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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The sequence dependence of Drosophila topoisomerase II supercoil relaxation and binding activities has been examined. The DNA substrates used in binding experiments were two fragments from Drosophila heat shock locus 87A7. One of these DNA fragments includes the coding region for the heat shock protein hsp70, and the other includes the intergenic non-coding region that separates two divergently transcribed copies of the hsp70 gene at the locus. The intergenic region was previously shown to have a much higher density of topoisomerase cleavage sites than the hsp70 coding region. Competition nitrocellulose filter binding assays demonstrate a preferential binding of the intergene fragment, and that binding specificity increases with increasing ionic strength. Dissociation kinetics indicate a greater kinetic stability of topoisomerase II complexes with the intergene DNA fragment. To study topoisomerase II relaxation activity, we used supercoiled plasmids that contained the same fragments from locus 87A7 cloned as inserts. The relative relaxation rates of the two plasmids were determined under several conditions of ionic strength, and when the plasmid substrates were included in separate reactions or when they were mixed in a single reaction. The relaxation properties of these two plasmids can be explained by a coincidence of high-affinity binding sites, strong cleavage sites, and sites used during the catalysis of strand passage events by topoisomerase II. Sequence dependence of topoisomerase II catalytic activity may therefore parallel the sequence dependence of DNA cleavage by this enzyme.
J Mol Biol 1987 Mar 20
PMID:Sequence dependence of Drosophila topoisomerase II in plasmid relaxation and DNA binding. 303 51

Binding of [3H]muscimol to mouse brain gamma-aminobutyric acid receptors has been assayed under more physiological conditions (never-frozen membranes, 22 degrees) than in previous studies (0-4 degrees on frozen-thawed membranes). Binding affinities were lower and agreed more closely with physiological dose response curves. In addition, heterogeneity in affinity was still present, apparently due to a mixture of non-interconvertible subpopulations and ligand-induced changes in kinetics. Super-high affinity sites (Kd less than 10 nM) observed in frozen membranes were not observed in fresh membranes under equilibrium binding conditions, and high affinity sites (Kd congruent to 25 nM) seen at 0 degree had lower affinity (Kd congruent to 250 nM) at 22 degrees. Despite an apparent best fit single-component Scatchard plot for the latter data, cold ligand displacement and association and dissociation rates demonstrated heterogeneity of affinities. Pentobarbital greatly increased the amount of high affinity sites at the expense of low affinity sites in equilibrium binding at 0 degree and slightly increased affinity at 22 degrees. The kinetics of [3H]muscimol association at 22 degrees (kapp = 1-2/min) were virtually independent of the ligand concentration, suggesting either negative cooperativity or an agonist binding-dependent receptor isomerization. Dissociation triggered by addition of excess cold ligand to membranes equilibrated with [3H]muscimol at different concentrations revealed two off-rates with t1/2 values of under 10 sec and 1-3 min; unexpectedly, the ratio of these two populations did not vary with ligand concentration but was constant at 50:50. Dissociation triggered by infinite dilution gave two off-rates, but the slowest component had a t1/2 of about 20 min; including cold muscimol in the dilution buffer increased the off-rate toward that observed in the excess cold ligand method (t1/2 = 1-2 min). The very slow off-rate was only observed following removal of agonist from a previously occupied receptor and suggests receptor isomerization into a high affinity state; this state is similar to that observed at 0 degree. Pentobarbital also favored the production of the receptor state showing very slow [3H]muscimol dissociation upon infinite dilution, opposing the action of high receptor occupancy with cold muscimol. Thus, [3H]muscimol-binding sites observed at 22 degrees with fresh membranes do not show artificially high affinity and have a Kd of 0.2-0.3 microM, closer to the EC50 for chloride channel activation (approximately 2-3 microM and 0.4 microM with pentobarbital).(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1987 Aug
PMID:gamma-Aminobutyric acid receptor binding in fresh mouse brain membranes at 22 degrees C: ligand-induced changes in affinity. 303 41

The range of allosteric interaction in the 24-meric hemocyanin from the tarantula Eurypelma californicum was studied by measuring the oxygen-binding properties of defined oligomeric fragments. Dissociation intermediates comprising 19, 12, 7 or 6 subunits were obtained by incubation of native hemocyanin with 10 mM-cysteine at pH 4.4, with 40 mM-dithiothreitol at pH 7 or 8, by short-term alkaline (pH 9.6) treatment or by treatment with 4 M-urea. These could be stabilized by returning to neutral buffer conditions and, in the case of the dodecamer, by carboxymethylation. Conditions were chosen so that part of the starting material remained intact to serve as control in the oxygen-binding measurements. Oxygen equilibrium curves were obtained by a very sensitive fluorimetric/polarographic method. Oxygen affinity and the magnitude of the Bohr effect remain constant from the hexamer up to the control four-hexamer. Co-operativity, in contrast, increases with aggregate size in two steps: n (hexamer) = n (heptamer) less than n (dodecamer) = n (19-mer) less than n (4-hexamer). The characteristic pH-dependence of nH is absent in the hexa- and heptamer but is weakly indicated in the dodecamer, and fully established in the four-hexamer. Results from different preparations are highly consistent, if nH is expressed as a percentage of the control values. Full co-operativity is reached only in the four-hexamer, disproving the dodecameric half-molecule (the smallest repeating unit) as the allosteric unit. The stepwise increase in co-operativity appears to be correlated with higher levels of symmetry in the hierarchy of quaternary structure.
J Mol Biol 1988 Nov 20
PMID:Hemocyanins in spiders. XXII. Range of allosteric interaction in a four-hexamer hemocyanin. Co-operativity and Bohr effect in dissociation intermediates. 322 91

The dissociation of daunomycin from DNA was examined using a sodium dodecyl sulfate-sequestered stopped flow procedure. Two dissociation processes were observed with calf thymus and bacterial DNA, with approximately 45% of the amplitude associated with the faster process. Both processes were largely independent of DNA sequence for bacterial DNA, comprising 30-70% (G + C) content. The rate of both processes increased by a similar amount with increasing ionic strength. The faster process decreased with decreasing drug loading, whereas the slower process was independent of drug loading. Only one dissociation event was observed for the dissociation of daunomycin from four different synthetic polydeoxynucleotides. All observations are consistent with a parallel model of sodium dodecyl sulfate-induced dissociation of daunomycin from DNA, where the two processes observed reflect two resolvable processes that may be comprised of a series of rate constants for the dissociation of drug from differing environments. The slower process observed with bacterial DNA (0.5-0.7 sec-1) is related to dissociation from preferential 5'-CA DNA-binding sites, whereas the faster process reflects dissociation of drug from lower affinity sites on heterogenous DNA (3.2-4.1 sec-1). Dissociation of daunomycin from four different synthetic polydeoxynucleotides (which did not contain the 5'-CA preferential daunomycin-binding site) exhibited dissociation rates characteristic of low affinity sites (3.3-4.8 sec-1).
Mol Pharmacol 1988 Feb
PMID:Daunomycin-DNA dissociation kinetics. 334 82

Brome mosaic virus reversibly dissociates into subunits in the pressure range of 600 x 10(5) to 1600 x 10(5) Pa, as demonstrated by studies of the spectral shift of intrinsic fluorescence, of filtration chromatography and of electron microscopy of samples fixed under pressure. Smaller shell particles (T = 1) were detected as intermediates in the dissociation pathway. Dissociation was facilitated by decreasing the concentration, as expected for a multimolecular reaction. The estimated change in volume upon dissociation into 90 dimer particles was -2960 ml/mol. Large increases in the intrinsic fluorescence intensity and in the binding of bis(8-anilinonaphthalene-1-sulfonate) occurred at pressures higher than 1400 x 10(5) Pa. The pressure-dependence profile of the different spectral properties shifted to lower pressures when 5 mM-MgCl2 was included in the buffer or when the pH was raised from 5.5 to 5.9. When the pressure was progressively increased above 1400 x 10(5) Pa, a value that led to 75% dissociation, the capsid subunits lost the ability to reassociate into regular shells and only amorphous aggregates were formed after decompression, as evidenced by both electron microscopy and gel filtration chromatography. The formation of these random aggregates of brome mosaic virus can be explained by a conformational drift of the separated subunits, similar in nature to that found in simpler oligomeric proteins.
J Mol Biol 1988 Jan 05
PMID:Pressure-induced dissociation of brome mosaic virus. 335 16

Rat adipocytes in primary culture have been used to study the intracellular processing of growth hormone (GH) receptors. Pretreatment of adipocytes with 20 micrograms/ml cycloheximide resulted in a rapid decline (t1/2 approximately 45 min) of the 125I-human growth hormone (hGH) binding capacity of the cells. This decline occurred at a faster rate in the presence of extracellular unlabeled hGH (400 ng/ml) and was not due to receptor occupancy. These data suggest that GH receptors turn over rapidly and constitutively on the plasma membrane and in the absence of protein synthesis are not replaced. Dissociation of GH-receptor complexes was shown not to occur at pH 5.5, the pH encountered in the acidic pre-lysosomal compartments (endosomes) where intracellular dissociation of many hormone-receptor complexes takes place. These data, together, suggest that the majority of GH receptors are not recycled but instead suffer the same fate as the majority of GH, i.e. degradation. To determine the rate of appearance of GH receptors at the cell surface, adipocytes were first treated with trypsin and then incubated at 37 degrees C to permit incorporation of any available GH receptors into the plasma membrane. Binding of 125I-hGH recovered to pre-trypsin levels by 2 h. This recovery was completely blocked by concomitant treatment with monensin, cytochalasin B, colchicine and 2,4-dinitrophenol. NH4Cl had no effect on receptor recovery. These data suggest that once GH receptors are synthesized in the rough endoplasmic reticulum, they travel via the Golgi apparatus to the plasma membrane (by processes involving both microfilaments and microtubules) and are then inserted into the plasma membrane in an energy-dependent step.
Mol Cell Endocrinol 1988 May
PMID:Intracellular processing of growth hormone receptors by adipocytes in primary culture. 339 60

G. Weber [(1984) Proc. Natl. Acad. Sci. USA 81, 7098-7102] has inferred that the Monod-Wyman-Changeux (MWC) model for ligand binding by hemoglobin would require (contrary to experimental evidence) that increased ligand binding must promote stabilization of alpha 2 beta 2 tetramers with respect to dissociation into alpha beta dimers. Reexamination of the MWC model, however, in the light of general linkage principles and the specific analysis by G. K. Ackers and M. L. Johnson [(1981) J. Mol. Biol. 147, 559-582] shows that the opposite relation must hold, in agreement with experiment. The T form of the tetramer, with low ligand affinity, must be destabilized and progressively dissociates into the high-affinity dimers, designated D, as ligand binding increases. Each ligand molecule bound shifts the standard Gibbs free energy delta G2T for the D-T equilibrium by approximately 3 kcal/mol in favor of the dimer. Thus, T must exist in (at least) five delta G levels of cooperative free energy as it becomes progressively destabilized by successive binding of ligand molecules. Dissociation of the R tetramer to dimers, in contrast, is independent of the amount of ligand bound, so long as dimers and R-state tetramers possess the same (high) affinity for ligand. While the intrinsic ligand-binding constants of the T and R states (KT and KR) remain unchanged throughout by the postulates of the model, the model should not be regarded as a strictly two-state system in view of the multiple free-energy levels indicated above. The present analysis gives approximate, though not precise, agreement with experimental findings on the dimer-tetramer equilibrium considered by Weber and provides a rationale for interpreting other recent experiments concerning this equilibrium.
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PMID:Linkage between ligand binding and the dimer-tetramer equilibrium in the Monod-Wyman-Changeux model of hemoglobin. 345 57

The synthesis of decidium and hexidium diiodides, their spectroscopic properties, and association with acetylcholinesterase from Torpedo californica are described and compared with those for propidium. Decidium, hexidium, and propidium, bisquaternary analogs of the fluorescent phenanthridinium ligand ethidium, contain 10, 6, and 3 methylene carbons, respectively, interposed between the exocyclic and endocyclic quaternary nitrogens. The three ligands exhibit linear competitive inhibition of enzyme carbamylation by N-methyl-7-dimethylcarbamoxyquinolinium. Dissociation constants for decidium, hexidium, and propidium are found by direct fluorescence titration to be 2.1 +/- 0.2 X 10(-8), 5.8 +/- 1.4 X 10(-7), and 3.7 +/- 0.4 X 10(-6) M, values in close accord with the inhibition constants obtained from kinetic analyses. Association of the three ligands is characterized by a stoichiometry of one fluorescent ligand per 80-kDa molecular weight subunit and occurs with respective 6.5-, 4.5-, and 3-fold increases in both quantum yield and fluorescence lifetime. Decidium and hexidium, in marked contrast with propidium, are dissociated by ligands selective for the active center and undergo pronounced reduction in affinity upon modification of the active center with pyrenebutyl methylphosphonofluoridate. Whereas the kinetics reveal no clear distinctions in inhibitory action of the three ligands, the fluorescence studies indicate that the alkyltrimethylammonium moiety of decidium and hexidium occludes the active center; propidium, in contrast, associates solely with the peripheral anionic site and does not occlude the active center. The temperature dependence of binding indicates that decidium association engenders a substantial increase (+55 eu) in entropy. The data indicate that the active center and peripheral anionic sites are separated by a crevice which can accommodate the hydrocarbon portion of extended n-alkyl cationic ligands, thereby affording entropic stabilization of complex formation. This stabilization is realized, however, only when the anionic subsite of the active center is not occluded, enabling electrostatic interaction between cationic ligand and the anionic active center.
Mol Pharmacol 1987 Jun
PMID:Site selectivity of fluorescent bisquaternary phenanthridinium ligands for acetylcholinesterase. 360 Jun 5

Dissociation kinetics of parvalbumin complexes with calcium and magnesium ions were studied by means of stopped-flow method employing intrinsic protein fluorescence registration. In the temperature range from 10 to 30 degrees C the kinetic curves of Ca2+ and Mg2+ dissociation are best fitted with a sum of two exponential terms, each term is ascribed to a dissociation process in one of two bindings sites of parvalbumin. Dissociation rate constants in this temperature range increase from 0.03 to 0.8 s-1 and from 0.18 to 5 s-1 for Ca2+, and from 0.9 to 4.5 s-1 and from 4 to 33 s-1 for Mg2+. Parvalbumin equilibrium binding constants of Ca2+ and Mg2+ were also measured in the same temperature range. It makes possible to estimate the rate constants of association of Ca2+ and Mg2+. In the case of Ca2+ the rate of association approaches the diffusion controlled limit.
Mol Biol (Mosk)
PMID:[Kinetics of dissociation of parvalbumin complexes with calcium and magnesium ions]. 365 79

We compared the binding of [N-methyl-3H]scopolamine methyl chloride [( 3H]NMS) and pirenzepine to muscarinic receptors in four regions of the rat central nervous system (cortex, hippocampus, striatum, and cerebellum) and in rat heart. Equilibrium binding studies suggested the existence of three classes of receptors: A, receptors with high affinity for pirenzepine and [3H] NMS (in cortex, hippocampus, and striatum); B, receptors with intermediate affinity for pirenzepine and high affinity for [3H]NMS (in the same brain regions); and C, receptors with low affinity for pirenzepine and [3H]NMS (in cerebellum and heart). Dissociation kinetic studies indicated that the receptor types A, B, and C had different koff values allowing, therefore, a separate study of their binding properties. We observed that: [3H]NMS recognized muscarinic receptors A, B, and C with the following order of potency: B greater than A much greater than C; and pirenzepine recognized these receptors with a different order of potency: A much greater than B greater than C. Thus, dissociation kinetics provide a useful tool to identify muscarinic receptor types.
Mol Pharmacol 1986 Oct
PMID:Kinetic studies of [3H]-N-methylscopolamine binding to muscarinic receptors in the rat central nervous system: evidence for the existence of three classes of binding sites. 376 20


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