Electron paramagnetic resonance spectroscopic measurement of Mn2+ binding affinities to the hammerhead ribozyme and correlation with cleavage activity

Efficient phosphodiester bond cleavage activity by the hammerhead ribozyme requires divalent cations. Toward understanding this metal ion requirement, the Mn2+-binding properties of hammerhead model ribozymes have been investigated under dilute solution conditions, using electron paramagnetic resonance spectroscopy (EPR) to detect free Mn2+ in the presence of added ribozyme. Numbers and affinities of bound Mn2+ were obtained at pH 7.8 (5 mM triethanolamine) in the presence of 0, 0.1, and 1.0 M NaCl for an RNA-DNA model consisting of a 13-nucleotide DNA "substrate" hybridized to a 34-nucleotide RNA "enzyme" [Pley, H. W., Flaherty, K. M., and McKay, D. B. (1994) Nature 372, 68-74]. In 0.1 M NaCl, two classes of Mn2+ sites are found with n1 = 3.7 +/- 0.4, Kd(1) = 4 +/- 1 microM (type 1) and n2 = 5.2 +/- 0.4, Kd(2) = 460 +/- 130 microM (type 2). The high-affinity type 1 sites are confirmed for an active RNA-RNA hybrid (34-nucleotide RNA enzyme:13-nucleotide RNA substrate) by EPR measurements at low Mn2+ concentrations. Decreasing NaCl concentration results in an increased number of bound Mn2+ per hammerhead. By contrast, a binding titration in 1 M NaCl indicates that a single Mn2+ site with apparent Kd approximately 10 microM is populated in low concentrations of Mn2+, and apparent cooperative effects at higher Mn2+ concentrations result in population of a similar total number of Mn2+ sites (n1 = 8-10) as found in 0.1 M NaCl. Mn2+-dependent activity profiles are similar for the active RNA-RNA hybrid in 0.1 and 1 M NaCl. Correlation with binding affinities determined by EPR indicates that hammerhead activity in 0.1 M NaCl is only observed after all four of the high-affinity Mn2+ sites are occupied, rises with population of the type 2 sites, and is independent of Mn2+ concentrations corresponding to > 8-9 Mn2+ bound per hammerhead. Equivalent measurements in 1 M NaCl demonstrate a rise in activity with the cooperative transition observed in the Mn2+ binding curve. These measurements indicate that, over this NaCl concentration range, hammerhead ribozyme activity is influenced by population of a specific set of divalent cation sites.     

Thermodynamics of binding of calcium, magnesium, and zinc to the N-methyl-D-aspartate receptor ion channel peptidic inhibitors, conantokin-G and conantokin-T

The binding isotherms of the divalent metal cations, Ca2+, Mg2+, and Zn2+, to the synthetic gamma-carboxyglutamic acid-containing neuroactive peptides, conantokin-G (con-G) and conantokin-T (con-T), have been determined by isothermal titration calorimetry (ITC) at 25 degreesC and pH 6.5. We have previously shown by potentiometric measurements that con-G contains 2-3 equivalent Ca2+ sites with an average Kd value of 2800 microM. With Mg2+ as the ligand, two separate exothermic sites are obtained by ITC, one of Kd = 46 microM and another of Kd = 311 microM. Much tighter binding of Zn2+ is observed for these latter two sites (Kd values = 0.2 microM and 1.1 microM), and a third considerably weaker binding site is observed, characterized by a Kd value of 286 microM and an endothermic enthalpy of binding. con-T possesses a single exothermic tight binding site for Ca2+, Mg2+, and Zn2+, with Kd values of 428 microM, 10.2 microM, and 0.5 microM, respectively. Again, in the case of con-T, a weak (Kd = 410 microM) endothermic binding site is observed for Zn2+. The binding of these cations to con-G and con-T result in an increase in the alpha-helical content of the peptides. However, this helix is somewhat destabilized in both cases by binding of Zn2+ to its weakest site. Since the differences observed in binding affinities of these three cations to the peptides are substantially greater than their comparative Kd values to malonate, we conclude that the structure of the peptide and, most likely, the steric and geometric properties imposed on the cation site as a result of peptide folding greatly influence the strength of the interaction of cations with con-G and con-T. Further, since the Zn2+ concentrations released in the synaptic cleft during excitatory synaptic activity are sufficiently high relative to the Kd of Zn2+ for con-G and con-T, this cation along with Mg2+, are most likely the most significant metal ion ligands of these peptides in neuronal cells.     

Bimodal action of alkaline earth cations on Dictyostelium discoideum ribonuclease P activity

The ribonucleoprotein ribonuclease P (RNase P) cleaves all tRNA precursors endonucleolitically to produce the mature 5'-end. Dictyostelium discoideum RNase P displays an absolute requirement for Mg2+. Only the alkaline earth cations Ca2+, Sr2+, and Ba2+, under appropriate conditions can substitute to some extent for Mg2+. The transition metals Mn2+, Co2+, Ni2+, and Cd2+ are efficient inhibitors of the enzyme activity. Ca2+, Sr2+ and Ba2+, in the presence of Mg2+, exhibit a bimodal action at the kinetic phase of the reaction. Kinetic analysis of the activation phase revealed that Ca2+, Sr2+, or Ba2+ attached on a specific site of RNase P act as nonessential-noncompetitive activators. Further additions of Ca2+, Sr2+, or Ba2+ cause noncompetitive inhibition on the RNase P reaction, indicating that RNase P possesses a second binding site responsible for the inhibitory effect of Ca2+, Sr2+, and Ba2+. Both activator and inhibitory sites can be occupied by Ca2+, Sr2+, or Ba2+ at the same time.     

Characteristics of cation binding to the I domains of LFA-1 and MAC-1. The LFA-1 I domain contains a Ca2+-binding site

The crystal structures of the I domains of integrins MAC-1 (alphaM beta2; CD11b/CD18) and LFA-1 (alphaL beta2; CD11a/CD18) show that a single conserved cation-binding site is present in each protein. Purified recombinant I domains have intrinsic ligand binding activity, and in several systems this interaction has been demonstrated to be cation-dependent. It has been proposed that the I domain cation-binding site represents a general metal ion-dependent adhesion motif utilized for binding protein ligands. Here we show that the purified recombinant I domain of LFA-1 (alphaLI) binds cations, but with significantly different characteristics compared with the I domain of MAC-1 (alphaMI). Both alphaLI and alphaMI bind 54Mn2+ in a conformation-dependent manner, and in general, cations with charge and size characteristics similar to Mn2+ most effectively inhibit 54Mn2+ binding. Surprisingly, however, physiological levels of Ca2+ (1-2 mM) inhibited 54Mn2+ binding to purified alphaLI, but not to alphaMI. Using 45Ca2+ and 54Mn2+ in direct binding studies, the dissociation constants (KD) for the interactions between these cations and alphaLI were estimated to be 5-6 x 10(-5) and 1-2 x 10(-5) M, respectively. Together with the available structural information, the data suggest differential affinities for Mn2+ and Ca2+ binding to the single conserved site within alphaLI. Antagonism of LFA-1, but not MAC-1, -mediated cell adhesion by Ca2+ may be related to the Ca2+ binding activity of the LFA-1 I domain.     

Change in conformation of plasma membrane phospholipid scramblase induced by occupancy of its Ca2+ binding site

Phospholipid (PL) scramblase is a 35.1 kDa plasma membrane protein that mediates the accelerated transbilayer migration of plasma membrane PL in activated, injured, or apoptotic cells exposed to elevated intracellular Ca2+. We recently identified a conserved segment in the PL scramblase polypeptide (residues Asp273 to Asp284) that is essential for its PL-mobilizing function and was presumed to contain the Ca2+ binding site of the protein (Zhou, Q., Sims, P. J., and Wiedmer, T. (1998) Biochemistry 37, 2356-2360). Whereas the sequence of this peptide segment resembles that of known Ca2+-binding loops within EF-hand containing proteins, it is unusual in being a single such loop in the entire protein and in being closely spaced to the predicted transmembrane helix (Ala291-Gly309). To gain insight into how Ca2+ activates the PL-mobilizing function of PL scramblase, we analyzed conformational changes associated with occupancy of this putative Ca2+ binding site. In addition to activation by Ca2+, the PL-mobilizing function of PL scramblase was found to be activated by other ions, with apparent affinities Tb3+, La3+ > Ca2+ > Mn2+ > Zn2+ > Sr2+ > Ba2+, Mg2+. Evidence for coordinate binding of metal ion by the polypeptide was provided by resonance energy transfer from protein Trp to Tb3+, which was competed by excess Ca2+. Metal binding to PL scramblase was accompanied by increased right-angle light scattering and by a prominent change in circular dichroism, suggesting that coordinate binding of the metal ion induces a conformational change that includes self-aggregation of the polypeptide. Consistent with this interpretation, addition of Ca2+ was found to protect PL scramblase from proteolysis by trypsin both in detergent solution as well as in situ, within the erythrocyte membrane. Mutation in the segment Asp273-Asp284 reduced Tb3+ incorporation and attenuated the change in CD spectrum induced by bound metal ligand, confirming that this suspected EF-hand loopike segment of the polypeptide directly contributes to the Ca2+ binding site.     

Chromium(III) modification of the first metal binding site of phosphoenolpyruvate carboxykinase

Chicken liver phosphoenolpyruvate carboxykinase (PEPCK) is activated by Cr2+ as the sole activator under anaerobic conditions. PEPCK was modified with Cr3+, starting with either Cr2+ or Cr3+. Cr3+ has the distinct advantage of being a paramagnetic cation that could serve as a paramagnetic probe. Activators Mn2+, Mg2+, and Co2+ protect against Cr3+ incorporation. EPR, CD, and fluorescence studies indicate that Cr3+ was incorporated into the cation binding site of PEPCK. The water proton relaxation rate (PRR) and fluorescence binding studies showed that Cr3+(n1)-PEPCK forms enzyme-substrate complexes similar to those observed for the Mn2+(n1)-PEPCK complex (n1 represents the metal "enzyme binding site" as opposed to the metal "nucleotide binding site"). Cr3+(n1)-PEPCK requires an additional divalent cation for activity, an indication of two metal sites on PEPCK. Cr3+(n1)-PEPCK retains 15% residual activity as compared to unmodified PEPCK and demonstrates normal Michaelis-Menten kinetics. This is the first report of an active Cr3+-modified enzyme complex.     

Ca2+ activation of wheat peroxidase: a possible physiological mechanism of control

Peroxidation of substrates such as ascorbic acid, pyrogallol, or ferulic acid, as well as indole acetic acid oxidation catalyzed by wheat germ peroxidase (WGP)2 C2, were found to be activated by Ca2+. This activation is independent of the stabilizing effect of structural Ca2+ reported for peroxidases. Steady state kinetics of ferulic acid oxidation catalyzed by WGP C2 showed an increase in the rate of compound I formation and of compound II decomposition in the presence of the ion, evidenced as an increase in rate constants k1, from 8.9 x 10(5) to 4.5 x 10(5) M-1 cm-1, and k3, from 4.4 x 10(5) to 1.1 x 10(6) M-1 cm-1. The dissociation constant Kd, for the cyanide derivative of the enzyme showed a marked decrease from 220 to 34 microM in the presence of Ca2+, thus implying an effect of the ion in the H2O2 binding step. In the presence of Ca2+, a conformational change in the protein was revealed by tryptophan fluorescence, providing a basis for the activation mechanism. Other peroxidases such as horseradish peroxidase and WGP C3 were not activated by Ca2+. The results suggest the existence of a physiological mechanism of control of peroxidase isozymes activity mediated by Ca2+.     

Characterization of native and recombinant forms of an unusual cobalt-dependent proline dipeptidase (prolidase) from the hyperthermophilic archaeon Pyrococcus furiosus

Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per subunit. Its catalytic activity also required the addition of Co2+ ions (Kd, 0.24 mM), indicating that the enzyme has a second metal ion binding site. Co2+ could be replaced by Mn2+ (resulting in a 25% decrease in activity) but not by Mg2+, Ca2+, Fe2+, Zn2+, Cu2+, or Ni2+. The prolidase exhibited a narrow substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the N terminus. Optimal prolidase activity with Met-Pro as the substrate occurred at a pH of 7.0 and a temperature of 100 degrees C. The N-terminal amino acid sequence of the purified prolidase was used to identify in the P. furiosus genome database a putative prolidase-encoding gene with a product corresponding to 349 amino acids. This gene was expressed in Escherichia coli and the recombinant protein was purified. Its properties, including molecular mass, metal ion dependence, pH and temperature optima, substrate specificity, and thermostability, were indistinguishable from those of the native prolidase from P. furiosus. Furthermore, the Km values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greater Vmax value than the native protein. The amino acid sequence of P. furiosus prolidase has significant similarity with those of prolidases from mesophilic organisms, but the enzyme differs from them in its substrate specificity, thermostability, metal dependency, and response to inhibitors. The P. furiosus enzyme appears to be the second Co-containing member (after methionine aminopeptidase) of the binuclear N-terminal exopeptidase family.     

A high affinity binding site for [3H]-Dofetilide on human leukocytes

Certain Class III anti-arrhythmic agents have been shown to interact with human leukocytes and after antigenic and mitogenic activation. We hypothesized that a binding site for the Class III anti-arrhythmic agent, dofetilide, would exist on human leukocytes. Analysis of binding isotherms defined the presence of a single high affinity binding site on mononuclear cells and neutrophils: Kd 26+/-4 nm, Bmax 61+/-14 fmol/10( 6) cells and Kd 33+/-14 nm, Bmax 163+/-45 fmol/10(6) cells, respectively. Other Class III drugs inhibited [3H]-dofetilide binding at physiologically relevant concentrations, but the IC50 values of E4031 and quinidine were significantly higher for leukocytes than for cardiac myocytes. Interestingly, verapamil inhibited [3H]-dofetilide binding to leukocytes, but not to cardiac myocytes at physiologic concentrations (10 microM). Charybdotoxin and tetraethlyammonium inhibited [3H]-dofetilide binding to leukocytes at microM mm concentrations, respectively, however, apamin did not inhibit binding even at 1 microM concentrations. These data suggest that a Ca2+-activated K+ channel, like K(Ca) mini (apamin-insensitive isoform), is a candidate for the leukocyte [3H]-dofetilide binding site. To assess the functional significance of defetilide binding to leukocyte biology, we evaluated fMLP-stimulated superoxide production in the presence or absence of dofetilide. Dofetilide, at 30 nm suppressed of superoxide production. In conclusion, dofetilide binds to human leukocytes at physiologic concentrations and this binding alters leukocyte function possibly through interaction with a Ca2+-activated K+ channel.     

Calcium ion binding to thrombospondin 1

We have quantified the binding of Ca2+ to platelet thrombospondin 1 (TSP1) using equilibrium dialysis with 45CaCl2. Ca2+ binding to TSP1 was found to be cooperative with 10% occupancy at 15-20 microM CaCl2, 90% occupancy at 100 microM CaCl2, and a Hill coefficient of 2.4 +/- 0.2 The average apparent Kd was 52 +/- 5 microM. Maximum binding, assuming Mr = 450,000 and epsilon = 0.918 (A280/mg/ml), was 35 +/- 3 Ca2+/TSP1. This value is close to the 33 sites (11 per subunit) predicted based on homology of the epidermal growth factor (1 site) and aspartate-rich (10 sites) regions to known Ca2+ binding sequences. Ca2+ protected the aspartate-rich region from trypsin proteolysis, but not until nearly all of the Ca2+ binding sites were filled. At lower occupancy of Ca2+ binding sites, several limited tryptic digest products were obtained. This finding and the previous demonstration of extensive thiol-disulfide isomerization within the aspartate-rich regions suggest that subregions of the aspartate-rich region are stabilized in different conformers. Zn2+, Cu2+, Mn2+, Mg2+, Co2+, Cd2+, and Ba2+ were tested for their ability to modulate Ca2+ binding and protease sensitivity of TSP1. Zn2+ inhibited 40% of the Ca2+ binding but neither protected TSP1 from trypsin proteolysis, nor labilized TSP1 toward trypsin proteolysis. These results provide direct evidence for high capacity, cooperative and specific binding of Ca2+ to conformationally labile aspartate-rich repeats of TSP1.     

Expression of adhesion molecules on circulating PMN during hyperdynamic endotoxemia

The activity of endopeptidase EC 3.4.24.15 (thimet oligopeptidase, EP 24.15), as measured by cleavage of a quenched fluorescent substrate, 7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-Pro-D-Lys (2,4-dinitrophenyl), was increased 2-3 fold by the addition of 1 mM Mn2+ or of 10 mM Ca2+. The inhibitory capability of a specific EP. 24.15 inhibitor, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate, was also increased at similar concentrations of these metal ions. However, the hydrolysis of naturally-occurring peptides, thought to be the physiological substrates for EP 24.15, was not affected by either Mn2+ or Ca2+. These results suggest that the binding of synthetic analogs to the enzyme may differ significantly from the binding, and thus hydrolysis, of natural peptide substrates and caution against drawing conclusions about substrate interactions with the active site from data obtained with modified peptide ligands.     

Interaction of manganese with bovine prothrombin and its thrombin-mediated cleavage products

The binding of the paramagnetic metal, Mn(II), to bovine prothrombin and the thrombin-mediated cleavage products of prothrombin, i.e. fragment 1 and the prethrombin 1 has been investigated. Analysis of the Scatchard plots of the binding data reveals that prothrombin has two high affinity Mn(II) binding sites with a Kd of 1.2 +/- 1.0 X 10(-5) M and approximately two to three lower affinity Mn(II) sites with a Kd of 1.3 +/- 1.0 X 10(-4) M. Positive cooperativity in Mn(II) binding to prothrombin was observed for the strong sites. Fragment 1, the phospholipid-binding region of prothrombin, possesses two high affinity Mn(II) sites with a Kd of 2.2 +/- 1.0 X 10(-5) M and at least two lower affinity sites with a Kd of approximately 2.5 +/- 1.0 X 10(-4) M. Positive cooperativity was not observed for the binding of Mn(II) to fragment 1. Prethrombin 1 binds one Mn(II) with a Kd of 3.2 +/- 1.0 X 10(-4) M. Using the values of free Mn(II) concentration, as determined by EPR measurements and the observed enhancements of the water proton relaxation rates at various concentrations of Mn(II) and protein, the binary enhancement values (epsilon b) of the metal-protein complexes were obtained. The extrapolated values are 11 +/- 0.4 for the initial prothrombin-binding sites, and 10 +/- 0.3 for the tight binding sites of fragment 1. The unique epsilon b value obtained for prethrombin 1 was 5.3 +/- 0.7. When Mn(II) was used in a Factor Xa-metal ion-phospholipid system for activation of prothrombin, the rate of generation of thrombin was less than or equal to 5% of that obtained when Ca(II) was employed in this activation system. Addition of Mn(II) to the same activation system containing Ca(II) resulted in a marked decrease in the rate of thrombin generation, suggesting that Mn(II) probably competes for the same sites on prothrombin as Ca(II). In agreement with this is the observation that the Mn(II) sites on prothrombin could be displaced by Ca(II) at high concentrations of Ca(II).     

Binding of different divalent cations to the active site of avian sarcoma virus integrase and their effects on enzymatic activity

Retroviral integrases (INs) contain two known metal binding domains. The N-terminal domain includes a zinc finger motif and has been shown to bind Zn2+, whereas the central catalytic core domain includes a triad of acidic amino acids that bind Mn2+ or Mg2+, the metal cofactors required for enzymatic activity. The integration reaction occurs in two distinct steps; the first is a specific endonucleolytic cleavage step called "processing," and the second is a polynucleotide transfer or "joining" step. Our previous results showed that the metal preference for in vitro activity of avian sarcoma virus IN is Mn2+ > Mg2+ and that a single cation of either metal is coordinated by two of the three critical active site residues (Asp-64 and Asp-121) in crystals of the isolated catalytic domain. Here, we report that Ca2+, Zn2+, and Cd2+ can also bind in the active site of the catalytic domain. Furthermore, two zinc and cadmium cations are bound at the active site, with all three residues of the active site triad (Asp-64, Asp-121, and Glu-157) contributing to their coordination. These results are consistent with a two-metal mechanism for catalysis by retroviral integrases. We also show that Zn2+ can serve as a cofactor for the endonucleolytic reactions catalyzed by either the full-length protein, a derivative lacking the N-terminal domain, or the isolated catalytic domain of avian sarcoma virus IN. However, polynucleotidyl transferase activities are severely impaired or undetectable in the presence of Zn2+. Thus, although the processing and joining steps of integrase employ a similar mechanism and the same active site triad, they can be clearly distinguished by their metal preferences.     

The role of chondroitin sulfate chains of urinary trypsin inhibitor in inhibition of LPS-induced increase of cytosolic free Ca2+ in HL60 cells and HUVEC cells

Preincubation of HL60 cells and HUVEC cells with urinary trypsin inhibitor (UTI) inhibited increase of cytosolic free Ca2+ induced by LPS. In contrast, an increase of cytosolic free Ca2+ induced by LPS was not inhibited by deglycosylated UTI, UTI treated with monoclonal antibody of chondroitin sulfate. 45Ca2+ binding showed that UTI binds 45Ca2+ dose-dependently. Scatchard plot analysis showed that UTI has two binding sites for Ca2+, a high affinity binding site (Kd=15 microM) and a low affinity site (Kd=150 microM), and that UTI has more than 70 Ca2+ binding sites per molecule. The Ca2+ binding capacity of deglycosylated UTI and UTI treated with monoclonal antibody of chondroitin sulfate was markedly depressed. Furthermore, UTI forms multi-polymers in the presence of Ca2+ as demonstrated by gel filtration and agarose gel electrophoresis. These results suggest that UTI is a physiological Ca2+ chelator on the cells and that the action is due to chondroitin sulfate chains of UTI.     

Calcium binding to tandem repeats of EGF-like modules. Expression and characterization of the EGF-like modules of human Notch-1 implicated in receptor-ligand interactions

The Ca(2+)-binding epidermal growth factor (cbEGF)-like module is a structural component of numerous diverse proteins and occurs almost exclusively within repeated motifs. Notch-1, a fundamental receptor for cell fate decisions, contains 36 extracellular EGF modules in tandem, of which 21 are potentially Ca(2+)-binding. We report the Ca(2+)-binding properties of EGF11-12 and EGF10-13 from human Notch-1 (hNEGF11-12 and hNEGF10-13), modules previously shown to support Ca(2+)-dependent interactions with the ligands Delta and Serrate. Ca2+ titrations in the presence of chromophoric chelators, 5,5'-Br2BAPTA and 5-NBAPTA, gave two binding constants for hNEGF11-12, Kd1 = 3.4 x 10(-5) M and Kd2 > 2.5 x 10(-4) M. The high-affinity site was found to be localized to hNEGF12. Titration of hNEGF10-13 gave three binding constants, Kd1 = 3.1 x 10(-6) M, Kd2 = 1.6 x 10(-4) M, and Kd3 > 2.5 x 10(-4) M, demonstrating that assembly of EGF modules in tandem can increase Ca2+ affinity. The highest affinity sites in hNEGF11-12 and hNEGF10-13 had 10 to 100-fold higher affinity than reported for EGF32-33 and EGF25-31, respectively, from fibrillin-1, a connective tissue protein with 43 cbEGF modules. A model of hNEGF11-12 based on fibrillin-1 EGF32-33 demonstrates electronegative potential that could contribute to the higher affinity of the Ca(2+)-binding site in hNEGF12. These data demonstrate that the Ca2+ affinity of cbEGF repeats can be highly variable among different classes of cbEGF containing proteins.     

Pharmacological characterization of a specific binding site for angiotensin IV in cultured porcine aortic endothelial cells

This study demonstrated the existence of a specific binding site for angiotensin IV in porcine aortic endothelial cells. Non-equilibrium kinetic analyses at 37 degrees C allowed the calculation of a kinetic Kd of 0.44 nM. Pseudo-equilibrium saturation binding studies at 37 degrees C for 90 min indicated the presence of a single high-affinity site (Kd = 3.87 +/- 0.60 nM), saturable and abundant (Bmax = 9.64 +/- 1.44 pmol/mg protein). Competitive binding studies demonstrated the following rank order of effectiveness: angiotensin IV > angiotensin III > angiotensin II > angiotensin I > angiotensin II-(1-7), while 2-n-butyl-4-chloro-5-hydroxymethyl-1 [(2'-(1H-tetrazol-5-yl) biphenyl-4-yl) methyl] imidazol (DuP 753: losartan), 1-(4-amino-3-methyl-phenyl) methyl-5-diphenylisoethyl-4,5,6,7-tetrahydro-1H-imidazo [4,5-C] pyridine-6-carboxylic acid (PD 123177) or nicotinic acid-Tyr-(N alpha -benzyl-oxycarbonyl-Arg) Lys-His-Pro-Ile-OH (CGP 42112A) were inactive at the concentration of 100 microM. This binding site is, therefore, distinct from angiotensin II receptors, AT1 and AT2. Addition of the divalent cations Mg2+, Mn2+ or Ca2+ to the incubation buffer resulted in 90-95% inhibition of the [125I]angiotensin IV-specific binding to porcine aortic endothelial cells. Furthermore, the chelator, EGTA, at 5 mM increased the number of binding sites (Bmax = 17.8 +/- 2.5 pmol/mg protein), with no change in affinity (Kd = 5.7 +/- 1.3 nM). Exposure of porcine aortic endothelial cell membranes to the non-hydrolyzable GTP analog, GTP gamma S, had no effect on [125I]angiotensin IV binding. The presence of a high concentration of binding sites for angiotensin IV in porcine aortic endothelial cells suggests that this peptide may play an important role in the modulation of the cardiovascular system.     

Metal ion/buffer interactions. Stability of binary and ternary complexes containing 2-[bis(2-hydroxyethyl)amino]-2(hydroxymethyl)-1,3-propanediol (Bistris) and adenosine 5'-triphosphate (ATP)

The acidity constant of protonated 2-[bis(2-hydroxyethyl)amino]-2(hydroxymethyl)-1,3-propanediol (Bistris) has been measured. The influence of hydroxo groups on the basicity of Bistris and related bases is discussed. The interaction of Bistris with the metal ions (M2+) Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Pb2+ was studied by potentiometry and spectrophotometry in aqueous solution (I = 1.0 M, KNO3; 25 degrees C) and the stability constants of the M(Bistris)2+ complexes were determined. Unexpectedly Ca(Bistris)2+ is the most stable among the alkaline earth ion complexes (log KCaCa(Bistris) = 2.25; the corresponding values for the Mg2+, Sr2+ and Ba2+ complexes are 0.34, 1.44 and 0.85, respectively). The ions of the 3d series follow the Irving-Williams sequence: log KMnMn(Bistris) = 0.70, for Cu2+, 5.27 and Zn2+ 2.38. Ternary complexes containing ATP4- as a second ligand were also investigated: the values for delta log KM (= log KM(ATP)M(ATP)(Bistris) -log KMM(Bistris) are in general negative (e.g. delta log KCa = -0.40 or delta log KCu = -1.65), thus indicating that the interaction of Bistris with M(ATP)2- is somewhat less pronounced tan with M2+. However, even in mixed-ligand systems, complex formation may still be considerable, hence great reservations should be exercised in employing Bistris as a buffer in systems containing metal ions. Moreover, in several cases delta log KM is relatively high [for Mg2+-ATP4- -Bistris even positive], indicating some cooperativity between the coordinated ligands, possibly hydrogen-bond formation. Distributions of the complexes in dependence on pH are given, and the structures of the binary M(Bistris)2+ and the ternary M(ATP) (Bistris)2- complexes are discussed. The participation of Bistris hydroxo groups in complex formation is evident.     

Calcium induces binding and formation of a spin-coupled dimanganese(II,II) center in the apo-water oxidation complex of photosystem II as precursor to the functional tetra-Mn/Ca cluster

Two new intermediates are described which form in the dark as precursors to the light-induced assembly of the photosynthetic water oxidation complex (WOC) from the inorganic components. Mn2+ binds to the apo-WOC-PSII protein in the absence of calcium at a high-affinity site. By using a hydrophobic chelator to remove Mn2+ and Ca2+ from the WOC and nonspecific Fe3+, a new EPR signal becomes visible upon binding of Mn2+ to this site, characterized by six-line 55Mn hyperfine structure (DeltaHpp = 96 +/- 1 G) and effective g = 8.3. These features indicate a high-spin electronic ground state (S = 5/2) for Mn2+ and a strong ligand field with large anisotropy. This signal is eliminated if excess Ca2+ or Mg2+ is present. A second Mn2+ EPR signal forms in place of this signal upon addition of Ca2+ in the dark. The yield of this Ca-induced Mn signal is optimum at a ratio of 2 Mn/PSII, and saturates with increasing [Ca2+] >/= 8 mM, exhibiting a calcium dissociation constant of KD = 1.4 mM. The EPR signal of the Ca-induced Mn center at 25 K is asymmetric with major g value of approximately 2.04 (DeltaHpp = 380 G) and a shoulder near g approximately 3.1. It also exhibits resolved 55Mn hyperfine splitting with separation DeltaHpp = 42-45 G. These spectral features are diagnostic of a variety of weakly interacting Mn2(II, II) pairs with electronic spins that are magnetic dipolar coupled in the range of intermanganese separations 4.1 +/- 0.4 A, and commonly associated with one or two carboxylate bridges. The calcium requirement for induction of the Mn2(II,II) signal matches the value observed for steady-state O2 evolution (Michaelis constant, KM approximately 1.4 mM), and for light-induced assembly of the WOC by photoactivation. The Ca-induced Mn2(II,II) center is a more efficient electron donor to the photooxidized tyrosine radical, TyrZ+, than is the mononuclear Mn center present in the absence of Ca2+. The Ca-induced Mn2(II,II) signal serves as a precursor for photoactivation of the functional WOC and is abolished by the presence of Mg2+. Formation of the Mn2(II,II) EPR signal by addition of Ca2+ correlates with reduction of flash-induced catalase activity, indicating that calcium modulates the accessibility or reactivity of the Mn2(II,II) core with H2O2. We propose that calcium organizes the binding site for Mn ions in the apo-WOC protein and may even interact directly with the Mn2(II,II) pair via solvent or protein-derived bridging ligands.     

The structure of the MnIIADP-nitrate-lyxose complex at the active site of hexokinase

The coordination scheme of Mn2+ in the hexokinase-MnIIADP-nitrate-lyxose complex has been determined by electron paramagnetic resonance (EPR) spectroscopy with 17O-enriched ligands. Nitrate binds to the active site of hexokinase when MnIIADP and a sugar substrate or analogue are present. The binding of nitrate enhances inhibition by glucose when ADP is present and narrows the EPR signals of the enzyme-bound MnIIADP complex in the presence of sugar substrates or analogues. Experiments using regiospecifically 17O-enriched ADP, 17O-enriched nitrate, and 17O-enriched water establish the coordination scheme of Mn2+. The EPR experiments show that ADP is a beta-monodentate ligand and that nitrate binds directly to Mn2+. Four water molecules complete the coordination sphere of the enzyme-bound Mn2+. The dissociation constant (Kd approximately 8 mM) of nitrate for the complex with enzyme, MnIIADP, and lyxose was obtained from titration experiments. These results suggest that nitrate-stabilized, dead-end complexes of hexokinase may be useful in stabilizing the closed conformation of this "hinge-bending" enzyme for crystallographic experiments.     

Synaptosomal binding of 125I-labelled daboiatoxin, a new PLA2 neurotoxin from the venom of Daboia russelli siamensis

Daboiatoxin (DbTx), the PLA2 neurotoxin from Daboia russelli siamensis venom, was shown to bind specifically and saturably to rat cerebrocortical synaptosomes and synaptic membrane fragments. Two families of binding sites were detected by equilibrium binding analysis in the presence and absence of Ca2+. Scatchard analysis of biphasic plateaus revealed Kdl 5 nM and Bmax1, 6 pmoles/mg protein, and Kd2 80 nM and Bmax2 20 pmoles/mg protein, respectively, for the high- and low-affinity binding sites. The binding of 125I-DbTx to synaptosomes did not show marked dependence on Ca2+, Mg2+, Co2+ and Sr2+. Native DbTx was the only strong competitor to 125I-DbTx synaptosomal binding (IC50 12.5 nM, KI 5.5 nM). Two other crotalid PLA2 neurotoxins, crotoxin CB and mojave toxin basic subunit, and nontoxic C. Atrox PLA2 enzyme, were relatively weaker inhibitors, while two viperid PLA2 neurotoxins, ammodytoxin A and VRV PL V, were very weak inhibitors. Crotoxin CA was a poor inhibitor even at microM concentrations, whereas no inhibitory effect at all was observed with crotoxin CACB, ammodytoxin C, VRV PL VIIIa, taipoxin, beta-bungarotoxin, or with PLA2 enzymes from N. naja venom, E. schistosa venom, bee venom and porcine pancreas. All other pharmacologically active ligands examined (epinephrine, norepinephrine, histamine, choline, dopamine, serotonin, GABA, naloxone, WB-4101, atropine, hexamethonium and alpha-bun-garotoxin) also failed to interfere with 125I-DbTx binding. As those competitors that showed partial inhibition were effective only at microM concentration range compared to the Kd (5 nM) of 125I-DbTx synaptosomal binding, DbTx could well recognize a different neuronal binding site. Rabbit anti-DbTx polyclonal antisera completely blocked the specific binding. When a range of Ca2+ and K+ channels modulators were examined, Ca2+ channel blockers (omega-conotoxins GVIA and MVIIC, taicatoxin, calciseptine and nitrendiprene) did not affect the binding even at high concentrations, while charybdotoxin was the only K+ channel effector that could partially displace 125I-DbTx synaptosomal binding amongst the K+ channel blockers tested (apamin, dendrotoxin-I, iberiotoxin, MCD-peptide, 4-aminopyridine and tetraethylammonium), suggesting that neither K+ nor Ca2+ channels are associated with DbTx binding sites.