Dipole-dipole interaction stabilizes the transition state of apurinic/apyrimidinic endonuclease--abasic site interaction

Human apurinic/apyrimidinic (AP) endonuclease (hAPE) initiates the repair of an abasic site (AP site). To gain insight into the mechanisms of damage recognition of hAPE, we conducted surface plasmon resonance spectroscopy to study the thermodynamics and kinetics of its interaction with substrate DNA containing an abasic site (AP DNA). The affinity of hAPE binding toward DNA increased as much as 6-fold after replacing a single adenine (equilibrium dissociation constant, K(D), 5.3 nm) with an AP site (K(D), 0.87 nm). The enzyme-substrate complex formation appears to be thermodynamically stabilized and favored by a large change in Gibbs free energy, DeltaG degrees (-50 kJ/mol). The latter is supported by a high negative change in enthalpy, DeltaH degrees (-43 kJ/mol) and also positive change in entropy, DeltaS degrees (24 J/(K mol)), and thus the binding process is spontaneous at all temperatures. Analysis of kinetic parameters reveals small enthalpy of activation for association, DeltaH degrees++(ass) (-17 kJ/mol), and activation energy for association (E(a), -14 kJ/mol) when compared with the enthalpy of activation for dissociation, DeltaH degrees++(diss) (26 kJ/mol), and activation energy in the reverse direction (E(d), 28 kJ/mol). Furthermore, varying concentration of KCl showed an increase in binding affinity at low concentration but complete abrogation of the binding at higher concentration, implying the importance of hydrophobic, but predominantly ionic, forces in the Michaelis-Menten complex formation. Thus, low activation energy and the enthalpy of activation, which are perhaps a result of dipole-dipole interactions, play critical roles in AP site binding of APE.     

Binding of the antibacterial peptide magainin 2 amide to small and large unilamellar vesicles

The thermodynamics of binding of the antibacterial peptide magainin 2 amide (M2a) to negatively charged small (SUVs) and large (LUVs) unilamellar vesicles has been studied with isothermal titration calorimetry (ITC) and CD spectroscopy at 45 degrees C. The binding isotherms as well as the ability of the peptide to permeabilize membranes were found to be qualitatively and quantitatively similar for both model membranes. The binding isotherms could be described with a surface partition equilibrium where the surface concentration of the peptide immediately above the plane of binding was calculated with the Gouy-Chapman theory. The standard free energy of binding was deltaG0 approximately -22 kJ/mol and was almost identical for LUVs and SUVs. However, the standard enthalpy and entropy of binding were distinctly higher for LUVs (deltaH0 = -15.1 kJ/mol, deltaS0 = 24.7 J/molK) than for SUVs (deltaH0 = -38.5 kJ/mol, deltaS0 = -55.3 J/molK). This enthalpy-entropy compensation mechanism is explained by differences in the lipid packing. The cohesive forces between lipid molecules are larger in well-packed LUVs and incorporation of M2a leads to a stronger disruption of cohesive forces and to a larger increase in the lipid flexibility than peptide incorporation into the more disordered SUVs. At 45 degrees C the peptide easily translocates from the outer to the inner monolayer as judged from the simulation of the ITC curves.     

Quantitative determination of binding affinity of delta-subunit in Escherichia coli F1-ATPase: effects of mutation, Mg2+, and pH on Kd

To study the stator function in ATP synthase, a fluorimetric assay has been devised for quantitative determination of binding affinity of delta-subunit to Escherichia coli F(1)-ATPase. The signal used is that of the natural tryptophan at residue delta28, which is enhanced by 50% upon binding of delta-subunit to alpha(3)beta(3)gammaepsilon complex. K(d) for delta binding is 1.4 nm, which is energetically equivalent (50.2 kJ/mol) to that required to resist the rotor strain. Only one site for delta binding was detected. The deltaW28L mutation increased K(d) to 4.6 nm, equivalent to a loss of 2.9 kJ/mol binding energy. While this was insufficient to cause detectable functional impairment, it did facilitate preparation of delta-depleted F(1). The alphaG29D mutation reduced K(d) to 26 nm, equivalent to a loss of 7.2 kJ/mol binding energy. This mutation did cause serious functional impairment, referable to interruption of binding of delta to F(1). Results with the two mutants illuminate how finely balanced is the stator resistance function. delta' fragment, consisting of residues delta1-134, bound with the same K(d) as intact delta, showing that, at least in absence of F(o) subunits, the C-terminal domain of delta contributes zero binding energy. Mg(2+) ions had a strong effect on increasing delta binding affinity, supporting the possibility of bridging metal ion involvement in stator function. High pH environment greatly reduced delta binding affinity, suggesting the involvement of protonatable side-chains in the binding site.     

The binding of a K+ competitive ligand, 2-methyl,8-(phenylmethoxy)imidazo(1,2-a)pyridine 3-acetonitrile, to the gastric (H+ + K+)-ATPase

2-Methyl,8-(phenylmethoxy)imidazo(1,2-a)pyridine 3-acetonitrile (SCH 28080) is a freely reversible K+ site inhibitor of the gastric (H+ + K+)-ATPase. In the presence of 2 mMMgSO4, [14C]SCH 28080 bound saturably to gastric vesicle preparations containing the (H+ + K+)-ATPase and was displaced by lumenal K+. A binding stoichiometry of 2.2 +/- 0.1 mol of SCH 28080/mol of catalytic phosphorylation sites was observed. The affinity of SCH 28080 binding was increased approximately 10-fold (to 45 nM) in the presence of 2 mM ATP. High affinity binding also occurred with 2 microM ATP but not with up to 200 microM D-[beta, gamma-CH2]ATP, suggesting that high affinity binding was to a phosphorylated form of the enzyme. In the presence of ATP, the association rate constant was linearly related to the concentration of SCH 28080. However, the association and dissociation rates of SCH 28080 binding were slow, especially at low temperature (at 1.5 degrees C half-maximal binding of 50 nM SCH 28080 was calculated to occur after 232 s). Binding appeared to be predominantly entropy driven with a high activation energy (40 kJ/mol at 37 degrees C). In the absence of ATP, the association rate constant was not linearly related to the concentration of SCH 28080, suggesting that a conformational change in the enzyme was required before binding could occur.     

Interacting binding sites of isoleucyl-tRNA synthetase from Escherichia coli studied by equilibrium partition.

The binding of tRNAIIe to isoleucyl-tRNA synthetase in the presence of isoleucine or ATP was investigated using the equilibrium partition method. Isoleucine decreased the affinity of tRNAIIe for the enzyme by a factor of about 5. For the free standard energy of interaction a value of about 1 kcal/mol (4.2 kJ/mol) was calculated. ATP exhibits qualitatively the same effect as isoleucine. A binding of two molecules isoleucine per molecule of enzyme could not be demonstrated even in the presence of ATP and pyrophosphatase.     

花青素主要成分与HER-2激酶区的分子对接

用分子对接方法预测天然植物化学物质与受体蛋白的相互作用位点并探究作用机制。利用MVD(Molecular Virtual Docker 5.5)软件,以HER-2激酶区为受体模板建立活性位点,与12种花青素成分进行分子对接。结果表明12种化合物均能在同一活性腔中与HER-2激酶区对接(MolDock Score:苷元–105 kJ/mol,单葡糖苷–130 kJ/mol),主要作用力是疏水作用和氢键;该活性腔也是ATP与HER-2激酶区的结合(MolDock Score=–161 kJ/mol)位点,花青素的结合可能会干扰ATP与HER-2之间氢键的形成。提示花青素可能以竞争性结合方式阻碍ATP与HER-2的结合,抑制HER-2磷酸化激活及下游信号通路的激活,从而发挥抑癌活性。     

Biochemical aspects of the visual process,XXXV. Calcium binding by cattle rod outer segment membranes studied by means of equilibrium dialysis

The calcium binding capacity of cattle rod outer segment membranes has been studied by means of an equilibrium dialysis technique. The binding is not affected by prior lyophilization of the membranes or by the presence of ionophore A23187, indicating that only passive binding to membranes is involved without active translocation.The amount of calcium bound to the membranes is influenced by the ionic composition of the medium. Both Na⁺ and K⁺ decrease binding to about the same degree, but the size of the effects suggests a rather high specificity of the calcium binding sites on the membrane.From Scatchard plots for the amount of calcium bound as a function of the free calcium concentration, it appears that two types of binding sites exist: high affinity sites which can accommodate 5 nmol calcium per mg protein (0.3 mol. calcium/mol rhodopsin) and low affinity sites which can accommodate 195 nmol calcium per mg protein (13 mol calcium/mol rhodopsin). Depending on the medium composition, the high affinity sites show dissociation constants between 8 and 40 μM, and the low affinity sites between 0.3 and 1.6 mM.Illuminated rod outer segment membranes show a slight decrease of calcium binding as compared to dark-kept membranes, but the effect is independent of the amount of calcium bound and does not appear to be significant.From these findings and the assumption of a free calcium concentration of approx. 1 μM in the extrasaccular space in rod outer segments in vivo, it is concluded that mere passive binding to the rod sac membranes must be insufficient to explain the high calcium contents in rod outer segments.     

Importance of Na,K-ATPase residue alpha 1-Arg544 in the segment Arg544-Asp567 for high-affinity binding of ATP, ADP, or MgATP

To identify residues involved in ATP binding in the N-domain of the alpha1-subunit of Na,K-ATPase, mutations were directed to the segment Arg(544)-Asp(567), a beta-strand-loop-helix structure with Arg(544) positioned at the mouth of the ATP-binding pocket near the interface to the P-domain. Substitution of Arg(544) with Gln abolished high-affinity binding of free ATP, while substitution with lysine reduced ADP affinity with minor effects on ATP binding. The contribution of Arg(544) to the change in free energy of ATP binding was estimated to 6.9 kJ/mol (DeltaDeltaG(b)) from double mutations with Asp(369) and to 7.8 kJ/mol from the MgATP dependence of phosphorylation. The phosphorylation data show that binding of Mg(2+) may increase the apparent affinity of wild-type enzyme for ATP [K(1/2)(ATP) 12 nM]. Moderately reduced affinities for ATP were seen after mutations of Asp(555), Glu(556), Asp(565), or Asp(567) with DeltaDeltaG(b) approximately equals 0.5-3 kJ/mol. Mutations of Cys(549) did not affect ATP binding. In conclusion, Arg(544) is important for binding of ATP or ADP, probably by stabilizing the beta- or gamma-phosphate moieties and aligning the gamma-phosphate for interaction with the carboxylate group of Asp(369).     

Homotropic cooperative binding of organic solvent vapors by solid trypsin

Homotropic cooperative binding was observed at vapor sorption of organic solvents (acetonitrile, propionitrile, ethanol, 1-propanol, 2-propanol, nitroethane) by dried solid trypsin from porcine pancreas (0.05 g H2O/g protein). The vapor sorption isotherms were obtained by the static method of gas chromatographic headspace analysis at 298 K for 'vapor solvent+solid trypsin' systems in the absence of the liquid phase. All isotherms have a sigmoidal shape with significant sorbate uptake only above the threshold of sorbate thermodynamic activity. On the sorption isotherms of non-hydroxylic sorbates the saturation of trypsin by organic solvent was observed above the sorbate threshold activity. The formation of inclusion compounds with phase transition between solvent-free and solvent-saturated trypsin is supposed. Approximation of obtained isotherms by the Hill equation gives the inclusion stoichiometry S, inclusion free energy, and the Hill constant N of clathrates. The inclusion stoichiometry S depends significantly on the size and shape of sorbate molecules and changes from S=31 mol of sorbate per mol of trypsin for ethanol to S=6 for nitroethane. The inclusion free energies determined for the standard states of pure liquid sorbate and infinitely dilute solution in toluene are in the range from -0.5 to -1.2 kJ/mol and from -3.1 to -8.1 kJ/mol, respectively, per 1 mol of sorbate. The Hill constants are relatively high: from N=5.6 for 1-propanol to N approximately equal to 10(3) for nitroethane. The implication of the obtained results for the interpretation of solvent effects on the enzyme activity and stability in low-water medium is discussed.     

Entropic and enthalpic contributions to annexin V-membrane binding: a comprehensive quantitative model

Annexin V binds to membranes with very high affinity, but the factors responsible remain to be quantitatively elucidated. Analysis by isothermal microcalorimetry and calcium titration under conditions of low membrane occupancy showed that there was a strongly positive entropy change upon binding. For vesicles containing 25% phosphatidylserine at 0.15 m ionic strength, the free energy of binding was -53 kcal/mol protein, whereas the enthalpy of binding was -38 kcal/mol. Addition of 4 m urea decreased the free energy of binding by about 30% without denaturing the protein, suggesting that hydrophobic forces make a significant contribution to binding affinity. This was confirmed by mutagenesis studies that showed that binding affinity was modulated by the hydrophobicity of surface residues that are likely to enter the interfacial region upon protein-membrane binding. The change in free energy was quantitatively consistent with predictions from the Wimley-White scale of interfacial hydrophobicity. In contrast, binding affinity was not increased by making the protein surface more positively charged, nor decreased by making it more negatively charged, ruling out general ionic interactions as major contributors to binding affinity. The affinity of annexin V was the same regardless of the head group present on the anionic phospholipids tested (phosphatidylserine, phosphatidylglycerol, phosphatidylmethanol, and cardiolipin), ruling out specific interactions between the protein and non-phosphate moieties of the head group as a significant contributor to binding affinity. Analysis by fluorescence resonance energy transfer showed that multimers did not form on phosphatidylserine membranes at low occupancy, indicating that annexin-annexin interactions did not contribute to binding affinity. In summary, binding of annexin V to membranes is driven by both enthalpic and entropic forces. Dehydration of hydrophobic regions of the protein surface as they enter the interfacial region makes an important contribution to overall binding affinity, supplementing the role of protein-calcium-phosphate chelates.     

Two Saturable Recognition Sites for (-)[125I]Iodo-N6-(4-Hydroxyphenyl-Isopropyl)-Adenosine Binding on Purified Cardiac Sarcolemma

Abstract

Analysis of (-)[¹²⁵]iodo-N⁶-(4-hydroxyphenylisopropyl)-adenosine ([¹²⁵I]HPIA) binding to purified sarcolemmal preparations of guinea pig and bovine hearts revealed two classes of binding sites when unlabeled iodo-HPIA (100 μmol/1) was used as non-specific binding marker. In the presence of 1 mmol/1 theophylline, however, only the high affinity component was detected. Adenosine receptor agonists caused biphasic displacement of [¹²⁵I]HPIA binding, with a high affinity potency rank order typical of interaction with A₁-adenosine receptors. Biphasic competition curves were also observed with 8-phenyltheophylline and isobutylmethylxanthine, whereas the theophylline curve was monophasic up to 1 mmol/1. In brain membranes, specific binding of [¹²⁵I]HPIA as well as of [³H]PIA was further reduced when unlabeled iodo-HPIA replaces theophylline as the non-specific binding marker. These results suggest the presence of two [¹²⁵I]HPIA binding sites on cardiac sarcolemma and brain membranes, but receptor function can only be ascribed to the high affinity sites. The low affinity site probably represents an artefact, which is often observed when non-specific binding is defined with the unlabeled counterpart or a structurally related ligand of the radioligand used.     

Expression and purification of recombinant and native calreticulin.

Calreticulin is a 60-kDa Ca(2+)-binding protein of the endo(sarco)plasmic reticulum membranes of a variety of cellular systems. The protein binds approximately 25 mol of Ca2+ with low affinity and approximately 1 mol of Ca2+ with high affinity and is believed to be a site for Ca2+ binding/storage in the lumen of the endo(sarco)plasmic reticulum. In the present study, we describe purification procedures for the isolation of recombinant and native calreticulin. Recombinant calreticulin was expressed in Escherichia coli, using the glutathione S-transferase fusion protein system, and was purified to homogeneity on glutathione-Sepharose followed by Mono Q FPLC chromatography. A selective ammonium sulfate precipitation method was developed for the purification of native calreticulin. The protein was purified from ammonium sulfate precipitates by diethylaminoethyl-Sephadex and hydroxylapatite chromatography procedures, which eliminates the need to prepare membrane fractions. The purification procedures reported here for recombinant and native calreticulin yield homogeneous preparations of the proteins, as judged by the HPLC reverse-phase chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified native and recombinant calreticulin were identified by their NH2-terminal amino acid sequences, by their Ca2+ binding properties, and by their reactivity with anticalreticulin antibodies.     

Specific recognition of the 3'-terminal adenosine of tRNAPhe in the exit site of Escherichia coli ribosomes

Ribosomes from Escherichia coli possess, in addition to A and P sites, a third tRNA binding site, which according to its presumed function in tRNA release during translocation has been termed the exit site. The exit site exhibits a remarkable specificity for deacylated tRNA; charged tRNA, e.g. N-AcPhe-tRNAPhe, is not bound significantly. To determine the molecular basis of this discrimination, we have measured the exit site binding affinities of a number of derivatives of tRNAPhe from E. coli, modified at the 3' end. Binding to the exit site of the tRNAPhe derivatives was measured fluorimetrically by competition with a fluorescent tRNAPhe derivative. We show here that removal of the 2' and 3' hydroxyl groups of the 3'-terminal adenosine decreases the affinity of tRNAPhe for the exit site 15 and 40-fold, respectively. Substitutions at the 3' hydroxyl group (aminoacylation, phosphorylation, cytidylation) as well as removal of the 3'-terminal adenosine (or adenylate) of tRNAPhe lower the affinity below the detection limit of 2 x 10(5) M-1, i.e. more than 100-fold. Modification of the adenine moiety (1,N6-etheno adenine) or replacement of it with other bases (cytosine, guanine) has the same dramatic effect. In contrast, the binding to both P and A sites is virtually unaffected by all of the modifications tested. These results suggest that a major fraction (at least -12 kJ/mol, probably about -17 kJ/mol) of the free energy of exit site binding of tRNAPhe (-42 kJ/mol at 20 mM-Mg2+) is contributed by the binding of the 3'-terminal adenine to the ribosome. The binding most likely entails the formation of hydrogen bonds.     

Characterization of prostaglandin E receptors in canine small intestine using [3H] prostaglandin E1 binding.

Prostaglandin E (PGE) receptors in canine small intestinal mucosal and muscle membrane preparations were labeled with [3H] PGE1. Saturable, high affinity binding of [3H] PGE1 was observed in both preparations. The density of binding sites (fmol/mg protein) was 39 for mucosal membranes and 60 for muscle membranes, with corresponding dissociation constants of 10.6 nM and 5.8 nM, respectively. [3H] PGE1 binding sites in both preparations showed stereospecificity and high affinity for natural PGE1 and PGE2, but not for I or F-type PGs. Synthetic PGEs such as misoprostol and enisoprost had lower affinity than PGE1 or PGE2. Several analogs of enisoprost bound weakly to the binding sites. A highly significant correlation (C.C. = 0.9) was demonstrated between mucosal and muscle binding potency for a series of enisoprost analogs. There was also a significant positive correlation between the receptor binding potency and rat diarrheagenic activity for these analogs. These results indicate that PGE receptors in canine intestinal mucosa and muscle can be directly studied with [3H] PGE1 binding. The mucosal and muscle PGE receptors may have similar ligand binding specificity. We speculate that these receptors are likely to be associated with the diarrheagenic activity of PGEs.     

Saturable binding to cell membranes of the presynaptic neurotoxin, beta-bungarotoxin.

Brief exposure to the protein neurotoxin, beta-bungarotoxin, is known to disrupt neuromuscular transmission irreversibly by blocking the release of transmitter from the nerve terminal. This neurotoxin also has a phospholipase A2 activity, although phospholipases in general are not very toxic. To determine if the toxicity of this molecule might result from specific binding to neural tissue, we have looked for high affinity, saturable binding using 125I-labelled toxin. At low membrane protein concentration 125I-labeled toxin binding was directly proportional to the amount of membrane; at fixed membrane concentration 125I-labeled toxin showed saturable binding. It was unlikely that iodination markedly changed the toxin's properties since the iodinated toxin had a comparable binding affinity to that of native toxin as judged by competition experiments. Comparison of toxin binding to brain, liver and red blood cell membranes showed that all had high affinity binding sites with dissociation constants between one and two nanomolar. This is comparable to the concentrations previously shown to inhibit mitochondrial function. However, the density of these sites showed marked variation such that the density of sites was 13.0 pmol/mg protein for a brain membrane preparation, 2.4 pmol/mg for liver and 0.25 pmol/mg for red blood cell membranes. In earlier work we had shown that calcium uptake by brain mitochondria is inhibited at much lower toxin concentrations than is liver mitochondrial uptake. Both liver and brain mitochondria bind toxin specifically, but the density of 125I-labeled toxin binding sites on brain mitochondrial preparations (3.3 +/- 0.3 pmol/mg) exceeded by a factor of ten the density on liver mitochondrial preparations (0.3 +/- 0.05 pmol/mg). It is also shown that labeled toxin does not cross synaptosomal membranes, suggesting that mitochondria may not be the site of action of the toxin in vivo. We conclude that beta-bungarotoxin is an enzyme which can bind specifically with high affinity to cell membranes.     

The glycocalicin portion of platelet glycoprotein Ib expresses both high and moderate affinity receptor sites for thrombin. A soluble radioreceptor assay for the interaction of thrombin with platelets

A soluble radioreceptor assay has been developed to characterize thrombin receptor activities of the human platelet membrane. 125I-Thrombin was added to platelet membranes solubilized in 1% Triton X-100, and thrombin bound to platelet receptors was separated from free thrombin by precipitation with wheat germ agglutinin (WGA) in the presence of alpha 1-acid glycoprotein as carrier. Both high affinity binding (Ki, 0.09 nM; R1, 0.30 pmol/mg protein) and moderate affinity binding (K2, 38 nM; R2, 72 pmol/mg protein) were detected in the detergent-solubilized membrane preparations and these binding parameters were in excellent agreement with values previously determined using intact platelets (Harmon, J. T., and Jamieson, G. A. (1985) Biochemistry 24, 58-64). Using the soluble radioreceptor assay, both high and moderate affinity binding was detected in highly purified preparations of glycoprotein Ib (GPIb) and glycocalicin, and the binding isotherms were identical with those of the crude detergent-solubilized membrane preparation. Treatment of detergent-solubilized membranes with increasing concentrations of a monospecific polyclonal antibody to glycocalicin resulted in the stepwise depletion of GPIb and concomitant reductions of thrombin binding activity. These results demonstrate that both high and moderate affinity binding of thrombin to platelets is completely expressed in the glycocalicin portion of GPIb.     

Thermodynamic parameters of the binding of retinol to binding proteins and to membranes

Retinol (vitamin A alcohol) is a hydrophobic compound and distributes in vivo mainly between binding proteins and cellular membranes. To better clarify the nature of the interactions of retinol with these phases which have a high affinity for it, the thermodynamic parameters of these interactions were studied. The temperature-dependence profiles of the binding of retinol to bovine retinol binding protein, bovine serum albumin, unilamellar vesicles of dioleoylphosphatidylcholine, and plasma membranes from rat liver were determined. It was found that binding of retinol to retinol binding protein is characterized by a large increase in entropy (T delta S degrees = +10.32 kcal/mol) and no change in enthalpy. Binding to albumin is driven by enthalpy (delta H degrees = -8.34 kcal/mol) and is accompanied by a decrease in entropy (T delta S degrees = -2.88 kcal/mol). Partitioning of retinal into unilamellar vesicles and into plasma membranes is stabilized both by enthalpic (delta H degrees was -3.3 and -5.5 kcal/mol, respectively) and by entropic (T delta S degrees was +4.44 and +2.91 kcal/mol, respectively) components. The implications of these finding are discussed.     

Interaction of synthetic HA2 influenza fusion peptide analog with model membranes.

The interaction of the synthetic 21 amino acid peptide (AcE4K) with 1-oleoyl-2-[caproyl-7-NBD]-sn-glycero-3-phosphocholine membranes is used as a model system for the pH-sensitive binding of fusion peptides to membranes. The sequence of AcE4K (Ac-GLFEAIAGFIENGWEGMIDGK) is based on the sequence of the hemagglutinin HA2 fusion peptide and has similar partitioning into phosphatidylcholine membranes as the viral peptide. pH-dependent partitioning in the membrane, circular dichroism, tryptophan fluorescence, change of membrane area, and membrane strength, are measured to characterize various key aspects of the peptide-membrane interaction. The experimental results show that the partitioning of AcE4K in the membrane is pH dependent. The bound peptide inserts in the membrane, which increases the overall membrane area in a pH-dependent manner, however the depth of insertion of the peptide in the membrane is independent of pH. This result suggests that the binding of the peptide to the membrane is driven by the protonation of its three glutamatic acids and the aspartic acid, which results in an increase of the number of bound molecules as the pH decreases from pH 7 to 4.5. The transition between the bound state and the free state is characterized by the Gibbs energy for peptide binding. This Gibbs energy for pH 5 is equal to -30.2 kJ/mol (-7.2 kcal/mol). Most of the change of the Gibbs energy during the binding of AcE4K is due to the enthalpy of binding -27.3 kJ/mol (-6.5 kcal/mol), while the entropy change is relatively small and is on the order of 6.4 J/mol.K (2.3 cal/mol.K). The energy barrier separating the bound and the free state, is characterized by the Gibbs energy of the transition state for peptide adsorption. This Gibbs energy is equal to 51.3 kJ/mol (12.3 kcal/mol). The insertion of the peptide into the membrane is coupled with work for creation of a vacancy for the peptide in the membrane. This work is calculated from the measured area occupied by a single peptide molecule (220 A(2)) and the membrane elasticity (190 mN/m), and is equal to 15.5 kJ/mol (3.7 kcal/mol). The comparison of the work for creating a vacancy and the Gibbs energy of the transition state shows that the work for creating a vacancy may have significant effect on the rate of peptide insertion and therefore plays an important role in peptide binding. Because the work for creating a vacancy depends on membrane elasticity and the elasticity of the membrane is dependent on membrane composition, this provides a tool for modulating the pH for membrane instability by changing membrane composition. The insertion of the peptide in the membrane does not affect the membrane permeability for water, which shows that the peptide does not perturb substantially the packing of the hydrocarbon region. However, the ability of the membrane to retain solutes in the presence of peptide is compromised, suggesting that the inserted peptide promotes formation of short living pores. The integrity of the membrane is substantially compromised below pH 4.8 (threshold pH), when large pores are formed and the membrane breaks down. The binding of the peptide in the pore region is reversible, and the pore size varies on the experimental conditions, which suggests that the peptide in the pore region does not form oligomers.     

Characteristics of the binding of high-density lipoprotein3 by intact cells and membrane preparations of rat intestinal mucosa

We have investigated the binding of high-density lipoprotein (HDL3, d = 1.12-1.21 g/ml), and apolipoprotein E-deficient human and rat HDL, obtained by heparin-Sepharose affinity chromatography, to intact cells and membrane preparations of rat intestinal mucosal cells. Binding of 125I-labeled HDL3 to the basolateral plasma membranes was characterised by a saturable, specific process (Kd = 21 micrograms of HDL3 protein/ml, Bmax = 660 ng HDL3 protein/mg membrane protein) and E-deficient human HDL demonstrated a similar affinity for the binding site. The basolateral plasma membranes isolated from proximal and distal portion of rat small intestine showed similar binding affinities for HDL3, whereas the interaction of HDL with brush-border membranes was characterised by mainly nonspecific and nonsaturable binding. The binding of 125I-labeled HDL3 to basolateral plasma membranes was competitively inhibited by unlabeled HDL3 but less efficiently by unlabeled human LDL. The putative HDL receptor was not dependent on the presence of divalent cations but was markedly influenced by temperature and sensitive to pronase treatment. We have also demonstrated, using whole intestinal mucosal cells, that lysine and arginine-modified HDL3 inhibited binding of normal 125I-labeled HDL3 to the same extent as normal excess HDL3. These data suggest that basolateral plasma membranes of rat intestinal mucosal cells possess a specific receptor for HDL3 which contains mainly apolipoprotein A-I and A-II, and the mechanisms of recognition of HDL3 differ from those involved in binding to the B/E receptor.