Equilibrium Competition Experiments Research Articles

Biochemical and pharmacological characterization of three opioid-nociceptin hybrid peptide ligands reveals substantially differing modes of their actions

In an attempt to design opioid-nociceptin hybrid peptides, three novel bivalent ligands, H-YGGFGGGRYYRIK-NH2, H-YGGFRYYRIK-NH2 and Ac-RYYRIKGGGYGGFL-OH were synthesized and studied by biochemical, pharmacological, biophysical and molecular modelling tools. These chimeric molecules consist of YGGF sequence, a crucial motif in the N-terminus of natural opioid peptides, and Ac-RYYRIK-NH2, which was isolated from a combinatorial peptide library as an antagonist or partial agonist that inhibits the biological activity of the endogenously occurring heptadecapeptide nociceptin. Solution structures for the peptides were studied by analysing their circular dichroism spectra. Receptor binding affinities were measured by equilibrium competition experiments using four highly selective radioligands. G-protein activating properties of the multitarget peptides were estimated in [35S]GTPγS binding tests. The three compounds were also measured in electrically stimulated mouse vas deferens (MVD) bioassay. H-YGGFGGGRYYRIK-NH2 (BA55), carrying N-terminal opioid and C-terminal nociceptin-like sequences interconnected with GGG tripeptide spacer displayed a tendency of having either unordered or β-sheet structures, was moderately potent in MVD and possessed a NOP/KOP receptor preference. A similar peptide without spacer H-YGGFRYYRIK-NH2 (BA62) exhibited the weakest effect in MVD, more α-helical periodicity was present in its structure and it exhibited the most efficacious agonist actions in the G-protein stimulation assays. The third hybrid peptide Ac-RYYRIKGGGYGGFL-OH (BA61) unexpectedly displayed opioid receptor affinities, because the opioid message motif is hidden within the C-terminus. The designed chimeric peptide ligands presented in this study accommodate well into a group of multitarget opioid compounds that include opioid-non-opioid peptide dimer analogues, dual non-peptide dimers and mixed peptide- non-peptide bifunctional ligands.

Differential recognition of snake venom proteins expressing specific Arg-Gly-Asp (RGD) sequence motifs by wild-type and variant integrin αIIbβ3: further evidence for distinct sites of RGD ligand recognition exhibiting negative allostery

Several studies have demonstrated that the amino acid residues flanking the Arg-Gly-Asp (RGD) sequence of high-affinity ligands modulate their specificity of interaction with integrin complexes. Because of the absence of structural data for integrin complexes with bound ligand, the molecular basis for this specificity modulation remains obscure. In a previous paper [Rahman, Lu, Kakkar and Authi (1995) Biochem. J. 312, 223-232] we demonstrated that two genetically distinct venom-derived RGD proteins, kistrin and dendroaspin (both containing the sequence PRGDMP), were simple competitors, indicating the recognition of an identical binding site on the alpha(IIb)beta(3) complex. Furthermore, both kistrin and dendroaspin inhibited the binding of the disintegrin elegantin (containing the sequence ARGDNP) via a non-competitive mechanism, suggesting that the binding of elegantin to the alpha(IIb)beta(3) complex was at a remote site and down-regulated via an allosteric mechanism. Here we present further evidence for distinct RGD ligand recognition sites on the alpha(IIb)beta(3) complex that exhibit a negative allosteric relationship. A panel of well-characterized recombinant dendroaspin and elegantin derivatives were employed for this study. These recombinant molecules were constructed as glutathione S-transferase fusion proteins with either an Ala or Pro residue N-terminal to the RGD sequence in combination with either a Met or an Asn residue immediately C-terminal. Equilibrium competition experiments showed that elegantin binding to ADP-treated platelets was inhibited by derivatives Eleg. AM (ARGDMP) and Eleg. PM (PRGDMP) via an allosteric competitive mechanism, providing direct evidence that modulation of the RGD motif can alter competitive behaviour. In addition, recombinant kistrin and dendroaspin both inhibited elegantin binding via a non-competitive mechanism, confirming our previous observations. Further evidence for distinct binding sites employing an independent approach was obtained by analysing the binding of the panel of venom proteins to the functionally defective heterodimer alpha(IIb)beta(3) Ser(123)-->Ala expressed on Chinese hamster ovary cells. These studies demonstrated that simple competitors kistrin and dendroaspin bound with high affinity to the variant integrin complex. In contrast, the binding of elegantin and most significantly, recombinant Dendro. PN (PRGDNP) and Dendro. AN (ARGDNP) were abolished. These observations, taken together, are consistent with a model depicting the presence of distinct sites of RGD ligand recognition on the alpha(IIb)beta(3) complex that show the preferential recognition of specific RGD motifs. Competition experiments demonstrate a negative allosteric relationship between these RGD recognition sites.

Differential recognition of snake venom proteins expressing specific Arg-Gly-Asp (RGD) sequence motifs by wild-type and variant integrin αIIbβ3: further evidence for distinct sites of RGD ligand recognition exhibiting negative allostery

Several studies have demonstrated that the amino acid residues flanking the Arg-Gly-Asp (RGD) sequence of high-affinity ligands modulate their specificity of interaction with integrin complexes. Because of the absence of structural data for integrin complexes with bound ligand, the molecular basis for this specificity modulation remains obscure. In a previous paper [Rahman, Lu, Kakkar and Authi (1995) Biochem. J. 312, 223-232] we demonstrated that two genetically distinct venom-derived RGD proteins, kistrin and dendroaspin (both containing the sequence PRGDMP), were simple competitors, indicating the recognition of an identical binding site on the αIIbβ3 complex. Furthermore, both kistrin and dendroaspin inhibited the binding of the disintegrin elegantin (containing the sequence ARGDNP) via a non-competitive mechanism, suggesting that the binding of elegantin to the αIIbβ3 complex was at a remote site and down-regulated via an allosteric mechanism. Here we present further evidence for distinct RGD ligand recognition sites on the αIIbβ3 complex that exhibit a negative allosteric relationship. A panel of well-characterized recombinant dendroaspin and elegantin derivatives were employed for this study. These recombinant molecules were constructed as glutathione S-transferase fusion proteins with either an Ala or Pro residue N-terminal to the RGD sequence in combination with either a Met or an Asn residue immediately C-terminal. Equilibrium competition experiments showed that elegantin binding to ADP-treated platelets was inhibited by derivatives Eleg. AM (ARGDMP) and Eleg. PM (PRGDMP) via an allosteric competitive mechanism, providing direct evidence that modulation of the RGD motif can alter competitive behaviour. In addition, recombinant kistrin and dendroaspin both inhibited elegantin binding via a non-competitive mechanism, confirming our previous observations. Further evidence for distinct binding sites employing an independent approach was obtained by analysing the binding of the panel of venom proteins to the functionally defective heterodimer αIIbβ3 Ser123 → Ala expressed on Chinese hamster ovary cells. These studies demonstrated that simple competitors kistrin and dendroaspin bound with high affinity to the variant integrin complex. In contrast, the binding of elegantin and most significantly, recombinant Dendro. PN (PRGDNP) and Dendro. AN (ARGDNP) were abolished. These observations, taken together, are consistent with a model depicting the presence of distinct sites of RGD ligand recognition on the αIIbβ3 complex that show the preferential recognition of specific RGD motifs. Competition experiments demonstrate a negative allosteric relationship between these RGD recognition sites.

Pharmacological characterization of adenosine A1 receptors and its functional role in brown trout ( Salmo trutta) brain

The adenosine receptor agonist N 6-cyclohexyl[ 3 H ]adenosine ([ 3 H ]CHA) was used to identify and pharmacologically characterize adenosine A1 receptors in brown trout ( Salmo trutta) brain. In membranes prepared from trout whole brain, the A1 receptor agonist [ 3 H ]CHA bound saturably, reversibly and with high affinity ( K d=0.69±0.04 nM; B max=0.624±0.012 pmol/mg protein) to a single class of binding sites. In equilibrium competition experiments, the adenosine agonists and antagonists all displaced [ 3 H ]CHA from high-affinity binding sites with the rank order of potency characteristic for an adenosine A1 receptors. A1 receptor density appeared not age-related (from 3 months until 4 years), and was similar in different brain areas. The specific binding was inhibited by guanosine 5′-triphosphate (IC 50=0.778±0.067 μM). GTP (5 μM) induced a low affinity state of A1 receptors. In superfused trout cerebral synaptosomes, 30 mM K + stimulated the release of glutamate in a calcium dependent manner. Glutamate-evoked release was dose-dependently reduced by CHA, and the inhibition was reversed by the A1 antagonist 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, 30 mM K + as well as 1 mM glutamate stimulated the release of adenosine in a Ca 2+-independent manner and tetrodotoxin insensitive. These findings show that in trout brain adenosine A1 receptors are present which are involved in the modulation of glutamate transmitter release. Moreover, the stimulation of adenosine release by K + depolarisation or glutamate support the hypothesis that, as in mammalian brain, a cross-talk between adenosine and glutamate systems exists also in trout brain.

Spectrophotometric Study of Mithramycin Complexes with Cu(II), Fe(III) and Tb(III)

The coordination of the anticancer drug mithramycin to Tb(III), Fe(III) and Cu(II) was studied in aqueous solution using absorption measurements. The stability constants were calculated from equilibrium competition experiments by means of the SQUAD program. For both first ions, the competitor was oxalic acid and for the latter the competitor was the Cu(II) ions. Cu(II) at pH 7.5, Fe(III) at pH 3.5 and Tb(III) at pH 5.5 formed respectively 1:2, 1:3 and 1:4 metal-to-ligand species.

Biochemical and pharmacological characterization of adenosine A1 receptors in eel (Anguilla anguilla) brain

N6-cyclohexyl[3H]adenosine ([3H]CHA) was used to label adenosine A1 receptors in membranes prepared from male and female eel whole brain. The A1 receptor agonist [3H]CHA bound saturably, reversibly and with high affinity (Kd = 0.91 ± 0.12 nM; Bmax = 120.36 ± 5.2 fmol mg−1 protein). In equilibrium competition experiments, the adenosine agonists and antagonists all displaced [3H]CHA from high-affinity binding sites with the rank order of potency in displacing, characteristics of an A1 adenosine receptor. Mg2+ dramatically increased the affinity of [3H]CHA without modifying the maximal binding capacity. The specific binding was inhibited by guanosine 5′-triphosphate (Ki = 2.54 ± 0.98 μM). The [3H]CHA binding sites are ubiquitously distributed with a maximum in cerebellum and a minimum in olfactory bulb. No difference was observed between male and female brain. In eel brain, synaptosomes (P2), stimulation of adenosine 3′,5′-monophosphate (cyclic AMP) accumulation with 10−5 M forskolin was markedly reduced (45.5%) by treatment with the adenosine A1 receptor agonist CHA (10−4 M), and the reduction was reversed in presence of the selective A1 receptor antagonist 8-cyclopentyltheophylline (10−5 M). In superfused eel cerebellar synaptosomes, K+ stimulated the release of adenosine in a partially Ca2+-dependent manner. The findings, taken together, suggest the hypothesis that adenosine A1 receptors present in eel brain could modulate synaptic transmission, as A1 receptors do in other vertebrates.

Characterization of adenosine receptors in Mullus surmuletus

The intent of the present study was to investigate adenosine receptor sites in brain membranes of the saltwater teleost fish, Mullus surmuletus, using the A 1 receptor selective agonist, [ 3H]CHA, and A 2a receptor selective agonist [ 3H]CGS 21680. The A 1 selective agonist, [ 3H]CHA, bound saturably, reversibly and with high affinity to a single-class of binding sites (K d 1.47 nM; B max 100–190 fmol/mg protein, dependent on fish length). The A 2a selective agonist, [ 3H]CGS 21680, also bound saturably, reversibly and with relative high affinity to a single-class of binding sites (K d 44.2 nM; B max 150–300 fmol/mg protein dependent on fish length). In equilibrium competition experiments, adenosine analogous, NECA, CGS 21680, CHA, CPA, S-PIA, R-PIA, CPCA, DPMA, and xanthine antagonists, DPCPX, XAC, and THEO all displaced [ 3H]CHA and [ 3H]CGS 21680 specifically bound to brain membranes from Mullus surmuletus. Specific binding of both [ 3H]CHA and [ 3H]CGS 21680 was inhibited by GDPβS. For [ 3H]CHA the IC 50 value was 2.5 ± 0.1 μM, while for [ 3H]CGS 21680 the IC 50 value was 7.7 ± 0.3 μM. Our results indicate that the high affinity binding sites for [ 3H]CHA have some pharmacological characteristics of mammalian A 1 adenosine receptors, while the binding sites for [ 3H]CGS 21680 appear to be virtually identical to the binding sites for [ 3H]CHA.

Characterization of the peripheral-type benzodiazepine receptors in cultured astrocytes: evidence for multiplicity.

In mammalian brain peripheral benzodiazepine (PBZD) receptors are predominantly localized on astroglial cells. Previous studies utilizing whole membrane preparations from brain and peripheral organs of various species have indicated several distinctions between the drug-receptor interactions of the two prototypic PBZD receptor ligands, PK 11195 and Ro5-4864. The present study was undertaken to determine whether putative differences in the binding of PBZD receptor ligands in homogenates of primary astrocyte cultures can be interpreted as the labeling of PBZD receptor subtypes. Equilibrium competition and saturation binding experiments in homogenate preparations of primary astrocytes from cerebral cortex of new born rats revealed that [3H]PK 11195 labels twice the number of [3H]Ro5-4864 binding sites. Unlabeled Ro5-4864 competes for [3H]PK 11195 binding in a manner suggesting the existence of multiple PK 11195 binding sites. The competition binding experiments, using various benzodiazepines, indicate that one binding component of PK 11195 corresponds to Ro5-4864 binding sites, whereas the second is different. The latter binding site does not correspond to the central BZD receptor but displays the pharmacological properties of the PBZD receptor. Further differences between the binding of PK 11195 and Ro5-4864 in astrocytes were detected in the presence of ethanol which was more effective in inhibiting the binding of the latter. Subcellular distribution studies indicated, however, that the binding of both [3H]PK 11195 and [3H]Ro5-4864 is associated primarily with the mitochondrial fraction of astrocytes. Taken together, the present study indicates the existence of non-overlapping PBZD binding sites in astrocytes and thus suggests the existence of PBZD receptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of A 1 adenosine receptors in membranes from whole goldfish brain

1. 1. A 1 adenosine receptor binding was investigated using the selective agonist [ 3H]-cyclohexl-adenosine ([ 3H]CHA) on membranes prepared from adult (12–14 cm length) goldfish whole brain. 2. 2. The A 1 receptor agonist [ 3H]-cyclohexyladenosine bound saturably, reversibly and with high affinity ( K d = 1.20 ± 0.08 nM; B max = 6.8 ± 3.02 fmol/mg protein). 3. 3. The specific binding was increased by Mg 2+ and inhibited by guanosine 5′-triphosphate ( K i = 5.81 ± 1.54 μM). 4. 4. In equilibrium competition experiments, the adenosine analogs R-phenylisopropyladenosine, cyclohexyladenosine, N-ethylcarboxamidoadenosine, 2-chloroadenosine, S-phenylisopropyladenosine and the antagonists 8-cyclopentyl-1,3-dipropylxanthine and theophylline all displaced [ 3H]-cyclohexyladenosine from high-affinity binding sites with the rank order of potency in displacing characteristics of an A 1 adenosine receptor.

Specificity and stringency in DNA triplex formation.

Triple-helix formation can in principle serve as a general method for sequence-specific recognition and physical separation of duplex DNA molecules. Realization of this goal depends on how much the triplex is destabilized by mismatches and other defects (specificity) and on finding conditions in which perfect complexes are stable and defect complexes are not (stringency). We have addressed the question of specificity by determining the difference in free energy between perfect and defect complexes by using UV melting curves and equilibrium competition experiments. We find that third strands that bind with either single-base bulges or single mismatches are destabilized relative to the perfect triplex by 2.5-2.9 and 3.2-4.0 kcal/mol (1 cal = 4.184 J), respectively, essentially equivalent to the corresponding values determined for duplex DNA and RNA. Also, we present a method, referred to as stringency clamping, which maintains specific binding under conditions far from normal stringency. To do this, we provide for the formation of a competing structure involving the third strand with stability between that of the perfect and imperfect complexes; the competitive interaction effectively prevents triplex formation at imperfect sites even far below their melting temperature. We illustrate the phenomenon with three different stringency clamps, two of which compete for the all-pyrimidine third strand through Watson-Crick pairing and one that competes through all-pyrimidine pairing at acidic pH. We demonstrate physical separation of two duplex DNA molecules differing by a single base pair in their target sequence for triple-helix formation.

Identification and initial characterization of high-affinity [ 3H]dextrorphan binding sites in rat brain

We have identified specific high-affinity [ 3H]dextrorphan binding sites in rat forebrain. [ 3H]Dextrorphan binds saturably and reversibly to an apparently homogeneous class of sites characterized by a B max of 2.62±0.06 pmol/mg protein and K D of 60±4 nM. Glycine and glutamate independently increase [ 3H]dextrorphan binding in a concentration-dependent manner. The pharmacological profile of [ 3H]dextrorphan binding characterized by equilibrium competition experiments, together with these data suggest that [ 3H]dextrorphan labels a site at or near the N-methyl-D-aspartate receptor.

Differential binding properties of the peripheral-type benzodiazepine ligands [3H]PK 11195 and [3H]Ro 5-4864 in trout and mouse brain membranes.

High-affinity binding sites for [3H]PK 11195 have been detected in brain membranes of rainbow trout (Salmo gairdneri) and mouse forebrain, where the densities of receptors were 1,030 and 445 fmol/mg of protein, respectively. Ro 5-4864 (4'-chlorodiazepam) was 2,200-fold less potent as a competitor of [3H]PK 11195 binding in the piscine than the murine membranes. Investigation of the regional distribution of these sites in trout yielded a rank order of density of spinal cord greater than olfactory bulb = optic tectum = rhombencephalon greater than cerebellum greater than telencephalon. This site in trout shared some of the characteristics of the peripheral-type benzodiazepine receptor (PTBR) (also known as the mitochondrial benzodiazepine receptor) in rodents, i.e., high affinity for PK 11195 and the endogenous ligand protoporphyrin IX, but was unique in the low affinity of Ro 5-4864 (41 microM) and diazepam and the relatively high affinity of the calcium channel ligand diltiazem and two central benzodiazepine ligands, CGS 8216 and CGS 9896. The differential affinity for the two prototypic PTBR ligands in trout is similar to that previously observed in calf and human brain membranes. Structural differences for the trout sites are indicated by the relative inability of diethyl pyrocarbonate to modify histidine residues of the binding site in trout as compared with mouse membranes. Heterogeneity of binding of the two prototypic PTBR ligands in mouse brain membranes was indicated by additivity studies, equilibrium competition experiments, and saturation isotherms, which together support the hypothesis that Ro 5-4864 discriminates between two [3H]PK 11195 binding sites having high (nanomolar) and low (micromolar) affinity, respectively.

Evolutionary Temperature Adaptation of Agonist Binding to the A1Adenosine Receptor

The effects of temperature on the binding of the agonist [3H]cyclohexyladenosine to A1 adenosine receptors were studied by equilibrium binding techniques in brain membranes from eight vertebrate species with average body temperatures from 1 to 40°C. Kd values for rat and chicken increased markedly as measurement temperature decreased. In contrast, the Kd values for six teleost species, including warm-adapted, cold-adapted, and deep-living species, were much less sensitive to temperature perturbation. At 5°C Kd values vary 30-fold among the species; however, at temperatures approximating the cell temperatures of the species there is only a four-fold range of values. Binding enthalpies varied in sign and magnitude among the species. Binding entropies were positive for all the species; values were largest for the warm-adapted species, and smallest for the deep-living fishes. Bmax values were relatively insensitive to temperature changes. MgCl2 significantly increased Bmax values, and for two of three species, lowered Kd values. MgCl2 did not alter the enthalpy changes. In equilibrium competition experiments at 5°C using brain membranes from the deep-living teleost Antimora rostrata, the adenosine analogs R-phenylisopropyladenosine, N-ethylcarboxamidoadenosine, and 2-chloroadenosine were approximately 23-fold more potent than S-phenylisopropyladenosine. Despite perturbation by low temperature of agonist binding to mammalian and avian A1 adenosine receptors, agonist recognition and binding properties of the A1 receptor have been retained in vertebrates adapted to different body temperatures. These adaptive trends mirror those noted in studies of soluble enzyme homologs and muscle actins from species adapted to different temperatures.

Calmodulin binding by calcineurin. Ligand-induced renaturation of protein immobilized on nitrocellulose.

The interaction of calmodulin with calcineurin, a calcium- and calmodulin-stimulated protein phosphatase, was investigated using a solid-phase assay. Binding of 125I-calmodulin by calcineurin immobilized on nitrocellulose membrane filters was of high affinity, reversible, and calcium-dependent. Complex binding kinetics reflected a time- and calcium/calmodulin-dependent conformational change of calcineurin which was shown to be ligand-induced renaturation. After renaturation and removal of calmodulin, immobilized calcineurin exhibited simple 125I-calmodulin binding kinetics with a single class of independent sites. The maximum stoichiometry of 125I-calmodulin binding to immobilized calcineurin was 0.1 mol/mol. The association rate (K1 = 8.9 x 10(3) M-1 S-1) and the dissociation rate (K-1 = 8.5 x 10(-5) s-1) yielded a dissociation constant of Kd = 10 nM. Equilibrium binding analyses gave a Kd value of 16 nM. The affinity of 125I-calmodulin for immobilized calcineurin was half that of unmodified calmodulin. Using equilibrium competition experiments, we determined, for the first time, the dissociation constant for the binding of native calmodulin by calcineurin in solution, Kd less than or equal to 0.1 nM (Kd for 125I-calmodulin = 0.23 +/- 0.09 nM). The effects of ionic strength and pH on 125I-calmodulin binding to immobilized calcineurin were characterized. The dissociation rate was dependent on free calcium concentration, with half-maximal rate at 700 nM calcium. 125I-Calmodulin equilibrium binding by the immobilized A subunit of calcineurin exhibited half the affinity of the holoenzyme, Kd = 30 nM. The described phenomenon, of reversible denaturation associated with immobilization of a protein on nitrocellulose, may be a general one open to exploitation in other systems.

Nuclear thyroid hormone receptors in cultured bone cells

Thyroid hormones influence bone metabolism, but a direct interaction of triiodothyronine with nuclear T 3 receptors in bone cells has not yet been reported. We investigated 125I-T 3 binding to nuclei isolated from the cloned osteoblastlike rat osteosarcoma cells ROS 17 2.8 . At 37°C, saturable 125I-T 3 binding to isolated nuclei reached equilibrium by 30 minutes and was completely displaced upon the addition of 500 nmol/L unlabeled T 3. Nonsaturable binding represented about 0.5% of the radioactivity added (20% of the total binding). Thyroxine and 3,3′,5′triiodothyronine competed with 125I-T 3 with a 20-fold and 400-fold lower affinity than T 3, respectively. Analysis of equilibrium competition experiments revealed the presence of a single class of homogeneous binding sites with an association constant of 5.0 ± 0.3 × 10 9 mol/L −1 and a maximum nuclear binding capacity of 0.13 ± 0.02 ng/mg DNA. A twofold increase of bone Gla protein (BGP) secretion was observed with T 3 treatment suggesting that these T 3 nuclear receptors are coupled with a biological response.

Properties of a DNA-binding protein in a culture medium of thymus cells.

A DNA-binding protein, which migrated as one major protein band, with a molecular weight of 14,000, on sodium dodecylsulfate polyacrylamide gel, was purified from a culture medium of mouse thymus cells. The interaction of the isolated protein with DNA in vitro was assayed by a nitrocellulose filter binding technique. Equilibrium competition experiments demonstrated that the DNA-binding protein had the ability to differentiate among sequences of polynucleotides, indicating that the DNA-binding protein-DNA interaction was at least partially specific. This protein increased the helix melting temperature of DNA and inhibited the incorporation of [3H]dTMP into DNA by the DNA polymerase of calf thymus in vitro.

Involvement of outside DNA sequences in the major kinetic path by which EcoRI endonuclease locates and leaves its recognition sequence.

We have examined the kinetics of the interaction between endodeoxyribonuclease EcoRI (EC 3.1.23.13) and nine linear DNA fragments that range in size between 34 and 6,200 base pairs and contain the EcoRI site of plasmid pBR322 in a central location. The kinetic parameters governing both formation and decay of specific endonuclease . DNA complexes increase 8-fold with increasing chain length over this size range. In contrast, equilibrium competition experiments demonstrated that the intrinsic affinity of endonuclease for its recognition sequence is independent of DNA chain length over this range. Thus, DNA sequences outside the recognition site enhance the rate at which EcoRI endonuclease locates or leaves its recognition site without affecting the intrinsic thermodynamic parameters of site-specific interaction. These results are consistent with a facilitated diffusion mechanism for specific DNA site location by this enzyme.

Fibronectin: a chromatin-associated protein?

We have previously reported that chromatin preparations from human cultured fibroblasts contain a single homologous serum protein. In this paper we present evidence, based on immunological identity and physicochemical properties, that this serum protein is fibronectin. Furthermore, using a radioimmunoassay system, we have estimated that fibronectin represents about 0.7% of the total protein in both chromatin preparations and whole fibroblasts. Using a nitrocellulose filter assay system, we also show that fibronectin is a DNA-binding protein having an equilibrium constant of 4.6 × 10 −6 M. Equilibrium competition experiments have demonstrated that fibronectin has the ability to differentiate among nucleotides, indicating that fibronectin-DNA interaction is at least partially specific, and that a minimum polymer length of 12–18 nucleotides is required for effective binding to occur. Fibronectin has been isolated readily from plasma using DNA-affinity chromatography. We do not have direct evidence that fibronectin is an actual nonhistone chromosomal protein, but fibronectin is a DNA-binding protein (at least under in vitro assay conditions) and appears to be a normal constituent of chromatin as chromatin is currently isolated from cell nuclei.

Binding specificity of the two major DNA-binding proteins in human serum.

The two major DNA-binding proteins of human serum (DNA-binding protein 1 and DNA-binding protein 2) were shown to bind preferentially to single-stranded polynucleotides rich in guanine residues. Equilibrium competition experiments using a nitrocellulose filter assay system containing labeled human lymphocyte DNA and various competing natural and synthetic polynucleotides indicated that both proteins recognized sequences of bases containing a keto group in either position 6 (purines) or 4 (pyrimidines) and that these keto groups must be readily accessible for effective binding to occur. Guanine was shown to be the preferred nucleotide through inhibition experiments using a series of synthetic homopolymers and a series of bacterial DNAs of differing G + C content. The relationship between protein affinity and G + C content was shown to be directly proportional. The equilibrium constants for the binding of the human lymphocyte DNA by both proteins were on the order of 10(-6) M, and the length of the nucleotide sequence necessary for effective binding was found to be 12 to 18 bases using a series of oligomers of poly(dG).

Estrogen receptor has enhanced affinity for bromodeoxyuridine-substituted DNA.

The estrogen receptor (E2R) from rabbit uterus has an enhanced affinity for BrdUrd-substituted DNA compared to unsubstituted DNA. Increasing levels of BrdUrd substitution (from 0 to 100%) in a DNA sample are associated with increasingly tighter binding of E2R to that DNA as measured by equilibrium competition experiments and by rates of dissociation (or receptor transfer experiments). Although the rates of dissociation of E2R-DNA complexes vary greatly, depending on the BrdUrd-substitution level in the DNA, no differences were detected between the rates of association of E2R with unsubstituted and fully BrdUrd-substituted DNA. The E2R-DNA complex dissociates more rapidly in 150 mM KCl than in 50 mM KCl; but, at both ionic strengths, BrdUrd substitution in the DNA confers enhanced stability on the complex. The demonstration that a specific mammalian regulatory protein has an enhanced affinity for BrdUrd-substituted DNA further strengthens the possibility that BrdUrd modulates gene expression through an altered binding of regulatory proteins.