High-affinity epidermal growth factor binding is specifically reduced by a monoclonal antibody, and appears necessary for early responses

We have tested the effects of an mAb directed against the protein core of the extracellular domain of the human EGF receptor (mAb108), on the binding of EGF, and on the early responses of cells to EGF presentation. We used NIH 3T3 cells devoid of murine EGF receptor, transfected with a cDNA encoding the full-length human EGF receptor gene, and fully responsive to EGF. The binding to saturation of mAb108 to the surface of these cells at 4 degrees C and at other temperatures specifically reduced high-affinity binding of EGF, but did not change the dissociation constant or the estimated number of binding sites for low-affinity binding of EGF. The kinetics of EGF binding to the transfected cells were measured to determine the effects of the mAb on the initial rate of EGF binding at 37 degrees C. Interestingly, high-affinity EGF receptor bound EGF with an intrinsic on-rate constant 40-fold higher (9.8 x 10(6) M-1.s-1) than did low-affinity receptor (2.5 x 10(5) M-1.s-1), whereas the off-rate constants, measured at 4 degrees C were similar. Cells treated with the mAb or with phorbol myristate acetate displayed single on-rate constants similar to that for the low-affinity receptors. At low doses of EGF ranging from 0.4 to 1.2 nM, pretreatment of cells with mAb108 inhibited by 50-100% all of the early responses tested, including stimulation of tyrosine-specific phosphorylation of the EGF receptor, turnover of phosphatidyl inositol, elevation of cytoplasmic pH, and release of Ca2+ from intracellular stores. At saturating doses of EGF (20 nM) the inhibition of these early responses by prebinding of mAb108 was overcome. On the basis of these results, we propose that the high-affinity EGF receptors are necessary for EGF receptor signal transduction.     

Novel type of very high affinity calcium-binding sites in beta-hydroxyasparagine-containing epidermal growth factor-like domains in vitamin K-dependent protein S

Vitamin K-dependent protein S is shown to contain four very high affinity Ca2(+)-binding sites. The number of sites and their affinities were determined from Ca2+ titration in the presence of the chromophoric chelator Quin 2. In 0.15 M NaCl, pH 7.5, the four macroscopic binding constants are K1 greater than or equal to 1 x 10(8) M-1, K2 = 3 +/- 2 x 10(7) M-1, K3 = 4 +/- 2 x 10(6) M-1, and K4 = 9 +/- 4 x 10(5) M-1. At low ionic strength, the corresponding values are K1 greater than or equal to 2 x 10(9) M-1, K2 = 9 +/- 4 x 10(8) M-1, K3 = 2 +/- 1 x 10(8) M-1, and K4 = 9 +/- 4 x 10(7) M-1. To localize the Ca2(+)-binding sites, protein S was subjected to proteolysis using lysyl endopeptidase. This yielded a 20-21-kDa fragment which comprised the third and fourth epidermal growth factor (EGF)-like domains and remained high affinity Ca2(+)-binding site(s). The susceptibility of the EGF-like domains to proteolysis increased when Ca2+ was removed from protein S indicating that the Ca2+ binding is important for the stability and/or conformation of the EGF domains. Three of the four EGF-like domains in protein S contain beta-hydroxyasparagine. In each of these domains there is a cluster of three or four negatively charged amino acid residues which are likely to contribute to the extraordinary high Ca2+ affinity. From sequence homology it is suggested that this novel type of high affinity Ca2(+)-binding site is present in several other proteins, e.g. in the EGF-like domains in the low sensity lipoproteins receptor, thrombomodulin, the Notch protein of Drosophila melanogaster, and transforming growth factor beta 1-binding protein.     

Competitive ion binding to low density lipoproteins: an electron spin resonance study

The electron spin resonance (ESR) technique was used to evaluate binding constants for Ca(II) and Mg(II) in interaction with low density lipoprotein (LDL). The Ca(II) or Mg(II) ions competed with the paramagnetic Mn(II) ions for the same binding sites of two different classes on the LDL surface. For each ion competing with Mn(II), the solutions of eight non-linear competition equations were fit to the experimental titration curves, with two adjustable parameters, the two binding constants. The derived "intrinsic" values (the values corrected for the electrolyte-induced change of the surface potential) for "strong" binding sites for Ca(II) (170 +/- 85 M-1) and Mg(II) (60 +/- 30 M-1) differ significantly from the respective value for Mn(II) (760 M-1). The values for the "weak" binding sites (18 M-1, 15 M-1 and 10 M-1 for Mn(II), Ca(II) and Mg(II), respectively are in the range of the binding constants for these ions in interaction with model membranes.     

Modes of mononucleotide binding to ribonuclease T1.

The binding of the mononucleotide inhibitors 2'-GMP, 3'-GMP, and 5'-GMP to genetically engineered ribonuclease T1 has been investigated by conventional inhibition kinetics, fluorimetric titrations, molecular modeling, and fast relaxation techniques. The fluorimetric titrations in conjunction with molecular modeling revealed that apart from the already known primary binding site, three to four additional sites are present on the enzyme's surface. The association constants obtained from the fluorimetric titrations and the temperature jump experiments range between 3.1 x 10(6) M-1 and 4.3 x 10(6) M-1, indicating that the binding of the mononucleotides to the specific binding site of ribonuclease T1 is at least one order of magnitude tighter than has been anticipated so far. The kinetics of binding are nearly diffusion controlled with a kon determined for 2'-GMP and 3'-GMP, as (5.0 +/- 0.5 x 10(9) and 6.1 +/- 0.5 x 10(9) M-1, s-1 and koff as 1.2 +/- 0.2 x 10(3) and 2.0 +/- 0.3 x 10(3) s-1, respectively. Molecular modeling studies indicate that all three nucleotides are able to bind via their phosphate group to a positively charged array of surface amino acids including His27, His40, Lys41, and most probably Lys25 without obvious stereochemical hindrance. We propose that RNA wraps around RNase T1 in a similar fashion via phosphate binding when enzymatic hydrolysis occurs.     

Biostructural chemistry of magnesium ion: characterization of the weak binding sites on tRNA(Phe)(yeast). Implications for conformational change and activity

The thermodynamics and kinetics of magnesium binding to tRNA(Phe)(yeast) have been studied directly by 25Mg NMR. In 0.17 M Na+(aq), tRNA(Phe) exists in its native conformation and the number of strong binding sites (Ka greater than or equal to 10(4)) was estimated to be 3-4 by titration experiments, in agreement with X-ray structural data for crystalline tRNA(Phe) (Jack et al., 1977). The set of weakly bound ions were in slow exchange and 25Mg NMR resonances were in the near-extreme-narrowing limit. The line shapes of the exchange-broadened magnesium resonance were indistinguishable from Lorentzian form. The number of weak magnesium binding sites was determined to be 50 +/- 8 in the native conformation and a total line-shape analysis of the exchange-broadened 25 Mg2+ NMR resonance gave an association constant Ka of (2.2 +/- 0.2) X 10(2) M-1, a quadrupolar coupling constant (chi B) of 0.84 MHz, an activation free energy (delta G*) of 12.8 +/- 0.2 kcal mol-1, and an off-rate (koff) of (2.5 +/- 0.4) X 10(3) s-1. In the absence of background Na+(aq), up to 12 +/- 2 magnesium ions bind cooperatively, and 73 +/- 10 additional weak binding sites were determined. The binding parameters in the nonnative conformation were Ka = (2.5 +/- 0.2) X 10(2) M-1, chi B = 0.64 MHz, delta G* = 13.1 +/- 0.2 kcal mol-1, and koff = (1.6 +/- 0.4) X 10(3) s-1. In comparison to Mg2+ binding to proteins (chi B typically ca. 1.1-1.6 MHz) the lower chi B values suggest a higher degree of symmetry for the ligand environment of Mg2+ bound to tRNA. A small number of specific weakly bound Mg2+ appear to be important for the change from a nonnative to a native conformation. Implications for interactions with the ribosome are discussed.     

The reduction of ferrate(VI) to ferrate(V) by ascorbate

The reduction of ferrate(VI) by ascorbate has been studied under anaerobic conditions in the pH range between 6.8 and 11.5 at 24 degrees C. A mechanism is proposed that is consistent with the observed rate constants k11 (HFeO4- + AH-) = (5.6 +/- 0.6) x 10(6) M-1 s-1, k12(FeO4(2-) + AH-) = (1.3 +/- 0.1) x 10(6) M-1 s-1 and the pK(HFeO4- in equilibrium with H(+) + FeO4(2-) = 7.9. Stoichiometric studies show that at high ratios of [AH-]/[FeO4(2-)], one ferrate(VI) oxidizes three molecules of ascorbate to the corresponding ascorbyl (A-) radicals.     

Kinetics of colchicine binding to purified beta-tubulin isotypes from bovine brain.

Tubulin, the constituent protein of microtubules, is an alpha beta heterodimer; both alpha and beta exist in several isotypic forms whose functional significance is not precisely known. The antimitotic alkaloid colchicine binds to mammalian brain tubulin in a biphasic manner under pseudo-first-order conditions in the presence of a large excess of colchicine (Garland, D. L. (1978) Biochemistry 17, 4266-4272). We have studied the kinetics of colchicine binding to purified beta-tubulin isotypes and find that each of the purified beta-tubulin isotypes binds colchicine in a monophasic manner. The apparent on-rate constants for the binding of colchicine to alpha beta II-, alpha beta III-, and alpha beta IV-tubulin dimers are respectively 132 +/- 5, 30 +/- 2, and 236 +/- 7 M-1 s-1. When the isotypes are mixed, the kinetics become biphasic. Scatchard analysis revealed that the isotypes differ significantly in their affinity constants (Ka) for binding colchicine. The affinity constants are 0.24 x 10(6), 0.12 x 10(6), and 3.31 x 10(6) M-1, respectively, for alpha beta II-, alpha beta III-, and alpha beta IV-tubulin dimers. Our results are in agreement with the hypothesis that the beta-subunit of tubulin plays a major role in the interaction of colchicine with tubulin. Our binding data raise the possibility that the tubulin isotypes might play important regulatory roles by interacting differently with other non-tubulin proteins in vivo, which in turn, may regulate microtubule-based functions in living cells.     

Manganese peroxidase from the lignin-degrading basidiomycete Phanerochaete chrysosporium. Transient state kinetics and reaction mechanism

Stopped-flow techniques were used to investigate the kinetics of the formation of manganese peroxidase compound I (MnPI) and of the reactions of MnPI and manganese peroxidase compound II (MnPII) with p-cresol and MnII. All of the rate data were obtained from single turnover experiments under pseudo-first order conditions. In the presence of H2O2 the formation of MnPI is independent of pH over the range 3.12-8.29 with a second-order rate constant of (2.0 +/- 0.1) x 10(6) M-1 s-1. The activation energy for MnPI formation is 20 kJ mol-1. MnPI formation also occurs with organic peroxides such as peracetic acid, m-chloroperoxybenzoic acid, and p-nitroperoxybenzoic acid with second-order rate constants of 9.7 x 10(5), 9.5 x 10(4), and 5.9 x 10(4) M-1 s-1, respectively. The reactions of MnPI and MnPII with p-cresol strictly obeyed second-order kinetics. The second-order rate constant for the reaction of MnPII with p-cresol is extremely low, (9.5 +/- 0.5) M-1 s-1. Kinetic analysis of the reaction of MnII with MnPI and MnPII showed a binding interaction with the oxidized enzymes which led to saturation kinetics. The first-order dissociation rate constants for the reaction of MnII with MnPI and MnPII are (0.7 +/- 0.1) and (0.14 +/- 0.01) s-1, respectively, when the reaction is conducted in lactate buffer. Rate constants are considerably lower when the reactions are conducted in succinate buffer. Single turnover experiments confirmed that MnII serves as an obligatory substrate for MnPII and that both oxidized forms of the enzyme form productive complexes with MnII. Finally, these results suggest the alpha-hydroxy acids such as lactate facilitate the dissociation of MnIII from the enzyme.     

Selective distinction at equilibrium between the two alpha-neurotoxin binding sites of Torpedo acetylcholine receptor by microtitration

The binding of the monoiodinated alpha-neurotoxin I from Naja mossambica mossambica to the membrane-bound acetylcholine receptor from Torpedo marmorata was investigated using a new picomolar-sensitive microtitration assay. From equilibrium binding studies a non-linear Scatchard plot demonstrated two populations of binding sites characterized by the two dissociation constants Kd1 = 7 +/- 4 pM and Kd2 = 51 +/- 16 pM and having equal binding capacities. These two populations differed in their rate of dissociation (k-1.1 = 25 x 10(-6) s-1 and k-1.2 = 623 x 10(-6) s-1 respectively), but not in their rate of formation of the toxin-receptor complex (k + 1 = 11.7 x 10(6) M-1 s-1). From these rate constants the same two values of dissociation constant were deduced (Kd1 = 2 pM and Kd2 = 53 pM). All the specific binding was prevented by the cholinergic antagonists alpha-bungarotoxin and d-tubocurarine. In addition, a biphasic competition phenomenon allowed us to differentiate between two d-tubocurarine sites (Kda = 103 nM and Kdb = 13.7 microM respectively). Evidence is provided indicating that these two sites are shared by d-tubocurarine and alpha-neurotoxin I, with inverse affinities. Fairly conclusive agreement between our equilibrium, kinetic and competition data demonstrates that the two high-affinity binding sites for this short alpha-neurotoxin are selectively distinguishable.     

Activation/inactivation and uni-site catalysis by the reconstituted ATP-synthase from chloroplasts

The proton-translocating ATP-synthase of chloroplasts, CF0F1, was isolated and reconstituted into asolectin liposomes. CF0F1 can exist in at least four different states, oxidized or reduced, either inactive or active. These states are characterized by different kinetics of ADP binding: There is no binding of ADP to the inactive, oxidized state, the rate constant for ADP binding to the inactive, reduced states is 7.10(2) M-1.s-1. ADP binding to the active, reduced state occurs under deenergized conditions with 10(5) M-1.s-1 and transforms the enzyme into the inactive, reduced state. Parallel to the ADP-dependent inactivation, the enzyme can also inactivate without ADP binding with a first-order rate constant of 7.10(-3) M-1.s-1. With the active, reduced enzyme ATP-hydrolysis was measured under uni-site conditions as has been carried out with MF1 (Grubmeyer, C., Cross, R.C. and Penefsky, H.S. (1982) J. Biol. Chem. 257, 12092-12100). The rate constant for ATP binding is 10(6) M-1.s-1, the 'equilibrium constant' on the enzyme EADPPi/EATP is 0.4. The rate constants for Pi release and ADP release are 0.2 s-1 and o.1 s-1, respectively. This indicates that the enzyme carries out a complete turnover under uni-site conditions with rates much higher than that reported for MF1.     

Kinetic study on chemical modification of taka-amylase A. I. Location and role of tryptophan residues

1. Five and four tryptophan residues in Taka-amylase A [EC 3.2.1.1] of A. oryzae (TAA) were modified with dimethyl(2-hydroxy-5-nitrobenzyl)-sulfonium bromide (K-IWS) in the absence and the presence of 15% maltose (substrate analog), respectively. Only one tryptophan residue was modified with dimethyl(2-methoxy-5-nitrobenzyl)-sulfonium bromide (K-IIWS) irrespective of the presence or absence of maltose. Kinetic parameters (molecular activity, k0, Michaelis constant, Km, and inhibitor constant, Ki) of the enzyme modified with K-IWS and K-IIWS were determined. The k0 value decreased with increase in the number of modified residues, but Km and Ki values and the type of inhibition were not altered by the modification. 2. The fluorescence quenching reaction of TAA with N-bromosuccinimide (NBS) proceeded in three phases. The second-order rate constants of the three phases were determined to be (4.3 +/- 0.5) x 10(5) M-1 . s-1, (2.1 +/- 0.3) x 10(3) M-1 . s-1 and (1.7 +/- 0.2) x 10(2) M-1 . s-1, respectively. In the presence of maltose, the first phase was further separated into two phases with rate constants of (4.6 +/- 0.6) x 10(6) M-1 . s-1 and (6.9 +/- 1.1) x 10(4) M-1 . s-1, respectively. On the basis of the results, it is estimated that five out of nine tryptophan residues are accessible to the solvent and among them, two tryptophan residues are substantially exposed: one is located in the maltose binding site near the catalytic site (its modification affects the catalytic function), and the other exists on the enzyme surface far from the active site.     

Binding of ellipticine base and ellipticinium cation to calf-thymus DNA. A thermodynamic and kinetic study

The acid-basic properties of ellipticine have been re-estimated. The apparent pK of protonation at 3 microM drug concentration is 7.4 +/- 0.1. The ellipticine free base (at pH 9, I = 25 mM) intercalates into calf-thymus DNA with an affinity constant of 3.3 +/- 0.2 X 10(5) M-1, and a number of binding sites per phosphate of 0.23. The ellipticinium cation (pH 5, I = 25 mM) binds also to DNA with a constant of 8.3 +/- 0.2 x 10(5) M-1 and at a number of binding sites (n = 0.19). It is postulated that the binding of the drug to DNA at pH 9 is driven by hydrophobic and/or dipolar effects. Even at pH 5, where ellipticine exists as a cation, it is thought that the hydrophobic interaction is the main contribution to binding. The neutral and cationic forms share common binding within DNA sites but yield to structurally different complexes. The free base has 0.04 additional specific binding sites per phosphate. As determined from temperature-jump experiments, the second-order rate constant of the binding of the free base (pH 9) is 3.4 x 10(7) M-1 s-1 and the residence time of the base within the DNA is 8 ms. The rate constant for the binding of the ellipticinium cation is 9.8 x 10(7) M-1 s-1 when it is assumed that drug attachment occurs via a pathway in which the formation of an intermediate ionic complex is not involved (competitive pathway).     

The stoichiometry and stability of the NADP complexes with manganese(II) ions as studied by electron paramagnetic resonance.

Magnetic resonance techniques have been applied to study the stability of the complexes formed between Mn(II) ions and NADP in aqueous solutions at a pH of 7.5 and 20 degrees C. The electron paramagnetic resonance (epr) data indicate that at low Mn(II) ion concentrations ([Mn(II)] less than 1 mM; [NADP] approximately 5 mM), a 1:1 complex is formed with an apparent stability constant K1 = 370 +/- 50 M-1 at an ionic strength of 0.22 in the presence of 0.20 M Cl-. At high Mn(II) ion concentrations, a Mn(II)2-NADP species, with an apparent stability constant K2 = 54 +/- 17 M-1, is present in significant amounts. When the epr data are corrected for the presence of the MnCl+ ion, the analysis of the new Scatchard plot yields stability constants for the two sites of K1 = 640 +/- 90 M-1 and K2 = 88 +/- 13 M-1, respectively. The presence of two metal ion binding sites on the NADP molecule has not been observed previously, and previous workers have always analyzed their data in terms of the 1:1 Mn(II)-NADP complex. An epr temperature study of K1 yields a value of delta H equal to 1.3 +/- 0.2 kcal/mol (1 cal = 4.187 J).     

Binding of latrotoxin to synaptosomes and synaptic membranes of the rat brain

The binding of [125I] alpha-latrotoxin to synaptosomes from the rat brain is studied. It is shown that the constant rate of toxin association with the synaptosome receptor at 37 degrees C is equal to 8.2 +/- 1.3 x 10(7) M-1.s-1, while that of synaptosomal membrane -7.6 +/- 2.7 x 10(6) M-1 s-1. Depolarization of the synaptosome membrane induced by 55 mM KCl decreases the binding rate of toxin to the receptor, the rate constant being equal to 3.9 +/- 1.5 x 10(7) m-1 s-1. The pattern of the dissociation process of the toxin-receptor complex of synaptosomes and of synaptosomal membrane is different. In the first case dissociation follows two stages with the rate constants 3.6 x 10(-3) s-1 and 1.2/10(-4) s-1, in the second case it follows one stage with the constant equalled 2.0 x 10(-5) s-1. The quantity of the toxin binding sites on synaptosomes may vary under the action of agents modifying the activity of calcium fluxes which are induced by alpha-latrotoxin. It is supposed that a decrease in the ATP level in synaptosomes as well as deenergy of the surface membrane leads to a change in the state of the alpha-latrotoxin receptor.     

Catalytic sites of Escherichia coli F1-ATPase. Characterization of unisite catalysis at varied pH.

Using manual rapid-mixing procedures in which small, equal volumes of Escherichia coli F1-ATPase and [gamma-32P]ATP were combined at final concentrations of 2 and 0.2 microM, respectively (i.e., unisite catalysis conditions), it was shown that greater than or equal to 66% of the 32P became bound to the enzyme, with the ratio of bound ATP/bound Pi equal to 0.4 and the rate of dissociation of bound [32P]Pi equal to 3.5 x 10(-3) s-1, similar to previously published values. Azide is known to inhibit cooperative but not unisite catalysis in F1-ATPase [Noumi, T., Maeda, M., & Futai, M. (1987) FEBS Lett. 213, 381-384]. In the presence of 1 mM sodium azide, 99% of the 32P became bound to the enzyme, with the ratio of bound ATP/bound Pi being 0.57. These experiments demonstrated that when conditions are used which minimize cooperative catalysis, most or all of the F1 molecules bind substoichiometric ATP tightly, hydrolyze it with retention of bound ATP and Pi, and release the products slowly. The data justify the validity of previously published rate constants for unisite catalysis. Unisite catalysis in E. coli F1-ATPase was studied at varied pH from 5.5 to 9.5 using buffers devoid of phosphate. Rate constants for ATP binding/release, ATP hydrolysis/resynthesis, Pi release, and ADP binding/release were measured; the Pi binding rate constant was inferred from the delta G for ATP hydrolysis. ATP binding was pH-independent; ATP release accelerated at higher pH. The highest KaATP (4.4 x 10(9) M-1) was seen at physiological pH 7.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinct binding sites of Ala48-hirudin1-47 and Ala48-hirudin48-65 on alpha-thrombin

The interaction of alpha-thrombin with Ala48-hirudin, Ala48-hirudin1-47, and Ala48-hirudin48-65 was analyzed. Mutations at Pro48 were found to cause only slight changes in the kon (human: 3.1 +/- 0.3 x 10(8) M-1 s-1; bovine: 1.03 +/- 0.3 x 10(8) M-1 s-1) and koff (human: 0.4 +/- 0.2 x 10(-3) s-1; bovine: 2.9 +/- 0.4 x 10(-3) s-1) rate constants for the formation of the thrombin-hirudin complex. The amino-terminal fragment Ala48-hirudin1-47 containing the three disulfide bridges and the carboxyl-terminal fragment Ala48-hirudin48-65 were derived from the Ala48 mutant by proteolysis with endoproteinase Lys-C. These fragments inhibit bovine alpha-thrombin clotting activity with IC50 values of 0.6 and 4.9 microM, respectively (2.4 nM for r-hirudin). By mapping the interaction of Ala48-hirudin-derived fragments with bovine alpha-thrombin by limited proteolysis with trypsin and pancreatic elastase distinct binding sites for each fragment were determined. The carboxyl-terminal fragment was found to bind to the proposed anion-binding exosite in the region B62-74, whereas the amino-terminal fragment binds to a region around the elastase cleavage site at residues 150-151 of the alpha-thrombin B-chain.     

Kinetics of inhibition of calf intestinal adenosine deaminase by (+)- and (-)-erythro-9-(2-hydroxy-3-nonyl)adenine.

The enantiomers of erythro-9-(2-hydroxy-3-nonyl)adenine [(+)- and (-)-EHNA) bound to adenosine deaminase (ADA) at pH 7 with concomitant changes in the optical properties of the enzyme. The association rate constant for (+)-EHNA was 2.9 x 10(6) M-1 s-1 and that for (-)-EHNA was 6.4 x 10(6) M-1 s-1. The dissociation of (-)-EHNA.ADA or (+)-EHNA.ADA in the presence of excess coformycin was monitored by the quenching of enzyme fluorescence as coformycin.ADA was formed. The dissociation rate constants of (+)- and (-)-EHNA.ADA were 0.0054 s-1 and 2.7 s-1, respectively. A similar value for the dissociation rate constant (0.005 s-1) for (+)-EHNA.ADA was calculated from the time course for the appearance of catalytic activity after dilution of (+)-EHNA.ADA into 100 microM adenosine. The Ki values of ADA for (+)- and (-)-EHNA were similar to the dissociation constants calculated from the ratio of the respective dissociation and association rate constants. The biphasic time-dependent inhibition of the catalytic activity of ADA by (+/- )-EHNA [Frieden, C., Kurz, L. C., & Gilbert, H. R. (1980) Biochemistry 19, 5303-5309] was confirmed. However, the catalytic activity of ADA was inhibited monophasically by (+)-EHNA. Thus, the biphasic nature of the time course for inhibition of ADA by (+/- )-EHNA was the result of the presence of both enantiomers of the inhibitor in this assay. These kinetic data were interpreted in terms of single-step mechanisms for binding of (+)- and (-)-EHNA.     

Calorimetric experiments of Mn2+ -binding to alpha-lactalbumin

We measured by batch microcalorimetry the standard enthalpy change delta H degrees of the binding of Mn2+ to apo-bovine alpha-lactalbumin; delta H degrees = -90 +/- k J.mol-1. The binding constants, KMn2+, calculated from the calorimetric and circular dichroism titration curves, are (4.6 +/- 1).10(5) M-1 and (2.1 +/- 0.4).10(5) M-1, respectively. Batch calorimetry confirms the competitive binding Ca2+, Mn2+ and Na+ to the same site. The relatively small enthalpy change for Mn2+ binding compared to Ca2+ binding favours a model of a rigid and almost ideal Ca2+-complexating site, different from the well-known EF-hand structures. Cation binding to the high-affinity site most probably triggers the movement of an alpha-helix which is directly connected to the complexating loop.     

Diffusion-controlled binding of a peptide neurotoxin to its K+ channel receptor.

Single Ca2(+)-activated K+ channels were reconstituted into planar lipid bilayer membranes, and the effect of charybdotoxin, a pore-blocking peptide from scorpion venom, was studied. In particular, the effect of solution viscosity on the kinetics of block was assessed in order to test the idea that toxin binding is diffusion-controlled. This idea is supported by the strictly inverse relation between solution viscosity and the rate constants of both association and dissociation of peptide with the K+ channel mouth. However, at an ionic strength high enough to suppress local electrostatic potentials, the diffusion-controlled on-rate constant is surprisingly low, 10(5) M-1 s-1. These slow, viscosity-dependent kinetics may be understood if charybdotoxin can attain the bound state only from a rare set of encounters with the K+ channel.     

Identification of the dehydroascorbic acid reductase and thioltransferase (Glutaredoxin) activities of bovine erythrocyte glutathione peroxidase

Bovine erythrocyte glutathione (GSH) peroxidase (GPX, EC 1.11.1.9) was examined for GSH-dependent dehydroascorbate (DHA) reductase (EC 1.8.5.1) and thioltransferase (EC 1.8.4.1) activities. Using the direct assay method for GSH-dependent DHA reductase activity, GPX had a kcat (app) of 140 +/- 9 min-1 and specificity constants (kcat/Km(app)) of 5.74 +/- 0.78 x 10(2) M-1s-1 for DHA and 1.18 +/- 0.17 x 10(3) M-1s-1 for GSH based on the monomer Mr of 22,612. Using the coupled assay method for thioltransferase activity, GPX had a kcat (app) of 186 +/- 9 min-1 and specificity constants (app) of 1. 49 +/- 0.14 x 10(3) M-1s-1 for S-sulfocysteine and 1.51 +/- 0.18 x 10(3) M-1s-1 for GSH based on the GPX monomer molecular weight. GPX has a higher specificity constant for S-sulfocysteine than DHA, and both assay systems gave nearly identical specificity constants for GSH. The DHA reductase and thioltransferase activities of GPX adds to the repertoire of functions of this enzyme as an important protector against cellular oxidative stress.