Non-enzymic activation of the covalent binding reaction of the complement protein C3.

The covalent binding of [3H]glycerol to C3 by the transfer of the acyl group of the internal thioester of C3 to the hydroxy group of glycerol can be activated either proteolytically by trypsin or by various chaotropes and denaturants. The activation of binding by trypsin or KBr showed similar dependence on the concentration of glycerol, indicating a similar activation mechanism. It is therefore concluded that the conformational change of the protein is the critical step in the binding reaction, and that the conversion of C3 into C3b under physiological conditions is only a means to induce the conformational change. Guanidinium chloride induces the binding of glycerol to C3 at concentrations of about 1 M. On increasing the concentration of guanidinium chloride the extent of binding declines and is accompanied by an increase in the autolytic cleavage reaction [Sim & Sim (1981) Biochem. J. 193, 129-141]. The autolytic cleavage reaction is therefore not independently activated with respect to the binding reaction. Its occurrence, however, is structurally restricted under physiological or limited denaturing conditions and is permissible only when C3 is brought to a higher denaturation state.     

Identification of cysteine-10 of protein S18 as part of the mRNA-binding site of Escherichia coli ribosomes by affinity-labeling studies with a chemically reactive A-U-G analog.

The reaction of a bromoacetamidophenyl derivative of the initiation codon A-U-G (A-U-G) with tight couples of Escherichia coli ribosomes leads to an exclusive crosslinking of label to protein S18. This crosslinking inhibits A-U-G-directed fMet-tRNAfMet binding into the puromycin-sensitive site of ribosomes and stimulates elongation-factor-dependent binding of Met-tRNAmMet. It is, therefore, concluded that protein S18 is located at or near the aminoacyl-tRNA binding site of E. coli ribosomes. Peptide as well as amino acid analysis shows that the reaction between A-U-G and ribosomes took place at cysteine-10 of protein S18. A-U-G could not be crosslinked to ribosomal proteins of the temperature-sensitive E. coli strain 258ts, where arginine-11 of protein S18 is replaced by a cysteine residue.     

The plasma membrane H(+)-ATPase from yeast. Effects of pH, vanadate and erythrosine B on ATP hydrolysis and ATP binding.

The H(+)-ATPase from the plasma membrane of Saccharomyces cerevisiae was isolated and purified. The rate of ATP hydrolysis and ATP binding was measured as a function of pH and the effect of the vanadate and erythrosine B inhibitors was investigated. The pH dependence of the rate of ATP hydrolysis forms a bell-shaped curve with a maximum at pH 6 and half-maximal rates at pH 5.0 and 7.4. Only the pH dependence between pH 6 and pH 7.6 is reversible. Above pH 7.6 and below pH 5.5, denaturation of the isolated enzyme is observed. The rate of ATP binding shows the same pH dependency as that of ATP hydrolysis. Both pH dependencies can be described by the dissociation of a monovalent acidic group with a pK of 7.4. It is concluded that the enzyme must be protonated before ATP binding. Vanadate does not inhibit ATP binding, ADP release or Pi release at concentrations where complete inhibition of ATP hydrolysis is observed. It is concluded that vanadate inhibits a step of the reaction cycle which occurs after Pi release. In contrast, erythrosine B inhibits ATP binding and thus affects the first step of the reaction cycle.     

Co-operativity and enzymatic activity in polymer-activated enzymes. A one-dimensional piggy-back binding model and its application to the DNA-dependent ATPase of DNA gyrase

The binding of a ligand to a one-dimensional lattice in the presence of a second ("rider") ligand, which binds only to the first ligand (piggy-back binding), is studied. A model derived from this study is used to analyze the effects of co-operativity on the reaction rates of enzymes activated by polymeric cofactors that provide multiple binding sites for the enzyme. It is found that in the presence of strong co-operativity, the steady-state reaction rates of polymer-activated enzymes can be very different from the Michaelis-Menten paradigm. By adjusting the co-operativity parameters and the binding constants of the ligands, the model can generate apparent auto-catalytic enhancement by substrates at low substrate concentrations and apparent substrate inhibition at high substrate concentrations. The model is shown to be able to explain the differences in the rates of ATP hydrolysis by DNA gyrase in the presence of long versus short DNA molecules and in the presence of long DNA molecules at different gyrase to DNA ratios.     

血管活性肠多肽结合核苷酸性质的研究

采用光亲和技术检测了血管活性肠多肽(VIP)与核苷酸之间的相互作用,发现VIP可以特异性结合同位素标记的GTP,还发现未标记的GTP以及其它三磷酸核苷抑制这种结合,这意味着VIP与上述三磷酸核苷之间的结合是一种典型的可逆性的结合反应.发现低浓度的GDP,GMP不但不抑制VIP与同位素标记GTP的结合反应,反而增强这种结合.联系到其它研究者关于GTP影响VIP与其受体结合反应的结果,可认为VIP能可逆性地连接一种三磷酸核苷,这种连接受反应系统中不同核苷酸比例影响,通过这种连接来调节VIP与其受体之间的反应.     

Stopped-flow studies of guanine binding by calf spleen purine nucleoside phosphorylase

The binding of guanine to calf spleen purine nucleoside phosphorylase at 20 degrees C, in 20 mM Hepes-NaOH buffer, pH 7.0, at several ionic strength between 5 and 150 mM was investigated using a stopped-flow spectrofluorimeter. The kinetic transients registered after mixing a protein solution with ligand solutions of different concentrations were simultaneously fitted by several association reaction models using nonlinear least-squares procedure based on numerical integration of the chemical kinetic equations appropriate for given model. It is concluded that binding of a guanine molecule by each of the binding sites is a two-step process and that symmetrical trimeric calf spleen purine nucleoside phosphorylase represents a system of (identical) interacting binding sites. The interaction is visible through relations between the rate constants and non-additivity of changes in "molar" fluorescence for different forms of PNP-guanine complexes. It is also probable that electrostatic effects in guanine binding are weak, which indicates that it is the neutral form of the ligand which is bound and dissociated by PNP molecule.     

An oxygenation-linked dye binding to Limulus polyphemus hemocyanin

The reaction of Limulus polyphemus hemocyanin with a dye, bromthymol blue, was examined by equilibrium dialysis, spectrophotometric titration and stopped-flow methods. Oxy-hemocyanin contained one binding site per hexamer unit. The dye binding was linked to oxygenation, and the affinity of the dye for the oxy form was about 10 times as high as that for the deoxy form. Conversely, the dye increased the O2 affinity of hemocyanin. Hemocyanin showed a simple hyperbolic binding curve in the bromthymol blue titration, whereas the time course of the reaction was generally biphasic. It was inferred from the kinetic analyses that the reaction proceeds in two steps. The first bimolecular step is characterized by an increase in the apparent pKa of the bound dye, while the second unimolecular step by a red shift of the absorption band of the unionized dye. The dye binding to partially oxygenated hemocyanin was examined spectrophotometrically; the fractional change in the binding was found to be ahead of the increase in the average degree of O2 saturation. It was concluded that the structural changes in hemocyanin which lead to the increased dye affinity take place at an early stage of the ligand binding sequence.     

Molecular recognition of taxol by microtubules. Kinetics and thermodynamics of binding of fluorescent taxol derivatives to an exposed site

We have determined the kinetic scheme and the reaction rates of binding to microtubules of two fluorescent taxoids, 7-O-[N-(4'-fluoresceincarbonyl)-l-alanyl]Taxol (Flutax-1) and 7-O-[N-(2,7-difluoro-4'-fluoresceincarbonyl)-l-alanyl]Taxol (Flutax-2). Flutax-1 and Flutax-2 bind to microtubules with high affinity (K(a) approximately 10(7) m(-1), 37 degrees C). The binding mechanism consists of a fast bimolecular reaction followed by at least two monomolecular rearrangements, which were characterized with stopped-flow techniques. The kinetic constants of the bimolecular reaction were 6.10 +/- 0.22 x 10(5) m(-1) s(-1) and 13.8 +/- 1.8 x 10(5) m(-1) s(-1) at 37 degrees C, respectively. A second slow binding step has been measured employing the change of fluorescence anisotropy of the probe. The reversal of this reaction is the rate-limiting step of dissociation. A third step has been detected using small angle x-ray scattering and involves a 2-nm increase in the diameter of microtubules. It is suggested that the first step entails the binding of the Taxol moiety and the second a relative immobilization of the fluorescent probe. The equilibrium and some kinetic measurements required the use of stabilized cross-linked microtubules, which preserved taxoid binding. The results indicate that the Taxol binding site is directly accessible, in contrast with its location at lumen in the current model of microtubules. An alternative structural model is considered in which the binding site is located between protofilaments, accessible from the microtubule surface.     

Vitelline envelope of Bufo arenarum: biochemical and biological characterization

Vitelline envelopes (VEs) of Bufo arenarum were isolated in order to study their composition and their role in fertilization. VEs are composed of four glycoproteins, with molecular masses of 120, 75, 41, and 38 kDa. To characterize its biological properties, we quantitatively determined sperm-VE binding and the induction of the acrosome reaction. Heterologous binding of B. arenarum sperm to Xenopus laevis VE components was observed with about one-third the efficiency of homologous binding. Equivalent binding of X. laevis sperm to the B. arenarum VE was observed. When B. arenarum sperm were incubated with fluorescein isothiocyanate-labeled VE, the labeled glycoproteins bound to the anterior end of the sperm head, showing a lateral distribution. Induction of the acrosome reaction was evaluated by incubating sperm in hypotonic saline media with VE glycoproteins. VEs induced the acrosome reaction in a time- and concentration-dependent manner. The acrosome reaction was maximal after 10 min. The half-maximal effect was obtained at a glycoprotein concentration of 1 microg/ml. Specificity was determined using fertilization envelope glycoproteins, which failed to induce the acrosome reaction. The B. arenarum VE is biochemically similar to other egg envelopes. It also seems that its biological properties are similar to other species in regard to sperm binding and induction of the acrosome reaction. However, as far as we are aware, this is the first observation of the VE inducing the sperm acrosome reaction in amphibians. The relatively small differences observed in heterologous sperm-VE binding in X. laevis and B. arenarum are inconsistent with the current paradigm that species specificity in fertilization is regulated at the sperm-VE binding step.     

Properties of the PriA helicase domain and its role in binding PriA to specific DNA structures

Primosome assembly protein PriA functions in the assembly of the replisome at forked DNA structures. Whereas its N-terminal DNA binding domain (DBD) binds independently to DNA, the affinity of DBD protein for forked structures is relatively weak. Although the PriA helicase domain (HD) is required for high affinity fork binding, HD protein had very low affinity for DNA. It had only low levels of ATPase activity, and it hydrolyzed ATP when DNA was absent whereas PriA did not. HD catalyzed unwinding of a minimal substrate composed of a duplex with a 3' single-stranded tail. Single-strand binding protein (SSB) bound to the tail of this substrate inhibited this reaction by full-length PriA but enhanced the reaction by HD. SSB stabilized binding of PriA but not of DBD or HD to duplexes with a 5' or 3' single-stranded tail. On forked substrates SSB enhanced helicase action on the lagging-strand arm by PriA but not by HD. The results indicate that synergy of the DBD and HD allows stable binding at the interface between duplex and single-stranded DNA bound by SSB. This mode of binding may be analogous to fork binding, which orients the helicase to act on the lagging-strand side of the fork.     

Binding of antibacterial magainin peptides to electrically neutral membranes: thermodynamics and structure.

Magainins are positively charged amphiphatic peptides which permeabilize cell membranes and display antimicrobial activity. They are usually thought to bind specifically to anionic lipids, and binding studies have been performed almost exclusively with negatively charged membranes. Here we demonstrate that binding of magainins to neutral membranes, a reaction which is difficult to assess with spectroscopic means, can be followed with high accuracy using isothermal titration calorimetry. The binding mechanism can be described by a surface partition equilibrium after correcting for electrostatic repulsion by means of the Gouy-Chapman theory. Unusual thermodynamic parameters are observed for the binding process. (i) The three magainin analogues that were investigated bind to neutral membranes with large exothermic reaction enthalpies DeltaH of -15 to -18 kcal/mol (at 30 degrees C). (ii) The reaction enthalpies increase with increasing temperature, leading to a large positive heat capacity DeltaC(p) of approximately 130 cal mol(-)(1) K(-)(1) (at 25 degrees C). (iii) The Gibbs free energies of binding DeltaG are between -6.4 and -8.6 kcal/mol, resulting in a large negative binding entropy DeltaS. The binding of magainin to small unilamellar vesicles is hence an enthalpy-driven reaction. The negative DeltaH and DeltaS and the large positive DeltaC(p) contradict the conventional understanding of the hydrophobic effect. CD experiments reveal that the membrane-bound fraction of magainin is approximately 80% helical at 8 degrees C, decreasing to approximately 60% at 45 degrees C. Since the random coil --> alpha-helix transition in aqueous solution is known to be an exothermic process, the same process occurring at the membrane surface is shown to account for up to 65% of the measured reaction enthalpy. In addition to membrane-facilitated helix formation, the second main driving force for membrane binding is the insertion of the nonpolar amino acid side chains into the lipid bilayer. It also contributes a negative DeltaH and follows the pattern for the nonclassical hydrophobic effect. Addition of cholesterol drastically reduces the extent of peptide binding and reveals an enthalpy-entropy compensation mechanism. Membrane permeability was measured with a dye assay and correlated with the extent of peptide binding. The level of dye efflux is linearly related to the amount of surface-bound peptide and can be traced back to a membrane perturbation effect.     

Inhibition of co-operative enzymes by substrate-analogues: Possible implications for the physiological significance of negative co-operativity illustrated by phenylalanine metabolism in higher plants

The “Hill” equation for co-operative binding-systems has been extended to describe the effect of substrate-analogue on the binding of substrate to an oligomeric protein. It is demonstrated that the more negatively co-operative the binding-system, the more sensitive is the binding of substrate to inhibition by increases in the relative concentration of substrate-analogue. It is proposed that the physiological significance of negative co-operativity for enzymes may be complementary to the physiological significance of positive co-operativity. The effect of negative co-operativity is to make substrate binding more sensitive to inhibition by relative increases in the concentration of substrate-analogue (e.g. for many enzymes product of the reaction) at the expense of decreased sensitivity of substrate binding to relative changes in substrate concentration compared to a system with equivalent, independent substrate binding sites. In contrast, the effect of positive co-operativity is to make the enzyme more sensitive to relative changes in substrate concentration at the expense of decreased sensitivity to inhibition by relative increases in product concentration, compared to an enzyme without co-operative binding.     

A circadian rhythm of the luciferin binding protein fromGonyaulax polyedra

Summary It has previously been shown that a protein extracted fromGonyaulax polyedra strongly and specifically binds luciferin, the substrate of the bioluminescent reaction. This binding is markedly dependent on pH with tight binding at pH 8.0 and almost no binding at pH 6.5, as measured by two independent methods. A procedure for the determination of the dissociation constant (K_d) of the luciferin binding protein (LBP) is presented, and K_d is estimated to be7×10^–9 M at pH 8.0, assuming an overall quantum yield of 0.1 for the bioluminescent reaction. With cells grown in a 12 h light — 12 h dark cycle, 5 to 10 times more LBP activity can be extracted from dark phase cells than from light phase cells. This rhythm persists in a circadian fashion in cultures maintained in constant dim light.Supported in part by a grant from the National Institutes of Health to J.W.H. (GM 19536)     

The effect of calmodulin inhibitors and the new cardiotonic drug DPI 201-106 on the formation of interprotein bonds in the cardiac troponin complex

Using the binding of labeled [125I]troponin C (TnC) to troponin I (TnI) and troponin (TnT) immobilized on a polyvinylchloride matrix, the Ca-dependent formation of interprotein bonds in the cardiac troponin complex and the effects of various drugs on the above reaction were studied. It has been found that in the absence of Ca2+ the dissociation constant, Kd, for the TnC-TnI complex in equal to (2.5 +/- 1.03).10(-7) M. In the presence of Ca2+ the number of binding sites increases twofold; the Kd value for the bonds formed thereby is (1.74 +/- 0.18).10(-7) M. The complex is stable to the effect of 5 M urea. TnC binding to immobilized TnT is nonspecific and is completely abolished by an addition of 5 M urea. DPI 201-106 used at concentrations up to 10(-3) M does not affect the Ca-dependent binding of TnC to TnI; trifluoperazine inhibits this interaction by 60%, whereas substance 48/80 inhibits the reaction by 50% when used at a concentration of 210 micrograms/ml. It is supposed that the compounds interacting with TnC affect, primarily, the cation-binding properties of troponin. These compounds can also inhibit the formation of interprotein bonds but only when used at much higher concentrations.     

The relation of ligand binding to redox state in the hexa-heme nitrite reductase of Wolinella succinogenes.

Ligand binding reactions and the relation between redox state and ligand binding in the hexa-heme nitrite reductase of Wolinella succinogenes have been studied using laser flash photolysis. On a picosecond time scale, a rapid excursion was observed corresponding to the breaking and reforming of an iron histidine bond. With the CO derivative, a geminate reaction was observed with a rate of 3 ns-1. On a nanosecond time scale, no slower geminate reactions were observed. For the cyanide derivative, no geminate reactions were observed at either time scale. The second order reaction of CO with the enzyme had a time course consisting of two distinct components. This time course changed in form as the enzyme came to equilibrium with CO, and the slower rebinding component was replaced by a faster rebinding component. It is suggested that CO binding enhances reduction of a heme with an unusually low redox potential and opens the structure of the active site to allow a faster second order reaction of CO. The proportion of the geminate CO reaction was unchanged, consistent with changes relatively remote from the ligand binding site. The second order reactions of cyanide also showed that redox effects influence its rebinding reaction. Adding cyanide to the CO complex of nitrite reductase showed that the two ligands have distinct heme binding sites.     

Adenosine binding sites in brain; relationship to endogenous levels of adenosine and to its physiological and regulatory roles

Adenosine is an endogenous component of brain tissue and is present at micromolar concentrations, well above those required to saturate the adenylate cyclase-linked receptors recently demonstrated by binding studies. It is suggested that a major binding site for adenosine may be the enzyme S-adenosyl homocysteinase, and modulation of the equilibrium catalysed by this reaction may regulate the concentration of adenosine available for stimulation of cyclic AMP production and its other physiological roles.     

Inhibition of mitochondrial ATPase by water-soluble carbodiimide]

Modification of the carboxyl group with pK 6,8-7,0 of isolated factor F1 by N-cyclohexyl-N'-beta-(4-xethylmorpholine) ethylcarbodiimide (CMCD) leads to the inhibition of the ATPase activity of the enzyme. The reaction rate of factor F1 with CMCD drops in the presence of ATP. It has been shown that during the first stage of the reaction reversible binding of CMCD with factor F1 occurs, forming a stable "enzyme--inhibitor" complex (Kdis=2.10(-4) M). N-cyclohexyl-N'-beta-(4-methylmorpholine) ethylcarbamide, a close analog of CMCD, is a competitive inhibitor of the ATPase reaction with Ki=9.10(-4) M. It is assumed that the carboxyl group, which reacts with CMCD, is located in the catalytic site of factor F1 and serves as the ligand of Me2+ in binding the substrate of the ATPase reaction Me-ATP. The reaction of factor F1, which was modified by CMCD, with proflavine, is accompanied by the covalent binding of the fluorescent label to the enzyme. The binding of proflavine to factor F1 leads to a sharp drop in the solubility of the enzyme in water.     

Conformational changes at benzodiazepine binding sites of GABA(A) receptors detected with a novel technique

Benzodiazepines are widely used for their anxiolytic, sedative, myorelaxant and anticonvulsant properties. They allosterically modulate GABA(A) receptor function by increasing the apparent affinity of the agonist GABA. We studied conformational changes induced by channel agonists at the benzodiazepine binding site. We used the rate of covalent reaction between a benzodiazepine carrying a cysteine reactive moiety with mutated receptor having a cysteine residue in the benzodiazepine binding pocket, alpha1H101Cbeta2gamma2, as a sensor of its conformation. This reaction rate is sensitive to local conformational changes. Covalent reaction locks the receptor in the conformation stabilized by positive allosteric modulators. By using concatenated subunits we demonstrated that the covalent reaction occurs either exclusively at the alpha/gamma subunit interface, or if it occurs in both alpha1 subunits, exclusively reaction at the alpha/gamma subunit interface can modulate the receptor. We found evidence for an increased rate of reaction of activated receptors, whereas reaction rate with the desensitized state is slowed down. The benzodiazepine antagonist Ro15-1788 efficiently inhibited the covalent reaction in the presence of 100 microm GABA but only partially in its absence or in the presence of 10 microm GABA. It is concluded that Ro15-1788 efficiently protects activated and desensitized states, but not the resting state.     

Optimization of electrostatic interactions in protein-protein complexes

In this article, we present a statistical analysis of the electrostatic properties of 298 protein-protein complexes and 356 domain-domain structures extracted from the previously developed database of protein complexes (ProtCom, http://www.ces.clemson.edu/compbio/protcom). For each structure in the dataset we calculated the total electrostatic energy of the binding and its two components, Coulombic and reaction field energy. It was found that in a vast majority of the cases (>90%), the total electrostatic component of the binding energy was unfavorable. At the same time, the Coulombic component of the binding energy was found to favor the complex formation while the reaction field component of the binding energy opposed the binding. It was also demonstrated that the components in a wild-type (WT) structure are optimized/anti-optimized with respect to the corresponding distributions, arising from random shuffling of the charged side chains. The degree of this optimization was assessed through the Z-score of WT energy in respect to the random distribution. It was found that the Z-scores of Coulombic interactions peak at a considerably negative value for all 654 cases considered while the Z-score of the reaction field energy varied among different types of complexes. All these findings indicate that the Coulombic interactions within WT protein-protein complexes are optimized to favor the complex formation while the total electrostatic energy predominantly opposes the binding. This observation was used to discriminate WT structures among sets of structural decoys and showed that the electrostatic component of the binding energy is not a good discriminator of the WT; while, Coulombic or reaction field energies perform better depending upon the decoy set used.     

Further studies on the temperature dependence of the binding of testosterone to human pregnancy plasma proteins

The binding of testosterone by pregnancy plasma proteins has been studied by a new equilibrium dialysis system. The temperature dependence on the association constant has been investigated and the enthalpy change ΔH and entropy change ΔS have been calculated.By a computer optimization program, the binding constant of the high affinity testosterone binding protein has been estimated from Scatchard plots. The binding reactions were carried out at 5°, 25° and 37° C. The corresponding values were 3.1.10 1.2.10⁹ and 7.2.10⁸ liter/mole. The resulting enthalpy and entropy changes were ?2.0 kcal/mole and 35.0 cal/(mole.degree) respectively.It can be concluded that the binding of testosterone to the specific binding protein is an exothermic reaction and is stabilized by hydrophobic binding forces.