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

The kinetics of specific [35S]-t-butylbicyclophosphorothionate (TBPS) binding was studied in rat brain synaptosomal membrane preparations. The effects of a representative depressant barbiturate, R-(-)-N-(1)-Me-5-Phe-5-Pr-barbituric acid [R(-)MPPB, and gamma-aminobutyric acid (GABA) were biphasic on TBPS binding, but the enhancements of binding by low concentrations of R(-)MPPB or GABA disappeared when binding equilibrium was reached. The slope factors of TBPS displacement and the IC50 values of R(-)MPPB and GABA decreased when TBPS binding approached equilibrium. Up to 70 min, binding in the presence of 300 microM R(-)MPPB exceeded, then remained below, the level of the control. R(-)MPPB decreased the apparent association half-life of TBPS binding from 41.5 min to 11.9 min. The GABA agonist, muscimol, in the concentration range of 20 and 200 nM, progressively accelerated the rate of TBPS dissociation. This effect was completely reversed by 20 microM bicuculline methochloride. GABA antagonists, bicuculline methochloride (20 microM) and R 5135 (20 nM), alone decelerated the rate of TBPS dissociation. Dissociation of TBPS was also initiated by dilution in the presence of various salts. Replacement of Cl- by Br- ions (0.5 molar) accelerated the rate of dissociation, whereas replacement of K+ by Na+ ions had no effect. This indicates the role of Eccles anions, not of cations, in TBPS binding and the possible involvement of the chloride inophore. A kinetic model is discussed for the allosteric modulation of TBPS binding by various GABAergic agents. Interconvertible populations of TBPS sites are proposed with rapid and slow kinetics. Model calculations involving modulation in the same direction of the on and off rates of binding can reproduce observed phenomena. The model predicts that GABA agonists, barbiturate and pyrazolopyridine depressants, as well as 1,4-benzodiazepine agonists, would allosterically increase the proportion of a rapid kinetic population of TBPS sites. In contrast, the model predicts that a decrease in the contribution of the rapid phase might be brought about by GABA antagonists and beta-carboline inverse agonists. The slow and rapid kinetic populations of TBPS sites might represent the closed and open states of the chloride ionophores, respectively.
Mol Pharmacol 1986 Oct
PMID:Kinetic regulation of convulsant (TBPS) binding by GABAergic agents. 376 21

Dissociation of turnip crinkle virus (TCV) at elevated pH and ionic strength produces free dimers of the coat protein and a ribonucleoprotein complex that contains the viral RNA, six coat-protein subunits, and the minor protein species, p80 (a covalently linked coat-protein dimer). This "rp-complex" is stable for several days in high salt at pH 8.5. Reassembly of TCV can be accomplished under physiological conditions, using isolated coat protein and either rp-complex or protein-free RNA. If rp-complex is used in reassembly, the same subunits remain bound to RNA on subsequent dissociation; if free RNA is used, rp-complex is regenerated. In both cases, the assembly is selective for viral RNA in competition experiments with heterologous RNA. Electron microscopy shows that assembly proceeds by continuous growth of a shell from an initiating structure, rather than by formation of distinct intermediates. We suggest that rp-complex is the initiating structure, suggest a model based on the organization of the TCV particle, and propose a mechanism for TCV assembly.
J Mol Biol 1986 Oct 20
PMID:Structure and assembly of turnip crinkle virus. II. Mechanism of reassembly in vitro. 380 77

Previous studies have documented that high affinity binding of [125I]angiotensin II to adrenal cortex receptors was modulated by guanine nucleotides. Since in other receptor systems, similar properties of hormone-receptor interactions were shown to be specific for agonists, we studied the differential binding characteristics of agonists and antagonists to this receptor using a new radiolabeled antagonist [125I] [Sar1,Ile8] angiotensin II. Receptor saturation studies indicate that the antagonist is binding to a homogeneous population of sites with a Kd of 0.6-2.0 nM and with a receptor density around 1 pmol/mg of protein. Competition curves using unlabeled antagonists are characterized by a slope factor of 1 and a single Kd of 1-3 nM. Addition of guanylylimidodiphosphate to the assay is absolutely without effect on radiolabeled antagonist binding. In contrast, competition curves using the full agonists angiotensin II, [Sar1]angiotensin II, angiotensin III, and [des-Arg]angiotensin III display slope factors of 0.79, 0.87, 0.70, and 0.84, respectively. These curves can be explained by two apparent forms of the receptor having high and low affinity for the agonist. The higher affinity form associated with these four agonists is characterized by a Kd of 1.2 nM, 0.25 nM, 0.8 nM, and 3 microM, and corresponds to 60, 56, 42, and 25% of angiotensin II-binding sites, respectively. The other form displays 13- to 33-fold lower affinity. Addition of guanine nucleotide to the assay results in a 2-4-fold shift to the right and a steepening (slope factor 0.9-1.0) of agonist competition curves. Angiotensin II receptors, occupied by the full agonist [131I] [Sar1] angiotensin II or by the antagonist [125I] [Sar1, Ile8]angiotensin II, were then solubilized with the nonionic detergent octylglucoside. Dissociation of the agonist [131I] [Sar1] angiotensin II from solubilized receptors is enhanced by guanylylimidodiphosphate or sodium acetate, while dissociation of the antagonist [125I] [Sar1, Ile8]angiotensin II displays little sensitivity towards guanine nucleotides or increased ionic strength. Inclusion of bile salts in the solubilization medium preferentially destabilizes receptor-bound agonist, presumably by interfering with protein-protein interactions required for high affinity agonist binding. Separation of radiolabeled agonist and antagonist-occupied solubilized receptor complexes by steric exclusion high performance liquid chromatography reveals that the agonist-occupied receptor complex behaves as a larger protein than the antagonist-occupied receptor complex.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1984 Nov
PMID:Evidence for agonist-induced interaction of angiotensin receptor with a guanine nucleotide-binding protein in bovine adrenal zona glomerulosa. 609 99

The kinetics of [3H]flunitrazepam binding to synaptosomal (P2) membranes from rat cerebral cortex was studied. The pseudo-first order association at 0 degrees was curvilinear and the association rate was increased with increasing concentrations of ligand. The association curve was resolved best as two linear exponentials, representing a fast and a slow component. The percentage of each association component varied with the ligand concentration. Association was slightly decreased in the presence of gamma-aminobutyric acid. Dissociation at 0 degrees, whether equilibrium binding had occurred at 0 degrees or 37 degrees, was also biphasic. The dissociation of the complexes was more rapid at higher concentrations of ligand. Dissociation was slower in the presence of gamma-aminobutyric acid, and this effect was more pronounced at higher concentrations of ligand. Prolonged preincubation at 37 degrees either in the presence or in the absence of [3H]flunitrazepam also decreased the dissociation rate. Dissociation was faster before equilibrium than after equilibrium had been reached. These results are discussed in terms of one homogeneous type of benzodiazepine receptor with two interconvertible conformations.
Mol Pharmacol 1982 Jan
PMID:Kinetics of [3H]flunitrazepam binding to membrane-bound benzodiazepine receptors. 612 22

The structure and expression of the Shrunken (Sh) locus have been examined in several maize strains with mutations at the locus caused by the controlling element Dissociation (Ds). Three of the strains (sh-m6233, sh-m5933, and sh-m6258) are of independent origin, and the fourth (sh-m6795) represents a spontaneous recessive sh allele derived from an Sh revertant of strain sh-m6258. The sh-m6233 and sh-m5933 strains produce undetectable levels of the Sh-encoded sucrose synthetase and very small amounts (less than 1%) of an apparently normal sucrose synthetase mRNA in immature endosperm tissue. Both strains have rearrangements affecting the structure of the locus near the 5' end of the transcription unit. The Sh locus in the related strains sh-m6258 and sh-m6795 is interrupted by an insertion or a rearrangement having one breakpoint in an intervening sequence near the 3' end of the mRNA coding sequence. The 5' end of the gene is transcribed in immature kernels of both mutants, giving aberrant poly (A)+ mRNAs that are homologous to the normal transcript up to the insertion or rearrangement breakpoint and lack homology to the 3' end of the gene. The aberrant transcripts from both strains are translated in vivo and in vitro, yielding 82- and 85-kD proteins that are immunologically related to the 92-kD Sh-encoded sucrose synthetase monomer.
J Mol Appl Genet 1983
PMID:Molecular studies on mutations at the Shrunken locus in maize caused by the controlling element Ds. 622 Oct 58

The kinetics of formation and dissociation of specific (open) complexes between active Escherichia coli RNA polymerase holoenzyme (RNAP) and the lambda PR promoter have been studied by selective nitrocellulose filter binding assays at two temperatures (25 degrees C, 37 degrees C) and over a range of ionic conditions. Competition with a polyanion (heparin) or stabilization of open promoter complexes at PR by incubation with specific combinations of nucleoside triphosphates was employed to obtain selectivity in the filter assay. This study provides a useful example of how information about mechanism may be obtained from the quantitative analysis of the effects of salt concentration and temperature on the rate constants of a protein-DNA interaction. The association reaction between RNAP and lambda PR was investigated under ionic conditions where the process is essentially irreversible, and under pseudo first-order conditions of excess polymerase. The pseudo first-order rate constant is directly proportional to the concentration of active polymerase over the entire range investigated (2 to 10 nM) at both 25 degrees C and 37 degrees C, within experimental uncertainty. Second-order association rate constants (ka), calculated from these data at standard ionic conditions (0.12 M-KCl, 0.01 M-MgCl2, 0.04 M-Tris (pH 8)), were strongly temperature-dependent: ka = (2.6 +/- 0.4) X 10(6) M-1 S-1 at 37 degrees C and ka = (7.2 +/- 1.4) X 10(5) M-1 s-1 at 25 degrees C, corresponding to an activation energy of the association reaction of approximately 20 +/- 5 kcal. In addition, ka decreases strongly with increasing KCl concentration, corresponding to the net release of the thermodynamic equivalent of at least nine monovalent ions prior to or during the rate-limiting step of the association reaction. This strong dependence of ka on the ionic environment suggests that inorganic cations should be considered as possible regulators of in vivo transcription initiation. Dissociation rate constants (kd) were also measured under irreversible reaction conditions. At the standard ionic conditions, kd = (2.2 +/- 0.3) X 10(-5) s-1 at 37 degrees C and kd = (4.0 +/- 0.4) X 10(-5) s-1 at 25 degrees C. The increase in kd with decreasing temperature corresponds to a negative activation energy of dissociation (-9 +/- 4 kcal). In addition, kd increases with increasing KCl concentration, corresponding to the net uptake of the thermodynamic equivalent of at least six monovalent ions in or prior to the rate-limiting step of the dissociation reaction.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1984 Jul 15
PMID:Kinetics and mechanism of the interaction of Escherichia coli RNA polymerase with the lambda PR promoter. 623 75

E. coli RNA polymerase is shown to be capable of catalyzing consecutive DNA-dependent pyrophosphorolysis of RNA in the presence of inorganic pyrophosphate and Mg2+. Active ternary complex of the enzyme with DNA and nascent RNA is needed for the reaction, the mixure of all the components can not carry out pyrophosphorolysis. The reaction was realized in the absence of added nucleoside triphosphates. Nucleoside triphosphates are low molecular mass products of the reaction. The rate of pyrophosphorolysis of the RNA synthesised for the AI promoter of the DNA of wild type T7 phage and delta D III T7 mutant phage was followed as a function of primary structure of the DNA, temperature, ionic strength and inorganic pyrophosphate concentration. The relative rate pyrophosphorolysis for particular nucleotides in different regions of the RNA can differ by several orders of magnitude depending on the primary structure of the RNA that undergoes pyrophosphorolysis. Ternary complex containing partially pyrophosphorilised RNA is active on the RNA synthesis when pyrophosphate is removed and nucleoside triphosphates are added to the reaction mixture. RNA as short as 70-8 nucleotides long can be produced at the conditions used. It seems that efficient dissociation in this region of RNA limits the pyrophosphorolysis to proceed up to the 5' end of RNA. Ternary complex of RNA polymerase with nascent RNA and DNA is shown to undergo site specific dissociation. The specificity of the dissociation is shown to be a function of the primary structure of RNA and the direction of the reaction. Dissociation occurs at different places along RNA sequence when the RNA is synthesised and when it is pyrophosphorilised.
Mol Biol (Mosk)
PMID:[Reaction of pyrophosphorolysis catalyzed by Escherichia coli RNA polymerase]. 626 61

The kinetics of the dissociation of thyrotropin (TSH) from human thyroid plasma membranes were studied in an attempt to further understand the molecular dynamics of the TSH--receptor interaction. Dissociation of bound [125I]TSH from thyroid plasma membranes was a biphasic process consisting of rapidly and slowly dissociable components, RDC and SDC, respectively. The dilution induced dissociation of bound [125I]TSH was enhanced by the addition of excess TSH (DEC). DEC was proportional to the dose of unlabeled TSH and its magnitude increased linearly with temperature. These results are in contrast to those reported for the kinetics of [125I]insulin dissociation. The functional significance of DEC remains largely unexplained. It was found that the fraction of SDC was dependent upon time of association in a temperature-dependent and apparently saturable process. It could not be attributed to alterations in the electrophoretic, immunologic or binding properties of [125I]TSH. Furthermore, no correlation was observed between generation of SDC and change in the Scatchard profile of TSH binding, in contrast to studies on growth hormone. These data suggest that, like some other polypeptide hormones, binding of TSH to its receptor does not proceed according to laws describing simple, rapidly reversible, bimolecular reactions. Furthermore, bound TSH undergoes a receptor-mediated conversion from a rapidly to a slowly dissociable state with time of incubation.
Mol Cell Endocrinol 1981 Nov
PMID:Dissociation kinetics of the thyrotropin-receptor complex. Characterization of a slowly dissociable component. 627 12

The binding kinetics of [3H]Ro 15-1788, a selective benzodiazepine receptor antagonist, to synaptosomal membranes of rat cerebral cortices was studied. [3H]Ro 15-1788 binds with high affinity (dissociation constant, 0.53 nM) to a single class of binding sites (maximal binding capacity, 1.97 pmoles/mg of protein). Equilibrium binding was not affected by gamma-aminobutyric acid (GABA), NaCl, pentobarbital, or pretreatment of the membranes at 37 degrees. Association at 0 degrees was identical whether measured in the absence or presence of GABA or bicuculline methiodide or after preincubation of the membranes at 37 degrees. The association rate under pseudo-first order conditions was curvilinear and consisted of a fast component and a slow component. Dissociation at 0 degrees with 1 X 10(-5)M clonazepam was also curvilinear and could best be fitted by two linear exponential components. The dissociation rate was not altered by GABA, NaCl, pentobarbital, or pretreatment of membranes at 37 degrees. The dissociation rate was similar for 0.1, 1, and 10 nM [3H]Ro 15-1788. The ratio of slow to fast dissociation component for 10 nM [3H]Ro 15-1788 was larger than that for 0.1 and 1 nM [3H]Ro 15-1788. In contrast, the dissociation rate for 20 nM [3H]flunitrazepam ( [3H]FNP) was much greater than that for 2 nM [3H]FNP. Using ligand concentrations occupying the same fraction of receptors, the ratio of slow to fast dissociation components was invariably greater for [3H]Ro 15-1788 than that for [3H]FNP. The rate of dissociation for [3H]Ro 15-1788 was faster under pre-equilibrium conditions than under equilibrium conditions. These results, discussed in terms of the cyclic model of interaction between receptors and benzodiazepines, suggest that [3H]Ro 15-1788 is a powerful ligand in inducing conformational changes in the initial, more labile, binary complex. They also suggest that different conformational states deduced from studies of in vitro binding kinetics may not correspond to the distinct pharmacological actions of benzodiazepines. It is speculated that intrinsic activities of benzodiazepines probably are determined by the step beyond the complex formation and conformational changes suggested to occur by these studies of binding kinetics.
Mol Pharmacol 1983 Mar
PMID:Conformational changes in benzodiazepine receptors induced by the antagonist Ro 15-1788. 630 Jun 40

The preceding article shows that there are eight highly protected amide protons in the S-peptide moiety of RNAase S at pH 5, 0 degrees C. The residues with protected NH protons are 7 to 13, whose amide protons are H-bonded in the 3 to 13 alpha-helix, and Asp 14, whose NH proton is H-bonded to the CO group of Val47. We describe here the exchange behavior of these eight protected protons as a function of pH. Exchange rates of the individual NH protons are measured by 1H nuclear magnetic resonance in D2O. A procedure is used for specifically labeling with 1H only these eight NH protons. The resonance assignments of the eight protons are made chiefly by partial exchange, through correlating the resonance intensities in spectra taken when the peptide is bound and when it is dissociated from S-protein in 3.5 M-urea-d4, in D2O, pH 2.3, -4 degrees C. The two remaining assignments are made and some other assignments are checked by measurements of the nuclear Overhauser effect between adjacent NH protons of the alpha-helix. There is a transition in exchange behavior between pH 3, where the helix is weakly protected against exchange, and pH 5 where the helix is much more stable. At pH 3.1, 20 degrees C, exchange rates are uniform within the helix within a factor of two, after correction for different intrinsic exchange rates. The degree of protection within the helix is only 10 to 20-fold at this pH. At pH 5.1, 20 degrees C, the helix is more stable by two orders of magnitude and exchange occurs preferentially from the N-terminal end. At both pH values the NH proton of Asp 14, which is just outside the helix, is less protected by an order of magnitude than the adjacent NH protons inside the helix. Opening of the helix can be observed below pH 3.7 by changes in chemical shifts of the NH protons in the helix. At pH 2.4 the changes are 25% of those expected for complete opening. Helix opening is a fast reaction on the n.m.r. time scale (tau much less than 1 ms) unlike the generalized unfolding of RNAase S which is a slow reaction. Dissociation of S-peptide from S-protein in native RNAase S at pH 3.0 also is a slow reaction. Opening of the helix below pH 3.7 is a two-state reaction, as judged by comparing chemical shifts with exchange rates. The exchange rates at pH 3.1 are predicted correctly from the changes in chemical shift by assuming that helix opening is a two-state reaction. At pH values above 3.7, the nature of the helix opening reaction changes. These results indicate that at least one partially unfolded state of RNAase S is populated in the low pH unfolding transition.
J Mol Biol 1983 Sep 05
PMID:Exchange behavior of the H-bonded amide protons in the 3 to 13 helix of ribonuclease S. 631 52


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