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PubMed1984

A structural concept for nucleoli of Dictyostelium discoideum deduced from dissociation studies.

We aimed to establish whether there is a matrix structure in the nucleolus to which the ribosomal DNA (rDNA) is strongly attached. To detect artifacts that might occur during the harsh histone extraction procedures frequently used for matrix preparation, we dissociated nucleoli of Dictyostelium discoideum with a range of NaCl or heparin concentrations. With heparin treatment significant amounts of rDNA were solubilized into the dissociating solution. When the residual nucleoli were digested with Eco RI, none of the Eco RI fragments of the rDNA remained preferentially bound to the residual nucleoli, indicating that there is no matrix attached to a specific site on the rDNA. When residual nucleoli were examined by electron microscopy, a correlation was found between the extent of solubilization of rDNA, the loss of nucleosomes, and, in heparin-treated nucleoli, the loss of ribonucleoprotein-bound components. These results suggest that the rDNA is released from the nucleoli as soon as nucleosomes have been dissociated and transcription complexes disrupted. Electron microscopy also showed that the NaCl concentration required for dissociation of nucleosomes was higher when divalent cations (Ca2+, Mg2+, Cu2+) were used during the isolation or the treatment of the nucleoli prior to dissociation in high salt. Furthermore, the residual, high-salt-resistant structures were much larger when nucleoli were pretreated with divalent cations or when they were purified in the presence of Ca2+ than when they were purified in its absence. Hence divalent cations, which induce chromatin condensation, prevented nucleolar dissociation whereas treatment with chelating agents, which loosen chromatin compaction, led to much smaller residual matrixlike structures. Nucleoli could be dissociated with heparin to a larger extent than with NaCl so that in Ca2+-free preparations no residual nucleolar matrixlike structures could be detected. Our results suggest that the nucleolar "matrix" seen in the electron microscope is due to incomplete dissociation of the nucleolar material. We propose that in nucleoli of Dictyostelium the rDNA is not attached to a tightly binding matrix structure, but that nucleoli are stabilized by side-to-side contacts between chromatin fibers and transcription complexes.

Cell Fractionation↗

PubMed1983

Possible implications of the comparative dissociative behavior of testosterone- and 5 alpha-dihydrotestosterone-receptor complexes formed by human skin fibroblasts.

We have studied the dissociative behavior of 5 alpha-dihydrotestosterone (DHT)- or testosterone (T)-receptor complexes extracted from normal and 5 alpha-reductase-deficient human genital skin fibroblasts, respectively. The rate at which each hormone dissociates from the androgen receptor is temperature-dependent (29-40 degrees C), but DHT does so in a first-order (monophasic) manner, while T yields complex (biphasic) dissociation kinetics. Prior exposure to 0.1 M sodium thiocyanate induces DHT-receptor complexes to dissociate with complex kinetics. The temperature dependence of these complex dissociation profiles can be simulated identically by a kinetic model involving three conformational states of the androgen-receptor complex. Neither the rate constants of the rapidly- or slowly-dissociating components of T-receptor dissociation, nor the magnitude of the latter are affected by sodium molybdate (5 mM). Similarly, neither sodium molybdate nor potassium chloride (0.1-0.3 M) alters the complex dissociative behavior of DHT-receptor complexes that is induced by 0.1 M sodium thiocyanate.

Dihydrotestosterone↗

PubMed1986

The effect of dithiothreitol on the kinetics of dissociation of dexamethasone from the non-transformed mammary cytosolic glucocorticoid receptor.

Sulfhydryl reducing agents such as dithiothreitol are required for maximum binding of dexamethasone to the mammary cytosolic glucocorticoid receptor, but little is known concerning the effects of dithiothreitol on the kinetics of the binding reaction. In this report we have examined the influence of dithiothreitol on the dissociation kinetics of dexamethasone from the non-transformed glucocorticoid-receptor complex at 0-4 degrees C under various experimental conditions. Without dithiothreitol, the rate of dissociation of dexamethasone remains essentially the same (t1/2 approximately 17 h) regardless of the method chosen to monitor dissociation. With dithiothreitol, however, there is a marked acceleration in the rate of dissociation of receptor-bound dexamethasone when an excess of unlabeled dexamethasone is used to study dissociation (t1/2 approximately 5 h) but not when dissociation is investigated by removal of free labeled dexamethasone by charcoal adsorption (t1/2 approximately 21 h); dithiothreitol also accelerates the observed rate of dissociation when a combination of these methods is used. An acceleration in the rate of receptor-bound dexamethasone is also observed when an excess of the synthetic progestin, R5020, is used in the dissociation assay. The possible reasons and importance underlying these findings have been discussed.

Animals↗

PubMed1983

Analysis of heterogeneous dissociation kinetics in polyclonal populations of rabbit anti-fluorescyl-IgG antibodies.

Hapten dissociation kinetics were used to characterize the expression of hyperimmune anti-fluorescyl-IgG antibodies within a population of 14 New Zealand White (NZW) rabbits. Anti-fluorescyl-IgG antibody preparations, obtained from individual hyperimmune NZW rabbits, exhibited either three or four discrete dissociative components. The fastest component was typically heterogeneous with respect to dissociation rate, exhibiting an average dissociative lifetime of 8 sec, where dissociative lifetime was the reciprocal dissociation rate constant. Longer-lifetime components exhibited lifetimes of several hundred seconds, and became progressively more homogeneous with increasing lifetime. Comparison of dissociative components, within the NZW rabbit population, revealed the presence of four distinct groupings of dissociative lifetimes, which were approximately Gaussian in their distribution. Average lifetimes of 8, 52.6, 313 and 1417 sec were observed for the four groupings, which corresponded to free-energy (delta G0) values of -9.6, -10.6, -11.6 and -12.42 kcal/mole, respectively (using an association rate value of 5 X 10(6) M-1 sec-1). These findings implied that affinity maturation of the anti-fluorescyl-IgG antibody response involved the generation of discrete high-affinity subpopulations of antibodies rather than a continuous spectrum of high-affinity antibodies.

Animals↗

PubMed1994

Endothelin receptor agonists and antagonists exhibit different dissociation characteristics.

Endothelins (ETs) are vasoconstricting peptides that bind to membrane receptors to initiate their physiological effects. This report compares the dissociation characteristics of selected ET agonists and antagonists, and studies the effects of any difference in dissociation characteristics on the potency of antagonists. Competition studies using various ET receptor ligands against [125I]ET-1 or [125I]ET-3 binding demonstrated that porcine cerebellum membranes contain predominantly ETB receptor. [125I]IRL1620 associated with the receptors in a time-dependent manner. Although bound [125I]IRL1620 was easier to dissociate than bound [125I]ET-3, both agonists exhibited a dissociation half life > 20 h. For non-radiolabeled ligands, bind-and-wash studies were employed in which membranes were pre-incubated with unlabeled ligand followed by extensive washing before assaying for [125I]ET-1 binding. Results from bind-and-wash studies confirmed that bound non-radiolabeled IRL1620 and ET were as difficult to dissociate as [125I]ligands. In contrast, bound PD142893 and Ro46-2005 were easily dissociated from ETB receptors. Consequently, the inhibitory effects of PD142893 and Ro46-2005 on [125I]agonist binding diminished following incubation time. In cloned human ETA and ETB receptors, bound ET-1 was also more difficult to dissociate than bound antagonists. These results suggest that the differences in the dissociation characteristics of ET receptor agonists vs. antagonists may account for the diminished potency of Ro46-2005 and PD142893 as a function of incubation time.

Amino Acid Sequence↗

PubMed1994

Different effects of antidepressants on dissociation of 3H-imipramine from solubilized binding sites of rat brain.

1. The effects of several antidepressants and 5-hydroxytryptamine on dissociation of 3H-imipramine from solubilized binding sites were investigated. 2. Binding sites were solubilized from rat brain membranes and gelfiltrated on a column of Sephacryl S-300. 3. Most of the agents used allowed biphasic dissociation with 1mM of displacing agent and without using dilution-induced dissociation. This biphasic dissociation without nonspecific effects of membranes may be due to the existence of low-affinity binding sites. 4. Dissociation of up to 40 min followed first-order kinetics. The dissociation half-life of 3H-imipramine with the various displacing agents was calculated at from 15.0 to 25.0 min, and the differences among the agents were not so significant as the attenuation or the acceleration of the dissociation was indicated. The lower concentration of the displacing agents may obscure the modulation of the dissociation.

Animals↗

PubMed1986

Studies on dissociation of mouse prolactin from mouse hepatic receptors.

The influence of pH, temperature, ethylene glycol, urea, chaotropic anions and excess unlabelled secreted mouse prolactin (smPRL) on the dissociation kinetics of 125I-iodosmPRL from mouse hepatic receptors was investigated. The destabilization of smPRL-receptor complexes by chaotropic anions followed the typical trend of the Hofmeister series: I- greater than Br- greater than Cl- greater than F-. Increasing the temperature of the dissociation reaction from 8 degrees C to 23 degrees C and 30 degrees C caused partial dissociation of 125I-iodosmPRL-receptor complexes. Dissociation of 125I-iodosmPRL from mouse hepatic receptors was pH dependent, with the slowest rate of dissociation occurring at pH 8 and the fastest rate of dissociation occurring at pH 5 and 6. Both ethylene glycol and urea accelerated the rate of dissociation of 125I-iodosmPRL from mouse hepatic receptors in a concentration-dependent manner. Dissociation of 125I-iodosmPRL from mouse hepatic receptors was 6-fold faster in the presence of excess unlabelled smPRL than in its absence. The results of these investigations suggest that both protonation/de-protonation reactions and hydrophobic interactions play important roles in stabilizing the smPRL-receptor complex. In addition, they suggest that cooperative interactions may be involved in the binding of smPRL to mouse hepatic receptors.

Animals↗

PubMed1996

GTP binding to Gs does not promote subunit dissociation.

The stimulatory G protein (Gs) mediates activation of adenylyl cyclase. Gs is a heterotrimeric protein (alpha beta gamma) that is activated when guanosine triphosphate (GTP) or a non-hydrolyzable GTP analogue displaces tightly bound guanosine diphosphate (GDP) from the guanine nucleotide-binding site of the alpha-subunit (Gs alpha). Divalent cations such as magnesium are also required for Gs activation. Subunit dissociation can accompany Gs activation and is thought to be critical for this process. We investigated the effects of MgCl2 and various purine nucleotides on Gs-subunit dissociation and activation. Subunit dissociation was assayed by measuring the amount of G protein beta-subunit that was co-precipitated by Gs alpha-specific antiserum. Gs activation was determined by its ability to reconstitute adenylyl cyclase activity in S49 cyc-membranes that lack Gs alpha. High concentrations of MgCl2 caused bound GDP to dissociate from Gs and inactivated the protein unless high concentrations of GDP or GTP were present in solution. MgCl2 caused a concentration-dependent dissociation of Gs subunits. GTP gamma S (a non-hydrolyzable GTP analogue) shifted the MgCl2 concentration-response curve for subunit dissociation to lower concentrations of MgCl2, suggesting that GTP gamma S promoted subunit dissociation. On the other hand, GDP and GTP were equally effective in shifting the curve to higher concentration of MgCl2. These results suggest that GTP, the compound that activates Gs in vivo, was no more effective at promoting Gs subunit dissociation than was GDP.

Adenosine Triphosphate↗

PubMed1994

The kinetics of [3H]SCH 23390 dissociation from rat striatal dopamine D1 receptors: effect of dopamine.

The present study investigated possible allosteric interactions between dopamine and [3H]SCH 23390 ((R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepi n-7-ol)- labelled dopamine D1 receptors in rat striatum. As previously described, dopamine prevented [3H]SCH 23390 binding in a mixed competitive/non-competitive manner, causing both a loss of ligand affinity and a decrease in Bmax. The effect of dopamine was largely reversed following pretreatment of the membranes with 100 microM Gpp(NH)p (5'-guanylylimidodiphosphate) and was significantly enhanced by omission of Na+ from the incubation buffer. In dissociation kinetic studies, two methods of initiating ligand dissociation were used: dilution into 100-fold volume excess of buffer or addition of a molar excess of drug. Both methods yielded similar rates of [3H]SCH 23390 dissociation. Inclusion of dopamine in the volume excess of buffer did not alter the k-1 for [3H]SCH 23390 dissociation. However, when 100 microM dopamine was used instead of 1 microM piflutixol to initiate dissociation, a significant slowing of the rate of dissociation of [3H]SCH 23390 occurred. This effect of dopamine on k-1 was Na(+)-dependent since in the absence of Na+ the dopamine-induced rate of dissociation was only slightly slower than control values. Under neither condition did dopamine accelerate the rate of ligand dissociation, indicating that dopamine does not interact allosterically with [3H]SCH 23390 binding sites. These data, therefore, preclude an allosteric mechanism to explain the dopamine-induced decrease in dopamine D1 receptor density and provide direct evidence that dopamine masks ligand binding by binding to a high affinity site which can be modulated by Gpp(NH)p and Na+.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed2006

Electron capture in spin-trap capped peptides. An experimental example of ergodic dissociation in peptide cation-radicals.

Electron capture dissociation was studied with tetradecapeptides and pentadecapeptides that were capped at N-termini with a 2-(4'-carboxypyrid-2'-yl)-4-carboxamide group (pepy), e.g., pepy-AEQLLQEEQLLQEL-NH(2), pepy-AQEFGEQGQKALKQL-NH(2), and pepy-AQEGSEQAQKFFKQL-NH(2). Doubly and triply protonated peptide cations underwent efficient electron capture in the ion-cyclotron resonance cell to yield charge-reduced species. However, the electron capture was not accompanied by backbone dissociations. When the peptide ions were preheated by absorption of infrared photons close to the dissociation threshold, subsequent electron capture triggered ion dissociations near the remote C-terminus forming mainly (b(11-14) + 1)(+)* fragment ions that were analogous to those produced by infrared multiphoton dissociation alone. Ab initio calculations indicated that the N-1 and N-1' positions in the pepy moiety had topical gas-phase basicities (GB = 923 kJ mol(-1)) that were greater than those of backbone amide groups. Hence, pepy was a likely protonation site in the doubly and triply charged ions. Electron capture in the protonated pepy moiety produced the ground electronic state of the charge-reduced cation-radical with a topical recombination energy, RE = 5.43-5.46 eV, which was greater than that of protonated peptide residues. The hydrogen atom in the charge-reduced pepy moiety was bound by >160 kJ mol(-1), which exceeded the hydrogen atom affinity of the backbone amide groups (21-41 kJ mol(-1)). Thus, the pepy moiety functioned as a stable electron and hydrogen atom trap that did not trigger radical-type dissociations in the peptide backbone that are typical of ECD. Instead, the internal energy gained by electron capture was redistributed over the peptide moiety, and when combined with additional IR excitation, induced proton-driven ion dissociations which occurred at sites that were remote from the site of electron capture. This example of a spin-remote fragmentation provided the first clear-cut experimental example of an ergodic dissociation upon ECD.

Cations↗

PubMed1998

Serotonin transporter gene variants in alcohol-dependent subjects with dissocial personality disorder.

BACKGROUND: We tested the hypothesis that a functional biallelic repetitive element in the 5' regulatory region of the human serotonin transporter gene (SLC6A4) confers susceptibility to serotonin-related personality traits underlying alcohol dependence with dissocial behavior. METHODS: The association study was focused on 64 alcohol-dependent subjects with a dissocial personality disorder (according to ICD-10) who were derived from 315 German alcohol-dependent subjects. The Tridimensional Personality Questionnaire (TPQ) was applied to assess personality dimensions in 101 alcohol-dependent men, including 39 dissocial alcoholics. RESULTS: Our association analyses revealed a trend towards a higher frequency of the short (S) allele of the SLC6A4 polymorphism in dissocial alcoholics compared to 216 German controls (chi 2 = 2.81, df = 1, p = 0.094). Dissocial alcoholics carrying the S/S genotype exhibited significant lower scores of harm avoidance compared to those lacking it (U-test, p = 0.015). Significantly higher novelty seeking scores were obtained in dissocial alcoholics carrying the S allele relative to those lacking it (U-test, p = 0.021). CONCLUSIONS: Our tentative association findings in dissocial alcoholics suggest that the S allele of the 5' regulatory SLC6A4 polymorphism confers susceptibility to a temperamental profile of high novelty seeking and low harm avoidance that has been postulated to underlie dissocial (type-2) alcoholism according to Cloninger's neurogenetic theory of personality.

Adult↗

PubMed1997

Delayed dissociation of in vitro moving actin filaments from heavy meromyosin induced by low concentrations of Triton X-100.

The in vitro motility of fluorescent actin filaments over heavy meromyosin (HMM) was studied in the presence of the nonionic detergent Triton X-100. Below 0.004% Triton X-100 concentration, motility was not affected. Above 0.007%, motility was not observed because actin filaments were dissociated from HMM. In the Triton X-100 concentration range of 0.004-0.007%, the sliding actin filaments dissociated from HMM with a delay. The dissociation delay time decreased with increasing Triton X-100 concentration, increasing ATP (adenosine-5'-triphosphate) concentration, and increasing temperature. The delayed acto-HMM dissociation was absent when weak-binding kinetic intermediates of the myosin ATPase cycle (M.ATP and M.ADP-Pi) were used. The presence of sliding movement was necessary to evoke the delayed acto-HMM dissociation. The acto-HMM dissociation delay was independent of actin filament length. For a given Triton X-100 concentration, the dissociation delay time was found to be inversely proportional to sliding velocity, indicating that actin filaments travel a more or less constant distance prior to dissociation from HMM. The actin-activated HMM ATPase activity was not inhibited by Triton X-100; rather, it was slightly enhanced. The results imply the presence of a motility-associated conformational change in acto-HMM.

Actins↗

PubMed2000

Analyte-receptor binding and dissociation kinetics for biosensor applications: a fractal analysis.

A fractal analysis of confirmative nature only is presented for analyte-receptor binding and dissociation kinetics for biosensor applications. Data taken from the literature may be modeled, in the case of binding using a single-fractal analysis or a dual-fractal analysis. The dual-fractal analysis represents a change in the binding mechanism as the reaction progresses on the surface. Relationships are presented for the binding and dissociation rate coefficients as a function of their corresponding fractal dimension, Df or the degree of heterogeneity that exists on the surface. When analyte-receptor binding or dissociation is involved, an increase in the heterogeneity on the surface (increase in Df) leads to an increase in the binding and in the dissociation rate coefficient. It is suggested that an increase in the degree of heterogeneity on the surface leads to an increase in the turbulence on the surface owing to the irregularities on the surface. This turbulence promotes mixing, minimizes diffusional limitations, and leads subsequently to an increase in the binding and in the dissociation rate coefficient (Martin S.J., Granstaff, V.E., Frye, G.C., Anal. Chem., 65, (1991) 2910). The binding and the dissociation rate coefficient are rather sensitive to the degree of heterogeneity, Df,bind and Df,diss respectively, that exists on the biosensor surface. For example, the order of dependence on Df,bind is 19.2 for the binding rate coefficient, kbind for the binding of 0.03-1.0 microM SH-2Ld in solution to 2C TCR immobilized on a surface plasmon resonance (SPR) biosensor (Corr, M., Salnetz, A.E., Boyd, L.F., Jelonek, M.T., Khilko, S., Al-Ramadi, B.K., Kim, Y.S., Maher, S.E., Bothwell, A.L.M., Margulies, D.H., Science, 265, (1994) 946). The order of dependence on Df,diss is -6.22 for the dissociation rate coefficient, kdiss for the dissociation of 250-1000 nM Sophora japonica agglutinin (SJA)-lactose complex from the SPR surface. In general, the technique is applicable to other reactions occurring on different types of surfaces, such as cell-surface reactions.

Biosensing Techniques↗

PubMed1997

Effects of charge state on fragmentation pathways, dynamics, and activation energies of ubiquitin ions measured by blackbody infrared radiative dissociation.

Blackbody infrared radiative dissociation spectra of the (M + 5H)5+ through (M + 11H)11+ ions of the protein ubiquitin (8.6 kDa) formed by electrospray ionization were measured in a Fourier-transform mass spectrometer. The 5+ ion dissociates exclusively by loss of water and/or ammonia, whereas the 11+ charge state dissociates only by formation of complementary y and b ions. These two processes are competitive for intermediate charge state ions, with the formation of y and b ions increasingly favored for the higher charge states. The y and b ions are formed by cleavage of the backbone amide bond on the C-terminal side of acidic residues exclusively, with cleavage adjacent to aspartic acid favored. Thermal unimolecular dissociation rate constants for the dissociation of each of these charge states were measured. From the temperature dependence of these rates, Arrhenius activation parameters in the rapid energy exchange limit are obtained. The activation energies (Ea) and preexponential factors (A) for the 5+, 8+, and 9+ ions are 1.2 eV and 10(12) s-1, respectively. These values for the 6+ and 7+ ions are 0.9-1.0 eV and 10(9) s-1, and those for the 10+ and 11+ ions are 1.6 eV and 10(16)-10(17) s-1. Thus, with the exception of the 5+ ion, the higher charge states of ubiquitin have larger dissociation activation energies than the lower charge states. The different A factors observed for production of y and b ions from different precursor charge states indicate that they are formed by different mechanisms, ranging from relatively complex rearrangements to direct bond cleavages. These results clearly demonstrate that the relative dissociation rates of large biomolecule ions by themselves are not necessarily a reliable indicator of their relative dissociation energies, even when similar fragment ions are formed.

Infrared Rays↗

PubMed1995

Dissociation of serum amyloid P from C4b-binding protein and other sites by lactic acid: potential role of lactic acid in the regulation of pentraxin function.

Serum amyloid P (SAP) and C-reactive protein (CRP) are two members of the pentraxin family of proteins. These proteins associate with a variety of other materials that are found in serum under normal or pathological circumstances. This study showed that carboxylated compounds, especially lactic acid, were capable of dissociating pentraxins from several macromolecular binding sites. When measured by sucrose density gradient ultracentrifugation, complete dissociation of the complex of hSAP (human SAP) with C4b-binding protein (C4BP) occurred at > or = 5 mM lactate. Lactate dissociated the hSAP-membrane complex and prevented hSAP self-association. The only interaction that was not dissociated by 10 mM lactate was the hSAP-heparin complex. The relative efficacies of several dissociating agents were O-phosphorylethanolamine > lactate > succinate > carbonate > epsilon-amino-n-caproic acid. This suggested that the carboxyl group plus a hydrogen-bonding site on the hydrocarbon chain was important, but a charged amino group was not a contributor to function when the anion was provided by a carboxyl group. The concentration of lactic acid needed to dissociate hSAP from C4BP was dependent on protein concentration in a manner suggesting the cooperative binding of lactate (coefficient = 2) to hSAP. Pure proteins, at concentrations found in normal serum, required about 12 mM lactate for half-dissociation of the hSAP-C4BP complex. Other pentraxins also interacted with lactic acid, but with lower affinities. An important observation was that lactic acid was capable of dissociating rat CRP from lipoproteins in rat serum. Human CRP bound very weakly to lactate, so that lactate probably is not a significant regulator of this pentraxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1993

Physical heterogeneity of muscle glycogen phosphorylase revealed by hydrostatic pressure dissociation.

Four independent methods that employ fluorescence spectroscopy show that the tetramer of glycogen phosphorylase A (GPA) from rabbit muscle is reversibly dissociated into monomers by hydrostatic pressures under 2.5 kbar, if aggregation of the monomers is prevented by the addition of 8% glycerol. The free energy of association at 20 degrees C (-32 kcal mol-1) depends upon a large entropy increase (T delta S = +65 kcal mol-1) that counteracts an unfavorable enthalpy of association of +33 kcal mol-1. The association volumes calculated from the pressure dependence of the dissociation are nearly 4-fold smaller than those calculated from the shift in dissociation pressure with concentration. The dimer obtained by dilution of GPA at atmospheric pressure differs from the hypothesized dimer intermediate in the pressure dissociation by the much larger monomer affinity of the former. Like other tetramers, GPA shows hysteresis of the pressure profile upon decompression and conformational drift of the dissociated monomers. By use of the energy transfer method it is demonstrated that the relaxation time for half-dissociation (5 min) is over an order of magnitude shorter than that for subunit exchange (118 min). In all three tetramers studied, lactate dehydrogenase, glyceraldehyde phosphate dehydrogenase, and glycogen phosphorylase, the deterministic character of the dissociation equilibrium under pressure and the anomalous concentration dependence of the pressure dissociation demonstrate that these tetramers are heterogeneous populations with regard to their free energy and/or volumes of association.

Animals↗

PubMed1993

Pressure-induced dissociation of fluorescein from the anti-fluorescein single-chain antibody 4-4-20.

Hydrostatic pressure was used to dissociate fluorescein (Fl) from the high-affinity anti-Fl single-chain antibody 4-4-20 (SCA 4-4-20). Fl dissociation was monitored by measuring (1) the shift in the Fl absorption peak, (2) the recovery in Fl fluorescence intensity, which is quenched upon SCA binding, or (3) the decrease in Fl fluorescence polarization. Pressure effects were studied at two different Fl:SCA 4-4-20 molar ratios: 1:1, at which Fl fluorescence quenching was ca. 35% at atmospheric pressure, and 1:5, at which quenching reached 95-97% under the same conditions. In both cases, pressure-induced dissociation was favored by concomitant dilution of protein and ligand. Dissociation constants (KD) at each pressure were calculated on the basis of measurements of Fl fluorescence polarization under pressure. The dependence of KD, and consequently of delta G of dissociation, on pressure permitted calculation of the magnitude of the standard volume change (delta V) involved in the dissociation process. According to this study, delta V of dissociation for the Fl-SCA complex is -50 mL/mol, which corresponds to a 10-times higher value than that found for dissociation of Fl from the intact IgG mAb 4-4-20 [Herron, J. N., Kranz, D. M., Jameson, D. M., & Voss, E. W., Jr. (1986) Biochemistry 25, 4602-4609]. This difference is explained in terms of a higher overall flexibility of unliganded SCA and of a less stable binding site in SCA relative to mAb.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies↗

PubMed1991

Kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum ATPase.

We investigated the kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum Ca2(+)-ATPase by combining fast filtration with stopped-flow fluorescence measurements. At pH 6 and 20 degrees C, in the absence of potassium and in the presence of 20 mM MgCl2, isotopic exchange of bound calcium exhibited biphasic kinetics, with two phases of equal amplitude, regardless of the initial extent of binding site saturation. The rapidly exchangeable site, whose occupancy by calcium controlled the rate constant of the slow phase, had an apparent affinity for calcium of about 3-6 microM. A similar high affinity was also deduced from measurements of the calcium dependence of the rate constant for ATPase fluorescence changes. This affinity was higher than the overall affinity for calcium deduced from the equilibrium binding measurements (dissociation constant of 15-20 microM); this was consistent with the occurrence of cooperativity (Hill coefficient of 1.6-1.8). The drop in intrinsic fluorescence observed upon chelation of calcium was always slightly faster than the dissociation of calcium itself, although the rates for both this drop in fluorescence and calcium dissociation varied slightly from one preparation to the other. This fluorescence drop was therefore mainly due to dissociation of the bound ions, not to slow transconformation of the ATPase. Dissociation of the two bound calcium ions in a medium containing EGTA exhibited monophasic kinetics in the presence of a calcium ionophore, with a rate constant about half that of the fast phase of isotopic exchange. This particular pattern was observed over a wide range of experimental conditions, including the presence of KCl, dimethyl sulfoxide, 4-nonylphenol, or a nucleotide analogue, at pH 6 or 7, and at various temperatures. The kinetics of calcium dissociation under the above various conditions were not correlated with the ATPase affinity for calcium deduced from equilibrium measurements under the same conditions. These results are consistent with sequential dissociation of calcium from a narrow binding pocket inside which a single calcium ion can move fairly easily. Escape of calcium might be controlled by a structural compartment acting as a gate.

Adenosine Triphosphate↗