Ca(2+) binding to both the heavy and light chains of factor VIII is required for cofactor activity

Previously, we demonstrated that Ca(2+) was necessary for the generation of cofactor activity following reconstitution of factor VIII from its isolated light chain (LC) and heavy chain (HC) but that Ca(2+) did not affect HC-LC binding affinity (Wakabayashi et al. (2001) Biochemistry 40, 10293-10300). Titration of EDTA-treated factor VIII with Ca(2+) followed by factor Xa generation assay showed a two-site binding pattern, with indicated high-affinity (K(d) = 8.9 +/- 1.8 microM) and low-affinity (K(d) = 4.0 +/- 0.6 mM) sites. Analysis by equilibrium dialysis using (45)Ca and <400 microM free Ca(2+) verified a high-affinity binding (K(d) = 18.9 +/- 3.7 microM). Preincubation of either HC or LC with 6 mM Ca(2+) followed by reassociation with the untreated complementary chain in the presence of 0.12 mM Ca(2+) failed to generate significant cofactor activity (<0.5 nM min(-1) (nM LC)(-1)). However, pretreatment of both HC and LC with 6 mM Ca(2+) followed by reassociation (at 0.12 mM Ca(2+)) generated high activity (7.5 +/- 0.4 nM min(-1) (nM LC)(-1)). Progress curves for activity regain following factor VIII-Ca(2+) association kinetics fitted well to a series reaction scheme rather than one of simple association (p < 0.0001), suggesting a multistep process which may include a Ca(2+)-dependent conformational change. These results suggest that factor VIII contains two Ca(2+) binding sites with different affinities and that active factor VIII can be reconstituted from HC and LC only when both chains are preactivated by Ca(2+).     

Metal ion-independent association of factor VIII subunits and the roles of calcium and copper ions for cofactor activity and inter-subunit affinity

Factor VIII circulates as a divalent metal ion-dependent heterodimer comprised of a light chain (LC) and a heavy chain (HC). Reassociation of factor VIII subunits was assessed using fluorescence energy transfer where LC and HC were labeled with acrylodan (Ac; fluorescence donor) and fluorescein-5-maleimide (Fl; fluorescence acceptor), respectively. The reduction of donor fluorescence due to the acceptor was used as an indicator of binding. Subunits associated with high affinity (K(d) = 53.8 nM) in the absence of metal ion and presence of EDTA. However, this product showed no cofactor activity, as measured by a factor Xa generation assay. In the presence of 25 mM Ca(2+), no increase in the intersubunit affinity was observed (K(d) = 48.7 nM) but specific activity of the cofactor was approximately 30% that of native factor VIII. At saturating levels of Fl-HC relative to Ac-LC, donor fluorescence decreased to 79.3 and 73.5% of its original value in the absence and presence of Ca(2+), respectively. Thrombin cleaved the heterodimers that were associated in the absence or presence of Ca(2+) with similar efficiency, indicating that the lack of activity was not the result of a defect in activation. Cu(2+) (0.5 microM) increased the intersubunit affinity by approximately 100 fold (K(d) = 0.52 nM) and the specific activity to approximately 60% of native factor VIII. The former effect was independent of Ca(2+), whereas the latter effect required Ca(2+). These results indicate that the intersubunit association in factor VIII is primarily metal-ion independent while divalent metal ions serve specific roles. Ca(2+) appears essential to promote the active conformation of factor VIII while Cu(2+) primarily enhances the intersubunit affinity.     

Residues 110-126 in the A1 domain of factor VIII contain a Ca2+ binding site required for cofactor activity

Generation of factor VIII cofactor activity requires divalent metal ions such as Ca2+ or Mn2+. Evaluation of cofactor reconstitution from isolated factor VIIIa subunits revealed the presence of a functional Ca2+ binding site within the A1 subunit. Isothermal titration calorimetry demonstrated at least two Ca2+ binding sites of similar affinity (K(d) = 0.74 microm) within the A1 subunit. Mutagenesis of an acidic residue-rich region in the A1 domain (residues 110-126) homologous to a putative Ca2+ binding site in factor V (Zeibdawi, A. R., and Pryzdial, E. L. (2001) J. Biol. Chem. 276, 19929-19936) and expression of B-domainless factor VIII molecules yielded reagents to probe Ca2+ and Mn2+ binding in a functional assay. Basal activity observed for wild type factor VIII in a metal ion-free buffer was enhanced approximately 2-fold with saturating Ca2+ or Mn2+ and yielded functional K(d) values of 1.2 and 1.40 microm, respectively. Ca2+ binding affinity was greatly reduced (or lost) in several mutants including E110A, E110D, D116A, E122A, D125A, and D126A. Alternatively, E113A, D115A, and E124A showed wild type-like activity with little or no reduction in Ca2+ affinity. However, Mn2+ affinity was minimally altered except for mutant D125A (and D116A). These results are consistent with region 110-126 serving a critical role for Ca2+ coordination with selected residues capable of contributing to a partially overlapping site for Mn2+, and that occupancy of either site is required for maximal cofactor activity.     

Derivatives of blood coagulation factor IX contain a high affinity Ca2+-binding site that lacks gamma-carboxyglutamic acid

We have examined the calcium-binding properties and metal ion-dependent conformational changes of proteolytically modified derivatives of factor IX that lack gamma-carboxyglutamic acid (Gla) residues. Equilibrium dialysis experiments demonstrated that a Gla-domainless factor IX species retained a single high affinity calcium ion-binding site (Kd = 85 +/- 5 microM). Ca2+ binding to this site was accompanied by a decrease in intrinsic fluorescence emission intensity (Kd = 63 +/- 15 microM). These spectral changes were reversed upon the addition of EDTA. Titration with Sr2+ resulted in little change in fluorescence intensity below 1 mM, while titration with Tb3+ caused fluorescence changes similar to those observed with Ca2+. Tb3+ and Ca2+ appear to bind to the same site because tryptophan-dependent terbium emission was reduced by the addition of Ca2+. Similar results were obtained with a Gla-domainless factor IX species lacking the activation peptide. Gla domain-containing factor IX species exhibited fluorescence changes similar to those of the Gla-domainless proteins at low Ca2+, but an additional structural transition was found at higher Ca2+ concentrations (apparent Kd greater than 0.8 mM). Thus, the conformations of factor IX proteins are nucleated and/or stabilized by calcium binding to a high affinity site which does not contain Gla residues. The binding of Ca2+ to lower affinity Gla domain-dependent metal ion-binding sites elicits an additional conformational change. The strong similarities between these results and those obtained with protein C (Johnson, A. E., Esmon, N. L., Laue, T. M. & Esmon, C. T. (1983) J. Biol. Chem. 258, 5554-5560), coupled with the remarkable sequence homologies of the vitamin K-dependent proteins, suggest that the high affinity Gla-independent Ca2+-binding site may be a common feature of vitamin K-dependent proteins.     

pH-dependent association of factor VIII chains: enhancement of affinity at physiological pH by Cu2+

Reconstitution of factor VIII from isolated heavy chain (HC) and light chain (LC) shows pH-dependence. In the presence of Ca2+, up to 80% of native factor VIII activity was recovered over a wide range of pH. In contrast, affinity of HC and LC was maximal at pH 6.5-6.75 (Kd approximately 4 nM), whereas a Kd approximately 20 nM was observed at physiological pH (7.25). The effect of Cu2+ (0.5 microM total Cu2+) on maximal activity regenerated was negligible at pH 6.25-8.0. However, this level of Cu2+ increased the inter-chain affinity by approximately 5-fold at pH 7.25. This effect resulted from an approximately 1.5-fold increased association rate constant (k(on)) and an approximately 3-fold reduced dissociation rate constant (k(off)). High affinity (Kd=5.3 fM) of the factor VIII heterodimer for Cu2+ was estimated by increases in cofactor activity. No significant increase in inter-chain affinity was observed when either isolated chain was reacted with Cu2+ followed by addition of the complementary chain. Together, these results suggest that the protonation state of specific residues modulates inter-chain affinity. Furthermore, copper ion contributes to the maintenance of the heterodimer at physiologic pH by a mechanism consistent with bridging the two chains.     

Factor VIII A3 domain residues 1954-1961 represent an A1 domain-interactive site

Factor VIIIa consists of subunits designated A1, A2, and A3C1C2. Reassociation of the A1 and A3C1C2 subunits monitored by the factor Xa generation assay and fluorescence resonance energy transfer yielded intersubunit affinity values (K(d)) of 58.0 +/- 12.5 and 58.8 +/- 16.8 nM, respectively. These affinity values were equivalent to that previously determined for factor VIII heavy and light chains [Wakabayashi, H., et al. (2001) Biochemistry 40, 10293-10300], suggesting that the A2 domain makes a minimal contribution to the interchain affinity in factor VIII. Ca(2+) showed no effect on A1/A3C1C2 intersubunit affinity (K(d) = 51.6 +/- 16.6 nM), while Cu(2+) enhanced the A1/A3C1C2 intersubunit affinity approximately 5-fold (K(d) = 12.5 +/- 2.3 nM). A synthetic peptide to A3 domain residues 1954-1961 inhibited association of the A1 and A3C1C2 subunits (K(i) = 65.8 +/- 11.9 microM). Three factor VIII point mutants, His1957Ala, Gly1960Val, and His1961Asp, were stably expressed in BHK cells and purified. All mutants exhibited reduced specific activity (39, 42, and 4%, respectively) compared with that of wild-type factor VIII, and their activity was less stable following heat denaturation analysis (t(1/2) values of 13.3 +/- 0.7, 8.7 +/- 0.3, and 8.1 +/- 0.1 min, respectively) compared with that of the wild type (18.8 +/- 0.8 min). This reduced stability appeared to result from an approximately 2-fold increased dissociation rate for the Gly1960Val and His1961Asp dimers as judged by solid-phase binding assays. We propose that residues 1954-1961 of the A3 domain contribute to interactions with the A1 domain, facilitating their association in factor VIII.     

Investigation of the binding of Ca2+, Mg2+, Mn2+ and K+ to the vitamin D-dependent Ca2+-binding protein from pig duodenum.

The cation-binding properties of the vitamin D-dependent Ca2+-binding protein from pig duodenum were investigated, mainly by flow dialysis. The protein bound two Ca2+ ions with high affinity, and Mg2+, Mn2+ and K+ were all bound competitively with Ca2+ at both sites. The sites were distinguished by their different affinities for Mn2+, the one with the higher affinity being designated A (Kd 0.61 +/- 0.02 microM) and the other B (Kd 50 +/- 6 microM). Competitive binding studies allied to fluorimetric titration with Mg2+ showed that site A bound Ca2+, Mg2+ and K+ with Kd values of 4.7 +/- 0.8 nM, 94 +/- 18 microM and 1.6 +/- 0.3 mM respectively, and site B bound the same three cations with Kd values of 6.3 +/- 1.8 nM, 127 +/- 38 microM and 2.1 +/- 0.6 mM. For the binding of these cations, therefore, there was no significant difference between the two sites. In the presence of 1 mM-Mg2+ and 150 mM-K+, both sites bound Ca2+ with an apparent Kd of 0.5 microM. The cation-binding properties were discussed relative to those of parvalbumin, troponin C and the vitamin D-dependent Ca2+-binding protein from chick duodenum.     

Ca2+-dependent structural changes in bovine blood coagulation factor Va and its subunits

The calcium dependence of the structures of bovine blood coagulation factor Va and its subunits (Vh and Vl) has been examined spectroscopically in order to characterize the conformational changes which accompany the binding of Ca2+ to Vh and Vl to form factor Va. The far-UV CD spectra of the isolated subunits indicate that the secondary structures of both Vh and Vl are predominantly beta-sheet (greater than 45%), with little alpha-helix content (less than 15%). No change in the far-UV CD spectrum was observed when factor Va was formed by the addition of Ca2+ to an equimolar mixture of Vl and Vh. Hence, no detectable change in secondary structure occurs during the formation of factor Va. In contrast, the addition of Ca2+ to an equimolar mixture of Vh and Vl caused a small (2%) increase in the total intrinsic fluorescence intensity and a blue shift in the emission spectrum that resulted from a tertiary structural change and/or the association of nonpolar surfaces at the subunit interface. This fluorescence change correlated closely with the appearance of functional factor Va, since the rate of the spectral change was the same as the rate of recovery of cofactor activity, and since both were half-maximal near 50 microM Ca2+. This fluorescence change required both subunits, was reversed by the addition of EDTA, and was observed only with metal ions that can substitute for Ca2+ in reconstituting factor Va activity from Vh and Vl (Mn2+ and Tb3+; not Mg2+). When a sample containing ANS (8-anilino-1-naphthalenesulfonate) and an equimolar mixture of calcium-free Vh and Vl was titrated with Ca2+, the ANS emission intensity decreased by about 30%, most likely because the association of Vl and Vh caused nonpolar regions at the subunit-subunit interface to become inaccessible for ANS binding. The calcium dependence of this spectral change yielded a Kd of 51 +/- 2 microM, and the rate of the decrease in ANS fluorescence occurred at nearly the same rate as the recovery of factor Va activity. Thus, both intrinsic and extrinsic fluorescence data, as well as other data, indicate that the calcium binding site in factor Va has an apparent Kd of 50 microM under our conditions and that the calcium-mediated binding between Vl and Vh involves hydrophobic interactions between the subunits.(ABSTRACT TRUNCATED AT 400 WORDS)     

The binding of metal ions to bovine factor IX

The binding isotherms of Ca2+ and Mn2+ to bovine factor IX have been determined at pH 6.5 and pH 7.4, at 25 degrees C. At pH 7.4, at least 2 strong Ca2+ sites, with an average KDISS of 0.1 +/- 0.02 mM, are found. An additional 10 to 11 weaker Ca2+ binding sites, with an average KDISS of 1.3 +/- 0.2 mM are noted, at high levels of Ca2+. At pH 6.5, again at least 2 strong Ca2+ sites on factor IX are evident, with an average KDISS of 0.11 +/- 0.02 mM; but slightly fewer (7 to 8) weaker sites, with an average KDISS of 1.9 +/- 0.3 mM, are obtained. Qualitatively, the binding of Mn2+ to bovine factor IX appears similar to that of Ca2+. At pH 6.5, approximately 2 strong Mn2+ binding sites, with an average KDISS of 13 +/- 1.5 micrometer, and an additional 5 to 6 weak sites, with an average KDISS of 160 +/- 15 micrometer, are present. Manganese does not completely displace Ca2+; and Ca2+ does not completely displace Mn2+ from their respective binding sites. On the other hand, Tb3+ and Sm3+ readily displace Ca2+, at pH 6.5, from its sites on factor IX. The rate and extent of activation of bovine factor IX, by bovine factor XIa, is dependent on the Ca2+ concentration, up to concentrations of Ca2+ which saturate its effect on the system. Substitution of Sr2+ for Ca2+ leads to approximately 50% of the maximum rate of factor IXa formation, and final yield of factor IXa, in this activation system. Manganese does not substitute for Ca2+ in this activation, but does inhibit the stimulatory effect of Ca2+. Both Tb3+ and Sm3+ are effective inhibitors of Ca2+ in factor IX activation by factor XIa.     

The interaction of a Ca2+-dependent monoclonal antibody with the protein C activation peptide region. Evidence for obligatory Ca2+ binding to both antigen and antibody

Protein C undergoes Ca2+-induced conformational changes required for activation by the thrombin-thrombomodulin complex. A Ca2+-dependent monoclonal antibody (HPC4) that blocks protein C activation was used to study conformational changes near the activation site in protein C. The half-maximal Ca2+ dependence was similar for protein C and gamma-carboxy-glutamic acid-domainless protein C for binding to HPC4 (205 +/- 23 and 110 +/- 29 microM Ca2+, respectively), activation rates (214 +/- 22 and 210 +/- 37 microM), and intrinsic fluorescence of gamma-carboxyglutamic acid-domainless protein C (176 +/- 34 microM). Protein C heavy chain binding to HPC4 was half-maximal at 36 microM Ca2+, although neither the heavy chain nor HPC4 separately bound Ca2+ with high affinity. The epitope was lost when the activation peptide was released. A synthetic peptide, P (6-17), which spans the activation site, exhibited Ca2+-dependent binding to HPC4 (half-maximal binding = 6 microM Ca2+). Thus, each decrease in antigen structure resulted in a reduced Ca2+ requirement for binding to HPC4. Tb3+ and Ca2+ binding studies demonstrated a Ca2+-binding site in HPC4 required for high affinity antigen binding. These studies provide the first direct evidence for a Ca2+-induced conformational change in the activation region of a vitamin K-dependent zymogen. Furthermore, Ca2+ binding to HPC4 is required for antigen binding. The multiple roles of Ca2+ described may be useful in interpretation of other metal-dependent antibody/antigen interactions.     

Terbium binding to rat brain acetylcholinesterase. A fluorescence probe of anionic sites

Studies are in progress to characterize the nature of ligand interactions at peripheral anionic sites on mammalian brain AChE, including the beta-anionic or "accelerator" anionic sites where enzyme activity is increased upon Ca2+ binding. Terbium was studied as a fluorescence probe of Ca2+ binding sites in partially purified AChE from whole rat brain. Scatchard analysis of Tb3+ binding in low ionic strength (2 mM) Pipes buffer revealed at least two populations of sites: high affinity sites with Kd(app) approximately 7.6 microM and low-affinity sites with a Kd(app) approximately 49.6 microM. Low-affinity binding was selectively inhibited by 50 mM NaCl; high-affinity binding was completely inhibited by 2 mM CaCl2; and all the bound Tb3+ could be displaced by 1 mM EDTA. The heterogeneity of Tb3+ binding sites is consistent with the multiple, concentration-dependent effects of Tb3+ on enzyme activity.     

Characterization of the Ca2+ coordination site regulating binding of Ca2+ channel inhibitors d-cis-diltiazem, (+/-)bepridil and (-)desmethoxyverapamil to their receptor site in skeletal muscle transverse tubule membranes

Ca2+ inhibits (-)[3H]desmethoxyverapamil, d-cis-[3H]diltiazem and (+/-)[3H]bepridil binding to skeletal muscle transverse-tubule membranes with a half-maximum inhibition constant, K0.5 = 5 +/- 1 microM. This value is close to that of the high affinity Ca2+ binding site which controls the ionic selectivity of the Ca2+ channel found in electrophysiological experiments suggesting that the Ca2+ coordination site which regulates the ionic selectivity is also the one which alters binding of the Ca2+ channel inhibitors investigated here. Ca2+ and (-)D888 bind to distinct sites. Occupation of the Ca2+ coordination site decreases the affinity of (-)D888 for its receptor by a factor of 5. Other divalent cations have the same type of inhibition behavior with the rank order of potency Ca2+ (K0.5 = 5 microM) greater than Sr2+ (K0.5 = 25 microM) greater than Ba2+ (K0.5 = 50 microM) greater than Mg2+ (K0.5 = 170 microM).     

Characterization of the cation-binding properties of porcine neurofilaments

In the presence of physiological levels of Na+ (10 mM), K+ (150 mM), and Mg2+ (2 mM), dephosphorylated neurofilaments contained two Ca2+ specific binding sites with Kd = 11 microM per unit consisting of eight low, three middle, and three high molecular subunits, as well as 46 sites with Kd = 620 microM. Only one class of 126 sites with Kd = 740 microM was detected per unit of untreated neurofilaments. A chymotryptic fraction enriched in the alpha-helical domains of neurofilament subunits contained one high-affinity Ca2+-binding site (Kd = 3.6 microM) per domain fragment of approximately 32 kDa. This site may correspond to a region in coil 2b of the alpha-helical domain, which resembles the I-II Ca2+-binding site in intestinal Ca2+-binding protein. Homopolymeric filaments composed of the low or middle molecular weight subunits contained low-affinity Ca2+-binding sites with Kd = 37 microM and 24 microM, respectively, while the Kd values for the low-affinity sites in heteropolymeric filaments were 8-10-fold higher. Competitive binding studies, using the chymotryptic fraction to assay the high-affinity Ca2+-binding sites and 22Na+ to monitor binding to the phosphate-containing low-affinity sites, yielded Kd values for Al3+ of 0.01 microM and 4 microM, respectively. This suggests that the accumulation of Al3+ in neurons may be due in part to its binding to neurofilaments.     

Terbium as a luminescent probe of metal-binding sites in protein kinase C

In the present report, we demonstrate that Tb3+ binds to protein kinase C and serves as a luminescent reporter of certain cationic metal-binding sites. Tb3+ titration of 50 nM protein kinase C results in a 20-fold enhancement of Tb3+ luminescence which is half-maximal at 12 microM Tb3+. A Kd of approximately 145 nM was determined for Tb3+ binding to the enzyme. The excitation spectrum of bound Tb3+ exhibits a peak at 280 nm characteristic of energy transfer from protein tryptophan or tyrosine residues. The luminescence of this complex can be markedly decreased by other metals, including Pb2+ (IC50 = 25 microM), La3+ (IC50 = 50 microM), Hg2+ (IC50 = 300 microM), Ca2+ (IC50 = 6 mM), and Zn2+ (IC50 greater than 10 mM), and chelation of Tb3+ by 2 mM EGTA. Tb3+ binding to protein kinase C is correlated with its inhibition of protein kinase activity (IC50 = 8 microM), r = 0.99) and phorbol ester binding (IC50 = 15 microM, r = 0.98). Tb3+ inhibition of protein kinase C activity cannot be overcome by excess Ca2+, but can be partially overcome with excess phosphatidylserine or by chelation of Tb3+ with EGTA. Tb3+ noncompetitively inhibits phorbol ester binding by decreasing the maximal extent of binding without significantly altering binding affinity. The results suggest that the Tb3(+)-binding site is at or allosterically related to the enzyme's phosphatidylserine-binding site, but is distinct from the phorbol ester-binding domain and the Ca2(+)-binding site that regulates enzyme activity.     

Kinetic and magnetic resonance studies of the role of metal ions in the mechanism of Escherichia coli GDP-mannose mannosyl hydrolase, an unusual nudix enzyme

Escherichia coli GDP-mannose mannosyl hydrolase (GDPMH), a homodimer, catalyzes the hydrolysis of GDP-alpha-D-sugars to yield the beta-D-sugar and GDP by nucleophilic substitution with inversion at the C1' carbon of the sugar [Legler, P. M., Massiah, M. A., Bessman, M. J., and Mildvan, A. S. (2000) Biochemistry 39, 8603-8608]. GDPMH requires a divalent cation for activity such as Mn2+ or Mg2+, which yield similar kcat values of 0.15 and 0.13 s(-1), respectively, at 22 degrees C and pH 7.5. Kinetic analysis of the Mn2+-activated enzyme yielded a K(m) of free Mn2+ of 3.9 +/- 1.3 mM when extrapolated to zero substrate concentration (K(a)Mn2+), which tightened to 0.32 +/- 0.18 mM when extrapolated to infinite substrate concentration (K(m)Mn2+). Similarly, the K(m) of the substrate extrapolated to zero Mn2+ concentration (K(S)(GDPmann) = 1.9 +/- 0.5 mM) and to infinite Mn2+ concentration (K(m)(GDPmann) = 0.16 +/- 0.09 mM) showed an order of magnitude decrease at saturating Mn2+. Such mutual tightening of metal and substrate binding suggests the formation of an enzyme-metal-substrate bridge complex. Direct Mn2+ binding studies, monitoring the concentration of free Mn2+ by EPR and of bound Mn2+ by its enhanced paramagnetic effect on the longitudinal relaxation rate of water protons (PRR), detected three Mn2+ binding sites per enzyme monomer with an average dissociation constant (K(D)) of 3.2 +/- 1.0 mM, in agreement with the kinetically determined K(a)Mn2+. The enhancement factor (epsilon(b)) of 11.5 +/- 1.2 indicates solvent access to the enzyme-bound Mn2+ ions. No cross relaxation was detected among the three bound Mn2+ ions, suggesting them to be separated by at least 10 A. Such studies also yielded a weak dissociation constant for the binary Mn2+-GDP-mannose complex (K1 = 6.5 +/- 1.0 mM) which significantly exceeded the kinetically determined K(m) values of Mn2+, indicating the true substrate to be GDP-mannose rather than its Mn2+ complex. Substrate binding monitored by changes in 1H-15N HSQC spectra yielded a dissociation constant for the binary E-GDP-mannose complex (K(S)(GDPmann)) of 4.0 +/- 0.5 mM, comparable to the kinetically determined K(S) value (1.9 +/- 0.5 mM). To clarify the metal stoichiometry at the active site, product inhibition by GDP, a potent competitive inhibitor (K(I) = 46 +/- 27 microM), was studied. Binding studies revealed a weak, binary E-GDP complex (K(D)(GDP) = 9.4 +/- 3.2 mM) which tightened approximately 500-fold in the presence of Mn2+ to yield a ternary E-Mn2+-GDP complex with a dissociation constant, K3(GDP) = 18 +/- 9 microM, which overlaps with the K(I)(GDP). The tight binding of Mn2+ to 0.7 +/- 0.2 site per enzyme subunit in the ternary E-Mn2+-GDP complex (K(A)' = 15 microM) and the tight binding of GDP to 0.8 +/- 0.1 site per enzyme subunit in the ternary E-Mg2+-GDP complex (K3 < 0.5 mM) indicate a stoichiometry close to 1:1:1 at the active site. The decrease in the enhancement factor of the ternary E-Mn2+-GDP complex (epsilon(T) = 4.9 +/- 0.4) indicates decreased solvent access to the active site Mn2+, consistent with an E-Mn2+-GDP bridge complex. Fermi contact splitting (4.3 +/- 0.2 MHz) of the phosphorus signal in the ESEEM spectrum established the formation of an inner sphere E-Mn2+-GDP complex. The number of water molecules coordinated to Mn2+ in this ternary complex was determined by ESEEM studies in D2O to be two fewer than on the average Mn2+ in the binary E-Mn2+ complexes, consistent with bidentate coordination of enzyme-bound Mn2+ by GDP. Kinetic, metal binding, and GDP binding studies with Mg2+ yielded dissociation constants similar to those found with Mn2+. Hence, GDPMH requires one divalent cation per active site to promote catalysis by facilitating the departure of the GDP leaving group, unlike its homologues the MutT pyrophosphohydrolase, which requires two, or Ap4A pyrophosphatase, which requires three.     

Localization of a fibrinogen calcium binding site between gamma-subunit positions 311 and 336 by terbium fluorescence

Calcium is required for effective fibrin polymerization. The high affinity Ca2+ binding capacity of fibrinogen was directly localized to the gamma-chain by autoradiography of nitrocellulose membrane blots of fibrinogen subunits incubated with 45Ca2+. Terbium (Tb3+) competitively inhibited 45Ca2+ binding to fibrinogen during equilibrium dialysis, accelerated fibrin polymerization, and limited fibrinogen fragment D digestion by plasmin. The intrinsic fluorescence of Ca2+-depleted fibrinogen was maximally enhanced by Ca2+ and Tb3+, but not by Mg2+, at about 3 mol of cation/mol of fibrinogen. Protein-bound Tb3+ fluorescence at 545 nm was maximally enhanced by resonance energy transfer from tryptophan (excitation at 290 nm) at about 2 mol of Tb3+mol of fibrinogen and about 1 mol of Tb3+/mol of plasmic fragment D94 (Mr 94,000). Fibrinogen fragments D78 (Mr 78,000) and E did not show effective enhancement of Tb3+ fluorescence, suggesting that the Ca2+ site is located within gamma 303 to gamma 411, the peptide which is absent in fragment D78 but present in D94. When CNBr fragments of the carboxyamidated gamma-subunit were assayed for enhancement of Tb3+ fluorescence, peptide CBi (gamma 311-336) bound 1 mol of Tb3+/mol of CBi. Thus, the Ca2+ site is located within this peptide. The sequence between gamma 315 and gamma 329 is homologous to the calmodulin and parvalbumin Ca2+ binding sites.     

Tb3+ binding to Ca2+ and Mg2+ binding sites on sarcoplasmic reticulum ATPase

The interactions of Tb3+ and sarcoplasmic reticulum (SR) were investigated by inhibition of Ca2+-activated ATPase activity and enhancement of Tb3+ fluorescence. Ca2+ protected against Tb3+ inhibition of SR ATPase activity. The apparent association constant for Ca2+, determined from the protection, was about 6 x 10(6) M-1, suggesting that Tb3+ inhibits the ATPase activity by binding to the high affinity Ca2+ binding sites. Mg2+ did not protect in the 2-20 mM range. The association constant for Tb3+ binding to this Ca2+ site was estimated to be about 1 x 10(9) M-1. No cooperativity was observed for Tb3+ binding. No enhancement of Tb3+ fluorescence was detected. A second group of binding sites, with weaker affinity for Tb3+, was observed by monitoring the enhancement of Tb3+ fluorescence (lambda ex 285 nm, lambda em 545 nm). The fluorescence intensity increased 950-fold due to binding. Ca2+ did not complete for binding at these sites, but Mg2+ did. The association constant for Mg2+ binding was 94 M-1, suggesting that this may be the site that catalyzes phosphorylation of the ATPase by inorganic phosphate. For vesicles, Tb3+ binding to these Mg2+ sites was best described as binding to two classes of binding sites with negative cooperativity. If the SR ATPase was solubilized in the nonionic detergent C12E9 (dodecyl nonaoxyethylene ether alcohol), in the absence of Ca2+, only one class of Tb3+ binding sites was observed. The total number of sites appeared to remain constant. If Ca2+ was included in the solubilization step, Tb3+ binding to these Mg2+ binding sites displayed positive cooperativity (Hill coefficient, 2.1). In all cases, the apparent association constant for Tb3+, in the presence of 5 mM MgCl2, was in the range of 1-5 x 10(4) M-1.     

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).     

Alamethicin channel permeation by Ca2+, Mn2+ and Ni2+ in bovine chromaffin cells.

Alamethicin causes a concentration-dependent increase of [Ca2+]i in suspensions of bovine adrenal chromaffin cells loaded with fura-2. The basal levels of Cai2+ (234 +/- 37 nM; n = 4) increased to a maximum of 2347 +/- 791 nM (n = 3) with 100 micrograms/ml alamethicin. In the presence of 1 mM Cae2+ the increase reached a plateau within about 2-5 s. This increase was due to Ca2+ entry into chromaffin cells, since in the absence of Cae2+ alamethicin did not modify [Ca2+]i. This contrasts with ionomycin (1 microM) which produced a Cai2+ transient even in the absence of Cae2+. Mn2+ ions also entered chromaffin cells in the presence of alamethicin, as measured by the quenching of fura-2 fluorescence following excitation at 360 nm. Resting chromaffin cells had a measurable permeability to Mn2+ which was drastically increased by cell depolarization by K+ (50 mM) addition. This suggests that Mn2+ is able to permeate voltage-dependent Ca2+ channels. Ni2+ uptake into either resting or K(+)-stimulated chromaffin cells was undetectable, but addition of alamethicin induced rapid uptake of this cation. The alamethicin-induced entry of Ni2+ was decreased by 50 mM K+. Overall, the results are compatible with the formation by alamethicin of ion channels in chromaffin cell plasma membranes.     

Ca2+ binding to chromaffin vesicle matrix proteins: effect of pH, Mg2+, and ionic strength

Recently we found that Ca2+ within chromaffin vesicles is largely bound [Bulenda, D., & Gratzl, M. (1985) Biochemistry 24, 7760-7765]. In order to explore the nature of these bonds, we analyzed the binding of Ca2+ to the vesicle matrix proteins as well as to ATP, the main nucleotide present in these vesicles. The dissociation constant at pH 7 is 50 microM (number of binding sites, n = 180 nmol/mg of protein) for Ca2+-protein bonds and 15 microM (n = 0.8 mumol/mumol) for Ca2+-ATP bonds. When the pH is decreased to more physiological values (pH 6), the number of binding sites remains the same. However, the affinity of Ca2+ for the proteins decreases much less than its affinity for ATP (dissociation constant of 90 vs. 70 microM). At pH 6 monovalent cations (30-50 mM) as well as Mg2+ (0.1-0.5 mM), which are also present within chromaffin vesicles, do not affect the number of binding sites for Ca2+ but cause a decrease in the affinity of Ca2+ for both proteins and ATP. For Ca2+ binding to ATP in the presence of 0.5 mM Mg2+ we found a dissociation constant of 340 microM and after addition of 35 mM K+ a dissociation constant of 170 microM. Ca2+ binding to the chromaffin vesicle matrix proteins in the presence of 0.5 mM Mg2+ is characterized by a Kd of 240 microM and after addition of 15 mM Na+ by a Kd of 340 microM.(ABSTRACT TRUNCATED AT 250 WORDS)