Effect of divalent cations on antiparallel G-quartet structure of d(G4T4G4)

The thermodynamic parameters of an antiparallel G-quartet formation of d(G4T4G4) with 1 mM divalent cation (Mg(2+), Ca(2+), Mn(2+), Co(2+), and Zn(2+)) were obtained. The thermodynamic parameters showed that the divalent cation destabilizes the antiparallel G-quartet of d(G4T4G4) in the following order: Zn(2+)>Co(2+)>Mn(2+)>Mg(2+)>Ca(2+). In addition, a higher concentration of a divalent cation induced a transition from an antiparallel to a parallel G-quartet structure. These results indicate that these divalent cations are a good tool for regulating the G-quartet structures.     

TRPM7 provides an ion channel mechanism for cellular entry of trace metal ions

Trace metal ions such as Zn(2+), Fe(2+), Cu(2+), Mn(2+), and Co(2+) are required cofactors for many essential cellular enzymes, yet little is known about the mechanisms through which they enter into cells. We have shown previously that the widely expressed ion channel TRPM7 (LTRPC7, ChaK1, TRP-PLIK) functions as a Ca(2+)- and Mg(2+)-permeable cation channel, whose activity is regulated by intracellular Mg(2+) and Mg(2+).ATP and have designated native TRPM7-mediated currents as magnesium-nucleotide-regulated metal ion currents (MagNuM). Here we report that heterologously overexpressed TRPM7 in HEK-293 cells conducts a range of essential and toxic divalent metal ions with strong preference for Zn(2+) and Ni(2+), which both permeate TRPM7 up to four times better than Ca(2+). Similarly, native MagNuM currents are also able to support Zn(2+) entry. Furthermore, TRPM7 allows other essential metals such as Mn(2+) and Co(2+) to permeate, and permits significant entry of nonphysiologic or toxic metals such as Cd(2+), Ba(2+), and Sr(2+). Equimolar replacement studies substituting 10 mM Ca(2+) with the respective divalent ions reveal a unique permeation profile for TRPM7 with a permeability sequence of Zn(2+) approximately Ni(2+) > Ba(2+) > Co(2+) > Mg(2+) >/= Mn(2+) >/= Sr(2+) >/= Cd(2+) >/= Ca(2+), while trivalent ions such as La(3+) and Gd(3+) are not measurably permeable. With the exception of Mg(2+), which exerts strong negative feedback from the intracellular side of the pore, this sequence is faithfully maintained when isotonic solutions of these divalent cations are used. Fura-2 quenching experiments with Mn(2+), Co(2+), or Ni(2+) suggest that these can be transported by TRPM7 in the presence of physiological levels of Ca(2+) and Mg(2+), suggesting that TRPM7 represents a novel ion-channel mechanism for cellular metal ion entry into vertebrate cells.     

Effect of divalent cations on ion fluxes and leaf photochemistry in salinized barley leaves

Photosynthetic characteristics, leaf ionic content, and net fluxes of Na(+), K(+), and Cl(-) were studied in barley (Hordeum vulgare L) plants grown hydroponically at various Na/Ca ratios. Five weeks of moderate (50 mM) or high (100 mM) NaCl stress caused a significant decline in chlorophyll content, chlorophyll fluorescence characteristics, and stomatal conductance (g(s)) in plant leaves grown at low calcium level. Supplemental Ca(2+) enabled normal photochemical efficiency of PSII (F(v)/F(m) around 0.83), restored chlorophyll content to 80-90% of control, but had a much smaller (50% of control) effect on g(s). In experiments on excised leaves, not only Ca(2+), but also other divalent cations (in particular, Ba(2+) and Mg(2+)), significantly ameliorated the otherwise toxic effect of NaCl on leaf photochemistry, thus attributing potential targets for such amelioration to leaf tissues. To study the underlying ionic mechanisms of this process, the MIFE technique was used to measure the kinetics of net Na(+), K(+), and Cl(-) fluxes from salinized barley leaf mesophyll in response to physiological concentrations of Ca(2+), Ba(2+), Mg(2+), and Zn(2+). Addition of 20 mM Na(+) as NaCl or Na(2)SO(4) to the bath caused significant uptake of Na(+) and efflux of K(+). These effects were reversed by adding 1 mM divalent cations to the bath solution, with the relative efficiency Ba(2+)>Zn(2+)=Ca(2+)>Mg(2+). Effect of divalent cations on Na(+) efflux was transient, while their application caused a prolonged shift towards K(+) uptake. This suggests that, in addition to their known ability to block non-selective cation channels (NSCC) responsible for Na(+) entry, divalent cations also control the activity or gating properties of K(+) transporters at the mesophyll cell plasma membrane, thereby assisting in maintaining the high K/Na ratio required for optimal leaf photosynthesis.     

Comparative studies on the iron chelators O-TRENSOX and TRENCAMS: selectivity of the complexation towards other biologically relevant metal ions and Al(3+)

Complexation constants have been determined by potentiometric titration and spectrophotometric measurements for several biologically relevant divalent metals (Ca(2+), Cu(2+), Zn(2+)) as well as Al(3+) with the sulfonated tris(8-hydroxyquinolinate) tripodal ligand O-TRENSOX. The values demonstrate great selectivity of O-TRENSOX for Fe(3+) according to the sequence Fe(3+) >Cu(2+)>Zn(2+)>Ca(2+). This selectivity is compared to that shown by tris(hydroxamate) and tris(catecholate) ligands. (1)H NMR spectroscopy of the diamagnetic complexes have been carried out in (2)H(2)O solutions.     

Physiological metal uptake by Nostoc punctiforme

Trace metals are required for many cellular processes. The acquisition of trace elements from the environment includes a rapid adsorption of metals to the cell surface, followed by a slower internalization. We investigated the uptake of the trace elements Co(2+), Cu(2+), Mn(2+), Ni(2+), and Zn(2+) and the non-essential divalent cation Cd(2+) in the cyanobacterium Nostoc punctiforme. For each metal, a dose response study based on cell viability showed that the highest non-toxic concentrations were: 0.5?μM Cd(2+), 2?μM Co(2+), 0.5?μM Cu(2+), 500?μM Mn(2+), 1?μM Ni(2+), and 18?μM Zn(2+). Cells exposed to these non-toxic concentrations with combinations of Zn(2+) and Cd(2+), Zn(2+) and Co(2+), Zn(2+) and Cu(2+) or Zn(2+) and Ni(2+), had reduced growth in comparison to controls. Cells exposed to metal combinations with the addition of 500?μM Mn(2+) showed similar growth compared to the untreated controls. Metal levels were measured after one and 72?h for whole cells and absorbed (EDTA-resistant) fractions and used to calculate differential uptake rates for each metal. The differences in binding and internalisation between different metals indicate different uptake processes exist for each metal. For each metal, competitive uptake experiments using (65)Zn showed that after 72?h of exposure Zn(2+) uptake was reduced by most metals particularly 0.5?μM Cd(2+), while 2?μM Co(2+) increased Zn(2+) uptake. This study demonstrates that N. punctiforme discriminates between different metals and favourably substitutes their uptake to avoid the toxic effects of particular metals.     

Molecular determinant for specific Ca/Ba selectivity profiles of low and high threshold Ca2+ channels

Voltage-gated Ca(2+) channels (VGCC) play a key role in many physiological functions by their high selectivity for Ca(2+) over other divalent and monovalent cations in physiological situations. Divalent/monovalent selection is shared by all VGCC and is satisfactorily explained by the existence, within the pore, of a set of four conserved glutamate/aspartate residues (EEEE locus) coordinating Ca(2+) ions. This locus however does not explain either the choice of Ca(2+) among other divalent cations or the specific conductances encountered in the different VGCC. Our systematic analysis of high- and low-threshold VGCC currents in the presence of Ca(2+) and Ba(2+) reveals highly specific selectivity profiles. Sequence analysis, molecular modeling, and mutational studies identify a set of nonconserved charged residues responsible for these profiles. In HVA (high voltage activated) channels, mutations of this set modify divalent cation selectivity and channel conductance without change in divalent/monovalent selection, activation, inactivation, and kinetics properties. The Ca(V)2.1 selectivity profile is transferred to Ca(V)2.3 when exchanging their residues at this location. Numerical simulations suggest modification in an external Ca(2+) binding site in the channel pore directly involved in the choice of Ca(2+), among other divalent physiological cations, as the main permeant cation for VGCC. In LVA (low voltage activated) channels, this locus (called DCS for divalent cation selectivity) also influences divalent cation selection, but our results suggest the existence of additional determinants to fully recapitulate all the differences encountered among LVA channels. These data therefore attribute to the DCS a unique role in the specific shaping of the Ca(2+) influx between the different HVA channels.     

Ionomycin, a carboxylic acid ionophore, transports Pb(2+) with high selectivity

Studies utilizing phospholipid vesicle loaded with chelator/indicators for polyvalent cations show that ionomycin transports divalent cations with the selectivity sequence Pb(2+) > Cd(2+) > Zn(2+) > Mn(2+) > Ca(2+) > Cu(2+) > Co(2+) > Ni(2+) > Sr(2+). The selectivity of this ionophore for Pb(2+) is in contrast to that observed for A23178 and 4-BrA23187, which transport Pb(2+) at efficiencies that are intermediate between those of other cations. When the selectivity difference of ionomycin for Pb(2+) versus Ca(2+) was calculated from relative rates of transport, with either cation present individually and all other conditions held constant, a value of approximately 450 was obtained. This rose to approximately 3200 when both cations were present and transported simultaneously. 1 microM Pb(2+) inhibited the transport of 1 mM Ca(2+) by approximately 50%, whereas the rate of Pb(2+) transport approached a maximum at a concentration of 10 microM Pb(2+) when 1 mM Ca(2+) was also present. Plots of log rate versus log ionomycin or log Pb(2+) concentration indicated that the transporting species is of 1:1 stoichiometry, ionophore to Pb(2+), but that complexes containing an additional Pb(2+) may occur. The species transporting Pb(2+) may include H.IPb.OH, wherein ionomycin is ionized once and the presence of OH(-) maintains charge neutrality. Ionomycin retained a high efficiency for Pb(2+) transport in A20 B lymphoma cells loaded with Indo-1. Both Pb(2+) entry and efflux were observed. Ionomycin should be considered primarily as an ionophore for Pb(2+), rather than Ca(2+), of possible value for the investigation and treatment of Pb(2+) intoxication.     

Protease activity of 80 kDa protein secreted from the apicomplexan parasite Toxoplasma gondii

This study describes the characterization of 80 kDa protease showing gelationlytic property among three proteases in the excretory/secretory proteins (ESP) from Toxoplasma gondii. The protease activity was detected in the ESP but not in the somatic extract of RH tachyzoites. This protease was active only in the presence of calcium ion but not other divalent cationic ions such as Cu(2+), Zn(2+), Mg(2+), and Mn(2+), implying that Ca(2+) is critical factor for the activation of the protease. The 80 kDa protease was optimally active at pH 7.5. Its gelatinolytic activity was maximal at 37 degrees C, and significant level of enzyme activity of the protease remained after heat treatment at 56 degrees C for 30 min or 100 degrees C for 10 min. This thermostable enzyme was strongly inhibited by metal chelators, i.e., EDTA, EGTA, and 1,10- phenanthroline. Thus, the 80 kDa protease in the ESP secreted by T. gondii was classified as a calcium dependent neutral metalloprotease.     

Cloning and characterization of a novel Mg(2+)/H(+) exchanger.

Cellular functions require adequate homeostasis of several divalent metal cations, including Mg(2+) and Zn(2+). Mg(2+), the most abundant free divalent cytoplasmic cation, is essential for many enzymatic reactions, while Zn(2+) is a structural constituent of various enzymes. Multicellular organisms have to balance not only the intake of Mg(2+) and Zn(2+), but also the distribution of these ions to various organs. To date, genes encoding Mg(2+) transport proteins have not been cloned from any multicellular organism. We report here the cloning and characterization of an Arabidopsis thaliana transporter, designated AtMHX, which is localized in the vacuolar membrane and functions as an electrogenic exchanger of protons with Mg(2+) and Zn(2+) ions. Functional homologs of AtMHX have not been cloned from any organism. Ectopic overexpression of AtMHX in transgenic tobacco plants render them sensitive to growth on media containing elevated levels of Mg(2+) or Zn(2+), but does not affect the total amounts of these minerals in shoots of the transgenic plants. AtMHX mRNA is mainly found at the vascular cylinder, and a large proportion of the mRNA is localized in close association with the xylem tracheary elements. This localization suggests that AtMHX may control the partitioning of Mg(2+) and Zn(2+) between the various plant organs.     

Cd2+ versus Zn2+ uptake by the ZIP8 HCO3--dependent symporter: kinetics, electrogenicity and trafficking

The mouse Slc39a8 gene encodes the ZIP8 transporter, which has been shown to be a divalent cation/HCO3- symporter. Using ZIP8 cRNA-injected Xenopus oocyte cultures, we show herein that: [a] ZIP8-mediated cadmium (Cd(2+)) and zinc (Zn(2+)) uptake have V(max) values of 1.8+/-0.08 and 1.0+/-0.08 pmol/oocyte/h, and K(m) values of 0.48+/-0.08 and 0.26+/-0.09 microM, respectively; [b] ZIP8-mediated Cd(2+) uptake is most inhibited by Zn(2+), second-best inhibited by Cu(2+), Pb(2+) and Hg(2+), and not inhibited by Mn(2+) or Fe(2+); and [c] electrogenicity studies demonstrate an influx of two HCO3- anions per one Cd(2+) (or one Zn(2+)) cation, i.e. electroneutral complexes. Using Madin-Darby canine kidney (MDCK) polarized epithelial cells retrovirally infected with ZIP8 cDNA and tagged with hemagglutinin at the C-terminus, we show that-similar to ZIP4-the ZIP8 eight-transmembrane protein is largely internalized during Zn(2+) homeostasis, but moves predominantly to the cell surface membrane (trafficking) under conditions of Zn(2+) depletion.     

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.     

Polyribosomes in protoplasts isolated from tobacco leaves

Polyribosomes (polysomes), active in an amino acid incorporation system in vitro, were isolated from tobacco leaf protoplasts. A comparison of polysome profiles indicated that the polysome/monosome ratio is greatly decreased in isolated protoplasts as compared to the intact leaf. In isolated protoplasts, a marked accumulation of ribosomal subunits was also found. The division of protoplasts, as investigated in the 8-cell and callus stages, was associated with a(n) (at least) partial regeneration of polysome profiles characteristic for leaves. Plasmolysis of leaves attached to the plant had no great effect on the polysome profile. However, leaf excision per se resulted in a dramatic loss of polysomes, even when the leaf tissue was floated on water. It is concluded that the isolation of the cell from its normal environment, and not the osmotic stress and associated increase in RNase activity, is the most important factor responsible for the loss of polysomes in isolated protoplasts.Abbreviations EGTA ethylene glycol bis (2-aminoethyl ether)-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - mRNA messenger ribonucleic acid - RNase ribonuclease - Tris tris(hydroxymethyl)aminomethane - TCA trichloroacetic acid     

Brain S100A5 is a novel calcium-, zinc-, and copper ion-binding protein of the EF-hand superfamily

S100A5 is a novel member of the EF-hand superfamily of calcium-binding proteins that is poorly characterized at the protein level. Immunohistochemical analysis demonstrates that it is expressed in very restricted regions of the adult brain. Here we characterized the human recombinant S100A5, especially its interaction with Ca(2+), Zn(2+), and Cu(2+). Flow dialysis revealed that the homodimeric S100A5 binds four Ca(2+) ions with strong positive cooperativity and an affinity 20-100-fold higher than the other S100 proteins studied under identical conditions. S100A5 also binds two Zn(2+) ions and four Cu(2+) ions per dimer. Cu(2+) binding strongly impairs the binding of Ca(2+); however, none of these ions change the alpha-helical-rich secondary structure. After covalent labeling of an exposed thiol with 2-(4'-(iodoacetamide)anilino)-naphthalene-6-sulfonic acid, binding of Cu(2+), but not of Ca(2+) or Zn(2+), strongly decreased its fluorescence. In light of the three-dimensional structure of S100 proteins, our data suggest that in each subunit the single Zn(2+) site is located at the opposite side of the EF-hands. The two Cu(2+)-binding sites likely share ligands of the EF-hands. The potential role of S100A5 in copper homeostasis is discussed.     

Understanding the multiple functions of Nramp1

Nramp1 regulates macrophage activation in infectious and autoimmune diseases. Nramp2 controls anaemia. Both are divalent cation (Fe(2+), Zn(2+), and Mn(2+)) transporters; Nramp2 a symporter of H(+) and metal ions, Nramp1 a H(+)/divalent cation antiporter. This provides a model for metal ion homeostasis in macrophages. Nramp2, localised to early endosomes, delivers extracellularly acquired divalent cations into the cytosol. Nramp1, localised to late endosomes/lysosomes, delivers divalent cations from the cytosol to phagolysosomes. Here, Fe(2+) generates antimicrobial hydroxyl radicals via the Fenton reaction. Zn(2+) and Mn(2+) may also influence endosomal metalloprotease activity and phagolysosome fusion. The many cellular functions dependent on metal ions as cofactors may explain the multiple pleiotropic effects of Nramp1, and its complex roles in infectious and autoimmune disease.     

The ionophore nigericin transports Pb2+ with high activity and selectivity: a comparison to monensin and ionomycin

The K(+) ionophore nigericin is shown to be highly effective as an ionophore for Pb(2+) but not other divalent cations, including Cu(2+), Zn(2+), Cd(2+), Mn(2+), Co(2+), Ca(2+), Ni(2+), and Sr(2+). Among this group a minor activity for Cu(2+) transport is seen, while for the others activity is near or below the limit of detection. The selectivity of nigericin for Pb(2+) exceeds that of ionomycin or monensin and arises, at least in part, from a high stability of nigericin-Pb(2+) complexes. Plots of log rate vs log Pb(2+) or log ionophore concentration, together with the pH dependency, indicate that nigericin transports Pb(2+) via the species NigPbOH and by a mechanism that is predominately electroneutral. As with monensin and ionomycin, a minor fraction of activity may be electrogenic, based upon a stimulation of rate that is produced by agents which prevent the formation of transmembrane electrical potentials. Nigericin-catalyzed Pb(2+) transport is not inhibited by physiological concentrations of Ca(2+) or Mg(2+) and is only modestly affected by K(+) and Na(+) concentrations in the range of 0-100 mM. These characteristics, together with higher selectivity and efficiency, suggest that nigericin may be more useful than monensin in the treatment of Pb intoxication.     

The effect of divalent cations on bovine spermatozoal adenylate cyclase activity.

The effect of divalent cations on bovine sperm adenylate cyclase activity was studied. Mn2+, Co2+, Cd2+, Zn2+, Mg2+ and Ca2+ were found to satisfy the divalent cation requirement for catalysis of the bovine sperm adenylate cyclase. These divalent cations in excess of the amount necessary for the formation of the metal-ATP substrate complex were found to stimulate the enzyme activity to various degrees. The magnitude of stimulation at saturating concentrations of the divalent cations was strikingly greater with M2+ than with either Ca2+, Mg2+, Zn2+, Cd2+ or Co2+. The apparent Km was lowest for Zm2+ (0.1 - 0.2 mM) than for any of the other divalent cations tested (1.2 - 2.3 mM). The enzyme stimulation by Mn2+ was decreased by the simultaneous addition of Co2+, Cd2+, Ni2+ and particularly Zn2+ and Cu2+. The antagonism between Mn2+ and Cu2+ or Zn2+ appeared to have both competitive and non-competitive features. The inhibitory effect of Cu2+ on Mn2+-stimulated adenylate cyclase activity was prevented by 2,3-dimercaptopropanol, but not by dithiothreitol, L-ergothioneine, EDTA, EGTA or D-penicillamine. Ca2+ at concentrations of 1-5 mM was found to act synergistically with Mg2+, Zn2+, Co2+ and Mn2+ in stimulating sperm adenylate cyclase activity. The Ca2+ augmentation of the stimulatory effect of Zn2+, Co2+, Mg2+ and Mn2+ appeared to be specific.     

Characterization of an integral protein of the brush border membrane mediating the transport of divalent metal ions

The transport of Fe(2+) and other divalent transition metal ions across the intestinal brush border membrane (BBM) was investigated using brush border membrane vesicles (BBMVs) as a model. This transport is an energy-independent, protein-mediated process. The divalent metal ion transporter of the BBM is a spanning protein, very likely a protein channel, that senses the phase transition of the BBM, as indicated by a break in the Arrhenius plot. The transporter has a broad substrate range that includes Mn(2+), Fe(2+), Co(2+), Ni(2+), Cu(2+), and Zn(2+). Under physiological conditions the transport of divalent metal ions is proton-coupled, leading to the acidification of the internal cavity of BBMVs. The divalent metal ion transporter can be solubilized in excess detergent (30 mM diheptanoylphosphatidylcholine or 1% Triton X-100) and reconstituted into an artificial membrane system by detergent removal. The reconstituted membrane system showed metal ion transport characteristics similar to those of the original BBMVs. The properties of the protein described here closely resemble those of the proton-coupled divalent cation transporter (DCT1, Nramp2) described by, Nature. 388:482-488). We may conclude that a protein of the Nramp family is present in the BBM, facilitating the transport of Fe(2+) and other divalent transition metal ions.     

Poliovirus RNA-dependent RNA polymerase (3D(pol)). Divalent cation modulation of primer, template, and nucleotide selection

We have analyzed the divalent cation specificity of poliovirus RNA-dependent RNA polymerase, 3D(pol). The following preference was observed: Mn(2+) > Co(2+) > Ni(2+) > Fe(2+) > Mg(2+) > Ca(2+) > Cu(2+), and Zn(2+) was incapable of supporting 3D(pol)-catalyzed nucleotide incorporation. In the presence of Mn(2+), 3D(pol) activity was increased by greater than 10-fold relative to that in the presence of Mg(2+). Steady-state kinetic analysis revealed that the increased activity observed in the presence of Mn(2+) was due, primarily, to a reduction in the K(M) value for 3D(pol) binding to primer/template, without any significant effect on the K(M) value for nucleotide. The ability of 3D(pol) to catalyze RNA synthesis de novo was also stimulated approximately 10-fold by using Mn(2+), and the enzyme was now capable of also utilizing a DNA template for primer-independent RNA synthesis. Interestingly, the use of Mn(2+) as divalent cation permitted 3D(pol) activity to be monitored by following extension of 5'-(32)P-end-labeled, heteropolymeric RNA primer/templates. The kinetics of primer extension were biphasic because of the enzyme binding to primer/template in both possible orientations. When bound in the incorrect orientation, 3D(pol) was capable of efficient addition of nucleotides to the blunt-ended duplex; this activity was also apparent in the presence of Mg(2+). In the presence of Mn(2+), 3D(pol) efficiently utilized dNTPs, ddNTPs, and incorrect NTPs. On average, three incorrect nucleotides could be incorporated by 3D(pol). The ability of 3D(pol) to incorporate the correct dNTP, but not the correct ddNTP, was also observed in the presence of Mg(2+). Taken together, these results provide the first glimpse into the nucleotide specificity and fidelity of the poliovirus polymerase and suggest novel alternatives for the design of primer/templates to study the mechanism of 3D(pol)-catalyzed nucleotide incorporation.     

Mouse spermatozoa contain a nuclease that is activated by pretreatment with EGTA and subsequent calcium incubation

We demonstrated that mouse spermatozoa cleave their DNA into approximately 50 kb loop-sized fragments with topoisomerase IIB when treated with MnCl(2) and CaCl(2) in a process we term sperm chromatin fragmentation (SCF). SCF can be reversed by EDTA. A nuclease then further degrades the DNA in a process we term sperm DNA degradation (SDD). MnCl(2) alone could elicit this activity, but CaCl(2) had no effect. Here, we demonstrate the existence of a nuclease in the vas deferens that can be activated by ethylene glycol tetraacetic acid (EGTA) to digest the sperm DNA by SDD. Spermatozoa were extracted with salt and dithiothreitol to remove protamines and then incubated with EGTA. Next, the EGTA was removed and divalent cations were added. We found that Mn(2+), Ca(2+), or Zn(2+) could each activate SDD in spermatozoa but Mg(2+) could not. When the reaction was slowed by incubation on ice, EGTA pretreatment followed by incubation in Ca(2+) elicited the reversible fragmentation of sperm DNA evident in SCF. When the reactions were then incubated at 37 degrees C they progressed to the more complete degradation of DNA by SDD. EDTA could also be used to activate the nuclease, but required a higher concentration than EGTA. This EGTA-activatable nuclease activity was found in each fraction of the vas deferens plasma: in the spermatozoa, in the surrounding fluid, and in the insoluble components in the fluid. These results suggest that this sperm nuclease is regulated by a mechanism that is sensitive to EGTA, possibly by removing inhibition of a calcium binding protein.