Large Differences In Affinity Research Articles

Nonradioactive methods for detecting activation of Ras-related small G proteins.

Ras-related small GTPases serve as critical regulators for a wide range of cellular signaling pathways and are activated by the conversion of the GDP-bound state to the GTP-bound conformation. Until recently, measurement of the GTP-bound active form of Ras-related G proteins involved immunoprecipitation of 32P-labeled protein followed by separation of the labeled GTP/GDP bound to GTPase. A new method based on the large affinity difference of the GTP- and GDP-bound form of Ras proteins for specific binding domains of effector proteins in vitro has been developed. By using glutathione S-transferase (GST) fusion proteins containing these binding domains, the GTP-bound form of the GTPase can be precipitated from cell lysates. In principle, this method can be used for all members of the Ras superfamily. Here we describe a general procedure to monitor the GTP-bound form of Ras-related GTPases.

Naturally occurring mutations at the acetylcholine receptor binding site independently alter ACh binding and channel gating.

By defining functional defects in a congenital myasthenic syndrome (CMS), we show that two mutant residues, located in a binding site region of the acetylcholine receptor (AChR) epsilon subunit, exert opposite effects on ACh binding and suppress channel gating. Single channel kinetic analysis reveals that the first mutation, ɛN182Y, increases ACh affinity for receptors in the resting closed state, which promotes sequential occupancy of the binding sites and discloses rate constants for ACh occupancy of the nonmutant αδ site. Studies of the analogous mutation in the δ subunit, δN187Y, disclose rate constants for ACh occupancy of the nonmutant αɛ site. The second CMS mutation, ɛD175N, reduces ACh affinity for receptors in the resting closed state; occupancy of the mutant site still promotes gating because a large difference in affinity is maintained between closed and open states. ɛD175N impairs overall gating, however, through an effect independent of ACh occupancy. When mapped on a structural model of the AChR binding site, ɛN182Y localizes to the interface with the α subunit, and ɛD175 to the entrance of the ACh binding cavity. Both ɛN182Y and ɛD175 show state specificity in affecting closed relative to desensitized state affinities, suggesting that the protein chain harboring ɛN182 and ɛD175 rearranges in the course of receptor desensitization. The overall results show that key residues at the ACh binding site differentially stabilize the agonist bound to closed, open and desensitized states, and provide a set point for gating of the channel.

Role of antigen receptor affinity in T cell-independent antibody responses in vivo.

To examine how B cell receptor affinity affects clonal selection in thymus-independent type 2 (TI-2) immune responses, we produced mice with antibodies that showed a 40-fold difference in affinity for the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP). The difference in the responses of high- and low-affinity B cells to NP-Ficoll was only twofold. However, in competition experiments only the high-affinity B cells responded to antigen. CD19 deficiency increased the affinity threshold of TI-2 responses, whereas Lyn deficiency enhanced clonal expansion but abrogated B cell terminal differentiation. Thus, in TI-2 immune responses, large differences in affinity produce only small differences in the intrinsic ability of B cells to respond to antigen, and selection for high-affinity clones is due to clonal competition during the earliest stages of the response.

Chromatin immunoprecipitation analysis fails to support the latency model for regulation of p53 DNA binding activity in vivo.

p53 can adopt two forms in vitro, a latent form that binds naked DNA poorly and an active form that binds DNA well. Conversion of the latent form to the active form is thought to occur by an allosteric mechanism induced by phosphorylation and acetylation. Despite the large differences in affinity produced by regulatory modifications in vitro, mutation of putative regulatory sites has not produced correspondingly large effects on transcription of p53 target genes in vivo. To determine whether genotoxic stress regulates DNA binding by p53 in vivo, we have performed quantitative chromatin immunoprecipitation (ChIP) assays on tumor and normal cell lines containing wild-type p53. ChIP recovers several hundredfold more p21 and MDM2 promoter DNA from p53 wild-type than p53-null cells, indicating that the assay is specific for p53. Genotoxic stress induces much smaller increases in chromatin precipitation, which are matched by changes in the p53 protein level. Thus, in the experimental systems tested, allosteric regulation of DNA binding is not a major level of regulation of p53 activity. The p53 target genes tested can be divided into a group showing high promoter occupancy in vivo (p21, MDM2, and PUMA) and a group giving substantially weaker or background p53 binding (bax, AIP1, and PIG3). Neither group shows selective recruitment of p53 to the promoter in cells undergoing apoptosis, indicating that the decision to undergo apoptosis or cell cycle arrest depends on other changes in the cell.

Streptokinase Triggers Conformational Activation of Plasminogen through Specific Interactions of the Amino-terminal Sequence and Stabilizes the Active Zymogen Conformation

Cleavage of Arg(561)-Val(562) in plasminogen (Pg) generates plasmin (Pm) through a classical activation mechanism triggered by an insertion of the new amino terminus into a binding pocket in the Pg catalytic domain. Streptokinase (SK) circumvents this process and activates Pg through a unique nonproteolytic mechanism postulated to be initiated by the intrusion of Ile(1) of SK in place of Val(562). This hypothesis was evaluated in equilibrium binding and kinetic studies of Pg activation with an SK mutant lacking Ile(1) (SK(2--414)). SK(2--414) retained the affinity of native SK for fluorescein-labeled [Lys]Pg and [Lys]Pm but induced no detectable conformational activation of Pg. The activity of SK(2--414) was partially restored by the peptides SK(1--2), SK(1--5), SK(1--10), and SK(1--15), whereas Pg(562--569) peptides were much less effective. Active site-specific fluorescence labeling demonstrated directly that the active catalytic site was formed on the Pg zymogen by the combination of SK(1--10) and SK(2--414), whereas sequence-scrambled SK(1-10) was inactive. The characterization of SK(1--10) containing single Ala substitutions demonstrated the sequence specificity of the interaction. SK(1--10) did not restore activity to the further truncated mutant SK(55-414), which was correlated with the loss of binding affinity of SK(55--414) for labeled [Lys]Pm but not for [Lys]Pg. The studies support a mechanism for conformational activation in which the insertion of Ile(1) of SK into the Pg amino-terminal binding cleft occurs through sequence-specific interactions of the first 10 SK residues. This event and the preferentially higher affinity of SK(2--414) for the activated proteinase domain of Pm are thought to function cooperatively to trigger the conformational change and stabilize the active zymogen conformation.

水溶性人工レセプターの設計と分子認識のメカニズム

Development of water-soluble synthetic receptors is described, particularly focusing on the mechanistic aspects of binding. Binding of guest by receptor is driven by (1) enthalpic stabilization owing to the host-guest attractive forces and (2) entropic stabilization upon desolvation. A number of thermodynamic data collected for synthetic receptors indicate that the binding in organic solvent is enthalpically driven while that in water both entropically and enthalpically driven. The average binding constant of cyclodextrins is 102.4M-1, while that of proteins 109.3M-1, indicating that understanding of the large difference in affinity between synthetic and native receptors will help further development of synthetic receptor chemistry. One important aspect of biological recognition is the switching between the R state and the T state of the receptor as suggested by the induced-fit binding model. Design of the receptor with a conformationally or solvationally unstable state will be the key to the higher-order functions of the synthetic receptors. The important role of synthetic organic chemistry in the development of these functional molecules is emphasized.

Evolution of the 14-3-3 protein family: does the large number of isoforms in multicellular organisms reflect functional specificity?

14-3-3 proteins constitute a family of eukaryotic proteins that are key regulators of a large number of processes ranging from mitosis to apoptosis. 14-3-3s function as dimers and bind to particular motifs in their target proteins. To date, 14-3-3s have been implicated in regulation or stabilization of more than 35 different proteins. This number is probably only a fraction of the number of proteins that 14-3-3s bind to, as reports of new target proteins have become more frequent. An examination of 14-3-3 entries in the public databases reveals 153 isoforms, including alleloforms, reported in 48 different species. The number of isoforms range from 2, in the unicellular organism Saccharomyces cerevisiae, to 12 in the multicellular organism Arabidopsis thaliana. A phylogenetic analysis reveals that there are four major evolutionary lineages: Viridiplantae (plants), Fungi, Alveolata, and Metazoa (animals). A close examination of the aligned amino acid sequences identifies conserved amino acid residues and regions of importance for monomer stabilization, dimer formation, target protein binding, and the nuclear export function. Given the fact that 53% of the protein is conserved, including all amino acid residues in the target binding groove of the 14-3-3 monomer, one might expect little to no isoform specificity for target protein binding. However, using surface plasmon resonance we show that there are large differences in affinity between nine 14-3-3 isoforms of A. thaliana and a target peptide representing a novel binding motif present in the C terminus of the plant plasma membrane H(+)ATPase. Thus, our data suggest that one reason for the large number of isoforms found in multicellular organisms is isoform-specific functions.

Effects of Site-directed Mutagenesis on Structure and Function of Recombinant Rat Liver S-Adenosylhomocysteine Hydrolase: CRYSTAL STRUCTURE OF D244E MUTANT ENZYME

A site-directed mutagenesis, D244E, of S-adenosylhomocysteine hydrolase (AdoHcyase) changes drastically the nature of the protein, especially the NAD(+) binding affinity. The mutant enzyme contained NADH rather than NAD(+) (Gomi, T., Takata, Y., Date, T., Fujioka, M., Aksamit, R. R., Backlund, P. S., and Cantoni, G. L. (1990) J. Biol. Chem. 265, 16102-16107). In contrast to the site-directed mutagenesis study, the crystal structures of human and rat AdoHcyase recently determined have shown that the carboxyl group of Asp-244 points in a direction opposite to the bound NAD molecule and does not participate in any hydrogen bonds with the NAD molecule. To explain the discrepancy between the mutagenesis study and the x-ray studies, we have determined the crystal structure of the recombinant rat-liver D244E mutant enzyme to 2.8-A resolution. The D244E mutation changes the enzyme structure from the open to the closed conformation by means of a approximately 17 degrees rotation of the individual catalytic domains around the molecular hinge sections. The D244E mutation shifts the catalytic reaction from a reversible to an irreversible fashion. The large affinity difference between NAD(+) and NADH is mainly due to the enzyme conformation, but not to the binding-site geometry; an NAD(+) in the open conformation is readily released from the enzyme, whereas an NADH in the closed conformation is trapped and cannot leave the enzyme. A catalytic mechanism of AdoHcyase has been proposed on the basis of the crystal structures of the wild-type and D244E enzymes.

Random chimeragenesis of G-protein-coupled receptors. Mapping the affinity of the cAMP chemoattractant receptors in Dictyostelium.

G-protein-coupled receptors mediate a wide variety of responses to extracellular stimuli in eucaryotic cells. Binding of the ligand to these receptors is thought to involve contacts within a pocket from the sequences of these genes. A family of four surface cAMP receptors that mediate responses to secreted cAMP coordinates the developmental program of Dictyostelium. A large difference in affinity for cAMP exists between cAR1 (25 and 230 nM) and cAR2 (> 5 microns). To understand the basis for this affinity difference, we generated an extensive series of cAR1/cAR2 and cAR2/cAR1 chimeras using a technique designated "random chimeragenesis." When a linearized plasmid was transformed into Escherichia coli, tandemly positioned cAR1 and cAR2 genes crossed over at homologous regions. The cAMP binding properties and EC50 values for agonist-induced phosphorylation of each of the chimeras were characterized in order to map the domains that determine the affinity. These studies implicated a domain in the second extracellular loop in which only 5 residues differ between the two receptors as the major determinant of affinity. A secondary domain including residues 110-147 (11 residue differences) was identified as a minor determinant of affinity.

Molecular Aspects and Mechanism of Action of Dihydrofolate Reductase Inhibitors

Derivatives of tetrahydrofolate (FH4) are involved in a variety of biosynthetic reactions. Inhibition of the enzyme dihydrofolate reductase (DHFR) depletes the available tetrahydrofolate and blocks the formation of thymidylate, purines, the amino acids methionine and glycine, and several other cell constituents. Lack of thymidylate disrupts DNA synthesis and cell growth ceases. Many folate analogues are competitive inhibitors of DHFR, and while some classes show no selectivity, suitably substituted diaminopyrimidines are several thousands times more active against bacterial than mammalian DHFR and are thus effective and safe antibiotics. The ease of isolation, stability and low molecular weight of DHFR has facilitated its study at a molecular level. Considerable knowledge has been accumulated about the interactions of inhibitors with the protein chain in and around the active site, but despite the availability of many sequences and several crystal structures the exact details of how subtle variations of inhibitor structure result in such large differences in affinity for the enzyme are not yet clear.

Resonance Raman spectroscopic evidence that carp deoxyhemoglobin remains in a T-like quaternary structure at high pH: implications for cooperativity.

Resonance Raman spectroscopy shows the Fe-proximal imidazole stretching band to shift from 215 to 219 cm-1 between human deoxyhemoglobin (deoxy-Hb) and a Hb sample which is 75% oxygenated, demonstrating that the T-R quaternary structure switch can be monitored by resonance Raman spectroscopy in native Hb at equilibrium. For deoxy-Hb from carp, the band is at 215 cm-1 at pH 9 as well as pH 6, contrary to previous reports of an elevated frequency at high pH. The invariance of this frequency over a large affinity difference is in contrast to a recent report of continuously varying vFe-ImH frequencies for human mutant deoxy-Hb's. The band shifts to 219 cm-1 for carp Hb at pH 9 when O2 is bound to only 20% of the hemes. The spectra are consistent with a T-R switch upon binding approximately 0.5 O2 per Hb, on the average, although the number may be higher if the binding affinity is higher for alpha- than for beta-chains. The 0.5 value, in conjunction with the weak cooperativity observed for carp Hb at pH 9, is incompatible with a value of the allosteric constant, L = (T0)/(R0), large enough to prevent the vFe-ImH band from shifting detectably at pH 9 in the absence of O2. The possibility of functionally important intermediate structures is discussed.

The two α2-Macroglobulin-bound trypsin molecules have different affinities for the basic pancreatic trypsin inhibitor

We have investigated the enzymatic properties of α 2-macroglobulin-bound porcine trypsin using a substrate: Z-Gly-Gly-Arg- p-nitroanilide and inhibotors; p-aminobenzamidine and basic pancreatic trypsin inhibitor. The ternary α 2-macroglobulin-(trypsin) 2 complex behaves like a mixture of two enzymes which bind basic pancreatic trypsin inhibitor with widely different ffinities ( K i = 0.11 μM and 23 μM). About one-half of the trypsin molecules of the ternary complex are covalently bound to α 2-macroglobulin. Preparation of the complex in the presence of hydroxylamine prevents covalent bond formation, but the two trypsins of this artificial complex still exhibit large differences in affinity for basic pancreatic trypsin inhibitor. The trypsin molecules of the ternary complex also exhibit small differences in their affinity for Z-Gly-Gly-Arg- p-nitroanilide and p-aminobenzamidine.

Oxygen affinity of sea turtle blood

The oxygen binding properties of whole blood in adult sea turtles (green, Chelonia mydas; loggerhead, Caretta caretta) were measured as a function of pH, and carbon dioxide partial pressure using the ‘Hemoscan Oxygen Dissociation Analyser’ with modifications that improved the performance of the method. Under approximately physiologicl conditions (25.7°C, P CO 2 = 37 Torr; pH 7.45) there is a marked affinity difference between the two species (green, P 50 = 29 Torr; loggerhead, 47 Torr). It is suggested that the large affinity difference between blood of green and loggerhead turtles may be related to differences in depths to which they typically dive. The Hill coefficient at 50% saturation is 2.4 in the green under these conditions, and 2.7 in loggerhead. Hill plots of both species curve upward and have significant regions with n approaching 1 at low saturation with the effect that the lower end of the binding curve is more hyperbolic than sigmoidal. The fixed acid and CO 2 Bohr effects in both species are strongly saturation dependent, being very low at low saturation and increasing markedly at higher saturation. The fixed acid Bohr effects are similar in the two species but the green is more sensitive to CO 2 at constant pH, and so has a larger CO 2 Bohr effect. The hyperbolic shape of the lower end of the binding curves, and low pH sensitivity in this region, would tend to favor oxygen loading during a dive, when lung Pa O 2 and blood pH are falling. These properties might therefore favor more complete utilization of the lung oxygen store. We suggest that diving vertebrates can usefully be divided into two groups, those that use the lung as the main oxygen store and those that use tissue (blood and myoglobin) stores.

Similarity of Ribulose-1,5-bisphosphate Carboxylases of Isogenic Diploid and Tetraploid Ryegrass (Lolium perenne L.) Cultivars

Partially purified ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) was isolated from diploid and tetraploid cultivars of ryegrass (Lolium perenne L.) using two separate methods. The apparent K(m) (CO(2)) values for the enzymes prepared by either method did not differ significantly between diploid and tetraploid when assayed by two separate techniques. The unpurified enzymes from freshly lysed (and fully functional) protoplasts of both diploid and tetraploid cultivars gave virtually identical apparent K(m) (CO(2)) values. There was no indication of large differences in affinity for CO(2) of illuminated intact protoplasts from the two cultivars.

Three classes of dopamine receptor (D-2, D-3, D-4) identified by binding studies with 3H-apomorphine and 3H-domperidone.

Dopamine receptors subtypes were studied in homogenates from rat brain areas, mainly the corpus striatum, using the two highly selective ligands 3H-apomorphine and 3H-domperidone. The clearly biphasic inhibition of the specific binding of these two ligands by some agents allowed us to define four distinct classes of binding site. 3H-apomorphine labels two classes of site displaying a large difference in affinity for domperidone, i.e. class I sites well recognized (IC50=5 nM) and class II sites poorly recognized (IC50=10 μM). 3H-domperidone also labels two distinet classes of site displaying a large difference in affinity for apomorphine and dopamine, i.e. class III sites well recognized by these agents (IC50=5 and 35 nM, respectively) and class IV sites poorly recognized (IC50=790 nM and 14 μM, respectively). The two classes I and III represent a single pharmacological class of dopaminergic receptors (labelled by either 3H-apomorphine or 3H-domperidone) as indicated by 1) their almost identical pharmacological specificities (high correlation between Kd or Ki values for a variety of dopaminergic agonists and antagonists); 2) their similar capacity in striatum as well as in other brain regions; 3) the identical decrease in capacity following kainate lesions; 4) their similar sensitivity to GTP and thermal denaturation. Because the pharmacological specificity of these sites excludes the possibility that they represent the recognition sites of the dopamine-sensitive adenylate cyclase, i.e. D-1 receptors, we propose to term them D-2 receptors. Class II and IV sites also differ from D-1 receptors as shown by drug specificity and the effect of kainate. We propose to term class II sites D-3 receptors and class IV sites D-4 receptors.

Metal-polysaccharide complexes—Part I

Many factors, including linear charge density, effective nuclear charge of the cation, basicity of the donor group, chelation, intermolecular binding of two or more donor groups by a single cation and steric fit, contribute—or possibly contribute—to the formation and stability of metal ion-polysaccharide complexes. In order to form a proper foundation for studying and interpreting metal ion-polysaccharide interactions, the author introduces into his treatise on polysaccharide complexes much current information concerning the interaction of metal ions with simple unidonor and simple multidonor ligands. For convenience of discussion, the subject of polysaccharide complexes is divided into two categories: interactions of neutral polysaccharides, such as cellulose and starch, and interactions of anionic polysaccharides, such as alginate, pectate, mucopolysaccharides and carrageenan. On the one hand, neutral polysaccharides form weak complexes with cations in neutral or non-alkaline media. Only in alkaline solution is there any great affinity between cation and donor. On the other hand, anionic polysaccharides have a strong affinity for metal counterions, even at low concentrations. Attempts are made to provide reasonable explanations for the formation of these complexes and for the often large differences in affinity between metals for a particular polysaccharide or between polysaccharides for a particular metal. In some instances, rheological behaviour of polysaccharide systems can be related to structural features of macromolecular complexes.

Phaseolain: A PLANT CARBOXYPEPTIDASE OF UNIQUE SPECIFICITY

Abstract The mode of action of phaseolain, a carboxypeptidase from French bean leaves, has been studied on a range of proteins and peptides of varying molecular weights. Critical experiments show that phaseolain contains no contaminant aminopeptidase or endopeptidase activity. Kinetic data on the release of COOH-terminal amino acids from polypeptide substrates showed that residues are released in a strictly sequential manner and that phaseolain will release proline and virtually the whole range of amino acids, with the possible exception of aspartic acid. Glutamic acid and glycine are released only slowly. The enzyme unexpectedly released glycinamide from the terminal position of oxytocin but did not release amidated residues from the termini of other peptides tested. The rate of release of COOH-terminal residues from N-benzyloxycarbonyl(Cbz)dipeptide substrates by phaseolain depends in part upon the nature of the COOH-terminal residue, a long chain aliphatic residue being preferred, but more particularly on the nature of the penultimate residue, where an aromatic residue greatly enhances enzyme action. Initial rates of release of leucine from N-Cbz-Phe-Leu are some 400-fold greater than from N-Cbz-Ile-Leu; similarly, proline is readily released from N-Cbz-Tyr-Pro, but is not released from N-Cbz-Gly-Pro under the experimental conditions used. Comparison of kinetic data for two substrates, N-Cbz-Phe-Leu and N-Cbz-Gly-Leu, indicate that the 40-fold difference in initial rate of leucine release by phaseolain (at 0.9 mm substrate) is not so much a consequence of large differences in affinity of enzyme for substrate (Km values are 1.22 ± 0.06 mm and 3.75 ± 0.80 mm, respectively) but is related more to the efficiency of catalysis (Vmax values, 0.82 ± 0.02 and 0.11 ± 0.01, respectively). Conversely, with N-acetyl-Gly-Leu as substrate, alteration of the blocking group hardly affects Vmax values (0.090 ± 0.001 for N-acetyl-Gly-Leu compared with 0.11 ± 0.01 for N-Cbz-Gly-Leu), whereas the lack of affinity of enzyme for the acetylated dipeptide is reflected in a very high Km value of 58.1 ± 0.5 mm. 3-Phenylpropionic acid is a competitive inhibitor of phaseolain (Ki = 1.78 mm with N-Cbz-Phe-Leu as substrate). Although there is general agreement between kinetic data obtained for polypeptide and N-blocked dipeptide substrates, there are differences, notably the very marked inhibitory effect of a penultimate threonine residue on release of leucine (a favorable COOH-terminal residue) from N-Cbz-Thr-Leu, whereas proline (relatively unfavorable COOH-terminal residue) is readily released from the COOH-terminal region of insulin B chain (partial sequence Tyr-Thr-Pro-Lys-Ala).