Preparation of 32P-labeled murine immune interferon and its binding to the mouse immune interferon receptor

Murine immune interferon (Mu-IFN-gamma) can be radiolabeled with [gamma-32P]ATP by the catalytic subunit of cAMP-dependent protein kinase. The resulting 32P-labeled Mu-IFN-gamma (32P-Mu-IFN-gamma) with high radiological specific activity (60-260 muCi/micrograms) retains biological activity. Acid hydrolysis of 32P-Mu-IFN-gamma or 32P-labeled human IFN-gamma leads to the release of [32P]phosphoserine but not phosphothreonine or phosphotyrosine. With 32P-Mu-IFN-gamma, we have demonstrated that there are 5 X 10(3) to 1.5 X 10(4) receptors per-cell on several murine cell lines of diverse origin and that the Kd at 24 degrees C for these cells is in the range of 1 X 10(-10) to 1 X 10(-9) M. Covalent binding of 32P-Mu-IFN-gamma to its receptor results in the formation of several specific high-molecular weight products, the major one of which has an apparent molecular weight of 90,000-100,000. If this represents a 1:1 complex of Mu-IFN-gamma and its receptor (or its binding subunit), the murine interferon gamma receptor has a molecular weight of 75,000-85,000.     

Characterization of specific high-affinity receptor for human lymphotoxin

The specific cell surface receptors for lymphotoxin (LT) which are expressed on murine fibroblast L.P3 cells, a subline of L929 cells, were found to consist of a single class of specific high-affinity receptors with a dissociation constant (Kd) of 3.8 X 10(-10) M and a density of 5.8 X 10(3) sites/cell. Similarly, murine fibroblast L929 cells, human melanoma A375 cells and human cervical carcinoma HeLa-S3 cells had about 7.2 X 10(3), 3.5 X 10(3), and 6.6 X 10(3) sites/cell with Kd values of 1.4 X 10(-10), 0.5 X 10(-10), and 1.1 X 10(-10) M, respectively. Among the LT receptor-positive cell lines, there was no direct correlation between the level of specific LT binding and the sensitivity to the cytotoxic or cytostatic effect of LT. Cross-linking of 125I-LT to the cell surface receptors with disuccinimidyl suberate, followed by two-dimensional gel electrophoresis of the cell lysate, revealed two kinds of LT-LT receptor complexes with molecular weights of 70 and 97 kDa, and having the same pI value of 6.8. Cell-bound 125I-LT was internalized within 1 h and degraded intracellularly, and finally secreted into the medium within a few hours. Appropriate concentrations of LT and interferon gamma (IFN gamma) showed synergistic cytotoxicity toward murine fibroblast L.P3 cells and human monocytoma U937 cells, but these cytokines were only slightly cytotoxic individually. Preincubation of these cells with IFN gamma increased the total number of LT receptors without any significant change in the dissociation constant or in the molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of platelet activating factor by isolated membranes from U937 cells

Platelet activating factor (PAF), 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acetyl-GPC) has been known to have biological effect on cells. The mechanisms of the effect of the potent phospholipid on cells has not been established. We have used 1-O-[3H]alkyl-2-acetyl-GPC [( 3H]PAF) to study the interaction on the isolated membranes of U937 cells. The binding process was time, protein concentration, temperature dependent and reversible. The binding of [3H]PAF to the U937 cell membranes was slightly inhibited by the addition of PAF analogue, 3-O-Hexadecyl-2-acetyl-sn-glycerol-1-phosphorylcholine. U937 cell membranes showed high affinity binding sites for PAF with equilibrium dissociation constant (Kd) of 5 x 10(-9) M. The displacement of bound [3H]PAF with 500-fold excess of nonlabeled PAF was not altered suggesting that the bound [3H]PAF was not degraded during the binding. Binding of [3H]PAF on U937 cell membranes was inhibited by PAF antagonist, 59227RP. The kinetic of the inhibition by PAF antagonist is competitive suggesting that PAF and PAF antagonist bind at the same site.     

Effect of a pentosan polysulphate upon thrombin and factor Xa inactivation by antithrombin III.

The kinetics of inhibition of human and bovine alpha-thrombin and human factor Xa by antithrombin III were examined under pseudo-first-order conditions as a function of the concentration of pentosan polysulphate [a fully sulphated (beta 1-4)-linked D-xylopyranose with a single laterally positioned 4-O-methyl-alpha-D-glucuronic acid]. Double-reciprocal plots of the observed first-order rate constant against concentration of pentosan polysulphate gave straight lines, intercepts on the axes giving values for maximum increase in second-order rate constant (by calculation) and apparent dissociation constant. These values were: for human alpha-thrombin 1.52 X 10(7) M-1 . min-1 and 3.6 microM respectively, for bovine alpha-thrombin 6.56 X 10(6) M-1 . min-1 and 0.16 microM and for factor Xa 6.86 X 106 M-1 . min-1 and 20 microM. In the presence of pentosan polysulphate the dissociation constant for the initial complex of antithrombin III and thrombin was shown to be reduced from approx. 2 X 10(-3) M to 61 X 10(-6) M without apparent change in the limiting rate constant of 750 min-1. An oligosaccharide (primarily 8-10 saccharide units) prepared from heparin and with high affinity for antithrombin III but low potency in the thrombin-antithrombin III interaction did not diminish the rate of interaction catalysed by pentosan polysulphate. The catalysis was shown to be due to a weak electrostatic interaction, since it was completely reversed by concentrations of NaCl greater than 0.3 M. It is concluded that the mechanism is independent of the heparin high-affinity binding site on antithrombin III and is probably due to binding of the high-charge-density polysaccharide to the proteinase. It is calculated that the acceleration in rate achieved, although lower than that of heparin, approaches that required to be of physiological significance and may be of importance in the anticoagulation role of antithrombin III at sites of high charge density which may occur in vivo.     

Characterization of binding of a specific antagonist, [3H]-SQ 29,548, to soluble thromboxane A2/prostaglandin H2 (TP) receptors in human platelet membranes.

Binding of [3H]-SQ 29,548 was characterized to soluble thromboxane A2/prostaglandin H2 (TP) receptors from human platelet membranes as a means of examining ligand-receptor interactions outside the lipophilic environment of the cell membrane. Kinetic determination revealed a rate of ligand-receptor association of 1.4 x 10(7) +/- 0.2 M-1 x min-1 and a rate of dissociation of 0.5 +/- 0.07 min-1. The resultant equilibrium affinity constant was 36.3 +/- 5.8 nM. Saturation binding analysis revealed a single class of [3H]-SQ 29,548 binding sites with an affinity constant of 39.7 +/- 4.3 nM and a B(max) of 1735.7 +/- 69.1 fmol/mg protein. Specific [3H]-SQ 29,548 binding was inhibited by specific TP receptor antagonists and agonists in a rank order of potency similar to that seen in platelet membranes: SQ 33,961 much greater than SQ 29,548 greater than BM 13,505 greater than or equal to U 46619 greater than BM 13,177. PGD2, PGE2 and PGI2 did not appreciably inhibit the specific binding of [3H]-SQ 29,548. These data indicate that [3H]-SQ 29,548 binding to soluble human platelet TP receptors was specific, saturable, and reversible.     

Leukotriene B4 binding to human neutrophils

[3H] Leukotriene B4 (LTB4) binds concentration dependently to intact human polymorphonuclear leukocytes (PMN's). The binding is saturable, reaches equilibrium in 10 min at 4 degrees C, and is readily reversible. Mathematical modeling analysis reveals biphasic binding of [3H] LTB4 indicating two discrete populations of binding sites. The high affinity binding sites have a dissociation constant of 0.46 X 10(-9)M and Bmax of 1.96 X 10(4) sites per neutrophil; the low affinity binding sites have a dissociation constant of 541 X 10(-9)M and a Bmax of 45.16 X 10(4) sites per neutrophil. Competitive binding experiments with structural analogues of LTB4 demonstrate that the interaction between LTB4 and the binding site is stereospecific, and correlates with the relative biological activity of the analogs. At 25 degrees C [3H] LTB4 is rapidly dissociated from the binding site and metabolized to 20-OH and 20-COOH-LTB4. Purification of neutrophils in the presence of 5-lipoxygenase inhibitors significantly increases specific [3H] LTB4 binding, suggesting that LTB4 is biosynthesized during the purification procedure. These data suggest that stereospecific binding and metabolism of LTB4 in neutrophils are tightly coupled processes.     

The Intact Human Neuroblastoma Cell (SH-SY5Y) Exhibits High-Affinity [3H]Pirenzepine Binding Associated with Hydrolysis of Phosphatidylinositols

The binding of [3H]pirenzepine to a human neuroblastoma cell line (SH-SY5Y) and its correlation with hydrolysis of phosphatidylinositols were characterized. Specific [3H]pirenzepine binding to intact cells was rapid, reversible, saturable, and of high affinity. Kinetic studies yielded association (k+1) and dissociation (k-1) rate constants of 5.2 +/- 1.4 X 10(6) M-1 min-1 and 1.1 +/- 0.06 X 10(-1) min-1, respectively. Saturation experiments revealed a single class of binding sites (nH = 1.1) for the radioligand with a total binding capacity of 160 +/- 33 fmol/mg protein and an apparent dissociation constant of 13 nM. The specific [3H]pirenzepine binding was inhibited by the presence of selected muscarinic drugs. The order of antagonist potency was atropine sulfate greater than pirenzepine greater than AF-DX 116, with K0.5 of 0.53 nM, 2.2 nM, and 190 nM, respectively. The binding properties of [3H](-)-quinuclidinyl benzilate and its quaternary derivative [3H](-)-methylquinuclidinyl benzilate were also investigated. The muscarinic agonist carbachol stimulated formation of inositol phosphates which could be inhibited by muscarinic antagonists. The inhibition constants of pirenzepine and AF-DX 116 were 11 nM and 190 nM, respectively. In conclusion, we show that the nonclassical muscarinic receptor antagonist [3H]pirenzepine identifies a high-affinity population of muscarinic sites which is associated with hydrolysis of phosphatidylinositols in this human neuroblastoma cell line.     

A variant of human transferrin with abnormal properties.

Normal human skin fibroblasts cultured in vitro exhibit specific binding sites for 125I-labelled transferrin. Kinetic studies revealed a rate constant for association (Kon) at 37 degrees C of 1.03 X 10(7) M-1 X min-1. The rate constant for dissociation (Koff) at 37 degrees C was 7.9 X 10(-2) X min-1. The dissociation constant (KD) was 5.1 X 10(-9) M as determined by Scatchard analysis of binding and analysis of rate constants. Fibroblasts were capable of binding 3.9 X 10(5) molecules of transferrin per cell. Binding of 125I-labelled diferric transferrin to cells was inhibited equally by either apo-transferrin or diferric transferrin, but no inhibition was evident with apo-lactoferrin, iron-saturated lactoferrin, or albumin. Preincubation of cells with saturating levels of diferric transferrin or apo-transferrin produced no significant change in receptor number or affinity. Preincubation of cells with ferric ammonium citrate caused a time- and dose-dependent decrease in transferrin binding. After preincubation with ferric ammonium citrate for 72 h, diferric transferrin binding was 37.7% of control, but no change in receptor affinity was apparent by Scatchard analysis. These results suggest that fibroblast transferrin receptor number is modulated by intracellular iron content and not by ligand-receptor binding.     

Evidence for a single forskolin-binding site in rat adipocyte membrane. Studies of [14,15-3H]dihydroforskolin binding and adenylate cyclase activation

[14,15-3H] Dihydroforskolin has been used as a tracer in the study of forskolin binding to adipocyte plasma membrane and the subsequent activation of adenylate cyclase (EC 4.6.1.1) of this membrane. The specific binding of radioactivity to the membrane was rapid, temperature-dependent, saturable, and readily reversible. The equilibrium dissociation reaction constant (KD) for the binding was 13 microM, with a maximum binding (Bmax) of 61 pmol of forskolin per mg of membrane protein. The Hill coefficient was 1.0. The bound [14,15-3H] dihydroforskolin was displaced by forskolin with rate constants of 0.07 X 10(6) M-1 min-1 and 1.2 min-1 for the association and dissociation reactions, respectively (30 degrees C). The equilibrium dissociation constant (KD) was approximately the same as the concentration that produced half-maximum activation (EC50) of the adenylate cyclase of rat adipocyte plasma membrane. There was a linear correlation between forskolin binding and adenylate cyclase activation. The results are consistent with the concept of a single class of binding site which binds forskolin. [14,15-3H] Dihydroforskolin appears to be a potentially useful tracer in the study of the mechanism of activation of the catalytic unit of adipocyte adenylate cyclase.     

Interactions of retinol with binding proteins: implications for the mechanism of uptake by cells

The kinetic parameters of the interaction of retinol with retinol binding protein (RBP) were studied. The rate constant for association of retinol with the protein (ka) was found to be 1.5 X 10(6) M-1 min-1. The rate constant for dissociation (kd) from the protein was determined by studying the transfer of retinol from RBP to lipid bilayers. It was found that such transfer proceeds via the aqueous phase and its rate-limiting step is the dissociation of retinol from the binding protein. The rate of transfer therefore represents the rate of dissociation. The kd was 0.112 min-1. These values were validated further by the following consideration. The equilibrium dissociation constant of RBP and retinol can be calculated from the expression Kd = kd/ka. The calculated value was 7.5 X 10(-8) M. Kd was also measured directly by fluorometric titration and was found to be 7 X 10(-8) M. The relative avidities of retinol for RBP, the complex RBP-transthyretin (RBP-TTR), and serum albumin were also studied. It was found that binding of RBP to TTR increased its avidity for retinol by about 2-fold. The avidity of albumin for retinol was 30-fold lower than that of RBP. The data imply that retinol spontaneously and rapidly dissociates from sites on binding proteins, which indicates that the vitamin can freely move in vivo between physiologic compartments with avidities for it.     

Binding of human interferon alpha to cells of different sensitivities: studies with internally radiolabeled interferon retaining full biological activity.

The characteristics of interferon binding to various cells with different interferon sensitivity were studied by using [3H]leucine-labeled, pure human interferon alpha from Namalwa cells. Scatchard analysis of the binding data on cells sensitive to interferon alpha (human FL and fibroblasts and bovine MDBK) indicated the presence of two kinds of binding sites with high and low affinities. The binding constants of the high-affinity sites in these cells were similar (4 X 10(10) to 11 X 10(10) M-1). Cells insensitive to human interferon alpha (human HEC-1 and mouse L cells) were shown to have only low-affinity sites, suggesting that high-affinity binding sites are indispensable for interferon sensitivity and represent interferon receptors. However, the number of sites in three human diploid fibroblast strains and one strain trisomic for chromosome 21 were not proportionally correlated to the interferon sensitivity of the cells. The high-affinity binding to human cells was completely inhibited by both nonradioactive human interferons alpha and beta in a similar manner, but binding to bovine MDBK cells, on which human interferon beta is practically inactive, was inhibited effectively only by interferon alpha and not by beta. These results suggest that the receptor for human interferon alpha is common to human interferon beta in human cells, whereas the receptor on bovine cells binds only human interferon alpha.     

A method for determining binding kinetics applied to thiamine-binding protein

A rapid and simple method for assaying the binding activity of thiamine-binding protein is described. By this assay method, the binding characteristics of rice bran thiamine-binding protein have been evaluated with [14C]thiamine as ligand. Analysis of these data by Scatchard plot resulted in linear plots giving a dissociation constant (Kd) for thiamine of 0.55 microM and a maximum binding (Bmax) of 14.5 pmol of ligand bound/microgram of protein. Thiamine binding to the binding protein was time dependent and reached equilibrium at approximately 20 min. The Kob was 0.18 min-1 and the k1 was 1.25 X 10(5) min-1 M-1. Reversibility of thiamine binding at equilibrium was completed at 60 min with a k2 value of 0.052 min-1. The Kd calculated from the reverse rate constant was 0.42 microM. These results indicated that this binding assay method was substantially reliable and accurate.     

Slow interaction of 5'-adenylyl-beta,gamma-imidodiphosphate with Escherichia coli DNA gyrase. Evidence for cooperativity in nucleotide binding.

We have examined the kinetics of interaction between Escherichia coli DNA gyrase and the nonhydrolyzable ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (ADPNP) in the presence and absence of ATP. In the absence of ATP, [alpha-32P]ADPNP binds extremely slowly to gyrase, with an apparent second-order rate constant (k1) of 120 M-1 min-1. Similarly, the limited negative supercoiling of closed-circular DNA caused by ADPNP binding is slow, requiring at least 2 h to reach completion in the presence of 100 microM ADPNP. A very slow but detectable rate of dissociation of ADPNP from gyrase was measured, with a rate constant of 3.5 x 10(-4) min-1. The calculated dissociation constant for ADPNP is thus 2.9 microM. ADPNP is a potent competitive inhibitor of ATP-dependent DNA supercoiling. Inhibition is established much more rapidly than can be accounted for by the slow rate of ADPNP binding in the absence of ATP. We have found that ATP can accelerate the rate of [32P]ADPNP binding by more than 15-fold (k1 = 1,850 M-1 min-1). The ATP-promoted rate enhancement requires the presence of DNA; in the absence of DNA, ATP has no effect on the rate of binding. Relaxed closed-circular, nicked-circular, and linear pBR322 DNA are all equally effective cofactors for ATP-stimulated binding of ADPNP. After a short lag, the presence of ATP also greatly speeds up ADPNP dissociation from gyrase bound initially to closed-circular DNA, with the restoration of DNA supercoiling activity. This effect is not observed in the presence of nicked-circular or linear DNA, suggesting that ADPNP dissociates more rapidly from gyrase bound to supercoiled DNA. The results of ADPNP binding provide evidence for cooperative interactions between the nucleotide binding sites. To account for these data, a model is proposed for the interaction of nucleotides at the two ATP binding sites on DNA gyrase.     

Evidence for muscarinic cholinergic receptors in dog portal vein: binding of [3H]quinuclidinyl benzilate

Muscarinic cholinergic receptor sites in dog portal veins were analyzed directly using [3H]quinuclidinyl benzilate (QNB) as a ligand. Specific [3H]QNB binding to crude membrane preparations from the isolated veins was saturable, reversible and of high affinity (KD = 15.5 +/- 2.8 pM) with a Bmax of 110 +/- 14.7 fmol/mg protein. Scatchard and Hill plot analyses of the data indicated one class of binding sites. From kinetic analysis of the data, association and dissociation rate constants of 1.91 X 10(9) M-1 min-1 and 0.016 min-1, respectively, were calculated. The dissociation constant calculated from the equation KD = K-1/K+1 was 8.3 pM, such being in good agreement with the Scatchard estimate of KD (15.5 pM). Specific binding of [3H]QNB was displaced by muscarinic agents. Nicotinic cholinergic agents, alpha-bungarotoxin, nicotine and hexamethonium, were ineffective in displacing [3H]QNB binding at 10 microM. Our findings provide direct evidence for the existence of muscarinic cholinergic receptors in dog portal veins.     

Benzodiazepine receptors along the nephron: [3H]PK 11195 binding in rat tubules

Binding of [3H]PK 11195, an isoquinoline carboxamide derivative, was measured in microdissected tubule segments of rat nephron. High specific binding capacities (1.1-1.8 fmol X mm-1) were found in the thick ascending limb of the Henle's loop and in the collecting tubule, whereas specific binding could not be detected in the proximal tubule. In the medullary collecting tubule, the association and dissociation rate constants at 4 degrees C were k1 = 3.0 X 10(6) M-1 X min-1 and k-1 = 0.021 min -1; the ratio k-1/k1 = 7.0 nM was in agreement with the estimated equilibrium dissociation constant (Kd = 2.4 nM). [3H]PK 11195 binding sites from medullary ascending limb and medullary collecting tubule revealed the following sequence of specificity: PK 11195 = Ro 5-4864 much greater than clonazepam, indicating that tubule binding sites might be the peripheral benzodiazepine receptors of the rat kidney.     

The binding of zinc to angiotensin-converting enzyme

The equilibrium constant for the dissociation of zinc ion from angiotensin-converting enzyme (ACE) was measured using zinc ion buffers of zinc chloride and nitrilotriacetic acid (NTA). The dissociation constant is 6.4 X 10(-10) M. The fraction of active enzyme at equilibrium is independent of the presence of substrate which indicates that hippuryl-histidylleucine binds equally well to the holoenzyme and apoenzyme. The rate constant for the dissociation of zinc from ACE was measured as 0.68 min-1 for the free enzyme; the rate constant for the enzyme substrate complex was roughly 0.18 min-1. The association of zinc ion and ACE is very fast; the rate constant is 1.06 X 10(9) M-1 min-1. Ethylenediaminetetraacetic acid (EDTA) and NTA rapidly remove zinc from ACE with rate constants of 1.27 X 10(3) and 2.2 X 10(3) M-1 min-1. The equilibrium constant for the reaction of NTA with ACE was measured as 4.6 X 10(-2) and was calculated for EDTA as 3.8 X 10(3).     

A simple and efficient method for the purification of human gamma interferon

A preparative, sequential chromatographic procedure has been developed for the purification of human gamma interferon (HuIFN-gamma). The four steps in the procedure are Controlled Pore Glass-adsorption chromatography, Concanavalin-A affinity chromatography, Heparin-Sepharose affinity chromatography and gel-filtration. By virtue of the development of a coordinated effluent-affluent buffer scheme, eluants can also serve as loading buffers for the succeeding column. Consequently, crude HuIFN-gamma preparations can be purified rapidly (approximately one week), easily, and is amenable to a semi-automated process. The procedure has also been shown to be efficient. Here, as an example, it is reported that an overall purification of greater than 75,000-fold can be achieved, yielding a specific activity of 5.2 X 10(7) units/mg, and a recovery of 95.5%. In addition, the peak fraction, representing 37.8% of the applied activity, had a specific activity of 1.0 X 10(8) units/mg protein and represents a purification of more than 145,000-fold. An SDS-PAGE analysis of one such fraction indicated that approximately 40% of the final material was HuIFN-gamma.     

Binding of N-[propionyl-3H]propionylated alpha-bungarotoxin and L-[benzilic-4,4'-3H] quinuclidinyl benzilate to CNS extracts of the cockroach Periplaneta americana

The nerve cord of the cockroach (Periplaneta americana) contains distinct saturable components of specific binding for the ligands N-[propionyl-3H]propionylated alpha-bungarotoxin and L-[benzilic-4,4'-3H]quinuclidinyl benzilate. N-[Propionyl-3H]propionylated alpha-bungarotoxin bound reversibly to homogenates with a Kd of 4.8 nM and Bmax of 910 fmol mg-1. The association rate constant (1.9 X 10(5) M-1 s-1) and dissociation rate constant (1.2 X 10(-4) s-1) yielded a Kd of 0.6 nM. Nicotinic ligands were found to displace toxin binding most effectively. The binding sites characterized in this way showed many similarities with the properties of the vertebrate neuronal alpha-bungarotoxin binding site. For a range of cholinergic ligands, inhibition constants calculated from toxin binding studies closely corresponded to their effectiveness in blocking the depolarizing response to acetylcholine recorded by electrophysiological methods from an identified cockroach motoneurone. The N-[propionyl-3H]propionylated alpha-bungarotoxin binding component therefore appears to be a constituent of a functional CNS acetylcholine receptor. Binding of L-[benzilic-4,4'-3H]quinuclidinyl benzilate was reversible with a Kd of 8 nM and Bmax of 138 fmol mg-1, determined from equilibrium binding experiments. The Kd calculated from the association rate constant (2.4 X 10(5) M-1 s-1) and dissociation rate constant (1.3 X 10(-4) s-1) was 1.9 nM. Muscarinic ligands were the most potent inhibitors of quinuclidinyl benzilate binding. The characteristics of this binding site resembled those of vertebrate CNS muscarinic cholinergic receptors. In contrast with vertebrate CNS, the nerve cord of Periplaneta americana contains more (approximately X 7) alpha-bungarotoxin binding sites than quinuclidinyl benzilate binding sites.     

Characterization of the binding of a rat somatomedin to receptors in human placental cell membranes.

The somatomedins presumably initiate their growth promoting effects by first binding to specific cell surface receptors in responsive tissues. The specific and high affinity binding of [¹²⁵I]-rat somatomedin to human placental membranes was saturable and reversible with a dissociation constant of 4.5 × 10^?9 M calculated from Scatchard analysis of competitive binding experiments. Competition for [¹²⁵I]-rat somatomedin binding to placental receptors by other somatomedins and growth factors suggest a close structural relationship between rat somatomedin and the human somatomedin, insulin-like growth factor I.     

Specificity and kinetics defining the interaction between a murine monoclonal autoantibody and DNA

Interactions between a murine monoclonal anti-DNA autoantibody (BV17-45) and DNA were examined by direct binding and competitive radioimmunoassays. Binding isotherms constructed by titration of purified BV17-45 with a series of distinct 32P-labeled double-stranded DNA ([32P]dsDNA) fragments were super-impossible, suggesting: 1) BV17-45/[32P]dsDNA binding is independent of dsDNA size using fragments greater than or equal to 192 base pairs in length, and 2) BV17-45 does not exhibit stringent sequence specificity. Single-stranded DNA-specific monoclonal antibody BV04-01 did not react with [32P]dsDNA, confirming its duplex character. In competition experiments, BV17-45 cross-reacted with phage (phi X174, M13) RF AND VIRION DNAS AT PICOMOLAR concentrations. Selectivity for B-form DNA was suggested by the ability of poly(dA) . poly(dT), but not other helical duplex forms, to block BV17-45/[32P] dsDNA binding. Among the four deoxyribohomopolymers, only deoxyadenylic acid polymers completely inhibited BV17-45/[32P]dsDNA complex formation. [32P]dsDNA binding was relatively insensitive to ionic strength, suggesting minimal contribution of electrostatic forces to the binding free energy. Measured BV17-45/[32P]dsDNA association and dissociation rate constants (4 degrees C) were 7.4 X 10(6) M-1 s-1 and 9.2 X 10(-5) s-1, respectively, yielding a functional affinity of 8 X 10(10) M-1. Results are discussed in terms of the relative contribution of B-DNA structural and substructural determinants to the mechanism of BV17-45 recognition.