The residual effect of chronic neuroleptic treatment on the neuroleptic binding assay in rats

Chronic chlorpromazine administration to rats (25 mg/Kg/day) for 30 days followed by a washout period of 10 days resulted in an increase in both the measured maximum number of binding sites, Bmax, and the apparent dissociation constant, Kd, for the binding of 3H-spiroperidol to neural membranes of the brain. When membrane suspensions were progressively diluted before the binding assay, it was found that the apparent Bmax did not change with dilution, remaining higher in membranes of chlorpromazine-treated rats than in controls. The apparent increase in Kd, on the other hand, was found to be an artifact of the assay. Thus extrapolation of the measured or apparent Kd value to infinite dilution resulted in identical value for Kd regardless of the treatment.     

Ca-binding properties of a unique ATPase inhibitor protein isolated from mitochondria of bovine heart and rat skeletal muscle

Previous studies showed that Ca²⁺ induced monomer to active dimer interconversion of a mitochondrial ATPase inhibitor protein from bovine heart or rat skeletal muscle (Yamada, E.W., Huzel, N.J. and Dickison, J.C. (1981) J. Biol. Chem. , 10203–10207). Initial equilibrium dialysis measurements of Ca²⁺ binding showed that this unique protein possesses three binding sites of high affinity with a maximum of one mol of Ca²⁺ bound/mol of protein monomer. Magnesium (1 mM) did not affect the first association constant but increased the second and third by about 1.2 and 1.5 fold, respectively. That the apparent association constants varied with concentration of protein monomer was in agreement with the self-associating nature of the protein. Scatchard plots at three concentrations of protein intersected at a molar ratio of about 0.5 (Ca²⁺/monomer). K_a1 and K_a2 values of 4.2 μM and 12.1 μM, respectively, were estimated by extrapolation of apparent constants to infinite dilution of protein. K_a3 (51.3 μM) was estimated by extrapolation of double reciprocal plots of apparent constants versus protein concentration to infinite levels of protein. A model for Ca²⁺ binding by this self-associating protein is described. Trifluoperazine had no effect on the activity of the inhibitor protein from either tissue.     

Isoelectric focusing of interacting systems. V. Determination of ligand-binding constants

A theory is formulated for an isoelectric focusing procedure which permits determination of intrinsic ligand-binding constants. The protein is first focused in the absence of ligand, after which ligand is added to the appropriate electrode compartment and then driven by the electric field into the focusing column where it complexes with the protein. The band of protein and its complexes moves to the constituent isoelectric point. An equation linearly relates the reciprocal of the overall distance moved to the reciprocal of the local concentration of ligand. The quotient of the intercept and slope gives the intrinsic binding constant. If the concentration of ligand in the electrode compartment is used in lieu of the local concentration, an apparent constant is obtained. Extrapolation of the apparent constant to infinite dilution of protein gives the intrinsic constant. For certain systems, conditions may be realized which give an apparent constant within 4% of the intrinsic constant.     

Characteristics of [3H]Hemicholinium-3 Binding to Rat Striatal Membranes: Evidence for Negative Cooperative Site-Site Interactions

The characteristics of [3H]hemicholinium-3 ([3H]HC-3) interactions with rat striatal membranes were investigated. Under the described assay conditions, [3H]-HC-3 binds with a saturable population of membrane binding sites having the following regional distribution: striatum much greater than hippocampus greater than or equal to cerebral cortex greater than cerebellum. The specific binding of [3H]HC-3 showed an obligatory requirement for NaCl; other halide salts of sodium or KCl failed to substitute for NaCl. The Scatchard transformation of saturation isotherm data generated a curvilinear plot with high- and low-affinity components of binding. The dissociation of [3H]HC-3 at infinite dilution was also multiexponential. The dissociation could, however, be accelerated if unlabeled HC-3 was included in the diluting buffer, and this increase in dissociation appeared to be dependent on the concentrations of unlabeled HC-3 used, with the maximal increase demonstrable at 100 nM. The dissociation was also dependent on the fractional saturation of binding sites with labeled HC-3, such that, at higher fractional saturation of binding sites, the overall dissociation was faster and the difference in the dissociation observed between "dilution only" and "dilution + unlabeled HC-3" was reduced. This occupancy-dependent change in dissociation could also be influenced by temperature and pH. Based on the results of these kinetic studies, the steady-state [3H]HC-3 binding data were analyzed for a homogeneous population of binding sites undergoing site-site interactions of the negative cooperative type. Such an analysis yielded a KD of 9.3 nM for the high-affinity state and a KD of 22.8 nM for the low-affinity state of binding sites, with a Bmax of 434 fmol/mg of protein. Competitive binding studies showed that unlabeled HC-3 was most potent in displacing [3H]HC-3, followed by choline. Other drugs known to have little influence on the synaptosomal sodium-dependent high-affinity choline uptake system (SDHACU) had no significant effect on [3H]HC-3 binding sites. Similarities in ionic dependencies, regional distributions, and pharmacological selectivities of [3H]HC-3 binding with synaptosomal SDHACU suggest that [3H]HC-3 selectively labels SDHACU sites located on presynaptic cholinergic neurons in rat CNS. We suggest that the two affinity states of [3H]HC-3 binding sites represent the different "functional" states of the SDHACU system. The binding of HC-3 (or choline) with the high-affinity state of the binding sites induces negative cooperative site-site interactions among the binding sites, resulting in the formation of a low-affinity binding state.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ca2+-binding properties of a unique ATPase inhibitor protein isolated from mitochondria of bovine heart and rat skeletal muscle

Previous studies showed that Ca²⁺ induced monomer to active dimer interconversion of a mitochondrial ATPase inhibitor protein from bovine heart or rat skeletal muscle (Yamada, E.W., Huzel, N.J. and Dickison, J.C. (1981) J. Biol. Chem. $\text{256}$, 10203–10207). Initial equilibrium dialysis measurements of Ca²⁺ binding showed that this unique protein possesses three binding sites of high affinity with a maximum of one mol of Ca²⁺ bound/mol of protein monomer. Magnesium (1 mM) did not affect the first association constant but increased the second and third by about 1.2 and 1.5 fold, respectively. That the apparent association constants varied with concentration of protein monomer was in agreement with the self-associating nature of the protein. Scatchard plots at three concentrations of protein intersected at a molar ratio of about 0.5 ($\text{Ca}{}^{\mathrm{2+}}\text{monomer}$). K_a1 and K_a2 values of 4.2 μM and 12.1 μM, respectively, were estimated by extrapolation of apparent constants to infinite dilution of protein. K_a3 (51.3 μM) was estimated by extrapolation of double reciprocal plots of apparent constants versus protein concentration to infinite levels of protein. A model for Ca²⁺ binding by this self-associating protein is described. Trifluoperazine had no effect on the activity of the inhibitor protein from either tissue.     

Interactions of the Escherichia coli Primosomal PriB Protein with the Single-stranded DNA. Stoichiometries, Intrinsic Affinities, Cooperativities, and Base Specificities

Quantitative analysis of the interactions of the Escherichia coli primosomal PriB protein with a single-stranded DNA was done using quantitative fluorescence titration, photocrosslinking, and analytical ultracentrifugation techniques. Stoichiometry studies were done with a series of etheno-derivatives of single-stranded (ss) DNA oligomers. Interactions with the unmodified nucleic acids were studied, using the macromolecular competition titration (MCT) method. The total site-size of the PriB dimer-ssDNA complex, i.e. the maximum number of nucleotides occluded by the PriB dimer in the complex, is 12 ± 1 nt. The protein has a single DNA-binding site, which is located centrally within the dimer and has a functionally homogeneous structure. The stoichiometry and photocrosslinking data show that only a single monomer of the PriB dimer engages in interactions with the nucleic acid. The analysis of the PriB binding to long oligomers was done using a statistical thermodynamic model that takes into account the overlap of potential binding sites and cooperative interactions. The PriB dimer binds the ssDNA with strong positive cooperativity. Both the intrinsic affinity and cooperative interactions are accompanied by a net ion release, with anions participating in the ion exchange process. The intrinsic binding process is an entropy-driven reaction, suggesting strongly that the DNA association induces a large conformational change in the protein. The PriB protein shows a dramatically strong preference for the homo-pyrimidine oligomers with an intrinsic affinity higher by about three orders of magnitude, as compared to the homo-purine oligomers. The significance of these results for PriB protein activity is discussed.     

Consequences of ligand bivalency in interactions involving particulate receptors: equilibrium and kinetic studies with Sephadex-concanavalin A, butylagarose-phosphorylase b, and Fc receptor-IgG dimer interactions as model systems

Theoretical consideration is given to the interaction of a bivalent ligand with particulate receptor sites, not only from the viewpoint of quantitatively describing the binding behavior but also from that of the kinetics of ligand release upon infinite dilution of a receptor-ligand mixture. In the latter regard, a general expression is derived that describes the time dependence of the amount of ligand bound as a function of two rate constants for the stepwise dissociation of cross-linked ligand-receptor complex and a thermodynamic parameter expressing the initial ratio of singly linked to doubly linked ligand-receptor complexes. An experimental study of the interaction between Sephadex and concanavalin A is then used to illustrate application of this recommended theoretical approach for characterizing the binding behavior and dissociation kinetics of a bivalent ligand for a system in which all ligand-receptor interactions may be described by a single intrinsic association constant. Published results on the interaction of phosphorylase b with butylagarose are also shown to comply with this simplest model of the bivalent ligand hypothesis; but those for the interaction between immunoglobulin G (IgG) dimers and Fc receptors require modification of the model by incorporation of different intrinsic association constants for the successive binding of receptor sites to the bivalent ligand. These results emphasize the need to consider ligand bivalency as a potential phenomenon in studies of interactions between protein ligands and particulate receptors and illustrate procedures by which the effects of ligand bivalency may be identified and characterized.     

Determination of ligand-binding constants by isoelectric focusing. Generalization and extension of theory

The theory of a proposed isoelectric focusing procedure for the determination of ligand binding constants has been generalized (via numerical calculations) to non-linear, electrophoretic velocity profiles and systems for which the change in velocity of the protein accompanying ligand binding depends upon pH. The theory has also been extended to include binding of ligand to heterogeneous sites. Determination of accurate values of the binding parameters depends upon extrapolation of apparent parameters to infinite dilution of protein.     

Multisite proteins and randomly coiled polymeric ligands. Chain length dependence of binding constants.

A model mechanism was developed for the binding of a rigid multisite protein with a randomly coiled multivalent ligand. Probabilities of the formation of chain loops between sites located at given distances at the protein were calculated by an extension of the concept of ring closure in coiled chain molecules. Expressions were derived for the dependence of overall equilibrium quantities, such as the binding constant between the protein and the ligand, on intrinsic parameters such as intrinsic binding constants, number of sites at the protein and their distances and on the chain length of the polymeric ligand. A pronounced chain length dependence of the overall binding constant was predicted even at chain lengths much longer than the size of the protein. Such a dependence was previously observed for the enzyme prolyl hydroxylase which acts on polymeric substrates like (ProProGly)n. This so far unexplained feature is quantitatively described by the model mechanism which is believed to be applicable to many other interactions of biological importance.     

Anion interactions with Na,K-ATPase: simultaneous binding of nitrate and eosin

Nucleotide binding affinity to Na,K-ATPase is reduced by a number of anions such as nitrate and perchlorate in comparison with affinity in the presence of chloride (all with sodium as the cation). The reduction correlates with the position of these anions in the Hofmeister series. Transient kinetic experiments using the fluorescent dye eosin—which binds to the nucleotide site of the Na,K-ATPase—show that simultaneous anion binding, exemplified with nitrate, and eosin binding is possible. The effect of nitrate on eosin binding is reflected in a decreased binding-rate constant and an increased dissociation rate constant, leading to a decreased equilibrium binding constant for eosin. Since eosin binding is analogous with nucleotide binding to Na,K-ATPase, the results suggest the simultaneous presence of nucleotide and anion binding sites.Abbreviations E₁ the protein conformation in Na⁺ - E₂ the enzyme conformation in K⁺ - Eo eosin (tetrabromofluorescein) - F fluorescence - I ionic strength - k_i rate constant - K_i equilibrium dissociation constant - K_i,0 equilibrium dissociation constant at zero ionic strength - N nitrate - z_i net charge - charge product z_i·z_j     

Multisite proteins and randomly coiled polymeric ligands. chain length dependence of binding constants

A model mechanism was developed for the binding of a rigid multisite protein with a randomly coiled multivalent ligand. Probabilities of the formation of chain loops between sites located at given distances at the protein were calculated by an extension of the concept of ring closure in coiled chain molecules. Expressions were derived for the dependence of overall equilibrium quantities, such as the binding constant between the protein and the ligand, on intrinsic parameters such as intrinsic binding constants, number of sites at the protein and their distances and on the chain length of the polymeric ligand. A pronounced chain length dependence of the overall binding constant was predicted even at chain lengths much longer than the size of the protein. Such a dependence was previously observed for the enzyme prolyl hydroxylase which acts on polymeric substrates like (ProProGly)_n. This so far unexplained feature is quantitatively described by the model mechanism which is believed to be applicable to many other interactions of biological importance.     

A general method of deriving the best binding site model consistent with experimental binding data

An analysis of binding data is presented which yields the best binding site model consistent with the experimental data. The analysis is applicable to homotropic binding and yields the number of independent sites, number of interacting sites (dimers and tetramers of sites), intrinsic association constants, and degree of interaction. The information is derived from the roots of a binding polynomial constructed by the fitted Adair constants.     

Size extensivity of elastic properties of alkane fragments

Using MP2, CCSD, and B3LYP methods of computational chemistry, we show length dependence in the intrinsic elastic properties of short alkane fragments. For isolated alkane fragments of finite length in the gas phase and zero temperature, the intrinsic elasticity constants are found to vary with the number of carbon atoms and its parity. From extrapolation of the elasticity constants calculations to infinite chain length, and by comparing with in-situ elasticity constant of single poly(ethylene) molecule obtained with atomic force microscopy, we estimate the softening effect of environment on the extension response of the polymer.     

Fluorescence resonance energy transfer for investigation of the interaction of Para Red with serum albumins

Para Red (PR) has been isolated from food additives, and shown to be toxic to humans. To facilitate examination of its toxicity, the interaction between PR and serum albumins (SA) was studied using fluorescence quenching and circular dichroism (CD) spectrophotometry. The experiments showed that the fluorescence intensity of serum albumins decreased with increasing concentrations of PR, which resulted from the binding of PR and SA. The binding constant, number of binding sites and thermodynamic parameters were calculated and hydrogen bond and van der Waals interactions were shown to play a key role in the binding process. Competition experiments indicated that PR mainly binds to Trp residues of SA within the site I. As the CD and three‐dimensional spectra revealed, the addition of PR induced a conformational change in SA. Copyright © 2015 John Wiley & Sons, Ltd.     

FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2

Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein. By eliminating its C-terminal region, a water soluble truncated version was obtained in our laboratory. Overall conformation of the mutant version resembles the wild-type protein. Considering these data and the fact that the mutant was obtained as an apo-protein, the truncated version is an ideal model to study the interaction between the enzyme and its cofactor. Here, the FAD binding properties of this version were characterized using far-UV circular dichroism (CD), differential scanning calorimetry (DSC), limited proteolysis, and steady-state and dynamic fluorescence spectroscopy. CD spectra, thermal unfolding and DSC profiles did not reveal any major difference in secondary structure between apo- and holo-protein. In addition, digestion site accessibility and tertiary conformation were similar for both proteins, as seen by comparable chymotryptic cleavage patterns. FAD binding to the apo-protein produced a parallel increment of both FAD fluorescence quantum yield and steady-state emission anisotropy. On the other hand, addition of FAD quenched the intrinsic fluorescence emission of the truncated protein, indicating that the flavin cofactor should be closely located to the protein Trp residues. Analysis of the steady-state and dynamic fluorescence data confirms the formation of the holo-protein with a 1:1 binding stoichiometry and an association constant K_A = 7.0(± 0.8) × 10⁴ M^− 1. Taken together, the FAD–protein interaction is energetically favorable and the addition of FAD is not necessary to induce the enzyme folded state. For the first time, a detailed characterization of the flavin:protein interaction was performed among alternative NADH dehydrogenases.     

Heterogeneous binding of oxygen and carbon monoxide to dissociated molluscan hemocyanin

Functional heterogeneity in O2 or CO binding of sites of dissociated molluscan hemocyanin polypeptide chains (Helix pomatia and Octopus vulgaris) has been estimated by an analysis of accurate noncooperative binding curves. Three types of experiments were performed: pure O2 or CO binding, competitive displacement of one ligand by the other, and simultaneous removal of both gases from protein partially saturated with O2 and CO. The data were analyzed in terms of a model which has two fractions of sites with different properties for O2 and CO. The relative proportion of the different binding sites and their affinity constant values were found by the combined use of the three different procedures. All species show a marked functional heterogeneity of sites for O2 binding, while for CO binding it has been observed only in the case of H. pomatia beta-hemocyanin. Moreover, in all three molluscan hemocyanins examined, the two classes of O2-binding sites, although present in different proportions within the polypeptide chains, display similar affinity constant values. The data reported show a good consistency with results obtained using digested and isolated domains, providing confidence in the analytical procedure used. From comparison of the O2/CO affinity ratios (KO2, KCO) of each class it may be suggested that the difference in O2 affinity of two kinds of binding sites is related to a different local structure of the active sites. The results, moreover, unequivocally confirm that binding and displacement of two gaseous ligands to hemocyanin occur by a simple competitive mechanism, although the binding site is structurally complex and the two ligands are bound with different geometries.     

Cloning and sequence analysis of a human type A/B hnRNP protein

A cDNA encoding a 284 residue long type A/B hnRNP protein has been cloned. This protein, previously referred to as type C [(1987) J. Biol. Chem. 262, 17126-17137], is an RNA unwinding protein from HeLa 40S hnRNP with a high affinity for G- followed by U-rich sequences. The N-terminal part of the protein contains two consensus RNA binding domains present in a number of other RNA binding proteins. The C-terminal part is glycine-rich and contains a potential ATP/GTP binding loop. The distribution of charged amino acids is highly uneven and there are multiple potential phosphorylation sites.     

Chain length-dependent binding of fatty acid anions to human serum albumin studied by site-directed mutagenesis

Human serum albumin is the most abundant protein in the circulatory system, and one of its principal functions is to transport fatty acids. Binding of octanoate, decanoate, laurate and myristate was studied by a rate-of-dialysis technique. The primary association constants increased, but not linearly, with chain length. The number of high-affinity sites also increased with chain length; octanoate and decanoate bind to one such site, whereas laurate and myristate most probably bind to two sites. Albumin is composed of three homologous helical domains (I-III), which can be subdivided into two subdomains (A and B). For getting information about the positions of the high-affinity sites we produced 13 recombinant isoforms mutated in four different subdomains. Results obtained with these albumins are in accordance with the following model: octanoate and decanoate bind to a single site in subdomain IIIA, laurate binds to sites in subdomains IIIA and IIIB, whereas myristate binds in subdomains IB and IIIB. The results also showed that primary fatty acid binding is sensitive to amino acid substitutions in other parts of the protein. This is in contrast to the effect of amino acid substitutions of genetic albumin variants (alloalbumins). Usually these substitutions, which are situated at the surface of the protein, have no effect on fatty acid binding. Binding of fatty acid anions to different high-affinity sites and the sensitivity of these sites to amino acid substitutions elsewhere in the protein (and perhaps also to other types of modifications) are important factors that could effect simultaneous binding of other ligands, e.g. in patients treated with albumin-binding drugs.