Identification of an octapeptide involved in homophilic interaction of the cell adhesion molecule gp80 of dictyostelium discoideum

During development of Dictyostelium discoideum, a surface glycoprotein of Mr 80,000 (gp80) is known to mediate EDTA-resistant cell-cell adhesion via homophilic interaction. Antibodies directed against a 13 amino acid sequence (13-mer) near the NH2 terminus of the protein were found to inhibit cell reassociation. This 13-mer also inhibited gp80-cell interaction and gp80-gp80 interaction. The cell binding site was mapped to the octapeptide sequence YKLNVNDS by using shorter peptide sequences to inhibit gp80 interaction. High salt concentrations inhibited homophilic interactions of both the 13-mer and gp80, suggesting that ionic interactions are involved in the forward binding reaction. Since disruption of homophilic interactions between the bound molecules required the presence of Triton X-100, hydrophobic interactions may occur after the initial ionic binding.     

Expression of multivalency in the affinity chromatography of antibodies. Appendix: Derivation and evaluation of equations for independent bivalent interacting systems in quantitative affinity chromatography.

The expression of multivalency in the interaction of antibody with immobilized antigen was evaluated by quantitative affinity chromatography. Zones of radioisotopically labeled bivalent immunoglobulin A monomer derived from the myeloma protein TEPC 15 were eluted from columns of phosphorylcholine-Sepharose both in the absence and presence of competing soluble phosphorylcholine. At sufficient immobilized phosphorylcholine concentration, the variation of elution volume of bivalent monomer with soluble ligand was found to deviate from that observed for the univalent binding of the corresponding Fab fragment. In addition, the apparent binding affinity of the bivalent monomer increased with immobilized antigen density. Use of equations relating the variation of elution volume with free ligand concentration for a bivalent binding protein allowed calculation of microscopic single-site binding parameters for the bivalent monomeric antibody to both immobilized and soluble phosphorylcholine. The chromatographic data not only demonstrate the effect of multivalency on apparent binding affinity but also offer a relatively simple means to measure microscopic dissociation constants for proteins participating in bivalent interactions with their ligands.     

Affinity chromatography of GroEL chaperonin based on denatured proteins: Role of electrostatic interactions in regulation of GroEL affinity for protein substrates

The chaperonin GroEL of the heat shock protein family from Escherichia coli cells can bind various polypeptides lacking rigid tertiary structure and thus prevent their nonspecific association and provide for acquisition of native conformation. In the present work we studied the interaction of GroEL with six denatured proteins (alpha-lactalbumin, ribonuclease A, egg lysozyme in the presence of dithiothreitol, pepsin, beta-casein, and apocytochrome c) possessing negative or positive total charge at neutral pH values and different in hydrophobicity (affinity for a hydrophobic probe ANS). To prevent the influence of nonspecific association of non-native proteins on their interaction with GroEL and make easier the recording of the complexing, the proteins were covalently attached to BrCN-activated Sepharose. At low ionic strength (lower than 60 mM), tight binding of the negatively charged denatured proteins with GroEL (which is also negatively charged) needed relatively low concentrations (approximately 10 mM) of bivalent cations Mg2+ or Ca2+. At the high ionic strength (approximately 600 mM), a tight complex was produced also in the absence of bivalent cations. In contrast, positively charged denatured proteins tightly interacted with GroEL irrespectively of the presence of bivalent cations and ionic strength of the solution (from 20 to 600 mM). These features of GroEL interaction with positively and negatively charged denatured proteins were confirmed by polarized fluorescence (fluorescence anisotropy). The findings suggest that the affinity of GroEL for denatured proteins can be determined by the balance of hydrophobic and electrostatic interactions.     

Accessibility of histone oligomers to the action of trypsin in a solution or in chromatin with different degrees of compactness

The accessibility to trypsin of "core" histones within the dimer (H2A-H2B), tetramer (H3-H4)2, octamer (H2A-H2B-H3-H4)2 and in chromatin was studied. It was shown that the hydrolysis of histones H2A and H2B within the dimer and octamer occurs in essentially the same way. The tetramer (H2-H4)2 becomes more compact with an increase in the ionic strength. Some of the tetramer (H3-H4)2 sites within the octamer are protected against trypsin. It was demonstrated that in terms of the histone accessibility to trypsin chromatin can exist in three states, i.e., tightly packed (in the presence of histone H1 and bivalent cations), intermediate (in the absence of histone H1 or bivalent cations) and folded (in the absence of histone H1 and bivalent cations). The folding of histones in neither of these chromatin states coincides with that within the octamer in 2M NaCl.     

The labeling with 8-azido-cyclic adenosine monophosphate of proteins in vesicles of sarcoplasmic reticulum from rabbit skeletal muscle

The technique of laser Doppler electrophoresis was applied for the study of the surface charge properties of (NA+, K+)-ATPase containing microsomal vesicles derived from guinea-pig kidney. The influence of pH, the screening and binding of uni- and divalent cations and the binding of ATP show: (1) one net negative charge per protein unit with a pK = 3.9; (2) deviation from the Debye relation between surface potential and ionic strength for univalent cations, with no difference in the effect of Na+ and K+; (3) Mg2+ binds with an association constant of Ka = 1.1. 10(2) M-1 while ATP binds with an apparent Ka = 1.1.10(4) M-1 for 1 mM NaCl, 0.2 mM KCI, 0.1 mM MgCl2, 0.1 mM Tris-HCl2, 0.1 mM Tris-HCl (pH 7.3). The binding is weaker at higher Mg2+ concentrations. There is no ATP binding in the absence of Mg2+. In addition, the average vesicle size derived from the linewidth of the quasielastic light scattering spectrum is 203.7 +/- 15.2 nm. In the presence of ATP a reduction in size is observed.     

The alkaline phospholipase A1 of rat liver cytosol.

1. Rat liver cytosol contains a heat-sensitive phospholipase A1 active against phosphatidylethanolamine, 1-acylglycerophosphoethanolamine and, to a very much lesser extent, phosphatidylcholine and phosphatidylinositol. 2. Activity towards a pure phosphatidylethanolamine substrate is invoked by the presence of water-soluble cations that do not precipitate at the pH optimum of the enzyme (9.5). In this activation bivalent cations, e.g. Mg2+, Ca2+, Mn2+, Sr2+ and Ba2+, are effective at much lower concentrations (2.5-5 mM) than univalent cations K+, Na+ and NH4+ (100 mM). 3. In the absence of such cations the enzyme can be activated by cationic amphiphiles containing quaternary nitrogen or by basic proteins. 4. It is concluded that these agents activate the enzyme by reducing the negative zeta potential on the substrate at the high pH optimum (9.5) and allow interaction with the enzyme whose isoelectric point is at 7.15. 5. The activated enzyme is markedly inhibited by mixing the phosphatidylethanolamine substrate with many other phospholipids that exist in cell membranes, e.g. phosphatidylcholine, phosphatidylinositol. On the other hand, both phosphatidylcholine and phosphatidylinositol can be hydrolysed much more readily if they are mixed with an excess of phosphatidylethanolamine. 6. Such results on the inhibition and substrate specificity of the enzyme, coupled with birefringence measurements, allow the tentative conclusion that phospholipid substrates are only attacked when they exist in a hexagonal or non-bilayer structure and not in the bilayer (lamellar) form.     

Acetylcholinesterase as polyelectrolyte in reaction with cationic substrates

It is shown that the salt effect in acetylcholinesterase-catalyzed hydrolysis of 2-(N-methylmorpholinium)-ethylacetate can be quantitatively described by the equation log(k2/KS) = log(k2/KS) degrees--psi log[M+Z] following from Manning's polyelectrolyte theory; the psi values for salts with univalent and bivalent cations at different pH values of the reaction medium were in accordance with the conclusions of the theory. Manning's polyelectrolyte theory seems to be a useful framework for studying salt effects in the reactions of charged substrates with enzymes as globular polyions.     

Interaction of bile salt monomer and cholesterol in the aqueous phase

The purpose of the present study was to evaluate the possible interaction of bile salt monomer and cholesterol in the intermicellar aqueous phase. Cholesterol and taurocholate monomer concentrations in the intermicellar aqueous phase were determined using 0-20 mM taurocholate solutions saturated with cholesterol. Maximal solubilities of cholesterol in aqueous solutions having various concentrations of taurocholate, especially below its intermicellar monomer concentration (critical micellar concentration), were determined and compared with the intermicellar cholesterol concentration. The intermicellar monomer concentration of taurocholate was constant (6 mM) and independent of taurocholate concentrations. The cholesterol concentration in the intermicellar aqueous phase gradually increased, depending upon taurocholate concentrations, and became constant (1,3 microM) above 10 mM taurocholate. The solubility of cholesterol increased linearly with the taurocholate concentration even below the critical micellar concentration, and was 0.3 microM at 6 mM taurocholate, which was approx. 20-times higher than the aqueous solubility of cholesterol, but a fifth of the maximal intermicellar cholesterol concentration. The results indicate that the higher cholesterol concentration in the intermicellar aqueous phase compared to its aqueous solubility can be primarily ascribed to the interaction of cholesterol with bile salt monomers possibly forming bile salt-cholesterol dimers, and partly to the sustaining forces induced by numerous micelles.     

Formation of D loops by the UvsX protein of T4 bacteriophage: a comparison of the reaction catalyzed in the presence or absence of gene 32 protein

The UvsX protein of T4 bacteriophage will catalyze the formation of D loops between linear single-stranded DNA (ssDNA) and homologous supercoiled double-stranded DNA (dsDNA) in the absence of T4 gene 32 protein (gp32). This reaction requires one monomer of UvsX protein per three nucleotides of ssDNA so that the ssDNA is completely covered with UvsX protein. Under these conditions, high rates of ATP hydrolysis are observed, and one-third of the products are joined paranemically. The reaction proceeds through a mechanism that creates homology-independent coaggregates of UvsX protein, dsDNA, and ssDNA. When UvsX protein is added to only 1 monomer per 8 nucleotides, but with 1 monomer of gp32 per 12 nucleotides, the rate of ATP hydrolysis is depressed, but D-loop formation is enhanced. Nearly all of the product is bound in plectonemic joints, and no coaggregated intermediates are formed. Coaggregate formation at high concentrations of UvsX protein is not inhibited by the presence of gp32; gp32 simply allows for efficient formation of D loops at such low concentrations of UvsX protein that coaggregates are not constructed. Electron microscopic visualization of the joint structures in this reaction reveals that both gp32 and UvsX protein are bound to the ssDNA. The single-stranded DNA binding (SSB) protein of Escherichia coli will substitute only partially for gp32: in the presence of SSB protein, D-loop formation can be catalyzed at one UvsX protein monomer per eight nucleotides, and it is accomplished without the formation of coaggregates, but a major portion of the product is joined paranemically.     

Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes

Analysis of the equilibrium binding of [3H]-neurotensin(1-13) at 25 degrees C to its receptor sites in bovine cortex membranes indicated a single population of sites with an apparent equilibrium dissociation constant (KD) of 3.3 nM and a density (Bmax) of 350 fmol/mg protein (Hill coefficient nH = 0.97). Kinetic dissociation studies revealed the presence of a second class of sites comprising less than 10% of the total. KD values of 0.3 and 2.0 nM were obtained for the higher and lower affinity classes of sites, respectively, from association-dissociation kinetic studies. The binding of [3H]neurotensin was decreased by cations (monovalent and divalent) and by a nonhydrolysable guanine nucleotide analogue. Competition studies gave a potency ranking of [Gln4]neurotensin greater than neurotensin(8-13) greater than neurotensin(1-13). Smaller neurotensin analogues and neurotensin-like peptides were unable to compete with [3H]neurotensin. Stable binding activity for [3H]neurotensin in detergent solution (Kd = 5.5 nM, Bmax = 250 fmol/mg protein, nH = 1.0) was obtained in 2% digitonin/1 mM Mg2+ extracts of membranes which had been preincubated (25 degrees C, 1 h) with 1 mM Mg2+ prior to solubilization. Association-dissociation kinetic studies then revealed the presence of two classes of sites (KD1 = 0.5 nM, KD2 = 3.6 nM) in a similar proportion to that found in the membranes. The solubilized [3H]-neurotensin activity retained its sensitivity to cations and guanine nucleotide.     

Measuring protein self-association using pulsed-field-gradient NMR spectroscopy: Application to myosin light chain 2

Summary At the millimolar concentrations required for structural studies, NMR spectra of the calcium-binding protein myosin light chain 2 (MLC2) showed resonance line widths indicative of extensive self-association. Pulsed-field-gradient (PFG) NMR spectroscopy was used to examine whether MLC2 aggregation could be prevented by the zwitterionic bile salt derivative 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). PFG NMR measurements indicated that CHAPS was capable of preventing MLC2 self-association, but only at concentrations well above the critical micelle concentration of 7.5 mM. CHAPS was most effective at a concentration of 22.5 mM, where the apparent molecular mass of MLC2 correponded to a protein monomer plus seven molecules of bound detergent. The resolution and sensitivity of 2D ¹⁵N-¹H HSQC spectra of MLC2 were markedly improved by the addition of 25 mM CHAPS, consistent with a reduction in aggregation following addition of the detergent. The average amide nitrogen T₂ value for MLC2 increased from 30 ms in the absence of CHAPS to 56 ms in the presence of 25 mM CHAPS. The results of this study lead us to propose that PFG NMR spectroscopy can be used as a facile alternative to conventional techniques such as analytical ultracentrifugation for examining the self-association of biological macromolecules.     

Effects of univalent cations on the activity of particulate starch synthetase

An investigation was made to determine the univalent cation requirements of starch synthetase from a variety of plant species of economic importance. The particulate enzyme from sweet corn was shown to have an absolute requirement for potassium, with the optimum activation occurring at 0.05 M KCl. Rubidium, cesium, and ammonium were 80% as effective as potassium while sodium and lithium were respectively 21% and 8% as effective as potassium. The K_A for potassium was determined to be 6 mM. In the case of the particulate starch synthetase from wheat, bush beans, field corn, soybeans, peas, or potatoes, considerable stimulation of enzyme activity was obtained by the addition of potassium to the reaction mixture. In these studies, low enzyme activity was observed in the absence of added potassium, but the content of endogenous univalent cations in the reactions may be sufficient to account for the activities observed. Anions of various types had no effect on starch synthetase activity. Divalent cations produced slight activation in the presence or absence of potassium. All efforts to show a potassium requirement for glycogen synthetase from rat liver have been negative.     

Cationic screening of charged surface groups (carboxylates) affects electron transfer steps in photosystem-II water oxidation and quinone reduction

The functional or regulatory role of long-distance interactions between protein surface and interior represents an insufficiently understood aspect of protein function. Cationic screening of surface charges determines the morphology of thylakoid membrane stacks. We show that it also influences directly the light-driven reactions in the interior of photosystem II (PSII). After laser-flash excitation of PSII membrane particles from spinach, time courses of the delayed recombination fluorescence (10 μs–10 ms) and the variable chlorophyll-fluorescence yield (100 μs–1 s) were recorded in the presence of chloride salts. At low salt-concentrations, a stimulating effect was observed for the S-state transition efficiency, the time constant of O₂-formation at the Mn₄Ca-complex of PSII, and the halftime of re-oxidation of the primary quinone acceptor (Q_a) by the secondary quinone acceptor (Q_b). The cation valence determined the half-effect concentrations of the stimulating salt effect, which were around 6 μM, 200 μM and 10 mM for trivalent (LaCl₃), bivalent (MgCl₂, CaCl₂), and monovalent cations (NaCl, KCl), respectively. A depressing high-salt effect also depended strongly on the cation valence (onset concentrations around 2 mM, 50 mM, and 500 mM). These salt effects are proposed to originate from electrostatic screening of negatively charged carboxylate sidechains, which are found in the form of carboxylate clusters at the solvent-exposed protein surface. We conclude that the influence of electrostatic screening by solvent cations manifests a functionally relevant long-distance interaction between protein surface and electron-transfer reactions in the protein interior. A relation to regulation and adaptation in response to environmental changes is conceivable.     

Influence of monovalent cations on the activity of T4 DNA ligase in the presence of polyethylene glycol.

Monovalent cations such as Na+ and K+ inhibit the activity of T4 DNA ligase. However, the extent of inhibition varies with the terminal sequence of the duplex DNA used as substrate; in many cases, ligation of DNA is completely inhibited at 200 mM. The activity of the ligase is stimulated by raising the concentration of polyethylene glycol 6000 from 0 to 15% (w/v) when NaC1 and KC1 were both absent. Ligation was reduced as the concentration of NaC1 or KC1 was raised in a mixture containing 5 or 15% PEG 6000. With 10% PEG 6000, both cohesive- and blunt-end ligation of this ligase increased at high concentrations of salt (150-200 mM NaC1, or 200-250 mM KC1). Further, with 10% PEG 6000, inter- and intramolecular ligation occurred at low salt concentrations (0-100 mM NaC1, or 0-150 mM KC1); only linear oligomers were formed by intermolecular ligation at the high concentrations.     

AMPHOTERIC COLLOIDS : IV. THE INFLUENCE OF THE VALENCY OF CATIONS UPON THE PHYSICAL PROPERTIES OF GELATIN.

1. A method is given by which the amount of equivalents of metal in combination with 1 gm. of a 1 per cent gelatin solution previously treated with an alkali can be ascertained when the excess of alkali is washed away and the pH is determined. The curves of metal equivalent in combination with 1 gm. of gelatin previously treated with different concentrations of LiOH, NaOH, KOH, NH₄OH, Ca(OH)₂, and Ba(OH)₂ were ascertained and plotted as ordinates, with the pH of the solution as abscissæ, and were found to be identical. This proves that twice as many univalent as bivalent cations combine with the same mass of gelatin, as was to be expected. 2. The osmotic pressure of 1 per cent solutions of metal gelatinates with univalent and bivalent cation was measured. The curves for the osmotic pressure of 1 per cent solution of gelatin salts of Li, Na, K, and NH₄ were found to be identical when plotted for pH as abscissæ, tending towards the same maximum of a pressure of about 325 mm. of the gelatin solution (for pH about 7.9). The corresponding curves for Ca and Ba gelatinate were also found to be identical but different from the preceding ones, tending towards a maximum pressure of about 125 mm. for pH about 7.0 or above. The ratio of maxi mal osmotic pressure for the two groups of gelatin salts is therefore about as 1:3 after the necessary corrections have been made. 3. When the conductivities of these solutions are plotted as ordinates against the pH as abscissæ, the curves for the conductivities of Li, Na, Ca, and Ba gelatinate are almost identical (for the same pH), while the curves for the conductivities of K and NH₄ gelatinate are only little higher. 4. The curves for the viscosity and swelling of Ba (or Ca) and Na gelatinate are approximately parallel to those for osmotic pressure. 5. The practical identity or close proximity of the conductivities of metal gelatinates with univalent and bivalent metal excludes the possibility that the differences observed in the osmotic pressure, viscosity, and swelling between metal gelatinates with univalent and bivalent metal are determined by differences in the degree of ionization (and a possible hydratation of the protein ions). 6. Another, as yet tentative, explanation is suggested.     

Purification and characterization of chromatin-bound DNA-dependent RNA polymerase I from parsley (Petroselinum crispum). Influence of nucleoside triphosphates.

The isolation and purification of DNA-dependent RNA polymerase I (EC 2.7.7.6) from parsley (Petroselinum crispum) callus cells grown in suspension culture is described. The enzyme was solubilized from isolated chromatin. Purification was achieved by using DEAE- and phospho-cellulose in batches, followed by column chromatography on DEAE- and phospho-cellulose (two columns) and density-gradient centrifugation. The highly purified enzyme was stable over several months. The properties of purified parsley RNA polymerase I were investigated. Optimum concentration for Mn2+ was 1 mM, and for Mg2+ 4-6 mM, Mn2+ was slightly more stimulatory than Mg2+. The enzyme was most active at low ionic strengths [10-20 mM-(NH4)SO4]. The influence of various phosphates was tested: pyrophosphate inhibited RNA polymerase at low concentrations, whereas orthophosphate had no effect on the enzyme activity. ADP was slightly inhibitory, and AMP had no effect on the enzyme reaction. Nucleoside triphosphates and bivalent cations in equimolar concentrations in the range 4-11 mM did not influence the RNA synthesis in vitro. Free nucleoside triphosphates in excess of this 1:1 ratio inhibited the enzyme activity, unlike free bivalent cations, which stimulated RNA polymerase I.     

Effect of propan-2-ol on enzymic and structural properties of elongation factor G.

Elongation factor G (EF-G) can support a GTPase activity in vitro even in the absence of ribosomes when propan-2-ol is present [GTPasep; De Vendittis, Masullo & Bocchini (1986) J. Biol. Chem. 261, 4445-4450]. In the present work the GTPasep activity of EF-G was further studied by investigating (i) the effect of ionic environment on GTPasep and (ii) the influence of propan-2-ol on the molecular structure of EF-G as determined by fluorescence and c.d. measurements. In the presence of 1-300 mM univalent cations (M+) alone, no detectable GTPasep activity was measured; however, in the presence of 1 mM-Mg2+ a considerable stimulation was observed at 40 mM-Li+ or 75 mM-NH4+. Among bivalent cations (M2+), 1 mM-Sr2+, 2-5 mM-Ca2+ and 1 mM-Ba2+ were the most effective, but, in the presence of 75 mM-NH4+, Mg2+ and Mn2+ became the most efficient, whereas the stimulation by other M2+ species was considerably decreased. C.d. measurements showed that the alcohol increased the mean molar residue ellipticity of EF-G at 285 nm, but not at 220 nm. As estimated from fluorescence measurements, in the presence of 20% (v/v) propan-2-ol the value of the dissociation constant of the complex formed between EF-G and 8-anilino-1-naphthalene-sulphonate decreased from 8 to 5 microM; similarly, the number of binding sites on EF-G for the fluorescent probe decreased from 13 to 6. Finally, the alcohol enhanced the quenching of the intrinsic fluorescence of EF-G caused by either acrylamide or KI. The data support the hypothesis that propan-2-ol induces moderate conformational changes of EF-G that make the catalytic centre accessible to the substrate even in the absence of ribosomes. Kinetics of GTPasep studied at different temperatures did not reveal additional structural changes of EF-G occurring with time or temperature.     

The interaction of cations with lipopolysaccharide from Escherichia coli C as shown by measurement of binding constants and aggregation reactions.

Lipopolysaccharide from Escherichia coli C interacts with polyvalent cations at low ionic strength at more than one site. The first site has high affinity with a KD value of 10(-8) M for Ca2+ and even stronger binding for [(NH3)5CoNH2Co(NH3)5]5+ and La3+. The high-affinity site for the latter cations is beyond the sensitivity of the assay method. The second, low-affinity, site for bivalent cations has a Km of 10(-3) M, whereas, for tervalent and quinquevalent metal cations and spermine and hexacyclen (1,4,7,10,13,16-hexa-azacyclo-octadecane), this constant has a value of 10(-5) M. Binding of cations to the high-affinity site does not alter the aggregation state of the lipopolysaccharide, but combination with the low-affinity site gives particles twice the size of those of the sodium salt. Very high Ca2+ concentrations (30 mM) give particles eight times the size of those of the sodium salt.     

Structural organisation of the head-to-tail interface of a bacterial virus

In tailed icosahedral bacteriophages the connection between the 5-fold symmetric environment of the portal vertex in the capsid and the 6-fold symmetric phage tail is formed by a complex interface structure. The current study provides the detailed analysis of the assembly and structural organisation of such an interface within a phage having a long tail. The region of the interface assembled as part of the viral capsid (connector) was purified from DNA-filled capsids of the Bacillus subtilis bacteriophage SPP1. It is composed of oligomers of gp6, the SPP1 portal protein, of gp15, and of gp16. The SPP1 connector structure is formed by a mushroom-like portal protein whose cap faces the interior of the viral capsid in intact virions, an annular structure below the stem of the mushroom, and a second narrower annulus that is in direct contact with the helical tail extremity. The layered arrangement correlates to the stacking of gp6, gp15, and gp16 on top of the tail. The gp16 ring is exposed to the virion outside. During SPP1 morphogenesis, gp6 participates in the procapsid assembly reaction, an early step in the assembly pathway, while gp15 and gp16 bind to the capsid portal vertex after viral chromosome encapsidation. gp16 is processed during or after tail attachment to the connector region. The portal protein gp6 has 12-fold cyclical symmetry in the connector structure, whereas assembly-na?ve gp6 exhibits 13-fold symmetry. We propose that it is the interaction of gp6 with other viral morphogenetic proteins that drives its assembly into the 12-mer state.     

Cooperativity and specificity in the interactions between DNA and the glucocorticoid receptor DNA-binding domain

We have employed fluorescence spectroscopy to study the chemical equilibrium between a 115 amino acid protein fragment containing the DNA-binding domain of the human glucocorticoid receptor (DBDr) and a 24-base-pair DNA oligomer containing the glucocorticoid response element (GRE) from the mouse mammary tumor virus promoter region and compared it with the binding to nonspecific DNA at various ionic conditions. We find that binding to both DNAs is cooperative but that DBDr shows a higher affinity for the GRE than for nonspecific DNA and that this difference is more pronounced at increased salt concentrations. Sequence-specific binding to the GRE sequence at 570 mM monovalent cations can be described by a two-site cooperative model, and this supports the notion that DBDr binding to the GRE is enhanced by dimer formation at the recognition site. The product between the (average) association constant for binding to a GRE half-site and the cooperativity parameter was estimated to be K omega = (1-4) x 10(7) M-1 at this salt concentration and 20 degrees C. The sequence-specific binding is not very sensitive to salt concentration in the interval 270-570 mM monovalent cations. However, at lower salt (70 mM) additional binding takes place, presumably nonspecific (cooperative) association to DNA adjacent to the GRE sequence. DBDr binding to nonspecific DNA can be described by the McGhee-von Hippel model for cooperative binding to a chain polymer and is very sensitive to ionic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)