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Showing papers on "Cooperative binding published in 1981"


Journal ArticleDOI
TL;DR: The activated 5S estrogen receptor is a homodimer and that its formation is associated with a positive cooperative estradiol-binding reaction, indicating that the cooperative interactions are dependent upon a monomer--dimer equilibrium.
Abstract: The equilibrium [3H]estradiol binding by the partially purified estrogen receptor from calf uteri was measured at 25 degrees C. The Scatchard plot of the binding data showed a convex curve characteristic of positive cooperativity and a Hill coefficient of 1.58 +/- 0.21, at receptor concentrations of 1 to 10 nM. Below a receptor concentration of approximately 0.3 nM the Scatchard plot approached linearity, suggesting that the cooperative interactions are dependent upon a monomer--dimer equilibrium. Trypsin pretreatment of the receptor resulted in a loss of dimer formation and of the cooperative interactions. The positive cooperative characteristics of the estrogen receptor were shown not to be produced by receptor inactivation, failure to complete the [3H]estradiol--receptor equilibrium reaction, or radioimpurity of the [3H]estradiol. These findings indicate that the activated 5S estrogen receptor is a homodimer and that its formation is associated with a positive cooperative estradiol-binding reaction.

172 citations


Journal Article
TL;DR: The heterogeneity of GABA binding would appear to involve two discrete populations of receptors on the grounds of noninterconvertible heterogeneity under the conditions tested, whereas the similarity in the drug specificity of the two populations would be more consistent with a model involving multiple coupled or conformational states of a single receptor.
Abstract: The binding of radioactive γ-aminobutyric acid (GABA) to receptor-like sites in mammalian brain membranes was analyzed by computer for comparison with models which might explain the observed apparent heterogeneity of ligand binding. The best fit was obtained with two independent binding sites. Binding was measured by centrifugation, using thoroughly washed, frozen, and thawed membranes without detergent treatment. Assays were carried out at 0° under sodium ion-free conditions which have previously been shown to allow detection only of those binding sites having the chemical specificity and other properties expected of receptor sites for the neurotransmitter GABA. Quantitative analysis of binding curves for several brain regions, subcellular fractions, and species revealed the general presence of two affinity classes for GABA receptors, one with KD Of 13 ± 6 nM ( B max = 0.33 pmole/mg of protein in bovine cortex) and the other with KD of 300 ± 150 nM ( B max 1.8 pmole/mg of protein in bovine cortex). The two-site model fit the data better than did models with one site, three sites, or negative cooperativity, but the fit to the mobile receptor-effector coupling hypothesis was almost as good as that of the two-site model. Consistent with the heterogeneity in equilibrium binding data, heterogeneity was also observed for association and dissociation rates of ligand binding and for rates of thermal inactivation of binding activity. The rate of heat denaturation was biphasic, with earlier times corresponding to selective loss of one of the two binding affinity subpopulations. Kinetics studies revealed two subpopulations of binding sites with respect to on- and off-rates. A slow component with k +1 = 1.9 x 107 M-1 min-1, and k -1 = 0.2 min-1, KD = 11 nM, and low B max corresponded to the high-affinity component of the equilibrium binding curve, and a rapidly dissociating population with lower affinity and higher B max corresponded to the lower-affinity component of the equilibrium binding curve. Independent IC50 values for the high-affinity and low-GABA site were determined for a series of analogues. All analogues tested were more effective inhibitors of the high-affinity GABA sites, and a marked similarity in the relative drug potency for the two sites was observed. Thus the heterogeneity of GABA binding would appear to involve two discrete populations of receptors on the grounds of noninterconvertible heterogeneity under the conditions tested, whereas the similarity in the drug specificity of the two populations would be more consistent with a model involving multiple coupled or conformational states of a single receptor. ACKNOWLEDGMENTS We thank N. Birdsall, E. Hulme, B. Meiners, W. B. Levy, and D. Ching for helpful discussions, and A. Snowman for expert technical assistance.

153 citations


Journal Article
TL;DR: It is concluded that a high-affinity binding site at which 1,4-dihydropyridines bind, has now been identified with radioligand binding techniques and may well represent the locus where the potent 1, 4-dhydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine carboxylic acid and nifedipine exert their pharmacological action.
Abstract: The tritiated calcium antagonist 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine carboxylic acid, 3-ethyl-5-methyl ester (nitrendipine, Bay e 5009), a potent analogue of nifedipine, binds in a reversible and saturable manner to partially purified guinea-pig heart membranes. The analyses of the equilibrium binding data with Scatchard plots is in accord with either negative cooperativity of binding or with the assumption of two classes of binding sites, where one site has an equilibrium dissociation constant (Kd value) of 0.1 nmol/l and a density of 300 fmol/mg of protein. The binding sites are stereoselective and discriminate between (+)-nitrendipine and (-)-nitrendipine. We conclude that a high-affinity binding site at which 1,4-dihydropyridines bind, has now been identified with radioligand binding techniques. This site may well represent the locus where the potent 1,4-dihydropyridines exert their pharmacological action.

126 citations


Journal ArticleDOI
TL;DR: A more realistic procedure is described, in which the estimate of extent of binding is based on a mean aqueous concentration related both to the external concentration and to the much higher internal concentration that exists in energized cells.
Abstract: Two of the commonly used probes for measuring membrane potential—lipophilic cations and the cyanine dye diS-C3(5)—indicated nominally opposite results when tetraphenylarsonium ion was added as a drug to suspensions of metabolizingBacillus subtilis cells. [3H]-Triphenylmethylphosphonium uptake was enhanced by the addition, indicating hyperpolarization, yet fluorescence of diS-C3(5) was also enhanced, indicating depolarization. Evidence is presented that both effects are artifactual, and can occur without any change in membrane potential, as estimated by86Rb+ uptake in the presence of valinomycin. The fluorescence studies suggest that tetraphenylarsonium ion displaces the cyanine dye from the cell envelope, or other binding site, into the aqueous phase. The uptake characteristics of the radiolabeled lipophilic cations were quite unusual: At low concentrations (e.g., less than 10 μm for triphenylmethylphosphonium) there was potential-dependent uptake of the label to a stable level, but subsequent addition of nonradioactive lipophilic cation caused further uptake of label to a new stable level. Labeled triphenylmethylphosphonium ion taken up to the first stable level could be displaced by 10mm magnesium ion, whereas86Rb+ uptake was unperturbed. Association of the lipophilic cations with the surface of de-energized cells was concentration-dependent, but there was no evidence for cooperative binding. This phenomenon of stimulated uptake inB. subtilis (which was not seen inEscherichia coli cells or vesicles) is consistent with a two-compartment model with access to the second compartment only being possible above a critical cation concentration. We tentatively propose such a model, in which these compartments are the cell surface and the cytoplasm, respectively. Triphenylmethylphosphonium up to 0.5mm exhibited linear binding to de-energized cells; binding of tetraphenylphosphonium and tetraphenylarsonium was nonlinear but was not saturated at the highest concentration tested (1mm). The usual assumption, that association of the cation with cell surfaces is saturated and so can be estimated on de-energized cells, therefore leads to undercorrected estimates of cytoplasmic uptake inB. subtilis, and hence to overestimates of membrane potential. We describe a more realistic procedure, in which the estimate of extent of binding is based on a mean aqueous concentration related both to the external concentration and to the much higher internal concentration that exists in energized cells. Using this procedure we estimate the membrane potential inB. subtilis to be 120 mV, inside-negative. The procedure is of general applicability, and should yield more accurate estimates of membrane potential in any system where there is significant potential-dependent binding.

101 citations


Journal ArticleDOI
TL;DR: The failure of the non-polymerizable tropomyosin to bind to F-actin, even at elevated MgCl2 concentrations, illustrates the importance of the head-to-tail interaction of tropomyOSin molecules in their cooperative binding to the thin filament structure.

88 citations


01 Nov 1981
TL;DR: Conformational coupling between subunits is complex and provides a mechanism for modulation of receptor affinity.
Abstract: Conformational changes induced or selected by drugs in receptors may be coupled to second sites on receptor molecules giving negative or positive cooperative binding. The structure-activity relationships may be changed depending on the predominant conformation. Some conformational changes can be identified by nuclear magnetic resonance spectroscopy. Conformational coupling between subunits is complex and provides a mechanism for modulation of receptor affinity.

80 citations


Journal ArticleDOI
02 Jul 1981-Nature
TL;DR: It is reported that binding of 125I-labelled factor VIII/von Willebrand factor to its platelet membrane receptors is inhibited by preincubation of washed human platelets with inhibitors of Ca2+–calmodulin complex.
Abstract: Interaction between factor VIII/von Willebrand factor, a plasma glycoprotein, and platelets is essential for platelet adhesion and subsequent platelet plug formation at sites of vascular injury during primary phase haemostasis1,2. We now report that binding of 125I-labelled factor VIII/von Willebrand factor to its platelet membrane receptors is inhibited by preincubation of washed human platelets with inhibitors of Ca2+–calmodulin complex (for example, 50 µM trifluoperazine or chlorpromazine) at 37 °C for 2 min. Scatchard analysis of the binding data indicates that the total number of accessible binding sites on each pretreated platelet is significantly reduced to ∼50% of the original value. There is also negative cooperativity of binding or reduced binding affinity in a major portion of binding sites. Platelet shape changes from diskoid to spherical after treatment. The results suggest that Ca2+–calmodulin complex modulates platelet shape and membrane receptor behaviour.

53 citations


Journal ArticleDOI
TL;DR: The pigeons cerebellum contains a unique kainic acid binding site characterized by a relatively low binding affinity and cooperative binding properties, and dry-mount autoradiographs of incubated tissue sections demonstrate that this type of binding site is exclusively localized in the molecular and Purkinje cell layers of the cerebellar cortex.

51 citations


Journal ArticleDOI
TL;DR: The changes in environment of the bound coenzyme produced by folinic acid, as revealed by 1H and 31P NMR, demonstrate clearly that the negative cooperativity shown by NADP+ and NADPH, respectively, arises by two structurally distinct mechanisms.
Abstract: The binding of folinic acid (5-formyl-5,6,7,8-tetrahydrofolate) to Lactobacillus casei dihydrofolate reductase has been measured. The natural 6S, alpha S diastereoisomer has a binding constant of 1.3 (+/- 0.6) X 10(8) M-1 at pH 6.0, 25 degrees C; the 6R, alpha S diastereoisomer binds approximately 10(4)-fold more weakly. The natural diastereoisomer of folinic acid binds negatively cooperatively with the coenzymes NADP+ and NADPH, binding 3 times more weakly in the presence of NADP+ and 600 times more weakly in the presence of NADPH than to the enzyme alone. Negative cooperativity has been unequivocally distinguished from competition by measurements of coenzyme binding as a function of folinic acid concentration, of the effects of folinic acid on the 1H and 31P chemical shifts of the bound coenzyme, and of the effects of folinic acid on the coenzyme dissociation rate constant. The latter experiments also give evidence for the coexistence of two slowly interconverting conformational forms of the ternary enzyme-coenzyme-folinic acid complex. Small changes in structure of the oxidized coenzymes have substantial effects on the cooperativity with folinic acid, with the thionicotinamide analogue showing positive rather than negative cooperativity. The changes in environment of the bound coenzyme produced by folinic acid, as revealed by 1H and 31P NMR, demonstrate clearly that the negative cooperativity shown by NADP+ and NADPH, respectively, arises by two structurally distinct mechanisms.

50 citations


Journal ArticleDOI
TL;DR: Polylysine is found to stimulate the release of tightly bound cy tochrome c from the cytochrome c-aa3 complex, which points to the existence of negative cooperativity between the two binding sites.

47 citations


Journal ArticleDOI
01 Jan 1981
TL;DR: Nicotine was bound saturably to crude particulate, and synaptosomal - mitochondrial fractions from mouse brain in this article, showing that nicotine binds in brain to non-cholinergic sites.
Abstract: (±)-[3H]Nicotine was bound saturably to crude particulate, and synaptosomal - mitochondrial fractions from mouse brain. Scatchard and Hill plots of the binding data are in agreement with the existence of two independent classes of binding sites with high (Kd of 0.1 - 0.4 μM) and low (kd is 20 μM) affinities, although negative cooperativity or a two-step model of ligand-receptor interaction cannot be ruled out. Nicotinic or muscarinic agonists and antagonists had little or no affinity for the nicotine binding sites, suggesting that nicotine binds in brain to noncholinergic sites. The binding did not display stereospecificity; this is consistent with the similarity in the pharmacological effects of (-)-and (+)-nicotine. Our results indicate that binding studies with [3H]nicotine should be interpreted with extreme caution.

Journal ArticleDOI
TL;DR: The cooperativity in the actinomycin D binding to calf thymus DNA increases with decreasing sodium chloride concentration, suggesting a role for DNA flexibility in allosteric binding.
Abstract: The equilibrium binding of the carcinogens N-hydroxy-N-acetyl-2-amino-fluorene (HAAF) and 4-nitroquinoline-1-oxide (NQO) to phi X174RF DNA have been studied by phase partition techniques. Both molecules bind in a cooperative manner with only a few carcinogen molecules binding to each phi X174RF DNA molecule. The binding data for both HAAF and NQO fit a model in which two carcinogens cluster into a small number of sites--four sites for HAAF and twelve sites for NQO. Phase partition techniques were also used to study the binding of actinomycin D to both calf thymus DNA and poly (dG-dC) . poly (dG-dC) at much lower r values than had been previously reported. These data exhibit humped Scatchard plots which are indicative of cooperative binding; the overall shape of the Scatchard plots are consistent with a model for drug induced allosteric transitions in the DNA structure. The cooperativity in the actinomycin D binding to calf thymus DNA increases with decreasing sodium chloride concentration, suggesting a role for DNA flexibility in allosteric binding.

Journal ArticleDOI
TL;DR: Results for binding on the inside vesicle surface indicate that the overall affinity for Pr 3+ is significantly greater and suggest that the site stoichiometry may be different, while a Hill analysis indicates that the binding data are more anti-cooperative than a realistic Langmuir isotherm, yet more cooperative than a Stern isotherms incorporating electrostatic considerations at the Debye-Huckel level.

Journal ArticleDOI
TL;DR: Comparative studies on the inhibition of [3H]P4S and [3h]GABA binding by various GABA analogues, strongly suggest that P4S binds to the GABA receptors.
Abstract: The binding of radioactive piperidine-4-sulphonic acid ([3H]P4S) to thoroughly washed, frozen, and thawed membranes isolated from cow and rat brains has been studied. Quantitative computer analysis of the binding curves for four regions of bovine brain revealed the general presence of two binding sites. In these brain regions less satisfactory computer fits were obtained for receptor models showing one or three binding sites or negative cooperativity. With the use of Tris-citrate buffer at 0 degree C the two affinity classes for P4S in bovine cortex membranes revealed the following binding parameters: KD = 17 +/- 7 nM (Bmax = 0.15 +/- 0.07 pmol/mg protein) and KD = 237 +/- 100 nM (Bmax = 0.80 +/- 0.20 pmol/mg protein). Heterogeneity was also observed for association and dissociation rates of [3H]P4S. The slow binding component (kon = 5.6 X 10(7) or 8.8 X 10(7) M-1 min-1, koff = 0.83 min-1, and KD = 14.7 or 9.4 nM, determined by two different methods in phosphate buffer containing postassium chloride) corresponds to the high-affinity component of the equilibrium binding curve (KD = 11 nM, Bmax = 0.12 pmol/mg protein in the same buffer system). The association and dissociation rates for the subpopulation of rapidly of dissociating sites, apparently corresponding to the low-affinity sites, were too rapid to be measured accurately. The binding of [3H]P4S appears to involve the same two populations of sites with Bmax values similar to those for [3H]GABA binding to the same tissue, although the kinetic parameters for the two ligands are somewhat different. Furthermore, comparative studies on the inhibition of [3H]P4S and [3H]GABA binding by various GABA analogues, strongly suggest that P4S binds to the GABA receptors. The different effects of P4S and GABA on benzodiazepine binding are discussed.

Journal ArticleDOI
TL;DR: Binding of 21 S dynein ATPase isolated from Tetrahymena cilia to B subfibers of microtubule doublets was used as a model system to study Dynein-tubulin interactions and their relationship to the microtubules-based sliding filament mechanism, and binding data suggest negative cooperativity or the presence of more than one class of dyne in binding sites on the micro Tubulin-dynein lattice.

Journal ArticleDOI
TL;DR: It was found that Pi inhibition of the light-induced dark binding of ADP can be reversed by the removal of the Pi, and a model for the roles of the adenine nucleotide tight binding site(s) and Pi in the modulation of the spinach CF1 ATPase activity is proposed.

Journal ArticleDOI
TL;DR: The data suggest that only the type I receptors are involved in the sequestration process, which is similar to that observed on sympathetic neurons and may be the first step in the internalization of NGF by responsive cells.
Abstract: Nerve growth factor (NGF) binds to two specific receptors on sensory nerve cells. These two receptors are characterized by different equilibrium dissociation constants. The higher affinity (type I) receptors have an equilibrium dissociation constant of 3.3 X 10(-11) M. The lower affinity (type II) receptors have an equilibrium dissociation constant of 1.7 X 10(-9) M. These two receptors are not a result of negative cooperativity, but apparently are different receptors. At 22 degrees C the rate of association is 1 X 10(7) M-1 S-1 and the rates of dissociation are 6.5 X 10(-4) S-1 (type I) and 3.2 X 10(-2) S-1 (type II). After binding, a time-dependent process occurs that makes that NGF inaccessible to the external milieu (sequestered). The sequestration process is energy-dependent, but apparently temperature-independent. The data suggest that only the type I receptors are involved in the sequestration process. This process is similar to that observed on sympathetic neurons and may be the first step in the internalization of NGF by responsive cells.

Journal ArticleDOI
TL;DR: The interactions of phosphorylase b with immobilized butyl residues are compared to those of proteins and it appears that the complex interactions of these proteins with cell-surface-binding sites of varying surface concentrations may be analyzed in a similar manner as the binding of proteins to alkyl agaroses.

Journal ArticleDOI
TL;DR: It is concluded that both the physiol state of teh micelle and the intrinsic behavior of the ATPase polypeptide affect the temperature dependence of ATPase activity.
Abstract: The interaction of Triton X-100 and other nonionic detergents with a delipidated preparation of the Ca2+ ATPase from sarcoplasmic reticulum has been studied Binding of radiolabeled Triton X-100 was determined by column chromatography at 6 degrees C, and two classes of binding sites were observed Below the critical micelle concentration (cmc), binding of Triton occurred at 35-40 equivalent sites on the delipidated ATPase with a binding constant of 27 X 10(4) M-1 Near the cmc cooperative binding of an additional 70 molecules of the detergent was observed The binding of monomeric Triton X-100 below the cmc was associated with a parallel activation of over half of the ATPase activity, and the remainder of the activity was recovered after the detergent concentration was increased to the cmc The ability to reactivate ATPase activity was more dependent on the polar poly(oxyethylene) portion of nonionic detergents than on the hydrocarbon portion Generalizing for all amphiphiles, these results suggest that there are discrete binding sites on the Ca2+ ATPase for phospholipid molecules in the native membrane and that the polar head groups of phospholipids interact more strongly with the protein than the hydrophobic acyl chains Perturbations in micelle structure were observed for several nonionic detergents by measurement of cis-parinaric acid fluorescence and differential scanning calorimetry, and discontinuities in Arrhenius plots occurred at the transition temperature of the detergent used for reactivation of ATPase activity It is concluded that both the physiol state of teh micelle and the intrinsic behavior of the ATPase polypeptide affect the temperature dependence of ATPase activity

Journal ArticleDOI
TL;DR: The affinity constants of magnesium for the active or inactive enzyme forms are much greater than those of fructose bisphosphate, which suggests that the metal ion gives to the enzyme the right conformation to bind the sugar phosphate.
Abstract: Fluorescence titration experiments of chloroplastic fructose-1,6-bisphosphatase by fructose bisphosphate and magnesium have been effected using the inactive dimeric, the inactive tetrameric and the active tetrameric enzyme forms. Magnesium binding to the inactive dimeric enzyme exhibits a positive cooperativity whereas fructose 1,6-bisphosphate exhibits no cooperativity at all. The binding of either magnesium or fructose bisphosphate to the inactive oxidized tetramer exhibits a succession of negative and positive cooperativities (mixed cooperativity). Upon reduction of the inactive tetramer by dithiothreitol and activation, the ligand binding properties of the enzyme are changed. Magnesium and fructose bisphosphate are bound to the active enzyme with a positive cooperativity. Non-linear least-square fitting allows one to estimate thea binding constants, and therefore the free energy of binding of either magnesium or fructose bisphosphate to the various forms of the enzyme. Whatever the state of fructose-1,6-bisphosphatase, one ligand is bound per subunit. The affinity constants of magnesium for the active or inactive enzyme forms are much greater than those of fructose bisphosphate. This suggests that the metal ion gives to the enzyme the right conformation to bind the sugar phosphate.

Journal ArticleDOI
TL;DR: The GH3 cells contain a heterogeneous population of saturable thyroliberin-binding sites, and that the higher affinity receptor may be associated with prolactin release and cyclic AMP formation.
Abstract: The binding of [3H]thyroliberin was studied in intact GH3 cells at 37°C and correlated to accumulation of adenosine 3′,5′-monophosphate (cyclic AMP) and prolactin release. Experiments which were performed at steady state, showed binding of [3H]thyroliberin which was specific and saturable, but the results could not be adequately described on the basis of one homogeneous population of receptors. When two classes of binding sites were assumed, average Kd values of 0.9 nM and 23 nM were found in experiments performed at steady state. The maximum binding capacity was calculated to be 780 fmol [3H]thyroliberin/mg cell protein. Of the calculated 82500 thyroliberin receptors per GH3 cell, about 1/3 represented the higher affinity binding site. The half-times for dissociation of [3H]thyroliberin were 160 min and 5 min for the higher and lower affinity site, respectively. Association and dissociation rate constants measured at 37 °C for the high and low affinity receptor sites gave the following average values, k+1/k−1: 1.1 × 105 M−1 s−1/0.7 × 10−4 s−1 and 0.7 × 105 M−1 s−1/ 12 × 10−4 s−1, respectively. The phenomenon of negative cooperativity was apparently not present in the GH3 cells since the rates of dissociation for [3H]thyroliberin were similar in the presence or the absence of a 100-fold excess thyroliberin at high and low receptor occupancy. There was no evidence for internalization of the [3H]thyroliberin-receptor complex during these experiments (duration ⋝ 4 h) since the binding was easily reversible and the rates of dissociation for the slowly dissociable binding site were similar in experiments using different times of association. At brief incubation periods (10–120 s) the binding of the tripeptide occurred simultaneously with the increase in cvclic AMP accumulation with a lag period of less than 10 s, and maximal concentrations of cyclic AMP occurred at about 10% receptor occupancy. The dose-response curve of thyroliberin for prolactin rciease showed half maximal stimulation at 0.4 nM while half-maximal binding occurred at 10 nM during equilibrium conditions. We conclude that the GH3 cells contain a heterogeneous population of saturable thyroliberin-binding sites, and that the higher affinity receptor may be associated with prolactin release and cyclic AMP formation.

Journal ArticleDOI
TL;DR: Three adenosine 3',5'-phosphate (cAMP) binding proteins were separated and partially purified from cytoplasmic extracts of Dictyostelium discoideum cells developed to aggregation competence and resemble the "adenosine analogue binding proteins" described in mammalian cells.
Abstract: Three adenosine 3',5'-phosphate (cAMP) binding proteins were separated and partially purified from cytoplasmic extracts of Dictyostelium discoideum cells developed to aggregation competence. Two species, A and B, representing respectively 50% and 20% of the total activity, bind cAMP with very rapid kinetics and high specificity. Species A (Kd = 7.5 nM) is a monomeric protein of 36 000 daltons with a sedimentation coefficient of 2.3 S. Species B, which binds cAMP with positive cooperativity, also displays a high affinity for the ligand (Kd = 3.2 nM). This protein is present in the extracts as an equilibrium between monomeric, dimeric, and tetrameric forms with respective sedimentation coefficients of 2.4, 4.5, and 6.5 S; binding of cAMP to the monomer induces the appearance of the multimeric forms. A third cAMP binding protein (species C, Kd - 9.5 nM) was characterized as a larger protein (Mr 190 000, sedimentation coefficient of 9.2 S) which also binds adenosine and adenosyl derivatives. Species C represents 30% of the activity in the extracts and resemble the "adenosine analogue binding proteins" described in mammalian cells. The relevance of the properties of these proteins to the developmental process of D. discoideum amoebas is discussed.

Journal ArticleDOI
TL;DR: Dialysis measurements indicate that anthramycin is very possibly binding at sites distant from MC sites and suggest a clustering of closely bound MC chromophores resulting from possible cooperative binding.
Abstract: Anthramycin and mitomycin C (MC) are two DNA reactive drugs, which bind covalently to GC pairs producing different effects on DNA: anthramycin stiffening and MC distorsion. This paper describes experiments in which we have used anthramycin as a probe to sense quantitatively the effects on DNA of MC binding. Saturation binding experiments show that both anthramycin and MC partially inhibit the binding of the other drug to DNA (maximum inhibition by MC and anthramycin, 22.4% and 19.7%, respectively) but by a mechanism other than direct site exclusion. This suggests that MC binds in the major groove of DNA, since anthramycin is known to bind in the minor groove. An abrupt reduction in the binding of anthramycin to DNA-MC complexes occurs between MC binding ratios of 0.030 and 0.035, which parallels and probably results from sudden intensification of a MC-induced DNA conformational change occurring between these binding ratios. Dialysis measurements indicate that anthramycin is very possibly binding at sites distant from MC sites and suggest a clustering of closely bound MC chromophores resulting from possible cooperative binding. S1 nuclease digest experiments demonstrate an initial enhancement of nuclease activity in DNA-MC complexes, the magnitude of which correlates well with the reduction of anthramycin binding, relative to the degree of MC binding. The enhanced nuclease activity in these complexes indicates regions of exposed DNA or helix base distortion which is related to or is the result of conformational change.

Journal ArticleDOI
TL;DR: The results demonstrate that acrosin-liposome binding is due in part to electrostatic charge interactions and indicate that the enzyme has properties of an extrinsic membrane protein.

Journal ArticleDOI
TL;DR: The results presented indicate that Cibacron Blue can bind strongly to dopamine β-monooxygenase, thus emphasizing the importance of using pure isomers of the dye when studying the interactions with proteins.

Journal ArticleDOI
TL;DR: None of the alterations in insulin binding observed here can explain the enlarged adipose cell's markedly decreased metabolic response to insulin.
Abstract: 125I-insulin binding and degradation have been studied in isolated rat adipose cells of increasing size. Binding at 24 degrees C reaches a steady state by 40-60 min in the absence of significant degradation. At 37 degrees C, binding reaches only a transient maximum by 10-15 min because insulin is rapidly degraded. Cellular enlargement is associated with increasing steady-state binding per cell at 24 degrees C, and increasing maximum binding and degradation per cell at 37 degrees C, in spite of increasing plasma insulin concentrations in the rats from which cells were prepared. Detailed steady-state studies at 24 degrees C, however, fail to delineate the mechanism of these alterations. Although dissociation experiments are consistent with the presence of a small degree of negatively cooperative binding site interaction, its magnitude is unchanged with increasing cell size. Furthermore, binding levels per unit cellular surface at 24 degrees C, at least at those insulin concentrations eliciting a biological response, remain relatively constant. None of the alterations in insulin binding observed here can explain the enlarged adipose cell's markedly decreased metabolic response to insulin.

Journal ArticleDOI
TL;DR: An alternative to the common lattice model for nonspecific DNA–protein interactions is presented by using ligands that translate freely along the polynucleotide instead of binding to distinct lattice sites along thePolyn nucleotide chain.
Abstract: We present an alternative to the common lattice model for nonspecific DNA–protein interactions by using ligands that translate freely along the polynucleotide instead of binding to distinct lattice sites along the polynucleotide chain. The general model we present corresponds to a one-dimensional continuum gas and is referred to as the “continuum model” to distinguish it from the general lattice model. Explicit expressions are obtained for the binding isotherm equation for two version of the continuum model, including the effects of binding-site exclusion and attractions between bound ligands. Theoretical results are compared to those obtained from the McGhee-von Hippel (1974) analysis of the lattice model with cooperative interactions between ligands occupying more than one lattice site. Practical applications of the continuum model are illustrated by analyzing (i) the noncooperative binding to single-stranded DNA by RNase (Jensen and von Hippel, 1976), and (ii) the highly cooperative binding to poly(rA) by a proteolyzed fragment of the gene 32 protein of phage T4 (Lonberg et al., 1981).

Journal ArticleDOI
01 Jan 1981
TL;DR: Dynein presumably binds with equal affinity to any protofilament on in vitro assembled microtubules, and, therefore, the MAPs may not be capable of effectively interfering with cooperative binding of dynein to these micro Tubulin dimers.
Abstract: Microtubule-associated proteins (MAPs), isolated from brain tubulin, bound to and saturated outer fibers of Chlamydomonas flagella. MAPs present on these microtubules prevented the subsequent recombination of dynein. MAPs also bound to intact axonemes and thus did not specifically bind to the dynein binding sites on the A subfiber. A molar ratio of 1 mole MAP2 per 27 moles tubulin dimers at saturation of the outer fibers with MAP2 suggested that MAPs could effectively interfere with dynein recombination only if the MAPs were near the dynein binding sites to sterically prevent binding. However, electron microscopic observations indicated that MAPs were not localized but, instead, were dispersed around the outer fibers. In addition, MAP2 present at saturating amounts on in vitro assembled brain microtubules had no significant effect on dynein binding. Dynein-decorated microtubules contained clusters of arms suggesting that there may be cooperative interaction between the arms during dynein binding. Because the A subfiber of axonemes contains sites to which dynein preferentially attaches, MAPs may prevent recombination by interfering with cooperative binding to these specific sites. Dynein presumably binds with equal affinity to any protofilament on in vitro assembled microtubules, and, therefore, the MAPs may not be capable of effectively interfering with cooperative binding of dynein to these microtubules.

Journal ArticleDOI
TL;DR: Interaction of four different asialotransferrins (human types 1, 2, and 3, and rabbit asialOTransferrin) with purified plasma membranes from the rat liver was studied by using a direct binding assay, consistent with the view that the binding sites involved in the binding of asialotsferrin are homogenous.

Journal ArticleDOI
TL;DR: The interactions of rat muscle glyceraldehyde-3-phosphate dehydrogenase purified from young and old animals with NADH and with the fluorescent analogue nicotinamide 1,N6-ethenoadenine dinucleotide were investigated and it was concluded that age-related modifications occur in the Nicotinamide binding sites, but not in the adenine binding sites of this enzyme.