Na+/Ca2+ antiport in cultured arterial smooth muscle cells. Inhibition by magnesium and other divalent cations

Cultured smooth muscle cells from rat aorta were loaded with Na+, and Na+/Ca2+ antiport was assayed by measuring the initial rates of 45Ca2+ influx and 22Na+ efflux, which were inhibitable by 2',4'-dimethylbenzamil. The replacement of extracellular Na+ with other monovalent ions (K+, Li+, choline, or N-methyl-D-glucamine) was essential for obtaining significant antiport activity. Mg2+ competitively inhibited 45Ca2+ influx via the antiporter (Ki = 93 +/- 7 microM). External Ca2+ or Sr2+ stimulated 22Na+ efflux as would be expected for antiport activity. Mg2+ did not stimulate 22Na+ efflux, which indicates that Mg2+ is probably not transported by the antiporter under the conditions of these experiments. Mg2+ inhibited Ca2+-stimulated 22Na+ efflux as expected from the 45Ca2+ influx data. The replacement of external N-methyl-D-glucamine with K+, but not other monovalent ions (choline, Li+), decreased the potency of Mg2+ as an inhibitor of Na+/Ca2+ antiport 6.7-fold. Other divalent cations (Co2+, Mn2+, Cd2+, Ba2+) also inhibited Na+/Ca2+ antiport activity, and high external potassium decreased the potency of each by 4.3-8.6-fold. The order of effectiveness of the divalent cations as inhibitors of Na+/Ca2+ antiport (Cd2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+) correlated with the closeness of the crystal ionic radius to that of Ca2+.     

An improved method for production of silica from rice hull ash

Biosorption of monovalent ions Na+ and K+, by deactivated protonated yeast (Saccharomyces cerevisiae) at controlled pH, was compared with biosorption of divalent ions Ca2+ and Mg2+ to help to understand the underlying bindingmechanisms. The adsorption for monovalent ions was accompanied by H+ release. Divalent ions were sorbed by proton displacement, and also an additional mode not accompanied by release of H+. The sorption uptake of both monovalent and divalent metal ions increased with pH in the range 3-7 peaking at 6.75. Equilibrium sorption isotherms at pH = 6.75 showed that the totalmaximum biosorptive capacity for metal ions decreased in the following order: Ca > Mg > Na > or = K.     

The hammerhead cleavage reaction in monovalent cations

Recently, Murray et al. (Chem Biol, 1998, 5:587-595) found that the hammerhead ribozyme does not require divalent metal ions for activity if incubated in high (> or =1 M) concentrations of monovalent ions. We further characterized the hammerhead cleavage reaction in the absence of divalent metal. The hammerhead is active in a wide range of monovalent ions, and the rate enhancement in 4 M Li+ is only 20-fold less than that in 10 mM Mg2+. Among the Group I monovalent metals, rate correlates in a log-linear manner with ionic radius. The pH dependence of the reaction is similar in 10 mM Mg2+, 4 M Li+, and 4 M Na+. The exchange-inert metal complex Co(NH3)3+ also supports substantial hammerhead activity. These results suggest that a metal ion does not act as a base in the reaction, and that the effects of different metal ions on hammerhead cleavage rates primarily reflect structural contributions to catalysis.     

Kinetics of sulfate transport by Penicillium notatum. Interactions of sulfate, protons, and calcium.

The active transport of inorganic sulfate by an ATP sulfurylase-negative strain of Penicillium notatum is promoted by H+ ions and metal ions (divalent metal ions being more effective than monovalent metal ions). Initial velocity studies suggest that H+ and SO4(2-) add to the carrier in an ordered sequence (H+ before SO4(2-)), with H+ at equilibrium with free carrier and carrier-H+ complex. The linear reciprocal plots and replots suggest a 1:1 stoichiometry between H+ and SO4(2-). Ca2+ and other divalent metal ions stimulate sulfate transport markedly in buffered suspensions of low ionic strength. The kinetics of the Ca2+/SO4(2-) interaction suggest that Ca2+ (like H+) adds to the carrier before SO4(2-) and is at equilibrium with free carrier and carrier-Ca2+ complex. The linear reciprocal plots and replots indicate a 1:1 stoichiometry between Ca2+ and SO4(2-). Thus the fully loaded carrier-SO4(2-) -Ca2+ -H+ complex has a net positive charge relative to that of the free carrier, a fact consistent with the chemiosmotic hypothesis of membrane transport. The kinetics of the H+/Ca2+ interaction point to a random A-B (rapid equilibrium), ordered C sequence with A = H+, B = Ca2+, and C = SO4(2-). Selenate (an alternate substrate competitive with sulfate) is an uncompetitive inhibitor with respect to Ca2+, in agreement with the suggested mechanism. Internal charge balance is not accomplished by a stoichiometric coaccumulation of Ca2+ and SO4(2-). Sulfate transport does, however, promote 45Ca2+ uptake. A significant fraction of the added Ca2+ is bound by the mycelial surface. Binding is extremely rapid, but reversible.     

Interactions of metal ions with mu-monothiopyrophosphate.

mu-Monothiopyrophosphate (MTP) binds monovalent and divalent metal ions with dissociation constants (Kd) similar to those for pyrophosphate (PPi). The values of Kd for metal-MTP complexes are the following, as measured kinetically in the hydrolysis of MTP (microM): Mg2+, 32 +/- 4; Mn2+, 5.4 +/- 1.4; and Co2+, 27 +/- 15. The thermodynamically measured (EPR) values for Mg2+ and Co2+ are 28 +/- 13 microns and 11 +/- 4 microM, respectively; and the Kd for the complex MnPPi is 3.4 +/- 0.5 microM. The metal-MTP complexes undergo hydrolysis at rates modestly faster or slower than the rate at which MTP itself reacts. The complexes MgMTP2-, CoMTP2-, and MnMTP2- undergo hydrolytic cleavage with release of thiophosphate with observed first-order rate constants of 1.6 x 10(-2) min-1, 2.3 x 10(-2) min-1, and 0.6 x 10(-2) min-1, respectively, at 35 degrees C, compared with 1.1 x 10(-2) min-1 for MTP4- under the same conditions. Alkali metal cations also stimulate or retard the hydrolysis of MTP. At 25 degrees C and pH 12.2, the observed rate constant for tetramethylammonium MTP4- is 2.1 x 10(-3) min-1, and the estimated rate constants (min-1) for saturating alkali metals under the same conditions are as follows: Li+, 0.25 x 10(-3); Na+, 3.9 x 10(-3), K+, 6.7 x 10(-3); and Cs+, 6.7 x 10(-3). Divalent metal ions markedly retard the hydrolysis of MTP at pH 7 and 8 because complexation shifts the pH rate profile more than 2 pH units toward the acid side.(ABSTRACT TRUNCATED AT 250 WORDS)     

Divalent cation-induced changes in conformation of protein kinase C

Using physical techniques, circular dichroism and intrinsic and extrinsic fluorescence, the binding of divalent cations to soluble protein kinase C and their effects on protein conformation were analyzed. The enzyme copurifies with a significant concentration of endogenous Ca2+ as measured by atomic absorption spectrophotometry, however, this Ca2+ was insufficient to support enzyme activity. Intrinsic tryptophan fluorescence quenching occurred upon addition to the soluble enzyme of the divalent cations, Zn2+, Mg2+, Ca2+ or Mn2+, which was irreversible and unaffected by monovalent cations (0.5 M NaCl). Far ultraviolet (200-250 nm) circular dichroism spectra provided estimations of secondary structure and demonstrated that the purified enzyme is rich in alpha-helices (42%) suggesting a rather rigid structure. At Ca2+ or Mg2+ concentrations similar to those used for fluorescence quenching, the enzyme undergoes a conformational transition (42-24% alpha-helix, 31-54% random structures) with no significant change in beta-sheet structures (22-26%). Maximal effects on 1 microM enzyme were obtained at 200 microM Ca2+ or 100 microM Mg2+, the divalent cation binding having a higher affinity for Mg2+ than for Ca2+. The Ca2(+)-induced transition was time-dependent, while Mg2+ effects were immediate. In addition, there was no observed energy transfer for protein kinase C with the fluorescent Ca2(+)-binding site probe, terbium(III). This study suggests that divalent cation-induced changes in soluble protein kinase C structure may be an important step in in vitro analyses that has not yet been detected by standard biochemical enzymatic assays.     

Ion regulation of homotypic vacuole fusion in Saccharomyces cerevisiae

Biological membrane fusion employs divalent cations as protein cofactors or as signaling ligands. For example, Mg2+ is a cofactor for the N-ethylmaleimide-sensitive factor (NSF) ATPase, and the Ca2+ signal from neuronal membrane depolarization is required for synaptotagmin activation. Divalent cations also regulate liposome fusion, but the role of such ion interactions with lipid bilayers in Rab- and soluble NSF attachment protein receptor (SNARE)-dependent biological membrane fusion is less clear. Yeast vacuole fusion requires Mg2+ for Sec18p ATPase activity, and vacuole docking triggers an efflux of luminal Ca2+. We now report distinct reaction conditions where divalent or monovalent ions interchangeably regulate Rab- and SNARE-dependent vacuole fusion. In reactions with 5 mm Mg2+, other free divalent ions are not needed. Reactions containing low Mg2+ concentrations are strongly inhibited by the rapid Ca2+ chelator BAPTA. However, addition of the soluble SNARE Vam7p relieves BAPTA inhibition as effectively as Ca2+ or Mg2+, suggesting that Ca2+ does not perform a unique signaling function. When the need for Mg2+, ATP, and Sec18p for fusion is bypassed through the addition of Vam7p, vacuole fusion does not require any appreciable free divalent cations and can even be stimulated by their chelators. The similarity of these findings to those with liposomes, and the higher ion specificity of the regulation of proteins, suggests a working model in which ion interactions with bilayer lipids permit Rab- and SNARE-dependent membrane fusion.     

EPR spectroscopic analysis of U7 hammerhead ribozyme dynamics during metal ion induced folding

Electron paramagnetic resonance (EPR) spectroscopy was used to examine changes in internal structure and dynamics of the hammerhead ribozyme upon metal ion induced folding, changes in pH, and the presence and absence of ribozyme inhibitors. A nitroxide spin-label was attached to nucleotide U7 of the HH16 catalytic core, and this modified ribozyme was observed to retain catalytic activity. U7 was shown by EPR spectroscopy to be more mobile in the ribozyme-product complex than in either the unfolded ribozyme or the ribozyme-substrate complex. A two-step divalent metal ion dependent folding pathway was observed for the ribozyme-substrate complex with a weak first transition observed at 0.25 mM Mg2+ and a strong second transition observed around 10 mM Mg2+, in agreement with studies using other biophysical and biochemical techniques. Previously, ribozyme activity was observed in the absence of divalent metal ions and the presence of high concentrations of monovalent metal ions, although the activity was less than that observed in the presence of divalent metal ions. Here, we observed similar dynamics for U7 in the presence of 4 M Na+ or Li+, which were distinctively different than that observed in the presence of 10 mM Mg2+, indicating that U7 of the catalytic core forms a different microenvironment under monovalent versus divalent metal ion conditions. Interestingly, the catalytically efficient microenvironment of U7 was similar to that observed in a solution containing 1 M Na+ upon addition of one divalent metal ion per ribozyme. In summary, these results demonstrate that changes in local dynamics, as detected by EPR spectroscopy, can be used to study conformational changes associated with RNA folding and function.     

Structures of the mono- and divalent metal nucleotide complexes in the myosin ATPase

The structure of both the mono- and the divalent metal nucleotide complexes active in the myosin subfragment 1 ATPase has been determined using the phosphorothioate analogs of ATP in the presence of various cations. Both the Sp and the Rp diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) were substrates in the presence of Mg2+, Ca2+, Mn2+, Co2+, Zn2+, and Cd2+ as well as with NH4+ and T1+. The Sp/Rp activity ratios obtained were largely independent of the cation. The simplest explanation of these results is that both mono- and divalent cations do not coordinate to the alpha-phosphate group. With adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), essentially only the Sp diastereomer was active with Mg2+ with Sp/Rp ratio of greater 3000. As the divalent metal ion was varied in the series given above, this ratio was progressively lowered to the value of 0.2 found with Cd2+. Similar changes in stereoselectivity were seen with monovalent cations. Thus, with NH4+, an Sp/Rp ratio of 8 was observed, whereas with T1+, this figure was reduced to 0.04. These data indicate that both mono- and divalent cations coordinate to the beta-phosphate group of the nucleoside triphosphate substrate. These results obtained with ATP alpha S and ATP beta S suggest that myosin uses the mono- or divalent cation delta, beta, gamma-bidentate nucleotide chelate as substrate.     

Comparative structural aspects of cation binding to phosphatidylserine bilayers

X-ray diffraction data recorded for monovalent and divalent cation complexes of a series of phosphatidylserines (PS) varying in chain length reveal a simple structural pattern. Only two bilayer structural types differing in hydrocarbon chain tilt but with similar polar group conformations are observed for (i) anhydrous acidic PS, (ii) anhydrous K+-PS, and (iii) Li+, Mg2+, Ca2+, Sr2+, Ba2+, and Pr3+ complexes of 'hydrated' PS. The X-ray diffraction data suggest that PS becomes dehydrated on complexing with Li+, Mg2+, Ca2+, and other divalent cations and adopts either the chain untilted (form I) or tilted (form II) bilayer structure.     

Genetic competence in Escherichia coli requires poly-beta-hydroxybutyrate/calcium polyphosphate membrane complexes and certain divalent cations.

In earlier studies of genetic competence in Escherichia coli induced with calcium-containing buffers, a strong correlation was found between transformation efficiency and the formation of poly-beta-hydroxybutyrate/calcium polyphosphate (PHB/Ca2+/PPi) complexes in the plasma membranes. In this study, we replaced Ca2+ with one of a number of other cations--monovalent, divalent, and trivalent--and found significant numbers of transformants (transformation efficiency, > 10(5)/micrograms of pBR322 DNA) only when the cells had high levels of PHB/Ca2+/PPi and the medium contained at least one of the divalent cations Ca2+, Mn2+, Sr2+, or Mg2+. Cells with high levels of the complexes were not competent when the medium did not contain these cations, but the cations were also ineffectual when the cells had few complexes. Surprisingly, Mn, Sr, and Mg were not incorporated into the complexes in place of Ca. These results indicate that PHB/Ca2+/PPi complexes and the above-mentioned divalent cations each have essential but disparate roles in genetic competence. Moreover, the strong selectivity of PHB/PPi for Ca2+ suggests the binding sites in the complexes are ionophoretic.     

Cationic modulation of follicle-stimulating hormone binding to granulosa cell receptor

Magnesium (Mg2+) increases binding of follicle-stimulating hormone (FSH) to membrane-bound receptors and increases adenylyl cyclase activity. We examined the effects of divalent and monovalent cations on FSH binding to receptors in granulosa cells from immature porcine follicles. Divalent and monovalent cations increased binding of [125I]iodo-porcine FSH (125I-pFSH). The divalent cations Mg2+, calcium (Ca2+) and manganese, (Mn2+) increased specific binding a maximum of 4- to 5-fold at added concentrations of 10 mM. Mg2+ caused a half-maximal enhancement of binding at 0.6 mM, whereas Ca2+ and Mn2+ had half-maximal effects at 0.7 mM and 0.8 mM, respectively. The monovalent cation potassium (K+) increased binding a maximum of 1.5-fold at an added concentration of 50 mM, whereas the monovalent cation (Na+) did not increase binding at any concentration tested. The difference between K+ and Na+ suggested that either enhancement of binding was not a simple ionic effect or Na+ has a negative effect that suppresses its positive effect. Ethylenediamine tetraacetic acid, a chelator of Mg2+, prevented binding of 125I-pFSH only in the presence of Mg2+, whereas pregnant mare's serum gonadotropin, a competitor with FSH for the receptor, prevented binding in both the absence and the presence of Mg2+. Guanyl-5-ylimidodiphosphate (Gpp[NH]p) inhibited binding of 125I-pFSH in the absence or presence of Mg2+, but only at Gpp(NH)p concentrations greater than 1 mM. We used Mg2+ to determine if divalent cations enhanced FSH binding by increasing receptor affinity or by increasing the apparent number of binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Concentration dependence of equivalent conductivity of polyelectrolyte

Equivalent conductivity of aqueous solutions of alternating copolymer of iso-butyl vinyl ether and maleic acid, [poly(iso BVE-co-MA)] was studied, especially its polymer-concentration dependence. Various species of counterions such as quaternary ammoniumions (NMe4+, NEt4+, NPr4+, NBu4+) and divalent ions (Ca2+, Sr2+, Ba2+) were employed besides alkali metal ions. The applicability of Manning's conductivity theory was examined for the case of univalent counterions at various degrees of neutralization (beta). A major discrepancy against the theory was observed at beta = 1.0, while a comparatively good agreement was found at beta around 0.5. This suggests that the rod-like polyion model, which is the basis of the theory, is applicable near beta = 0.5, where polyions are most expanded. The low conductivities in the case of quaternary ammonium counterions suggested the ion-binding due to hydrophobic interaction with alkyl side chains. Molecular weight dependence was not appreciably observed near beta = 0.5 similarly to usual polyelectrolytes, but it appeared slightly at beta = 1.0.     

Solution conformation of various uridine diphosphoglucose salts as probed by NMR spectroscopy

The solution conformations of uridine diphosphoglucose (UDP-Glc) under a variety of conditions (solvent, ionic strength, various mono- and divalent cations) have been studied by NMR spectroscopy (1H, 13C, 31P, and 25Mg). In the case of divalent cations (Ca2+, Mg2+, Mn2+) the phosphate oxygens are the preferred coordination sites and analysis of the 25Mg linewidths of solutions with various [Mg2+]/[UDP-Glc] ratios, indicates that the 1:1 Mg2+ UDP-Glc complex is the major species. From 13C relaxation data and hydrodynamic theory, it has been demonstrated that under all conditions UDP-Glc adopts a fairly extended overall shape and that magnesium ions lead to a significant increase in the average length of the UDP-Glc molecule as compared to monovalent cations. Thus, one of the roles of the metal ion in enzymic reactions involving nucleotide sugars may be to preorganize the nucleotide sugar.     

Ion selectivity predictions from a two-site permeation model for the cyclic nucleotide-gated channel of retinal rod cells.

We developed a two-site, Eyring rate theory model of ionic permeation for cyclic nucleotide-gated channels (CNGCs). The parameters of the model were optimized by simultaneously fitting current-voltage (IV) data sets from excised photoreceptor patches in electrolyte solutions containing one or more of the following ions: Na+, Ca2+, Mg2+, and K+. The model accounted well for 1) the shape of the IV relations; 2) the binding affinity for Na+; 3) reversal potential values with single-sided additions of Ca2+ or Mg2+ and biionic KCl; and 4) the K1 and voltage dependence for divalent block from the cytoplasmic side of the channel. The differences between the predicted K1's for extracellular block by Ca2+ and Mg2+ and the values obtained from heterologous expression of only the alpha-subunit of the channel suggest that the beta-subunit or a cell-specific factor affects the interaction of divalent cations at the external but not the internal face of the channel. The model predicts concentration-dependent permeability ratios with single-sided addition of Ca2+ and Mg2+ and anomalous mole fraction effects under a limited set of conditions for both monovalent and divalent cations. Ca2+ and Mg2+ are predicted to carry 21% and 10%, respectively, of the total current in the retinal rod cell at -60 mV.     

Self-cleavage activity of the genomic HDV ribozyme in the presence of various divalent metal ions.

To identify the divalent metal ions that can support the self-cleavage activity of the genomic ribozyme of human hepatitis delta virus (HDV), we tested the activity of various divalent metal ions in the ribozyme reactions catalyzed by HDV88 (683-770 nt) and 88DI3 (HDV88 with the sequence from 740-752 nt deleted). Among various metal ions tested, Mg2+, Mn2+, Ca2+ and Sr2+ efficiently supported the self-cleavage reactions of the HDV88 and 88DI3 ribozymes. In the case of the 88DI3 ribozyme, other divalent metal ions, such as Cd2+, Ba2+, Co2+, Pb2+ and Zn2+, were also able to support the self-cleavage reaction to some extent (< 10%). In the presence of spermidine (0.5 mM), the cleavage reaction was promoted at lower concentrations of effective divalent metal ions. The HDV ribozyme represents the only example of ribozyme to date of a ribozyme that catalyzes the self-cleavage reaction in the presence of Ca2+ ions as efficiently as it does in the presence of Mg2+ ions.     

Chelation of divalent cations by ATP, studied by titration calorimetry.

Thermodynamic parameters and stoichiometry for the formation of complexes of ATP with Mg2+, Ca2+, and Sr2+ were determined by titration calorimetry. In each case, 1:1 stoichiometry was observed and complex formation was entropy driven. Binding constants for formation of complexes decreased in the order of Mg2+ greater than Ca2+ greater than Sr2+, as expected from charge density considerations. Monovalent cations hindered complex formation with Mg2+, apparently by competing with the divalent cation for complexation with ATP. Analysis of this competitive effect provided estimates of the binding constants for complexes of ATP with monovalent cations, which decreased in the order expected from charge density considerations (Li+ greater than Na+ greater than K+).     

Regeneration and inhibition of proton pumping activity of bacteriorhodopsin blue membrane by cationic amine anesthetics

Bacteriorhodopsin (bR) is the prototype of an integral membrane protein with seven membrane-spanning alpha-helices and serves as a model of the G-protein-coupled drug receptors. This study is aimed at reaching a greater understanding of the role of amine local anesthetic cations on the proton transport in the bR protein, and furthermore, the functional role of "the cation" in the proton pumping mechanism. The effect of the amine anesthetic cations on the proton pump in the bR blue membrane was compared with those by divalent (Ca2+, Mg2+ and Mn2+) and monovalent metal cations (Li+, Na+, K+ and Cs+), which are essential for the correct functioning of the proton pumping of the bR protein. The results suggest that the interacting site of the divalent cation to the bR membrane may differ from that of the monovalent metal cation. The electric current profile of the bR blue membrane in the presence of the amine anesthetic cations was biphasic, involving the generation and inhibition of the proton pumping activity in a concentration-dependent manner. The extent of the regeneration of the proton pump by the additives increased in the order of monovalent metal cation相似文献   

Interaction of divalent cations with beta-galactosidase (Escherichia coli).

Although the addition of various divalent metals to beta-galactosidase resulted in apparent activation, only Mg2+ and Mn2+ actually did activate. The apparent activation by the other divalent metals was shown to be due to Mg2+ impurities. Calcium did not activate, but experiments suggested that it did bind. Other divalent metals which were studied failed to bind. The dissociation constants for Mg2+ and Mn2+ were 2.8 X 10(-7) and 1.1 X 10(-8) M, respectively, and in each case one ion bound per monomer. These constants corresponded very closely to apparent values which were obtained from activation studies. The apparent binding constant for Ca2+, obtained from competition studies, was 1.5 X 10(-5) M. Data were obtained which showed that Mg2+, Mn2+, and Ca2+ all compete for binding at a single site. Of interest and of possible molecular biological importance was the observation that, while Mg2+ bound noncooperatively (n = 1.0), Mn2+ did so in a highly cooperative manner (n = 3.4). The binding of Mn2+ (as compared to Mg2+) resulted in a twofold drop in the Vmax for the hydrolysis and transgalactosylis reactions of lactose but had little effect on the Vmax of hydrolysis of allolactose, p-nitrophenyl beta-D-galactopyranoside (PNPG), or o-nitrophenyl beta-D-galactopyranoside (ONPG); Km values were not effected differently for any of the substrates by Mn2+ as compared to Mg2+. When very low levels of divalent metal ions were present (0.01 M EDTA added) or when Ca2+ was bound with lactose as the substrate, a greater decrease was observed in the rate of the transgalactosylic reaction than in the rate of the hydrolytic reaction, and the Km values for lactose and ONPG were increased. Of the three divalent metal ions which bound to beta-galactosidase, only Mn2+ had significant stabilizing effects toward denaturing urea and heat conditions.