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PubMed2005

Zonula occludens-1 (ZO-1) redistribution is involved in the regulation of cell dissociation in pancreatic cancer cells.

In our previous study, dissociation factor (DF) and mitogen-activated protein kinase kinase 2 (MEK2) were isolated as factors relating to cancer cell dissociation in pancreatic cancer cells. On the other hand, tight junction protein zonula occludens 1 (ZO-1) has been indicated to be involved in carcinogenesis. In this study, the expression of ZO-1 and a downstream kinase of MEK2, extracellular signal-regulated kinase 2 (ERK2), was analyzed to clarify the regulatory mechanism of cell dissociation in pancreatic cancer cells. Two hamster (PC-1.0 and PC-1) and two human (AsPC-1 and CAPAN-2) pancreatic cancer cell lines were used. Immunocytochemical study was performed using anti-ZO-1, ERK2, and phosphorylated ERK1/2 (p-ERK1/2) antibodies. DF treatment obviously disrupted ZO-1 expression at the sites of cell-cell contact and markedly induced ERK2 and p-ERK1/2 expression, as well as the dissociation of cell clones in PC-1 and CAPAN-2 cells. In contrast, U0126 (a MEK1/2 inhibitor) treatment significantly induced the peripheral distribution of ZO-1 as well as cell aggregation in PC-1.0 and AsPC-1 cells, which usually grew as single cells, but seriously suppressed ERK2 and p-ERK1/2 expression. We conclude that redistribution of ZO-1 is closely correlated with cell dissociation status in pancreatic cancer cells through activation of ERK2.

Animals↗
PubMed1987

The pressure-induced inactivation of mammalian enolases is accompanied by dissociation of the dimeric enzyme.

The effects of exposure to pressure on both the activity and the quaternary structure of rabbit brain enolases, forms alpha alpha, alpha gamma, and gamma gamma were studied in the pressure range of 1 to 3400 bar. Effects on quaternary structure were determined by subunit scrambling (the formation of alpha alpha and gamma gamma from alpha gamma or vice versa). All three dimers are stable up to pressures of 1200 bar. The dissociation of gamma gamma begins at 1200 bar, yielding a stable monomer; inactivation of gamma gamma does not begin until the pressure is greater than 2000 bar. Dissociation of gamma gamma is not accompanied by changes in the tryptophan fluorescence of the protein. However, the fluorescence does decrease when the pressure is greater than 2000 bar, the point at which inactivation of gamma gamma starts. The alpha monomer, on the other hand, is unstable in the pressure range that produces dissociation of alpha alpha. This process, which also begins at 1200 bar, is paralleled by inactivation. Crosslinking the enzyme with glutaraldehyde demonstrated that the inactive form of the enzyme is monomeric. The pressure-induced inactivation of these forms of enolase is thus clearly a two-step process, with both dissociation and inactivation occurring. The difference in pressure sensitivity of rabbit brain alpha alpha and gamma gamma is due to a difference in stability of the alpha and gamma monomers and not due to a difference in the pressures required for dissociation.

Animals↗
PubMed1975

A dissociation factor from embryos of Xenopus laevis.

A dissociating factor has been extracted from the ribosomal KCl wash and from the cytosol of developing embryos of Xenopus laevis. No dissociating activity could be detected in the KCl wash of ribosomes from full grown oocytes and unfertilized eggs. As in bacteria, the acitivity of the dissociation factor seems to be correlated with the rate of protein synthesis suggesting a physiological role of the dissociation factor. The possibility that the dissociation factor might be one of the components which limits the rate of protein synthesis in the oocytes is discussed.

Animals↗
PubMed1981

Subunit structure and dissociation of Callinectes sapidus hemocyanin.

The hemocyanin of the blue crab, Callinectes sapidus has two major components sedimenting with approximate sedimentation coefficients of 17 S and 25 S. Molecular weight data based on light scattering and sedimentation equilibrium measurements at pH 7.8 suggest that the two components have molecular weights of approximately 450 000 and 900 000 in the presence of stabilizing Ca2+. In the absence of Ca2+, the molecular weights are found to be about 5% lower, suggesting some dissociation of the hemocyanin components. Circular dichroism and optical rotatory dispersion measurements in the far-ultraviolet region gave nearly identical spectra for the two components. Based on the reference parameters of Chen et al. (Chen, Y.H., Yang, J.T. and Martinez, H.M. (1972) Biochemistry 11, 4120--4131 and Chen, Y.H., Yang, J.T. and Chan, K.H. (1974) Biochemistry 13, 3340--3359), estimates of 16--20% alpha-helix, 40--60% beta-structure, and 30--40% random organization were obtained for the two hemocyanin components. Exposure to 6 M Gdn HCl gave light scattering molecular weights of approx. 68 000 and 77 000, which is close to one-sixth of the molecular weight of the 17 S component. These results support the view that the two components of C. sapidus hemocyanin share the hexameric and dodecameric organization common to arthropod hemocyanins. The salts of the Hofmeister series and the ureas are found to dissociate the dodecameric component with the former exhibiting the usual order of effectiveness of NaCl, NaBr, NaI, and NaClO4 dissociation, while the ureas show an inverse order of decreasing effectiveness in going from urea to methyl-, ethyl- and propylurea. This suggests that polar and ionic interactions are relatively more important than hydrophobic interactions for the stabilization of the dodecameric form of C. sapidus hemocyanin. The dissociation behavior of the 17 S hexameric species by GdnHCl in the 0--1.5 M concentration region (where essentially no denaturation occurs), based on light scattering molecular-weight measurements, is satisfactorily accounted for by equations describing the dissociation of hexamers to monomers.

Animals↗
PubMed1996

Visualising the dissociation of sequence selective ligands from individual binding sites on DNA.

We have used a modification of the footprinting technique to measure the dissociation of mithramycin, echinomycin and nogalamycin from their binding sites in a natural DNA fragment. Complexes with radiolabelled DNA were dissociated by addition of unlabelled DNA. Samples were removed at various times and subjected to DNase I digestion, and the rate of dissociation from each site was estimated from the time-dependent disappearance of the footprints. For echinomycin the slowest rate of dissociation is from ACGT, while the slowest site for mithramycin contains four contiguous guanines. The dissociation of nogalamycin is extremely slow, even from its weaker sites; the slowest rate was from ACGTA, which took longer than 4 h, even at 37 degrees C.

Base Sequence↗
PubMed1985

Specific uptake, dissociation, and degradation of 125I-labeled insulin in isolated turtle (Chrysemys dorbigni) thyroid glands.

Thyroid glands from turtles (Chrysemys dorbigni) pretreated with potassium iodide were incubated with 125I-insulin in the presence or absence of unlabeled insulin, in order to study its specific uptake. At 24 degrees, the specific uptake reached a plateau at 180 min of incubation. The dose of bovine insulin that inhibited 50% of the 125I-insulin uptake was 2 micrograms/ml of incubation medium. Most of the radioactive material (71%) extracted from the gland, after 30 min incubation with 125I-insulin, eluted in the same position as labeled insulin on Sephadex G-50. Only 24% eluted in the salt position. After 240 min incubation, increased amount of radioactivity appeared in the Na125I position. When bovine insulin was added together with the labeled hormone, a substantial reduction of radioactivity was observed in the insulin and Na125I elution positions. Dissociation studies were performed at 6 degrees in glands preincubated with 125I-insulin either at 24 or 6 degrees. The percentage of trichloroacetic acid (TCA)-soluble radioactive material in the dissociation medium increased with incubation time at both temperatures. However, the degradation activity was lower at 6 than at 24 degrees. The addition of bovine insulin to the incubation buffer containing 125I-insulin reduced the radioactive degradation products in the dissociated medium. Chloroquine or bacitracin inhibited the degradation activity. Incubation of thyroid glands with 125I-hGH or 125I-BSA showed values of uptake, dissociation, and degradation similar to those experiments in which an excess of bovine insulin was added together with the labeled hormone. Thus, by multiple criteria, such as specific uptake, dissociation, and degradation, the presence of insulin-binding sites in the turtle thyroid gland may be suggested.

Animals↗
PubMed1990

Dissociation rate constant of the biotin-streptavidin complex.

We measured the dissociation rate constants of the biotin/streptavidin and biotin/egg avidin complexes by following the release of radiolabeled biotin from the preformed complexes in the presence of excess unlabeled biotin. For separation of bound and free labeled biotin we employed ultrafiltration with disposable microconcentrators. The dissociation rate constant for underivatized streptavidin was 2.4 x 10(-6) s-1, or approximately 30-fold higher than that observed for egg avidin 7.5 x 10(-8) s-1). The value for streptavidin was further increased after derivatization with an acridinium ester label. Both biotin binding proteins exhibited a faster initial phase, suggesting binding site heterogeneity due to partial subunit dissociation or denaturation. The convenience of the method and the relatively fast dissociation of biotin from streptavidin render the dissociation rate constant a practical experimental criterion for monitoring the integrity of the binding site during purification and derivatization procedures.

Bacterial Proteins↗
PubMed1994

Measurement of the dissociation rate constant of antigen/antibody complexes in solution by enzyme-linked immunosorbent assay.

A reliable, convenient ELISA based method has been developed for measuring the dissociation rate constants of antigen/antibody complexes in solution. Its rationale is as follows: a solution containing the preformed antigen/antibody complex is diluted well below the equilibrium dissociation constant to initiate the dissociation and, at various times after the dilution, the amount of dissociated antibody contained in an aliquot is determined by a classical ELISA, using a brief incubation of the solution in antigen coated wells. To test the validity of this method, the dissociation rate constants for several antigen/antibody complexes were compared with those obtained by classical fluorescence based methods. The good agreement between both sets of data validates the ELISA procedure. The present method offers several advantages. It uses only minute amounts of sample which need not be purified; it requires no radioactive or fluorescent labelling of the antibody or antigen, and it can, in principle, be applied to any type of complex between macromolecules if an ELISA test can be set up to detect quantitatively one of the macromolecules.

Animals↗
PubMed1988

Pressure-induced dissociation of brome mosaic virus.

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.

Chromatography, High Pressure Liquid↗
PubMed1985

Serotonin uptake inhibitors differentially modulate high affinity imipramine dissociation in human platelet membranes.

The influence of selective serotonin uptake inhibitors on the dissociation rate of 3H-imipramine from its high affinity binding site in human platelet membranes was studied. Of the uptake inhibitors tested, one group of compounds attenuated dissociation, another group accelerated it, while a third group had little effect on the dissociation process. Drugs unrelated to the serotonin uptake system were ineffective. Removal of sodium ions markedly increased imipramine dissociation. Dose response curves of the active compounds indicated that micromolar concentrations were required to exert an effect on imipramine dissociation. These results can be adequately explained by an allosteric model which includes effector binding sites and distinct conformational states of the high affinity imipramine binding site.

Blood Platelets↗
PubMed1992

The in vitro dissociation kinetics of (R,R)-[125I]4IQNB is reflected in the in vivo washout of the radioligand from rat brain.

We have determined the kinetics of dissociation of (R)-3-Quinuclidinyl (R)-4-[125I]Iodobenzilate ((R,R)-[125I]4IQNB) from muscarinic acetylcholine receptor preparations from the cortex, hippocampus, caudate/putamen, thalamus, pons and colliculate bodies. The dissociation curves are well described by a biexponential function and are consistent with subtype selectivity favoring slow dissociation from the M1, M3, and M4 receptors with a 20-fold faster dissociation rate for the M2 receptor. Following intravenous injection, (R,R)-[125I]4IQNB binds to receptor in the rat brain in concentrations which reflect the receptor concentration present in a structure. We determined the extent of radioligand present at two times, 2 and 24 hrs, as an indication of the relative proportions of m-AChR which exhibits rapid vs. slow dissociation of (R,R)-[125I]4IQNB. A good correlation between in vitro and in vivo results suggests that the relative populations of receptor subtypes can be imaged using in vivo pharmacokinetics of (R,R)-[125I]4IQNB.

Animals↗
PubMed1992

Presence and some characterization of GDP dissociation inhibitors for a low Mr GTP-binding protein, ram p25, in rat spleen cytosol.

Two proteinous factors, designated here as ram p25 GDP dissociation inhibitor (I) and (II) (ram-GDI (I) and (II)), were detected in the cytosolic fraction of rat spleen, which inhibited the initial dissociation of GDP from ram p25 produced by E. coli by causing characteristic lag. They had very weak effects on the rate of dissociation of GDP after the lag, and did not affect the mode of the dissociation of 5'-(3-O-thio)triphosphate (GTP gamma S) from ram p25. By gel filtration, the molecular masses of ram-GDI (I) and (II) were calculated to be 90 KDa and 40 KDa, respectively. These ram-GDIs did not affect the GDP-dissociation of Ha-ras protein produced in E. coli.

Animals↗
PubMed1987

Anti-IgM antibody decreases dissociability of cell bound IgM anti-hapten antibodies as measured with fluid phase hapten.

We studied the effect of an IgG anti-IgM on the dissociation of cell bound IgM anti-hapten antibody, as measured with fluid phase hapten. Methotrexate (MTX) covalently bound to the red cells surface was used as hapten; rabbit anti-MTX IgM and rabbit anti-allotype IgG reactive with the IgM were used as antibodies. The amount of cell bound antibodies was measured with 125I-labelled protein A. In the absence of the anti-allotype antibody, most of the anti-hapten IgM was prevented from reassociation by fluid phase hapten. In the presence of the anti-allotype IgG most of the anti-hapten IgM was non-dissociable, with about a 1000-fold increase in the apparent binding constant. We found that (1) dissociability of the anti-hapten IgM depended on the density of the cell bound hapten, (2) decrease in dissociability was a function of the anti-allotype antibody concn and (3) even in the presence of excess anti-allotype antibody dissociability was an inverse function of anti-hapten IgM concn.

Animals↗
PubMed1989

Dissociation kinetics of antigen-antibody interactions: studies on a panel of anti-albumin monoclonal antibodies.

Kinetic parameters and equilibrium association constants (K) are reported for a panel of anti-bovine serum albumin (BSA) monoclonal antibodies (MAb) immobilized onto agarose particles. For 12 covalently immobilized MAb of moderate affinity (K = 0.25 x 10(8)-1.2 x 10(8) M-1) measured dissociation time constants varied two orders of magnitude, from 2.1 to 410 min. Directly measured association rate parameters agree with values calculated from measured equilibrium and dissociation rate parameters. Dissociation time constants and equilibrium association constants were also determined for eight MAb immobilized biospecifically (via their Fc regions). A significantly lower K was observed with those MAb which were covalently immobilized as opposed to biospecifically immobilized. These decreases in K appear to reflect decreased association rates rather than increased dissociation rates. The data suggest that, for the MAb described herein, dissociation rates do not correlate with equilibrium association constants.

Animals↗
PubMed1990

Uptake of 67copper complexed to 3H-histidine by brain hypothalamic slices: evidence that dissociation of the complex is not the only factor determining 67copper uptake.

It was previously shown that complexation of 67Cu with His facilitates 67Cu uptake by hypothalamic slices and that His, in a concentration that is 1000-fold greater than Cu(His)2, inhibits 67Cu uptake (D. E. Hartter and A. Barnea, J. Biol. Chem. 263, 799-805 (1988)). We addressed the question: Does dissociation of the Cu(His)2 complex occur during the process of Cu2+ uptake and if so, is dissociation the only factor determining uptake? Rat hypothalamic slices were incubated with 67Cu(3H-His)2 and the kinetic profiles of 67Cu and 3H-His uptake were evaluated. 67Cu uptake was linear for up to 60 min, Vo vs S [0.1-160 microM Cu(His)2] was sigmoidal, Lineweaver-Burk plot was non-linear, Scatchard plot was bell-shaped, and Hill plot had multiple slopes. In contrast, 3H-His uptake was linear for up to 30 min, Vo vs S was biphasic, Lineweaver-Burk plot was linear, Scatchard plot was biphasic, and Hill plot had a single slope. Keeping [67Cu] constant and increasing [3H-His] resulted in a dose-dependent inhibition of 67Cu uptake which was not accompanied by an inhibition of 3H-His uptake. Substituting His in the complex with Phe or Lys resulted in a marked shift to the right in Vo vs S for 67Cu uptake and at S less than 40 microM, only His facilitated 67Cu uptake relative to ionic 67Cu2+. However, Vo vs S for 3H-His, 3H-Phe, and 3H-Lys uptake were superimposeable, indicating comparable dissociation of the complexes. In summary, we demonstrate that, although complexation of Cu2+ is essential for 67Cu uptake by hypothalamic tissue, 67Cu and 3H-His are taken up by distinct processes, which implies dissociation of the complex at the level of the membrane. Moreover, even though dissociation occurs, it is not the only factor that determines Cu2+ uptake by the hypothalamic tissue. It is suggested that the physicochemical properties of the Cu complex is an important factor determining Cu uptake by brain tissue.

Animals↗
PubMed1985

Subunit dissociation of Busycon canaliculatum hemocyanin.

The hemocyanin of the channeled whelk, Busycon canaliculatum, is a multisubunit protein with a molecular weight close to 9 X 10(6). The increase in pH above neutrality and the addition of 0-5 M urea and 0-2 M GdnHCl is found to dissociate the whole molecules to half-molecules and smaller dimeric and monomeric fragments of one-tenth and one-twentieth mass of the parent hemocyanin. The molecular weight transitions investigated at constant protein concentration of 5 X 10(-2) g X l-1 show no clearly discernible plateau regions, where essentially only half-molecules and one-tenth molecules are present. The ultracentrifugation patterns in much of the dissociation region produced by urea at pH 6.9 suggests the presence of three distinct components consisting of whole molecules, half-molecules and largely one-tenth molecular weight fragments. At pH 8.2 and higher, where whole molecules are largely absent, the effects of urea on the dissociation of half-molecules to tenths and tenth-molecules to twentieth molecule was investigated by means of light scattering. Analysis of the urea data based on a decamer to dimer and dimer to monomer scheme of dissociation used in our earlier studies gave apparent estimates of about 90 amino acid groups at the contact areas of the dimers in the half-molecules and 110 groups at the monomer contacts forming the dimers. The latter relatively large estimate of groups suggests that the dissociation of the tenth molecules or dimers must occur by longitudinal splitting of the contact areas along both the folded domains and the connecting chain segments of the twentieth molecules. Circular dichroism, absorbance and viscosity data suggest that the secondary structure and conformation of the folded domains of the hemocyanin subunits are largely retained at both high pH and in 3-8 M urea solutions. The molecular weights at pH 9.0-10.6 and in 3-8 M urea are found to be (4.2-4.7) X 10(5), close to one-twentieth of the mass of the parent hemocyanin. Denaturation and unfolding of the subunit domains is observed between 3 and 6 M GdnHCl solutions, as evidenced by the abolition of the characteristic copper absorbance in the neighborhood of 346 nm and the relatively pronounced changes in circular dichroism at 222 nm and intrinsic viscosity. The further decrease in molecular weights to about (2.6-3.2) X 10(5), below one-twentieth of the mass of hemocyanin suggests the presence of hidden breaks or scissions in the polypeptide chains suffered during isolation, which become exposed as a result of complete unfolding in GdnHCl solutions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Specific dissociation of alpha B subunits from alpha-crystallin.

Exposure of bovine alpha-crystallin to 0.1 M glycine at pH 7 decreases the average molar mass of the protein from 700 to 420 kDa. When the pH is lowered to 2.5, in the same buffer, the alpha B chains specifically dissociate from the aggregates, leaving a particle of 290 kDa containing only alpha A chains. The decrease in the molar mass corresponds to the mass of the alpha B chains in the original aggregate. The pH-dependent dissociation is fully reversible. Similar changes were observed with rat and kangaroo alpha-crystallins but the dogfish protein was not affected. Sedimentation velocity analyses and fluorescence spectroscopy yielded a pK, for the dissociation, of 3.7 for alpha-crystallin and 4.0 for a homopolymer constructed from purified alpha B2 polypeptides. An alpha A2 homopolymer was virtually unaffected by the lowering of pH. The products from the dissociation were isolated and their properties studied by sedimentation analysis and acrylamide quenching of tryptophan fluorescence. The alpha B chains were found to be completely denatured, whereas the structure of the alpha A chains, in the 290 kDa, particle, were only slightly altered. Comparisons of the sequences of the various proteins examined suggested that decreased ionization of aspartic acid 127 in the alpha B chain was responsible for the specific dissociation of this polypeptide.

Animals↗
PubMed1990

Dissociation and aggregation of D-glyceraldehyde-3-phosphate dehydrogenase during denaturation by guanidine hydrochloride.

The inactivation of lobster muscle D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) (GAPDH) during guanidine hydrochloride (GdnHCl) denaturation has been compared with its state of aggregation and unfolding, by light scattering and fluorescence measurements. The enzyme first dissociates at low concentrations of GdnHCl, followed by the formation of a highly aggregated state with increasing denaturant concentrations, and eventually by complete unfolding and dissociation to the monomer at concentrations of greater than 2 M GdnHCl. The aggregation and final dissociation correspond roughly with the two stages of fluorescence changes reported previously (Xie, G.-F. and Tsou, C.-L. (1987) Biochim. Biophys. Acta 911, 19-24). Rate measurements show a very rapid inactivation, the extents of which increase with increasing concentrations of GdnHCl. This initial rapid phase of inactivation which takes place before dissociation and unfolding of the molecule is in agreement with the results obtained with other enzymes, that the active site is affected before noticeable conformational changes can be detected for the enzyme molecule as a whole. A scheme for the steps leading to the final denaturation, and dissociation of the enzyme to the inactive and unfolded monomer, is proposed.

Animals↗