Inhibition of Na+/K(+)-ATPase and Mg(2+)-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na(+)/K(+)-ATPase and Mg(2+)-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu(2+), Zn(2+), Fe(2+) and Co(2+)) and heavy metals (Hg(2+) and Pb(2+)) ions were studied. All investigated metals produced a larger maximum inhibition of Na(+)/K(+)-ATPase than Mg(2+)-ATPase activity. The free concentrations of the key species (inhibitor, MgATP(2-), MeATP(2-)) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu(2+)/Fe(2+) or Hg(2+)/Pb(2+) caused additive inhibition, while Cu(2+)/Zn(2+) or Fe(2+)/Zn(2+) inhibited Na(+)/K(+)-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu(2+)/Fe(2+) or Cu(2+)/Zn(2+) inhibited Mg(2+)-ATPase activity synergistically, while Hg(2+)/Pb(2+) or Fe(2+)/Zn(2+) induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na(+)/K(+)-ATPase activity by reducing the maximum velocities (V(max)) rather than the apparent affinity (Km) for substrate MgATP(2-), implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg(2+)-ATPase activity by Zn(2+), Fe(2+) and Co(2+) as well as kinetic analysis indicated two distinct Mg(2+)-ATPase subtypes activated in the presence of low and high MgATP(2-) concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na(+)/K(+)-ATPase with various potencies. Furthermore, these ligands also reversed Na(+)/K(+)-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg(2+)-induced inhibition was not obtained.     

Studies on the pathogenesis of liver necrosis by α-amanitin. Effect of α-amanitin on ribonucleic acid synthesis and on ribonucleic acid polymerase in mouse liver nuclei

1. Injection of alpha-amanitin to mice causes a decreased incorporation of [6-(14)C]-orotic acid into liver RNA in vivo. 2. The activity of RNA polymerase activated by Mn(2+) and ammonium sulphate is greatly impaired in liver nuclei isolated from mice poisoned with alpha-amanitin, and is inhibited by the addition of the same toxin in vitro. 3. The activity of the Mg(2+)-activated RNA polymerase is only slightly affected by alpha-amanitin either administered to mice or added in vitro.     

Response of catalase activity to Ag+, Cd2+, Cr6+, Cu2+ and Zn2+ in five tissues of freshwater fish Oreochromis niloticus

Catalase (CAT, EC 1.11.1.6) is an important enzyme in antioxidant defense system protecting animals from oxidative stress. Freshwater fish Oreochromis niloticus were exposed for 96 h to different concentrations of Ag(+), Cd(2+), Cr(6+), Cu(2+) and Zn(2+), known to cause oxidative stress, and subsequently CAT activities in liver, kidney, gill, intestine and brain were measured. In vivo, CAT was stimulated by all metals except Ag(+) in the liver and the highest increase in CAT activity (183%) resulted from 1.0 mg Cd(2+)/L exposure, whereas 0.5 mg Ag(+)/L exposure resulted in a sharp decrease (44%). In tilapia kidney, cadmium and zinc had no significant effects on CAT activity, whereas 0.1 mg Cr(6+)/L exposure caused a decrease (44%). Cadmium and zinc did not significantly affect the CAT activity in gill; however, 0.5 mg Ag(+)/L exposure caused an increase (66%) and 1.5 mg Cr(6+)/L exposure caused a decrease (97%) in CAT activity. All metals, except Cu(2+)(41% increase), caused significant decreases in CAT activity in the intestine. In brain, 1.0 mg Zn(2+)/L resulted in an increase in CAT activity (126%), while 1.5 mg Ag(+)/L exposure caused a 54% decrease. In vitro, all metals -- except Ag(+) and Cu(2+) in kidney -- significantly inhibited the CAT activity in all tissues. Results emphasized that CAT may be considered as a sensitive bioindicator of the antioxidant defense system.     

Experimental conditions affecting ribonucleic acid polymerase in isolated rat liver nuclei. Effect of nucleoside triphosphate concentration, temperature, ammonium sulphate and heparin

1. The conditions affecting the activity of RNA polymerase in isolated rat liver nuclei were studied with Mg(2+) or Mn(2+) as activating ions. 2. The enzyme assayed with Mg(2+) and at low ionic strength is saturated by a lower concentration of nucleotide substrates than if assayed with Mn(2+) at low ionic strength or with either ion at high ionic strength. 3. At low and at high ionic strength the incorporation of AMP is affected in a similar way by variations in the temperature of incubation. Preincubation at 37 degrees impairs the AMP incorporation. 4. Heparin stimulates the RNA polymerase activity in the presence of Mn(2+). 5. Both ammonium sulphate and heparin ;restart' the reaction if added after 15min., the effect being more marked with ammonium sulphate than with heparin, and also more marked in the presence of Mn(2+) than of Mg(2+). 6. alpha-Amanitin abolishes the effect of ammonium sulphate and of heparin.     

Characterization of cadmium uptake in Lactobacillus plantarum and isolation of cadmium and manganese uptake mutants.

Two different Cd(2+) uptake systems were identified in Lactobacillus plantarum. One is a high-affinity, high-velocity Mn(2+) uptake system which also takes up Cd(2+) and is induced by Mn(2+) starvation. The calculated K(m) and V(max) are 0.26 microM and 3.6 micromol g of dry cell(-1) min(-1), respectively. Unlike Mn(2+) uptake, which is facilitated by citrate and related tricarboxylic acids, Cd(2+) uptake is weakly inhibited by citrate. Cd(2+) and Mn(2+) are competitive inhibitors of each other, and the affinity of the system for Cd(2+) is higher than that for Mn(2+). The other Cd(2+) uptake system is expressed in Mn(2+)-sufficient cells, and no K(m) can be calculated for it because uptake is nonsaturable. Mn(2+) does not compete for transport through this system, nor does any other tested cation, i.e., Zn(2+), Cu(2+), Co(2+), Mg(2+), Ca(2+), Fe(2+), or Ni(2+). Both systems require energy, since uncouplers completely inhibit their activities. Two Mn(2+)-dependent L. plantarum mutants were isolated by chemical mutagenesis and ampicillin enrichment. They required more than 5,000 times as much Mn(2+) for growth as the parental strain. Mn(2+) starvation-induced Cd(2+) uptake in both mutants was less than 5% the wild-type rate. The low level of long-term Mn(2+) or Cd(2+) accumulation by the mutant strains also shows that the mutations eliminate the high-affinity Mn(2+) and Cd(2+) uptake system.     

Effect of metal ions on the activity of green crab (Scylla serrata) alkaline phosphatase

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The present paper deals with the study of the effect of some kinds of metal ions on the enzyme. The positive monovalent alkali metal ions (Li(+), Na(+) and K(+)) have no effect on the enzyme; positive bivalent alkaline-earth metal ions (Mg(2+), Ca(2+) and Ba(2+)) and transition metal ions (Mn(2+), Co(2+), Ni(2+) and Cd(2+)) activate the enzyme; heavy metal ions (Hg(2+), Ag(+), Bi(2+), Cu(2+) and Zn(2+)) inhibit the enzyme. The activation of magnesium ion on the enzyme appears to be a partial noncompetitive type. The kinetic model has been set up and a new plot to determine the activation constant of Mg(2+) was put forward. From the plot, we can easily determine the activation constant (K(a)) value and the activation ratio of Mg(2+) on the enzyme. The inhibition effects of Cu(2+) and Hg(2+) on the enzyme are of noncompetitive type. The inhibition constants have been determined. The inhibition effect of Hg(2+) is stronger than that of Cu(2+).     

Purification and characterization of agarases from a marine bacterium <Emphasis Type="Italic">Vibrio</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> F-6

Marine bacterium Vibrio sp. F-6, utilizing agarose as a carbon source to produce agarases, was isolated from seawater samples taken from Qingdao, China. Two agarases (AG-a and AG-b) were purified to a homogeneity from the cultural supernatant of Vibrio sp. F-6 through ammonium sulfate precipitation, Q-Sepharose FF chromatography, and Sephacryl S-100 gel filtration. Molecular weights of agarases were estimated to be 54.0 kDa (AG-a) and 34.5 kDa (AG-b) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH values for AG-a and AG-b were about 7.0 and 9.0, respectively. AG-a was stable in the pH range of 4.0-9.0 and AG-b was stable in the pH range of 4.0-10.0. The optimum temperatures of AG-a and AG-b were 40 and 55 degrees C, respectively. AG-a was stable at temperature below 50 degrees C. AG-b was stable at temperature below 60 degrees C. Zn(2+), Mg(2+) or Ca(2+) increased AG-a activity, while Mn(2+), Cu(2+) or Ca(2+) increased AG-b activity. However, Ag(+), Hg(2+), Fe(3+), EDTA and SDS inhibited AG-a and AG-b activities. The main hydrolysates of agarose by AG-a were neoagarotetraose and neoagarohexaose. The main hydrolysates of agarose by AG-b were neoagarooctaose and neoagarohexaose. When the mixture of AG-a and AG-b were used, agarose was mainly degraded into neoagarobiose.     

Dimerization of human growth hormone in the presence of metal ions

Zn(2+) and Co(2+) ions are known to promote human growth hormone reversible dimerization. In these studies, dimerization was also shown to be initiated by nine other metal ions: Cd(2+), Hg(2+), Cu(2+), Ag+, Au(3+), Au+, Pd(2+), Ni(2+), and Pt(4+). In some cases (Hg(2+), Ag(+), Au(3+), and Ni(2+)) formation of higher oligomers also took place. In addition further detailed investigation of dimerization in the presence of Zn(2+) ions was carried out.     

Inhibition of hepatic deoxyribonucleic acid-dependent ribonucleic acid polymerases by the exotoxin of Bacillus thuringiensis in comparison with the effects of α-amanitin and cordycepin

The action of Bacillus thuringiensis exotoxin, a structural analogue of ATP, on mouse liver DNA-dependent RNA polymerases was studied and its effects were compared with those of alpha-amanitin and cordycepin. (1) Administration of exotoxin in vivo caused a marked decrease in RNA polymerase activity of isolated nuclei at various concentrations of Mg(2+), Mn(2+) and (NH(4))(2)SO(4). A similar action was recorded after addition of exotoxin to isolated nuclei from control or exotoxin-treated mice. (2) Chromatographic separation of nuclear RNA polymerases from mice treated in vivo with exotoxin showed a drastic decrease of the peak of nucleoplasmic RNA polymerase, whereas the peak of nucleolar RNA polymerase remained unaltered. The same effect was observed after administration of alpha-amanitin in vivo, but cordycepin did not alter the relative amounts of the two main RNA polymerase peaks. (3) Administration of exotoxin in vivo did not alter the template activity of isolated DNA or chromatin tested with different fractions of RNA polymerase from control or exotoxin-treated mice. (4) Addition of exotoxin to isolated liver RNA polymerases inhibited both enzyme fractions. However, the alpha-amanitin-sensitive RNA polymerase was also 50-100-fold more sensitive to exotoxin inhibition than was the alpha-amanitin-insensitive RNA polymerase. Kinetic analysis indicated the exotoxin produces a competitive inhibition with ATP on the nucleolar enzyme, but a mixed type of inhibition with nucleoplasmic enzyme. The results obtained indicate that the B. thuringiensis exotoxin inhibits liver RNA synthesis by affecting nuclear RNA polymerases, showing a preferential inhibition of the nucleoplasmic alpha-amanitin-sensitive RNA polymerase.     

The requirement for bivalent cations in formation of nicotinamide–adenine dinucleotide by nicotinamide mononucleotide adenylyltransferase of pig-liver nuclei

1. The requirement for bivalent cations in catalysis of NAD formation from ATP and NMN in the presence of NMN adenylyltransferase of pig-liver nuclei was studied. Rates of NAD formation in the presence of the activating cations Cd(2+), Mn(2+), Mg(2+), Zn(2+), Co(2+) and Ni(2+) were approximately a linear function of heats of hydration of the corresponding ions. Ba(2+), Sr(2+), Ca(2+), Cu(2+) and Be(2+) did not activate the enzyme; Be(2+) inhibited the reaction in the presence of Mg(2+) and, to a greater extent, in the presence of Ni(2+). 2. Michaelis constants for NAD formation, measured in a coupled assay with NMN adenylyltransferase and alcohol dehydrogenase at pH8.0 and 25 degrees , in the presence of 3mm concentrations of the unvaried reactants, were 88+/-7mum-ATP, 42+/-4mum-NMN and 85+/-4mum-Mg(2+). The results at this pH and at pH7.5 were consistent with mechanisms in which Mg(2+)-ATP complex is a reactant and free ATP a competitive inhibitor. 3. Formation of nicotinamide-hypoxanthine dinucleotide from NMN and ITP in the presence of the transferase was also more rapid with Ni(2+) and Co(2+) than with Mg(2+).     

Characterization of the PIB-Type ATPases present in Thermus thermophilus

P(IB)-type ATPases transport heavy metals (Cu(2+), Cu(+), Ag(+), Zn(2+), Cd(2+), Co(2+)) across biomembranes, playing a key role in homeostasis and in the mechanisms of biotolerance of these metals. Three genes coding for putative P(IB)-type ATPases are present in the genome of Thermus thermophilus (HB8 and HB27): the TTC1358, TTC1371, and TTC0354 genes; these genes are annotated, respectively, as two copper transporter (CopA and CopB) genes and a zinc-cadmium transporter (Zn(2+)/Cd(2+)-ATPase) gene. We cloned and expressed the three proteins with 8His tags using a T. thermophilus expression system. After purification, each of the proteins was shown to have phosphodiesterase activity at 65°C with ATP and p-nitrophenyl phosphate (pNPP) as substrates. CopA was found to have greater activity in the presence of Cu(+), while CopB was found to have greater activity in the presence of Cu(2+). The putative Zn(2+)/Cd(2+)-ATPase was truncated at the N terminus and was, surprisingly, activated in vitro by copper but not by zinc or cadmium. When expressed in Escherichia coli, however, the putative Zn(2+)/Cd(2+)-ATPase could be isolated as a full-length protein and the ATPase activity was increased by the addition of Zn(2+) and Cd(2+) as well as by Cu(+). Mutant strains in which each of the three P-type ATPases was deleted singly were constructed. In each case, the deletion increased the sensitivity of the strain to growth in the presence of copper in the medium, indicating that each of the three can pump copper out of the cells and play a role in copper detoxification.     

The loss of rapidly labelled ribonucleic acid from isolated HeLa cell nuclei

1. The loss of nucleic acids and protein from isolated HeLa-cell nuclei was studied. During 4hr. incubation at 37 degrees DNA was conserved, but appreciable amounts of RNA and protein were lost. 2. Two classes of nuclear RNA were distinguished: at least 75% of the RNA was lost from the nuclei relatively slowly through degradation to acid-soluble fragments; the rest of the RNA was lost much more rapidly, not only through degradation to acid-soluble fragments but also through diffusion of RNA out of the nuclei into the incubation medium. 3. The RNA that was preferentially lost was the fraction of nuclear RNA that was rapidly labelled when intact HeLa cells were grown in a medium containing radioactive precursors of RNA. 4. The RNA appearing in the incubation medium was apparently partially degraded and had a sedimentation coefficient of about that of transfer RNA. 5. Both the degradation of RNA and the loss of RNA from the nuclei were sensitive to bivalent cations. Low concentrations of Mg(2+) and Mn(2+) greatly increased the rate of degradation of the rapidly labelled RNA to acid-soluble fragments, and produced a corresponding decrease in the amount of RNA diffusing into the medium. At higher concentrations they suppressed both degradation and diffusion of RNA. The cations Ca(2+), Cu(2+), Zn(2+) and Ni(2+) all progressively inhibited both forms of loss of RNA. 6. Salts of univalent cations produced appreciable effects only at ionic strengths of about 0.2, when degradation to acid-soluble fragments was preferentially inhibited. 7. Both ADP and ATP inhibited loss of RNA at about 30mm. 8. It was concluded that the diffusion of rapidly labelled RNA out of the isolated nuclei was not related to the movement of RNA from nucleus to cytoplasm in vivo, but reflected the ease with which the rapidly labelled RNA detached from the chromatin and the permeability of the membranes of isolated nuclei.     

Cd2+ -promoted mitochondrial permeability transition: a comparison with other heavy metals

We compared action of Cd(2+), Hg(2+), and Cu(2+) on isolated rat liver mitochondria in the absence of added Ca(2+) and P(i). The heavy-metal ions produced dose-dependently: (1) enhanced membrane permeabilization manifested in mitochondrial swelling and activation of basal respiration, (2) inhibition of uncoupler-stimulated respiration, and (3) membrane potential dissipation. Among the metals, Cu(2+) exhibited maximal stimulatory effect on basal respiration and minimal inhibitory action on DNP-uncoupled respiration whilst Cd(2+) promoted the strongest depression of uncoupled respiration and the largest swelling in NH(4)NO(3) medium. Dithiothreitol induced a basal respiration release if added after high [Cd(2+)] and [Hg(2+)], and the stimulation was CsA-insensitive.     

Mannose-Escherichia coli interaction in the presence of metal cations studied in vitro by colorimetric polydiacetylene/glycolipid liposomes

Supramolecular assemblies of liposomes (vesicles) made of diacetylenic lipids and synthetic mannoside derivative glycolipid receptors were successfully used to mimic the molecular recognition occurring between mannose and Escherichia coli. This specific molecular recognition was translated into visible blue-to-red color transition (biochromism) of the polymerized liposomes, readily quantified by UV-visible spectroscopy. Some transition metal cations (Cd(2+), Ag(+), Cu(2+), Fe(3+), Zn(2+) and Ni(2+)) and alkali earth metal cations (Ca(2+), Mg(2+) and Ba(2+)) were introduced into the system to analyze their effects on specific biochromism. Results showed that the presence of Cd(2+), Ag(+), Ca(2+), Mg(2+) and Ba(2+) enhanced biochromism. A possible enhancement mechanism was proposed in the process of bacterial adhesion to host cells. However, Cu(2+), Fe(3+), Zn(2+) and Ni(2+) exhibited inhibitory effects that cooperated with diacetylene lipid with a carboxylic group and increased the rigidity of the liposomal outer leaflet, blocking changes in the side chain conformation and electrical structure of polydiacetylene polymer during biochromism.     

Decreased ribonucleic acid synthesis in isolated rat liver nuclei during starvation

1. Isolated nuclei from starved rats showed a lowered incorporation of [(14)C]UMP into RNA. 2. The Mg(2+)-dependent incorporation was decreased by 30% after 1 day of starvation, but incorporation in the presence of Mn(2+) and ammonium sulphate decreased only after longer periods of starvation. 3. RNA synthesis by nuclei in the presence of excess of added RNA polymerase was unchanged after 1 day of starvation and was inhibited by 20% after 4 days. 4. The capacity of nuclei to bind actinomycin D was unchanged after 1 day and was decreased by 20% after 4 days of starvation.     

Differential effect of copper (II) on the cytochrome P450 enzymes and NADPH-cytochrome P450 reductase: inhibition of cytochrome P450-catalyzed reactions by copper (II) ion

Inhibitory effects of Cu(2+) on the cytochrome P450 (P450)-catalyzed reactions of liver microsomes and reconstituted systems containing purified P450 and NADPH-P450 reductase (NPR) were seen. However, Zn(2+), Mg(2+), Mn(2+), Ca(2+), and Co(2+) had no apparent effects on the activities of microsomal P450s. Cu(2+) inhibited the reactions catalyzed by purified P450s 1A2 and 3A4 with IC(50) values of 5.7 and 8.4 microM, respectively. Cu(2+) also inhibited reduction of cytochrome c by NPR (IC(50) value of 5.8 microM). Copper caused a decrease in semiquinone levels of NPR, although it did not disturb the rate of formation of semiquinone. P450 reactions supported by an oxygen surrogate, tert-butyl hydroperoxide, instead of NPR and NADPH, were inhibited by the presence of Cu(2+). The results indicate that Cu(2+) inhibits the P450-catalyzed reactions by affecting both P450s and NPR. It was also found that the inhibition of catalytic activities of P450s by Cu(2+) involves overall conformational changes of P450s and NPR, investigated by CD and intrinsic fluorescence spectroscopy. These results suggest that the inhibitory effect of Cu(2+) on the P450-catalyzed reactions may come from the inability of an efficient electron transfer from NPR to P450 and also the dysfunctional conformation of NPR and P450.     

Characterization of metalloproteinase-like activities in barnacle (Balanus amphitrite) nauplii

The presence of extracellular matrix (ECM) degrading enzymes was investigated in naupliar stages of the barnacle Balanus amphitrite Darwin. The results of substrate gel-zymography and quantitative assays demonstrated that naupliar extracts contain several protease activities that are specific towards gelatin substrates; some caseinolytic activity was also detected. Substrate specificity was observed in all naupliar stages (II-VI). The gelatinolytic activities showed dependence on both Ca(2+) and Zn(2+) and inhibition by EDTA, EGTA, and 1,10-phenanthroline. Also Mg(2+) partially activated the enzymes, whereas Cd(2+), Cu(2+), Hg(2+) and Pb(2+) were inhibitory. The thermal denaturation profile was significantly different in the presence and absence of Ca(2+) and Zn(2+). Overall, the results indicate that the Ca(2+)/Zn(2+)-dependent gelatinase activities in barnacle nauplii belong to the subfamily of matrix metalloproteases. Barnacle larvae MMPs showed biochemical characteristics different from those of vertebrate MMPs but common to other gelatinases from marine invertebrates: they were unaffected by several protease inhibitors and insensitive to specific activators/inhibitors of vertebrate MMPs. The presence of MMP-like activities in different naupliar stages suggests a constitutive role for these enzymes in ECM remodeling during barnacle larvae growth and development.     

Studies on the solubilized ribonucleic acid polymerase from rat ventral prostate gland

1. By using ultrasonic treatment in media of high ionic strength, the RNA polymerase activities associated with prostatic nuclei and nucleoli can be completely solubilized. Such enzyme preparations are entirely dependent on the provision of added DNA for full activity. 2. The solubilized enzymes from the nucleolar and extranucleolar regions can be separated by ion-exchange chromatography. 3. Based on differences in the optimum DNA templates, pH optima and the effects of ammonium sulphate on the activities in vitro, Mn(2+)- and Mg(2+)-specific enzymes are associated with both the nucleolar and extranucleolar regions of prostatic nuclei. 4. Androgenic hormones administered in vivo have a particularly pronounced effect on the activity of Mg(2+)-dependent enzyme associated with the isolated prostatic nucleolus. 5. Time-course experiments in vivo show that androgens induce a rapid stimulation of the solubilized Mg(2+)-dependent nucleolar enzyme before a pronounced activation of nucleolar chromatin can be measured. 6. The implications of these findings to the mechanism of action of androgenic steroids are discussed.     

Calcium is a cofactor of polymerization but inhibits pyrophosphorolysis by the Sulfolobus solfataricus DNA polymerase Dpo4

Y-Family DNA polymerase IV (Dpo4) from Sulfolobus solfataricus serves as a model system for eukaryotic translesion polymerases, and three-dimensional structures of its complexes with native and adducted DNA have been analyzed in considerable detail. Dpo4 lacks a proofreading exonuclease activity common in replicative polymerases but uses pyrophosphorolysis to reduce the likelihood of incorporation of an incorrect base. Mg(2+) is a cofactor for both the polymerase and pyrophosphorolysis activities. Despite the fact that all crystal structures of Dpo4 have been obtained in the presence of Ca(2+), the consequences of replacing Mg(2+) with Ca(2+) for Dpo4 activity have not been investigated to date. We show here that Ca(2+) (but not Ba(2+), Co(2+), Cu(2+), Ni(2+), or Zn(2+)) is a cofactor for Dpo4-catalyzed polymerization with both native and 8-oxoG-containing DNA templates. Both dNTP and ddNTP are substrates of the polymerase in the presence of either Mg(2+) or Ca(2+). Conversely, no pyrophosphorolysis occurs in the presence of Ca(2+), although the positions of the two catalytic metal ions at the active site appear to be very similar in mixed Mg(2+)/Ca(2+)- and Ca(2+)-form Dpo4 crystals.