Equilibria between ionophore A23187 and divalent cations: stability of 1:1 complexes in solutions of 80% methanol/water

The cation complexation equilibria between ionophore A23187 and several alkaline earth and first transition series divalent cations have been investigated. Absorption and fluorescence spectroscopy were used to monitor the reactions which were studied in solutions of 80% methanol/water, at 25 degrees C, and under conditions of controlled ionic strength and pH. Titration of the ionophore with divalent cations results first in formation of the dimeric species MA2 and subsequently in the formation of MA+ by disproportionation of the first product. With Zn2+, Ni2+, and Co2+ (above pH approximately 6), a third species is detected which is postulated to be MA.OH. The existence of this species with Mn2+ and alkaline earth cations is uncertain. For formation of MA2, the second stepwise stability constant is similar to or exceeds the first value with all cations studied. However, it is possible to isolate the first reaction and determine accurate stability constants by working at an ionophore concentration of 3 X 10(-8) M or less and by employing pH values which preclude interference by the mixed ionophore/hydroxide species. Under these conditions, the relationship between log KMA' and pH is linear and displays a slope of 1.0. pH-independent stability constants were calculated by using pH-dependent stability constants and the known value of the ionophore's protonation constant in this solvent. The logarithms of the values obtained ranged from 7.54 +/- 0.06 for Ni2+ to 3.60 +/- 0.06 for Ba2+. The selectivity sequence and relative affinities (in parentheses) for the species MA+ are as follows: Ni2+ (977) greater than Co2+ (331) greater than Zn2+ (174) greater than Mn2+ (34) greater than Mg2+ (1.00) approximately equal to Ca2+ (0.89) greater than Sr2+ (0.20) greater than Ba2+ (0.11). Data are discussed in comparison to other studies on the complexation properties of A23187 and in terms of their significance to interpreting the transport properties of this ionophore.     

Polyphasic character of ATP hydrolysis in actomyosin system.

The interaction of 2-amino-2(hydroxymethyl)-1,3-propanediol (Tris) with the metal ions (M2+) Mg2+, Ca2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Pb2+ was studied by potentiometry and spectrophotometry in aqueous solution (I = 0.1 or 1.0 M, KNO3, 25 degrees C). Stability constants of the M(Tris)2+ complexes were determined; those constants which were measured by both methods agreed well. Ternary complexes containing ATP4- as a second ligand were also investigated and it is shown that in the presence of Tris, mixed-ligand complexes of the type M(ATP)(Tris)2- are formed. The values for delta log KM, where delta log KM = log KM(ATP)M(ATP)Tris--log KMM(Tris), are all negative, thus indicating that the interaction of Tris with M(ATP)2- is somewhat less pronounced than with M2+. However, it should be noted that even in mixed-ligand systems complex formation with Tris may still be considerable, hence great reservations should be exercised in employing Tris as a buffer in systems which also contain metal ions. Distributions of the complex species in dependence on pH are shown for several systems, and the structures of the binary M(Tris)2- and the ternary M(ATP)(Tris)2- complexes are discussed. The participation of a Tris-hydroxo group in complex formation is, at least for the M(Tris)2- species, quite evident.     

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+).     

Estimation of the ferrous-transferrin binding constants based on thermodynamic studies of nickel(II)-transferrin

The equilibrium constants for the binding of Ni2+ to human serum transferrin in 0.01 M hepes containing 5 mM sodium bicarbonate at 25 degrees C and pH 7.4 have been measured. The effective binding constants are log K1 = 4.10 +/- 0.15 and log K2 = 3.23 +/- 0.31 for the reactions Ni2+ + apoTr (K1) in equilibrium Ni2+-Tr. Ni2+ + Ni2+-Tr (K2) in equilibrium Ni2+-Tr-Ni2+ where the explicit terms for bicarbonate and hydrogen ion have been incorporated into the effective binding constants. Titration of both forms of mono(ferric)transferrin indicates that unlike other metal ions, Ni2+ binds preferentially to the N-terminal binding site, but that the site preference is rather small. A linear-free-energy relationship (LFER) for the complexation of Ni2+ and Fe2+ has been prepared. This LFER has been used to estimate effective binding constants of log K1 = 3.2 and log K2 = 2.5 for the ferrous-transferrin complex. These ferrous constants have been combined with the literature binding constants for ferric-transferrin to estimate formal reduction potentials of -340 mV vs. NHE for the C-terminal site and -280 mV for the N-terminal site.     

Role of second metal ion in establishing active conformations of concanavalin A

The stoichiometry of Mn2+ binding to concanavalin A was found to be influenced by temperature, pH, and the presence or absence of saccharide. Demetalized concanavalin A binds one Mn2+ (S1 site) at 5 degrees C, pH 6.5, and two Mn2+ at 25 degrees C (S1 and S2 sites). The association constants for Mn2+ are 6.2 x 10(5) and 3.7 x 10(4) M-1 for the S1 and S2 sites, respectively, at 25 degrees C. Concanavalin A with one Mn2+ bound per monomer remains in an open conformation and exhibits a relatively high water proton relaxation rate. Concanavalin A with two Mn2+ ions remains in a closed conformation characterized by a lower relaxation rate. The rate of binding of the second Mn2+ to concanavalin A as determined by ESR and the rate of conversion of open form to closed form (folding over) as determined by proton relaxation rate measurements gave an identical rate constant of 80.0 +/- 5.8 M-1 h-1 at 17 degrees C. Ca2+, Sr2+, and high levels of methyl alpha-D-mannopyranoside also induce folding of concanavalin A. Ca2+ is not catalytic but stoichiometric in causing the folding. Mn2+ in the S1 site can be displaced by Ni2+, Co2+, and Zn2+, and Mn2+ in the S2 site can be displaced by Ca2+ and Sr2+. Concanavalin A with Ni2+, Co2+, Zn2+, or Mn2+ in the S1 site and Ca2+ or Sr2+ in the S2 site has a higher affinity for methylumbelliferyl alpha-D-mannopyranoside than Ni-Mn-, Co-Mn-, Zn-Mn-, and Cd-Cd-concanavalin A.     

Monensin-mediated transports of H+, Na+, K+ and Li+ ions across vesicular membranes: T-jump studies.

Theoretical expression for the rate of decay of delta pH across vesicular membrane due to carrier-mediated ion transports, 1/tau, has been modified taking note of carrier states (such as mon- and mon-H-M+) for which the translocation rate constants in the membrane are small. The rates of delta pH decay due to monensin-mediated H+ and M+ transports (M+ = Na+, K+, Li+) observed in our experiments in the pH range 6-8, and [M+] range 50-250 mM at 25 degrees C have been analysed with the help of this expression. delta pH across soybean phospholipid vesicular membranes were created by temperature jump in our experiments. The following could be inferred from our studies. (a) At low pH (approximately 6) 1/tau in a medium of Na+ is greater than that in a medium of K+. In contrast with this, at higher pH (approximately 7.5) 1/tau is greater in a medium of K+. Such contradictory observations could be understood with the help of our equation and the parameters determined in this work. The relative concentrations of the rate-limiting species (mon-H, mon-K, and mon-Li at Ph approximately 7 in vesicle solutions having Na+, K+ and Li+, respectively) can explain such behaviours. (b) The proton dissociation constant KH for mon-H in the lipid medium (pKH approximately 6.55) is larger than the reported KH in methanol. (c) The concentrations of mon- and mon-H-Na+ are not negligible under the conditions of our experiments. The latter species cause a [Na+]-dependent inhibition of ion transports. (d) The relative magnitudes of metal ion dissociation constants KHM (approximately 0.05 M) for mon-H-Na+ and KM (approximately 0.03 M) for mon-Na suggest that the carboxyl group involved in the protonation may not be dominantly involved in the metal ion complexation. (e) The estimates of KM (approximately 0.03 M for Na+, 0.5 M for K+ and 2.2 M for Li+) follow the ionophore selectivity order. (f) The rate constants k1 and k2 for the translocations of mon-H and mon-M (M+ = Na+, K+ and Li+) are similar in magnitude (approximately 9 x 10(3) s-1) and are higher than that for nig-H and nig-M (approximately 6 x 10(3) s-1) which can be expected from the relative molecular sizes of the ion carriers.     

Kinetics and mechanisms of the recombination of Zn2+, Co2+, and Ni2+ with the metal-depleted catalytic site of horse liver alcohol dehydrogenase

The kinetics of the recombination of the metal-depleted active site of horse liver alcohol dehydrogenase (LADH) with metal ions have been studied over a range of pH and temperature. The formation rates were determined optically, by activity measurements, or by using the pH change during metal incorporation with a pH-indicator as monitor. The binding of Zn2+, Co2+, and Ni2+ ions occurs in a two-step process. The first step is a fast equilibrium reaction, characterized by an equilibrium constant K1. The spectroscopic and catalytic properties of the native or metal-substituted protein are recovered in a slow, monomolecular process with the rate constant k2. The rate constants k2 5.2 X 10(-2) sec-1 (Zn2+), 1.1 X 10(-3) sec-1 (Co2+), and 2 X 10(-4) sec-1 (Ni2+). The rate constants increase with increasing pH. Using temperature dependence, the activation parameters for the reaction with Co2+ and Ni2+ were determined. Activation energies of 51 +/- 2.5 kJ/mol (0.033 M N-Tris-(hydroxymethyl)methyl-2-aminomethane sulfonic acid (TES), pH 6, 9) for Co2+ and 48.5 +/- 4 kJ/mol (0.033 M TES, pH 7, 2) for Ni2+ at 23 degrees C were found. The correspondent activation entropies are - 146 +/- 10 kJ/mol K for Co2+ and - 163 +/- 9 kJ/mol K for Ni2+. Two protons are released during the binding of Zn2+ to H4Zn(n)2 LADH in the pH range 6.8-8.1. The binding of coenzyme, either reduced or oxidized, prevents completely the incorporation of metal ions, suggesting that the metal ions enter the catalytic site via the coenzyme binding domain and not through the hydrophobic substrate channel.     

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)     

Selective transport of Pb2+ and Cd2+ across a phospholipid bilayer by a cyclohexanemonocarboxylic acid-capped 15-crown-5 ether

A cyclohexanemonocarboxylic acid-capped 15-crown-5 ether was synthesized and found to be effective as an ionophore for Pb2+ and Cd2+, transporting them across a phospholipid bilayer membrane. Transport studies were carried out using 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylcholine (POPC) vesicles containing the chelating indicator 2-([2-bis(carboxymethyl)amino-5-methylphenoxy]methyl)-6-methoxy-8-bis(carboxymethyl)aminoquinoline (Quin-2). Data obtained at pH 7.0 using this system, show that the synthetic ionophore transports divalent cations with the selectivity sequence Pb2+ > Cd2+ > Zn2+ > Mn2+ > Co2+ > Ni2+ > Ca2+ > Sr2+. Selectivity factors, based on the ratio of individual initial cation transport rates, are 280 (Pb2+/Ca2+), 62 (Pb2+/Zn2+), 68 (Cd2+/Ca2+), and 16 (Cd2+/Zn2+). Plots of log initial rate versus logM(n+) or log ionophore concentration suggest that Pb2+ and Cd2+ are transported primarily as a 1:1 cation-ionophore complex, but that complexes with other stoichiometries may also be present. The ionophore transports Pb2+ and Cd2+ by a predominantly electrogenic mechanism, based upon an enhanced rate of transport that is produced by agents which dissipate transmembrane potentials. The rate of Pb2+ transport shows a biphasic pH dependence with the maximum occurring at pH approximately 6.5. The high selectivity for Pb2+ and Cd2+ displayed by the cyclohexanecarboxylic acid-capped 15-crown-5 ether suggests potential applications of this ionophore for the treatment of Pb and Cd intoxication, and removal of these heavy metals from wastewater.     

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+).     

Interaction of guanosine cyclic 3',5'-phosphate dependent protein kinase with lin-benzoadenine nucleotides

Using the activated cGMP-dependent protein kinase in the presence of the phosphorylatable peptide [[Ala34]histone H2B-(29-35)], we found that lin-benzoadenosine 5'-diphosphate (lin-benzo-ADP) was a competitive inhibitor of the enzyme with respect to ATP with a Ki (22 microM) similar to the Kd (20 microM) determined by fluorescence polarization titrations. The Kd for lin-benzo-ADP determined in the absence of the phosphorylatable peptide, however, was only 12 microM. ADP bound with lower affinity (Ki = 169 microM; Kd = 114 microM). With [Ala34]histone H2B-(29-35) as phosphoryl acceptor, the Km for lin-benzo-ATP was 29 microM, and that for ATP was 32 microM. The Vmax with lin-benzo-ATP, however, was only 0.06% of that with ATP as substrate [0.00623 +/- 0.00035 vs. 11.1 +/- 0.17 mumol (min.mg)-1]. Binding of lin-benzo-ADP to the kinase was dependent upon a divalent cation. Fluorescence polarization revealed that Mg2+, Mn2+, Co2+, Ni2+, Ca2+, Sr2+, and Ba2+ supported nucleotide binding to the enzyme; Ca2+, Sr2+, and Ba2+, however, did not support any measurable phosphotransferase activity. The rank order of metal ion effectiveness in mediating phosphotransferase activity was Mg2+ greater than Ni2+ greater than Co2+ greater than Mn2+. Although these results were similar to those observed with the cAMP-dependent protein kinase [Hartl, F. T., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 2347], major differences in the Vmax with lin-benzo-ATP as substrate and the effect of peptide substrates on nucleotide (both lin-benzo-ADP and ADP) binding were observed.     

Acid-base properties of ionophore A23187 in methanol-water solutions and bound to unilamellar vesicles of dimyristoylphosphatidylcholine

The acid-base properties of ionophore A23187 in methanol-water solutions (0--95% w/w) and bound to unilamellar vesicles of dimyristoylphosphatidylcholine were examined by ultraviolet and fluorescence spectroscopy, and the spectral properties for the acidic and basic forms were defined under these conditions. Standard mixed-solvent buffers were employed to calibrate pH measurement in the methanol-water solvents. In 65% methanol-water, two protonation equilibria were observed, the most basic of which displayed a value for the logarithm of the protonation constant (log KH) of 7.19 +/- 0.05 at 25 degrees C and 0.05 M ionic strength. Instability of A23187 was encountered below pH approximately 4; however, decomposition was slow enough to allow log KH for the more acidic equilibrium to be estimated as 1.28. Comparison of these results to those obtained with the methyl ester of A23187 (log KH = 1.32) and literature values for other model compounds allowed assignment of the more basic equilibrium to the carboxylic acid moiety and the more acidic one to the N-methylamino substituent of the benzoxazole ring. log KH of the carboxylic acid increased from 5.69 +/- 0.05 to 9.37 +/- 0.05 over the range of solvent polarity encompassed by water to 95% methanol-water. Values for the ground state (absorption) and first excited state (fluorescence) were equal within experimental error. The logarithm of the protonation constant for the membrane-bound ionophore, measured under conditions where the surface potential generated by ionization did not significantly alter the equilibrium, was found to be 7.85 +/- 0.05 at 25 degrees C and at ionic strength of 0.05 M in the aqueous phase. The value agrees with that observed in 80% methanol-water, as does the wavelength of maximum fluorescence emission for the membrane-bound free acid. An interfacial location for the monoprotonated form of the benzoxazolate moiety is proposed, both above and below the membrane phase transition temperature. The location of other regions of the A23187 molecule could not be assessed from these data.     

Evaluation of the metal-ion-coordinating differences between the 2'-, 3'- and 5'-monophosphates of adenosine

The stability constants of the 1:1 complexes formed between Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+ or Cd2+ and 2'AMP2-, 3'AMP2- or 5'AMP2- were determined by potentiometric pH titration in aqueous solution (I = 0.1 M, NaNO3; 25 degrees C). The experimental conditions were carefully selected such that self-association of the nucleotides and their complexes is negligibly small; i.e. it was made certain that the properties of the monomeric divalent-metal-ion--AMP [M(AMP)] complexes were studied. Based on recent measurements with simple phosphate monoesters, R-MP2- where R is a non-coordinating residue [Massoud, S. S. & Sigel, H. (1988) Inorg. Chem. 27, 1447-1453], it is shown that all the M(AMP) complexes of the alkaline earth ions, with the possible exception of Mg(5'AMP), have exactly the stability expected for a sole-phosphate coordination of the metal ion. The same property is revealed for the complexes with Mn2+, Co2+, Zn2+ or Cd2+ and 3'AMP2-; in case of Ni(3'AMP) and Cu(3'AMP) a slight stability increase just at the edge of the experimental-error limits is indicated. This slight stability increase is attributed to the formation of a macrochelate (possibly with N-3); in fact, additional information confirms macrochelation for Cu(3'AMP). About 45% of Cu(2'AMP) exists in aqueous solution as a macrochelate (probably involving N-3); the other M(2'AMP) complexes (M2+ = Mn2+, Co2+, Ni2+, Zn2+, Cd2+) form (if at all) only traces of a base-backbound species. Most pronounced is macrochelate formation with 5'AMP2-: all mentioned 3d ions and Zn2+ or Cd2+ form to some extent macrochelates via N-7 (the structures of these closed species are indicated). In case of M(5'AMP) the base-binding site is certain: replacement of N-7 by a CH unit (tubercidin 5'-monophosphate) eliminates any increased complex stability, whereas formation of the 1,N6-etheno bridge to form 1,N6-ethenoadenosine 5'-monophosphate results in the phenanthroline-like N-6,N-7 site which facilitates macrochelation significantly.     

The influence of methanol on the interactions of calcium with the pyridine nucleotides

The interactions of calcium with NAD+, NADH, NADP+ and NADPH in a 50% (by volume) methanol/water mixture (pH 7, 25 degrees C) were studied by calorimetry. The association constants for 1:1 complex formation were found to be 6.6 +/- 0.2, 270 +/- 76, 18 +/- 3 and 98 +/- 10 for NAD+, NADH, NADP+ and NADPH, respectively. Comparing these to the association constants for an aqueous system reveals that as the polarity of the solvent system is decreased the interactions involving NAD+, NADP+ and NADPH are all decreased. In contrast, the interaction involving NADH is markedly increased. All the interactions were found to be endothermic.     

A calorimetric study of 3d metal ions-acyclovir interactions. The 2-hydroxyethoxymethyl group of acyclovir mimics the role of ribose in deoxy-guanosine and guanosine promoting the coordination through N(7)

The equilibrium constants and enthalpic values of metal acyclovir complexes have been determined by calorimetry for Co(II) (log K=0.96+/-0.05, DeltaH (kJ/mol)=-19.7+/-1.3), Ni(II) (log K=1.39+/-0.03, DeltaH (kJ/mol)=-21.5+/-1.0), Cu(II) (log K=1.83+/-0.03, DeltaH (kJ/mol)=-23.2+/-0.8) and Zn(II) (log K=0.71+/-0.06, DeltaH (kJ/mol)=-18.6+/-1.5). The equilibrium constants are similar to those of the divalent ions with guanosine and 2,9-dimethylpurine. By comparison with previous thermodynamic data, it can be shown that the 2-hydroxyethoxymethyl group promotes coordination through N(7) versus N(1) of the guanine ring for 3d metal ions. These results reveal that the 2-hydroxyethoxymethyl group placed on the purine ring of guanine in acyclovir causes a greater effect than that of the 9-methyl in purines and similar to or greater than that of the ribose moiety in guanosine. The 2-hydroxyethyoxymethyl group of acyclovir mimics the role of ribose in deoxy-guanosine and guanosine promoting a similar coordination chemistry (with very close log K and DeltaH values) for acyclovir, deoxy-guanosine and guanosine with divalent metals.     

Microcalorimetric investigation of the interaction of calmodulin with seminalplasmin and myosin light chain kinase

Flow microcalorimetric titrations of calmodulin with seminalplasmin at 25 degrees C revealed that the high affinity one-to-one complex in the presence of Ca2+ (Comte, M., Malnoe, A., and Cox, J. A. (1986) Biochem. J. 240, 567-573) is entirely enthalpy-driven (delta H0 = -50 kJ.mol-1; delta S0 = O J.K-1.mol-1; delta Cp0 = O J.K-1.mol-1) and is not influenced by the proton or Mg2+ concentration. The Sr2+- and Cd2+-promoted high affinity complexes are also exothermic for -49 and -45 kJ.mol-1, respectively. The observed low affinity interaction in the absence of divalent ions displays no enthalpy change. No enthalpy changes are observed when calmodulin and seminalplasmin are mixed in the presence of millimolar concentrations of Mg2+, Zn2+, or Mn2+. Enthalpy titrations of the 1:1 calmodulin-seminalplasmin complex with Ca2+ and of partly Ca2+-saturated calmodulin with seminalplasmin revealed that only the species calmodulin.Can greater than or equal to 2 is fully competent for high affinity interaction with seminalplasmin. Binding of the second Ca2+ is strongly enhanced (K2 greater than or equal to 5 X 10(7) M-1) as compared to that in free calmodulin (K2 = 2.6 X 10(5) M-1). This is essentially due to the concomitant strongly exothermic step of isomerization of the calmodulin-seminalplasmin complex from its low to its high affinity form. Binding of the remaining two Ca2+ to the high affinity seminalplasmin-calmodulin complex displays the same affinity constants and endothermic enthalpy change as in free calmodulin. A microcalorimetric study on the complex formation between Ca2+-saturated calmodulin and turkey gizzard myosin light chain kinase revealed that the interaction is strongly exothermic with an important overall gain of order (delta H0 = -85 kJ.mol-1; delta S0 = -122 J.K-1.mol-1) and occurs with significant proton uptake (0.44 H+ per mol at pH 7.5). The observed low affinity interaction (K = 2.2 X 10(5) M-1) in the absence of Ca2+ (Mamar-Bachi, A., and Cox, J. A. (1987) Cell Calcium 8, 473-482) displays neither a change in enthalpy nor in protonation.     

3H]nitrendipine receptors in skeletal muscle

The richest source of receptors for the organic calcium channel blocker [3H]nitrendipine in muscle is the transverse tubule membrane. The tubular membrane preparation binds [3H]nitrendipine with a high affinity and has a very high number of [3H]nitrendipine binding sites. For example, for the transverse tubule membrane preparation from rabbit muscle, the dissociation constant of the nitrendipine-receptor complex is 1.8 +/- 0.3 nM and the maximum binding capacity Bmax = 50 +/- 6 pmol/mg of protein. Similar results have been found with a membrane preparation from frog muscle. The dissociation constant found at equilibrium is near that determined from the ratio of rate constants for association (kappa 1) and dissociation (kappa-1). Binding of [3H] nitrendipine is pH-dependent and reveals the presence of an essential ionizable group with a pK of 5.4 on the nitrendipine receptor. The binding is destroyed by proteases showing that the receptor is a protein. Three different classes of Ca2+ channel blockers inhibit [3H]nitrendipine to its specific site. (i) The dihydropyridine analogs of nitrendipine which are competitive inhibitors of [3H]nitrendipine. These molecules form tight complexes with the nitrendipine receptor with dissociation constants between 1.4 and 4.0 nM. (ii) Other antiarrhythmic molecules like verapamil, amiodarone, bepridil, and F13004 which are noncompetitive inhibitors of [3H]nitrendipine binding with dissociation constants between 0.2 and 1 microM. (iii) Divalent cations like Ni2+, Co2+, Mn2+, or Ca2+ which are noncompetitive inhibitors of [3H]nitrendipine binding with the following rank order of potency: Ni+ (K0.5 = 1.8 mM) greater than Co2+ (K0.5 = 2.7 mM) greater than Mn2+ (K0.5 = 4.8 mM) greater than Ca2+ (K0.5 = 65 mM).     

A role for Na+/Ca2+ exchange in the generation of superoxide radicals by human neutrophils

A Na+/Ca2+ exchange mechanism has been recently described in human neutrophils that constitutes the principal pathway for Ca2+ influx into resting cells. The potential role of this system in regulating the respiratory burst in response to activation by the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine was explored. In the presence of 1 mM Ca2+, a variety of di- and trivalent cations suppressed the generation of O(-2) radicals in a series of decreasing efficacy: La3+ approximately Zn2+ much greater than Sr2+ approximately Cd2+ greater than Ba2+ greater than Co2+ greater than Ni2+ approximately Mg2+. This sequence is similar to their rank order of activity in inhibiting 45Ca2+ influx via Na+/Ca2+ counter-transport. Benzamil, phenamil, and 2',4'-dichlorobenzamil, analogues of amiloride which selectively block Na+/Ca2+ exchange in neutrophils, likewise suppressed the release of O(-2) with apparent Ki values of approximately 30 microM. The effect of the cations was competitive with Ca2+, while the interaction between the benzamil derivatives and Ca2+ appeared to be noncompetitive in nature. Both the divalent cations and benzamil also inhibited the rise in cytoplasmic Ca2+ as monitored by fura-2 fluorescence: these agents reduced peak cytosolic Ca2+ levels after N-formyl-methionyl-leucyl-phenylalanine stimulation to values seen in the absence of extracellular Ca2+. These results are compatible with the hypothesis that the influx of Ca2+ via Na+/Ca2+ exchange contributes to the transient elevation in intracellular free Ca2+. The polyvalent cations block the entry of critical Ca2+ ions by competing with Ca2+ for binding to the translocation site on the exchange carrier, while benzamil acts by lowering the maximal transport rate. These studies emphasize that Na+/Ca2+ exchange through its effects on cytoplasmic Ca2+ plays a major regulatory role in activation of the respiratory burst in chemotactic factor-stimulated neutrophils.     

Potentiometric measurements of stoichiometric and apparent affinity constants of EGTA for protons and divalent ions including calcium

The stoichiometric affinity constants of H+, Ca2+, Mg2+ and Sr+ for the ligand EGTA were determined using a modified version of the pH metric method developed by Moisescu and Pusch (Moisescu, D.G. and Pusch, H. (1975) Pfluegers Arch. 355, 243). The values obtained were slightly higher than those previously published. In addition, the shift in the H+ and Ca2+ stoichiometric constants with ionic strength was found to fit an empirical relationship if the total ionic content of the titration solutions was measured in terms of ionic equivalents, Ie (Johansson, L. (1975) Acta Chem. Scand. A29, 365-373), rather than the formal ionic strength, If. Finally, the apparent affinity of EGTA for Ca2+ ions was measured using an abbreviated form of the titration technique. The measured apparent affinity constant agreed with published results only if calculated with respect to pH (measured) of 7.0, rather than pH (concentration).