was greater in plants cultivated in iron limited conditions than in plants where iron was not limiting. Ferric chelate reductase activity in alfalfa roots was inhibited in both iron limited and iron sufficient growth by 0.5 to 2.5 μM Co2+. While the addition of Cr3+ at 2.0 μM slightly inhibited ferric chelate reductase in roots of plants grown under iron limited conditions, Cr3+ at 10 μM stimulated ferric chelate reductase in roots from both iron limited and iron sufficient media. Cu2+ additions at 5 uM to hydroponic media greatly inhibits ferric chelate reductase in plants with iron limited growth while shoot growth was inhibited in both iron sufficient and iron limited growth. The addition of Ni2+ at levels greater than 5 μM to alfalfa cultures inhibits both root and shoot growth; however, at these concentrations of Ni2+ ferric chelate reductase is stimulated in iron sufficient media but reduced in iron stressed plants. Acidification of the media was observed only with iron stressed cultures and did not closely correlate with levels of ferric chelate reductase of the roots.

Reactions of N-methyl-N-nitrosotoluene-P-sulfonamide (MNTS) with 3,6-dibromocarbazole and its corresponding nitranion

Reaction of N-methyl-N-nitrosotoluene-p-sulfonamide (MNTS, 1) with 3,6-dibromocarbazole (2) in dry acetonitrile gave N-methyl-p-toluenesulfonamide (4) and N-nitroso-3,6-dibromocarbazole (5), whereas with the corresponding nitranion (3) gave N-(p-toluenesulfonyl)-3,6-dibromocarbazole (6). The results are rationalized in terms of direct nucleophilic substitution mechanism occurring on respective sites. The effects of solvents and added metal ions are also discussed.

Distinct Ca2+ Binding Properties of Novel C2 Domains of Plant Phospholipase Dα and β

Of the isoforms of plant phospholipase D (PLD) that have been cloned and characterized, PLDalpha requires millimolar levels of Ca(2+) for optimal activity, whereas PLDbeta is most active at micromolar concentrations of Ca(2+). Multiple amino acid sequence alignments suggest that PLDalpha and PLDbeta both contain a Ca(2+)-dependent phospholipid-binding C2 domain near their N termini. In the present study, we expressed and characterized the putative C2 domains of PLDalpha and PLDbeta, designated PLDalpha C2 and PLDbeta C2, by CD spectroscopy, isothermal titration calorimetry, and phospholipid binding assay. Both PLD C2 domains displayed CD spectra consistent with anticipated major beta-sheet structures but underwent spectral changes upon binding Ca(2+); the magnitude was larger for PLDbeta C2. These conformational changes, not shown by any of the previously characterized C2 domains of animal origin, occurred at micromolar Ca(2+) concentrations for PLDbeta C2 but at millimolar levels of the cation for PLDalpha C2. PLDbeta C2 exhibited three Ca(2+)-binding sites: one with a dissociation constant (K(d)) of 0.8 microm and the other two with a K(d) of 24 micrometer. In contrast, isothermal titration calorimetry data of PLDalpha C2 were consistent with 1-3 low affinity Ca(2+)-binding sites with K(d) in the range of 590-470 micrometer. The thermodynamics of Ca(2+) binding markedly differed for the two C2 domains. Likewise, PLDbeta C2 bound phosphatidylcholine (PC), the substrate of PLD, in the presence of submillimolar Ca(2+) concentrations, whereas PLDalpha C2 did so only in the presence of millimolar levels of the metal ion. Both C2 domains bound phosphatidylinoistol 4,5-bisphosphate, a regulator of PC hydrolysis by PLD. However, added Ca(2+) displaced the bound phosphatidylinoistol 4,5-bisphosphate. Ca(2+) and PC binding properties of PLDalpha C2 and PLDbeta C2 follow a trend similar to the Ca(2+) requirements of the whole enzymes, PLDalpha and PLDbeta, for PC hydrolysis. Taken together, the results suggest that the C2 domains of PLDalpha and PLDbeta have novel structural features and serve as handles by which Ca(2+) differentially regulates the activities of the isoforms.