Anion binding to liver alcohol dehydrogenase

1. Complex formation at the general anion-binding site of the liver alcohol dehydrogenase subunit has been characterized by transient-state kinetic methods, using NADH as a reporter ligand. Equilibrium dissociation constants for anion binding at the site are reported. They conform basically to the lyotropic series of affinity order, with exceptionally tight binding of sulphate. The particular specificity for sulphate might be a general characteristic of anion-binding enzymic arginyl sites. 2. Anionic species of phosphate and pyrophosphate buffer solutions do not interact significantly with the general anion-binding site over the pH range 8-10. At lower pH, phosphate binding becomes significant due to complex formation with the monovalent H2PO4 species. The latter interaction corresponds to a dissociation constant of about 60 mM, indicating that phosphate binding is comparatively weak also at low pH. 3. It is concluded that previously reported pH dependence data for coenzyme binding to liver alcohol dehydrogenase cannot be much affected by coenzyme-competitive effects of buffer anion binding. Kinetic parameter estimates now determined for NADH binding in weakly buffered solutions agree within experimental precision with those obtained previously from measurements made in buffer solutions of 0.1 M ionic strength.     

Demonstration and chemical modification of a specific phosphate binding site in the phosphate-starvation-inducible outer membrane porin protein P of Pseudomonas aeruginosa

The interaction of phosphate ions with the Pseudomonas aeruginosa anion-specific protein P channel was probed. The single-channel conductance of protein P incorporated into planar lipid bilayer membranes in the presence of 0.3 M H2PO-4 was shown to be 6.0 pS, demonstrating that protein P channels allowed the permeation of phosphate. When large numbers of protein P channels were incorporated into lipid bilayer membranes in the presence of 40 mM Cl-, addition of small concentrations of phosphate resulted in reduction of macroscopic Cl- conductance in a dose- (and pH-) dependent fashion. This allowed calculation of an I50 value of e.g. 0.46 mM at pH 7.0, suggesting that the affinity of protein P for its normal substrate phosphate was at least 60-100-fold greater than the affinity of the channel for other ions such as chloride. Pyrophosphate and the phosphate analogue, arsenate, also inhibited macroscopic Cl- conductance through protein P with I50 values at pH 7.0 of 4.9 mM and 1.3 mM, respectively. To probe the nature of the phosphate binding site, the epsilon-amino groups of available lysine residues of protein P were chemically modified. Acetylation and carbamylation which produced uncharged, modified lysines destroyed both the anion (e.g. Cl-) binding site and the phosphate binding site as determined by single-channel experiments and macroscopic conductance inhibition experiments respectively. Nevertheless, the modified proteins still retained their trimeric configuration and their ability to reconstitute single channels in lipid bilayer membranes. Methylation, which allowed retention of the charge on the modified lysine residues, increased the Kd of the channel for Cl- 33-fold and the I50 for phosphate inhibition of macroscopic Cl- conductance 2.5-4-fold. A molecular model for the phosphate binding site of the protein P channel is presented.     

Thermodynamic control of D-amino acid oxidase by benzoate binding

The redox properties of D-amino acid oxidase (D-amino-acid: O2 oxidoreductase (deaminating) EC1.4.3.3) have been measured at 18 degrees C in 20 mM sodium pyrophosphate, pH 8.5, and in 50 mM sodium phosphate, pH 7.0. Over the entire pH range, 2 eq are required per mol of FAD in D-amino acid oxidase for reduction to the anion dihydroquinone. The red anion semiquinone is thermodynamically stable as indicated by the separation of the electron potentials and the quantitative formation of the semiquinone species. The first electron potential is pH-independent at -0.098 +/- 0.004 V versus SHE while the second electron potential is pH-dependent exhibiting a 0.060 mV/pH unit slope. The redox behavior of D-amino acid oxidase is consistent with that observed for other oxidase enzymes. On the other hand, the behavior of the benzoate-bound enzyme under the same conditions is in marked contrast to the thermodynamics of free D-amino acid oxidase. Spectroelectrochemical experiments performed on inhibitor-bound (benzoate) D-amino acid oxidase show that benzoate binding regulates the redox properties of the enzyme, causing the energy levels of the benzoate-bound enzyme to be consistent with the two-electron transfer catalytic function of the enzyme. Our data are consistent with benzoate binding at the enzyme active site destroying the inductive effect of the positively charged arginine residue. Others have postulated that this positively charged group near the N(1)C(2) = O position of the flavin controls the enzyme properties. The data presented here are the clearest examples yet of enzyme regulation by substrate which may be a general characteristic of all flavoprotein oxidases.     

Phosphate Activates the Phosphoenolpyruvate Carboxylase from the C4 Plant Amaranthus viridis L.

Phosphoenolpyruvate carboxylase from Amaranthus viridis leaves was activated by inorganic orthophosphate in a concentration- and pH-dependent manner. Maximal activation at pH 7.0 was achieved at phosphate concentrations above 20 mM, and a positive cooperativity was observed for the binding of the anion at this pH. At pH 8.0 the maximum of activity was achieved at 10 mM phosphate; higher concentrations reduced the activation. K_M for phosphoenolpyruvate-Mg at pH 7.0 was lowered by phosphate in all concentrations tested up to 30 mM. While at pH 8.0 the K_M values were lower than that of the control up to 10 mM phosphate; higher anion concentrations raised the minimum value of K_M at this pH. V_MAX increased at pH 7.0, and remained unchanged at pH 8.0. A K_A value of 0.41 mM was calculated for phosphate at the alkaline pH. The phosphate analogue arsenate also behaved as an activating agent, while other anions (e.g. nitrate, nitrite, sulfate, tetraborate) were ineffective. The phosphate-activated enzyme was shown to be insensitive to glucose-6-phosphate, but was inhibited by l -malate to the same extent as the control.     

The interaction of the trp repressor from Escherichia coli with L-tryptophan and indole propanoic acid

The binding of the corepressor, L-tryptophan, and an inducer, indole propanoic acid, to the trp repressor from Escherichia coli was studied by absorbance, fluorescence, circular dichroic and proton NMR spectroscopy. The two ligands bind to the same site on the repressor in the same orientation; they are molecular competitors. The binding site is of relatively low polarity and contains at least one methyl group that lies 0.3 nm over the indole moiety near the C5 proton of the bound ligand, and an aromatic residue, probably tyrosine. The dissociation constant was determined as a function of temperature and pH. At 25 degrees C in 0.1 M phosphate buffer, pH 7.6, the dissociation constant is 18 +/- 2 microM for both ligands. In the same buffer system, the van't Hoff enthalpy for dissociation is 35.5 +/- 1 kJ/mol for tryptophan, and 30.5 +/- 2 kJ/mol for indole propanoic acid. The affinity of the repressor for indole propanoic acid is independent of pH in the range 7 less than 10, but decreases four fold for tryptophan in the same range. The amino group of tryptophan makes a significant contribution to its binding affinity. Difference NMR spectra showed that there are few changes of protein resonances on binding ligands. The NMR signals of the bound resonances were assigned by difference and nuclear Overhauser effect spectroscopy. The properties of the bound resonances are consistent with the ligands being largely immobilised within the binding site. The difference spectra, and the known functional differences of the two ligands, suggest that tryptophan induces a slightly different conformational state in the repressor from that induced by indole propanoic acid. There is no evidence for a global transition. The rate of dissociation of ligands is relatively large, being in the range 400-600 s-1.     

Reversible DIDS binding to band 3 protein in human erythrocyte membranes

Reversible binding of DIDS [4,4'-diisothiocyanato-2,2'-stilbenedisulphonate] to Band 3 protein, the anion exchanger located in erythrocyte plasma membrane, was studied in human erythrocytes. For this purpose, the tritiated form of DIDS ([3H]DIDS) has been synthesized and the filtering technique has been used to follow the kinetics of DIDS binding to the sites on Band 3 protein. The obtained results showed monophasic kinetics both for dissociation and association of the 'DIDS--Band 3' complex at 0 degree C in the presence of 165 mM KCl outside the cell (pH 7.3). A pseudo-first order association rate constant k+1 was determined to be (3.72 +/- 0.42) x 10(5) M-1 s-1, while the dissociation rate constant K-1 was determined to be (9.40 +/- 0.68) x 10(-3) s-1. The dissociation constant KD, calculated from the measured values of k-1 and k+1, was found to be 2.53 x 10(-8) M. The standard thermodynamics parameters characterizing reversible DIDS binding to Band 3 protein at 0 degree C were calculated. The mean values of the activation energies for the association and dissociation steps in the DIDS binding mechanism were determined to be (34 +/- 9) kJ mole-1 and (152 +/- 21) kJ mole-1, respectively. The results provide, for the first time, evidence for the reversibility of DIDS binding to Band 3 protein at 0 degree C. The existence of a stimulatory site is suggested, nearby the transport site on the Band 3 protein. The binding of an anion to this site can facilitate (through electrostatic repulsion interaction between two anions) the transmembrane movement of another anion from the transport site.     

Endothelin binding sites in porcine aortic and rat lung membranes

High-affinity binding sites for endothelin were identified on porcine aortic and rat lung membranes. Interaction of 125I-labelled endothelin with its binding site was specific, saturable, time- and temperature-dependent but dissociation of receptor-bound ligand was minimal. Maximal binding was observed at pH 7.0 in porcine aorta and at pH 3.1 in the rat lung. Treatment of membranes with trypsin destroyed the binding site in both tissues. Porcine endothelin showed a higher affinity for receptors in both tissues compared to rat endothelin. Vasoactive peptides and Ca2+ channel antagonists did not interact with this site suggesting high specificity of binding. Analysis of saturation binding showed that the number of binding sites was 1250 +/- 104 and 1650 +/- 170 fmol/mg protein and the affinity of binding sites was 0.47 +/- 0.15 and 0.16 +/- 0.07 nM in the aorta and the lungs respectively (n = 5). Presence of protease inhibitors did not alter binding suggesting that the label was stable under the incubation conditions. This was further confirmed by HPLC. Removal of the endothelium from the aorta did not change the binding characteristics of this tissue. Ca2+ and Mg2+ ions caused an increase in binding by increasing the affinity. Binding was completely abolished in the presence of Triton and dithiothreitol. The binding sites identified in this study may be responsible for the actions of endothelin in the aorta and the lung.     

Spectral and kinetic studies of phosphate and magnesium ion binding to yeast inorganic pyrophosphatase

Inorganic pyrophosphatase must bind two phosphate molecules in order to catalyze pyrophosphate synthesis. In this report it is shown that Pi causes marked effect on the absorption spectrum of baker's yeast inorganic pyrophosphatase and this effect can be used to analyze Pi binding to this enzyme. A series of absorbance versus Pi concentration curves in the presence of 0.5-20 mM free Mg2+ were obtained at pH 7.2 and computer-fitted to 19 models. The dissociation constant of magnesium phosphate (8.5 +/- 0.4 mM) used in this analysis was measured with a Mg2+-sensitive electrode. The best model implies successive binding of two magnesium phosphate molecules or random-order binding of magnesium phosphate and free phosphate molecules. The first route predominates at physiological concentrations of Mg2+. The Pi-inhibition pattern of pyrophosphate hydrolysis confirmed that Pi adds to the active site and provided further evidence for the existence of an activating Pi-binding site. The possibility is raised that the pathways of pyrophosphate synthesis and hydrolysis by inorganic pyrophosphatase may differ in the sense that the binding of the fourth metal ion/subunit may facilitate the synthesis and inhibit the hydrolysis.     

Thyrotropin receptors in normal human thyroid. Nonclassical binding kinetics not explained by the negative cooperativity model

Saturation analysis of equilibrium binding of iodinated thyrotropin (125I-TSH) to normal human thyroid preparations yielded linear Scatchard plots under non-physiological conditions of pH 6.0 or 20 mM Tris/acetate buffer, pH 7.4. The apparent equilibrium dissociation constant of this binding was approximately 10(-8) M. By contrast, nonlinear plots were obtained under standard conditions of pH 7.4 and 40 mM Tris/acetate buffer. Resolution of the components of these curves by computer analysis revealed the presence of at least two classes of binding sites, one of which is of a low capacity and high affinity (approximately 10(-10) M) consistent with receptor binding. The other component is of a high capacity and lower affinity. Binding to non-target tissues of muscle, parathyroid, mammary carcinoma, and placenta was only demonstrable at pH 6.0 or in 20 mM Tris/acetate buffer, pH 7.4, yielding linear Scatchard plots with similar binding affinity (approximately 10(-8)M) to normal thyroid but much reduced capacity. Preincubation of thyroid tissue at 50 degrees C resulted in an apparent selective loss of the high affinity component of binding measured under standard conditions. Kinetic experiments on the dissociation of bound 125I-TSH were undertaken to determine whether the non-linearity of Scatchard plots was due to two or more classes of binding sites or negative cooperativity. It was found that the experimental determinant that is presently ascribed to a negative cooperativity phenomenon regulating receptor affinity (i.e. an enhanced dilution-induced dissociation rate in the presence of excess native hormone), although apparently hormone-specific, was demonstrated under nonphysiological binding conditions and in non-target tissue. Significantly, the phenomenon was found under conditions of pH 6.0 or 20 mM Tris where a linear Scatchard plot was obtained. The evidence thus suggests that 125I-TSH binds to heterogeneous binding sites (of which the high affinity is probably the receptor for TSH) and that the enhanced dilution-induced dissociation of bound hormone by native hormone for this system, is only a characteristic of the low affinity binding site (maybe gangliosides).     

Interactions of inorganic phosphate and sulfate anions with collagen

We use direct infrared measurements to determine the number of binding sites, their dissociation constants, and preferential interaction parameters for inorganic phosphate and sulfate anions in collagen fibrils from rat tail tendons. In contrast to previous reports of up to 150 bound phosphates per collagen molecule, we find only 1-2 binding sites for sulfate and divalent phosphate under physiological conditions and approximately 10 binding sites at low ionic strength. The corresponding dissociation constants depend on NaCl concentration and pH and vary from approximately 50 microM to approximately 1-5 mM in the physiological range of pH. In fibrils, bound anions appear to form salt bridges between positively charged amino acid residues within regions of high excess positive charge. In solution, we found no evidence of appreciable sulfate or phosphate binding to isolated collagen molecules. Although sulfate and divalent phosphate bind to fibrillar collagen at physiological concentrations, our X-ray diffraction and in vitro fibrillogenesis experiments suggest that this binding plays little role in the formation, stability and structure of fibrils. In particular, we demonstrate that the previously reported increase in the critical fibrillogenesis concentration of collagen is caused by preferential exclusion of "free" (not bound to specific sites) sulfate and divalent phosphate from interstitial water in fibrils rather than by anion binding. Contrary to divalent phosphate, monovalent phosphate does not bind to collagen. It is preferentially excluded from interstitial water in fibrils, but it has no apparent effect on critical fibrillogenesis concentration at physiological NaCl and pH.     

Nuclear magnetic resonance studies of inorganic phosphate binding to yeast inorganic pyrophosphatase.

Yeast inorganic pyrophosphatase is a dimer of identical subunits. Previous work (Rapoport, T.A., et al. (1973) Eur. J. Biochem. 33, 341) indicated the presence of two different Mn2+ binding sites per subunit. In the present work, the binding of inorganic phosphate to the Mn2+-inorganic pyrophosphatase complex has been studied by 1H and 31P nuclear magnetic resonance. Two distinct phosphate sites have been found, having dissociation constants of 0.24 mM and 18 mM. The Mn2+-31P distance from tightly bound Mn2+ to phosphate bound in the low affinity site (6.2 A) is consistent with outer sphere binding. Binding to both phosphate sites can be simultaneously inhibited by the pyrophosphate analogue, hydroxymethanebisphosphonate, providing evidence for the physical proximity of these two sites. The weaker Mn2+ site is apparently far from both phosphate sites. From the magnitudes of the dissociation constants found for both phosphate and analogue binding and the recent work of P.D. Boyer and his co-workers (private communication) on enzyme-catalyzed phosphate-water exchange, it appears unlikely that the hydrolysis of enzyme-bound pyrophosphate is the rate-determining step in the overall enzymatic catalysis of pyrophosphate hydrolysis, at least when Mn2+ is the required divalent metal ion cofactor.     

Effects of external pH on substrate binding and on the inward chloride translocation rate constant of band 3

To test the hypothesis that amino acid residues in band 3 with titratable positive charges play a role in the binding of anions to the outside-facing transport site, we measured the effects of changing external pH (pH(O)) on the dissociation constant for binding of external iodide to the transport site, K(O)(I). K(O)(I) increased with increasing pH(O), and a significant increase was seen even at pH(O) values as low as 9.9. The dependence of K(O)(I) on pH(O) can be explained by a model with one titratable site with pK 9.5 +/- 0.2 (probably lysine), which increases anion affinity for the external transport site when it is in the positively charged form. A more complex model, analogous to one recently proposed by Bjerrum (1992), with two titratable sites, one with pK 9.3 +/- 0.3 (probably lysine) and another with pK > 11 (probably arginine), gives a slightly better fit to the data. Thus, titratable positively charged residues seem to be functionally important for the binding of substrate anions to the outward-facing anion transport site. In addition, analysis of Dixon plot slopes for L inhibition of Cl- exchange at different pH 0 values, coupled with the assumption that pH(O) has parallel effects on external I- and Cl- binding, indicates that k', the rate-constant for inward translocation of the complex of Cl- with the extracellular transport site, decreases with increasing pH(O). The data are compatible with a model in which titration of the pK 9.3 residue decreases k to 14 +/- 10% of its value at neutral pH(O). This result, however, together with Bjerrum's (1992) observation that the maximum flux J(M)) increases 1.6- fold when this residue is deprotonated, makes quantitative predictions that raise significant questions about the adequacy of the two titratable site ping-pong model or the assumptions used in analyzing the data.     

Anion transport in red blood cells. II. Kinetics of reversible inhibition by nitroaromatic sulfonic acids.

The anion exchange system of human red blood cells is highly inhibited and specifically labeled by isothiocyano derivatives of benzene sulfonate (BS) or stilbene disulfonate (DS). To learn about the site of action of these irreversibly binding probes we studied the mechanism of inhibition of anion exchange by the reversibly binding analogs p-nitrobenzene sulfonic acid (pNBS) and 4,4'-dinitrostilbene-disulfonic acid (DNDS). In the absence of inhibitor, the self-exchange flux of sulfate (pH 7.4, 25 degrees C) at high substrate concentration displayed self-inhibitory properties, indicating the existence of two anion binding sites: one a high-affinity transport site and the other a low-affinity modifier site whose occupancy by anions results in a noncompetitive inhibition of transport. The maximal sulfate exchange flux per unit area was JA = (0.69 +/- 0.11) X 10(-10) moles . min-1 . cm-2 and the Michaelis-Menten constants were for the transport site KS = 41 +/- 14 mM and for the modifier site Ks' = 653 +/- 242 mM. The addition to cells of either pNBS at millimolar concentrations or DNDS at micromolar concentrations led to reversible inhibition of sulfate exchange (pH 7.4, 25 degrees C). The relationship between inhibitor concentration and fractional inhibition was linear over the full range of pNBS or DNDS concentrations (Hill coefficient n approximately equal to 1), indicating a single site of inhibition for the two probes. The kinetics of sulfate exchange in the presence of either inhibitor was compatible with that of competitive inhibition. Using various analytical techniques it was possible to determine that the sulfate transport site was the target for the action of the inhibitors. The inhibitory constants (Ki) for the transport sites were 0.45 +/- 0.10 microM for DNDS and 0.21 +/- 0.07 mM for pNBS. From the similarities between reversibly and irreversibly binding BS and DS inhibitors in structures, chemical properties, modus operandi, stoichiometry of interaction with inhibitory sites, and relative inhibitory potencies, we concluded that the anion transport sites are also the sites of inhibition and of labeling of covalent binding analogs of BS and DS.     

Expression of functional Na,K-ATPase isozymes in normal human cardiac biopsies.

In human heart failure, disturbances in Ca2+ homeostasis are well known but the fate of the Na,K-ATPase isoforms (alpha1beta1, alpha2beta1 and alpha3beta1), the receptors for cardiac glycosides, still remains under study. Microsomes have been purified from non-failing human hearts. As judged by the sensitivities of Na,K-ATPase activity to ouabain (IC50 values: 7.0 +/- 2.5 and 81 +/- 11 nM), 3H-ouabain-binding measurements at equilibrium with and without 10 mM K+ and by a biphasic ouabain dissociation process, at least two finctionally active Na,K-ATPase isozymes coexist in normal human hearts. These are demonstrated as a very high- and a high affinity ouabain-binding site. The KD values are 3.6 +/- 1.6 nM and 17 +/- 6 nM, respectively. The two dissociation rate constants are 42 x 10(4) min(-1) and 360 x 10(-4) min(-1). Addition of 10 mM K+ ions shifted the respective KD values for ouabain from 3.6 +/- 1.6 to 20 +/- 5 nM and from 17 +/- 6 nM to 125 +/- 25 nM, respectively. The isozymes involved are identified by comparing these three pharmacological parameters to those of each alpha/beta-isozyme separately expressed in Xenopus oocytes (9). In human heart, the very high affinity site for ouabain is the alpha1beta1 dimer and the high affinity site is alpha2beta1.     

Characterisation of phosphate binding to mitochondrial and bacterial membrane-bound ATP synthase by studies of inhibition with 4-chloro-7-nitrobenzofurazan

The effect of phosphate on the inhibition by 4-chloro-7-nitrobenzofurazan of the ATPase activity of the proton-translocating ATP synthase in heart submitochondrial particles was investigated. Binding of phosphate protected strongly against the inhibition. A dissociation constant of 0.2 mM was determined for the enzyme X Pi complex and shown to be independent of pH in the range 7.0-8.0. The protective effect of phosphate was mimicked by arsenate but not by sulphate or malonate. Similar results were obtained for the enzyme from Paracoccus denitrificans. 2,4-Dinitrophenol enhanced phosphate binding to the mitochondrial enzyme since the protective effect of phosphate was increased. The data are compatible with protection arising from binding of phosphate to a catalytic site.     

Adenine nucleotides affect the binding of 3-phosphoglycerate to pig muscle 3-phosphoglycerate kinase

Pig muscle 3-phosphoglycerate kinase was complexed with 1-anilino-8-naphthalenesulfonate (ANS) in order to monitor the binding of substrates to the enzyme. The enzyme-dye interaction did not influence the enzymic activity under the experimental conditions used. By measuring the substrate-dependent change in the fluorescence emission of ANS molecules tightly bound to the enzyme (Kd less than or equal to 0.05 mM), fluorimetric titrations were carried out in 0.1 M Tris/HCl buffer pH 7.5, containing 5 mM mercaptoethanol, at 20 degrees C. The dissociation constants obtained for the separate bindings of 3-phosphoglycerate, MgATP, 1,3-bisphosphoglycerate and MgADP were 0.03 +/- 0.01 mM, 0.15 +/- 0.10 mM, 0.00005 +/- 0.00001 mM and 0.15 +/- 0.10 mM respectively. binding of 3-phosphoglycerate is weakened when MgATP is also bound to the enzyme: the dissociation constant of 3-phosphoglycerate in this ternary complex (0.25 +/- 0.08 mM) is comparable to its Km value (0.38 +/- 0.10 mM). The same weakening can be observed in the non-productive ternary complexes where MgATP is replaced by MgADP (Kd = 0.20 +/- 0.10 mM) or AMP (Kd = 0.12 +/- 0.05 mM), whereas adenosine has no such effect. This indicates the importance of the negatively charged phosphate(s) of nucleotides in influencing the binding of 3-phosphoglycerate. In contrast to 3-phosphoglycerate, the binding of the substrate analogue, glycerol 3-phosphate is practically not affected by the presence of MgATP: the dissociation constant to the free enzyme (0.40 +/- 0.10 mM) is comparable to its inhibitory constant (0.70 +/- 0.20 mM). This finding and the similarity of the dissociation constant of glycerol 3-phosphate binding (0.40 +/- 0.10 mM) and the Km value of 3-phosphoglycerate (0.38 +/- 0.10 mM) suggest that, during the enzymic reaction, binding of 3-phosphoglycerate occurs probably without involvement of the carboxyl group.     

Crystallographic and energetic analysis of binding of selected anions to the yellow variants of green fluorescent protein

The fluorescence emission of yellow fluorescent proteins (YFPs) has been shown to respond rapidly and reversibly to changes in the concentration of some small anions such as halides; this allows for the use of YFPs as genetically encodable Cl(-) sensors that may be targeted to specific organelles in living cells. Fluorescence is suppressed due to protonation of the chromophore upon anion binding, with a stronger level of interaction at low pH values. At pH 6.0, the apparent dissociation constant (K(app)) for Cl(-) is 32 mM for YFP and 22 mM for YFP-H148Q, whereas at pH 7.5, K(app) is 777 mM and 154 mM, respectively. In the cytosol, YFP-H148Q appears most promising as a halide sensor due to its high degree of sensitivity towards I(-) (K(app)=23 mM at pH 7.5). To aid in the design of variants with improved levels of specificity and affinity for Cl(-), we solved apo and I(-)-bound crystal structures of YFP-H148Q to 2.1 A resolution. The halide-binding site is found near van der Waals contact with the chromophore imidazolinone oxygen atom, in a small buried cavity adjacent to Arg96, which provides electrostatic stabilization. The halide ion is hydrogen bonded to the phenol group of T203Y, consistent with a mutational analysis that indicates that T203Y is indispensible for tight binding. A series of conformational changes occurs in the amphiphilic site upon anion binding, which appear to be propagated to the beta-bulge region around residue 148 on the protein surface. Anion binding raises the chromophore pK(a) values, since delocalization of the phenolate negative charge over the chromophore skeleton is suppressed. Extraction of microscopic binding constants for the linked equilibrium between anion and proton binding indicates that anion selectivity by YFP is related to hydration forces. Specific suggestions to improve Cl(-) binding to YFP-H148Q based on size and hydration energy are proposed.     

t-[3H]Butylbicycloorthobenzoate: New Radioligand Probe for the γ-Aminobutyric Acid–Regulated Chloride Ionophore

t-[3H]Butylbicycloorthobenzoate [( 3H]TBOB; 22 Ci/mmol) was prepared by reductive dechlorination of its 4-chlorophenyl analog with tritium gas. This new radioligand binds reversibly to fresh washed rat brain P2 membranes in 500 mM NaCl plus 50 mM sodium-potassium phosphate buffer (pH 7.4) at 25 degrees C, with 80-90% specific relative to total binding, a KD of 61 +/- 15 nM, and a Bmax of 1.6 +/- 0.5 pmol/mg of protein. [3H]TBOB association with its binding site(s) is monophasic, but its dissociation is biphasic. The binding characteristics of [3H]TBOB are essentially identical to those of t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) with respect to pH dependence, stimulation by anions, regional distribution in the brain, and pharmacological profile. Saturation analyses and dissociation studies further indicate that TBOB and TBPS have a common binding site. However, binding of the two radioligands differs in respect to temperature effects. In contrast to [35S]TBPS, which exhibits negligible binding at 0 degrees C, [3H]TBOB binds to rat brain membranes at 0, 25, and 37 degrees C with similar KD values. [3H]TBOB with its long radioactive half-life and temperature-independent KD is a valuable supplement to [35S]TBPS in further biochemical and pharmacological characterization of the gamma-aminobutyric acid receptor-ionophore complex.     

Phosphotyrosyl-specific protein phosphatase activity of a bovine skeletal acid phosphatase isoenzyme. Comparison with the phosphotyrosyl protein phosphatase activity of skeletal alkaline phosphatase

A partially purified bovine cortical bone acid phosphatase, which shared similar characteristics with a class of acid phosphatase known as tartrate-resistant acid phosphatase, was found to dephosphorylate phosphotyrosine and phosphotyrosyl proteins, with little activity toward other phosphoamino acids or phosphoseryl histones. The pH optimum was about 5.5 with p-nitrophenyl phosphate as substrate but was about 6.0 with phosphotyrosine and about 7.0 with phosphotyrosyl histones. The apparent Km values for phosphotyrosyl histones (at pH 7.0) and phosphotyrosine (at pH 5.5) were about 300 nM phosphate group and 0.6 mM, respectively, The p-nitrophenyl phosphatase, phosphotyrosine phosphatase, and phosphotyrosyl protein phosphatase activities appear to be a single protein since these activities could not be separated by Sephacryl S-200, CM-Sepharose, or cellulose phosphate chromatographies, he ratio of these activities remained relatively constant throughout the purification procedure, each of these activities exhibited similar thermal stabilities and similar sensitivities to various effectors, and phosphotyrosine and p-nitrophenyl phosphate appeared to be alternative substrates for the acid phosphatase. Skeletal alkaline phosphatase was also capable of dephosphorylating phosphotyrosyl histones at pH 7.0, but the activity of that enzyme was about 20 times greater at pH 9.0 than at pH 7.0. Furthermore, the affinity of skeletal alkaline phosphatase for phosphotyrosyl proteins was low (estimated to be 0.2-0.4 mM), and its protein phosphatase activity was not specific for phosphotyrosyl proteins, since it also dephosphorylated phosphoseryl histones. In summary, these data suggested that skeletal acid phosphatase, rather than skeletal alkaline phosphatase, may act as phosphotyrosyl protein phosphatase under physiologically relevant conditions.     

Characterization of indo-1 and quin-2 as spectroscopic probes for Zn2(+)-protein interactions

1-[2-Amino-5-(6-carboxyindol-2-yl)phenoxyl]-2-(2'- amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid (indo-1) and 2-[2-(bis(carboxymethyl)amino-5-methylphenoxy) methyl]-6- methyl-8-[bis-(carboxymethyl)amino]quinoline (quin-2) are sensitive, spectral indicators for Zn2+. Additions of subsaturating Zn2+ to 10-80 microM indo-1 or quin-2 at pH 7.0 produce uv difference spectra with isosbestic wavelengths at 342 and 282 nm or at 342, 317, and 252 nm, respectively. Formation of 1:1 Zn2+:indicator complexes at pH 7.0 and 20 degrees C in the absence (presence) of 100 mM KCl gives delta epsilon max = -2.4 +/- 0.2 X 10(4) M-1 cm-1 at 367 nm (-2.1 +/- 0.2 X 10(4) M-1 cm-1 at 365 nm) for indo-1 and delta epsilon max = -2.7 +/- 0.1 X 10(4) M-1 cm-1 at 266 nm (-2.6 +/- 0.1 X 10(4) M-1 cm-1 at 265 nm) for quin-2. Competition experiments at pH 7.0 and 20 degrees C with indo-1 and quin-2 and also 4-(2-pyridylazo)resorcinol (PAR) as the second chelator in the absence (presence) of 100 mM KCl yield apparent affinity constants: K'A = 2.5 +/- 1.0 X 10(10) M-1 (6.2 +/- 0.5 X 10(9) M-1) for indo-1 binding Zn2+ and K'A = 9.4 +/- 3.3 X 10(11) M-1 (2.7 +/- 0.1 X 10(11) M-1) for quin-2 binding Zn2+. The above constants provide the basis for rapid steady-state spectrophotometric determinations of the affinity of a protein for Zn2+ with K'A approximately 10(10) - 10(13) M-1.(ABSTRACT TRUNCATED AT 250 WORDS)