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PubMed1988

Association-dissociation and denaturation-renaturation of high-molecular-weight protein: carmin from safflower seed (Carthamus tinctorius L.) in alkaline solution.

The effect of alkaline pH on the association, dissociation, and denaturation of carmin, the high-molecular-weight protein from safflower seed was investigated in the pH range 7-12, using various biophysical techniques. The results indicate that the multimeric protein carmin dissociates at pH 8.0 where denaturation has not set in. The association-dissociation of the protein can be represented schematically as 11S in equilibrium 7S in equilibrium 4S----2S. Above pH 10, the protein undergoes simultaneous dissociation and denaturation. The denaturation process appears to be complete at approximately pH 12.5. The protein undergoes conformational change and covalent modifications and cleavage during the denaturation process. A reversibility study shows that the process of dissociation is reversible to a large extent, whereas denaturation appears to be irreversible. These results are discussed in terms of association-dissociation, denaturation and alkaline-catalyzed covalent modifications and cleavage of seed proteins.

Hydrogen-Ion Concentration↗
PubMed1981

Effect of dissociative methods on cortisol binding and glutamyltransferase inducibility in chick embryo retina.

The ability of the isolated embryonic chick retina (12 days) to bind a steroid (cortisol) decreases when the tissue is dissociated; the extent of this decrease depends upon the method of dissociation. Trypsin and mechanical dissociation decreased cortisol binding slightly; papain dissociation essentially eliminated it. Cortisol binding decreased with time in culture in both whole retina and monolayer cultures; this decrease may reflect, in part, a similar development decrease in ovo. Inducibility of glutamine synthetase in whole retinas and retinal monolayers prepared with either trypsin or papain also decreased with time in culture. For whole and trypsin-dissociated retinas, the drop in inducibility correlates with the drop in cortisol-binding capacity. This was not the case for monolayer cultures prepared by papain dissociation.

Animals↗
PubMed2003

Development of a quantification system of ionic dissociative metabolites using an FT-IR/ATR method.

A simultaneous quantification system of ionic dissociative metabolites was developed using a Fourier transform mid-infrared spectroscopic method by focusing our attention on the enzyme reaction from glucose 6-phosphate to fructose 6-phosphate with phosphoglucose isomerase (PGI). We studied the pH dependency of the infrared spectra of the mixture solution for which the PGI reaction was assumed. The infrared spectra of ionic dissociative components in the mixture solution were extracted by multiple linear regression analysis under the assumption of ionic dissociation equilibrium. Additionally, we constructed a simultaneous quantification system using the extracted spectra of the ionic dissociative components on the basis of the ionic dissociation equilibrium. We could accurately estimate the pH value and the concentrations of the ionic dissociative materials in their mixture solution by using this quantification system. In addition, the stability of quantification results for a pK shift was also verified.

Cell Culture Techniques↗
PubMed1988

Kinetics of vanadate dissociation: estimation of the rate by inhibitor inactivation.

Vanadate (+5) is a potent inhibitor of a variety of ATPases including dynein ATPase. We describe a method useful for estimating the functional dissociation rate of vanadate from the active site which does not rely on classical physical separation techniques. The method involves spectrophotometrically monitoring the enzymatic activity as the inhibitor dissociates from the enzyme and is inactivated by norepinephrine. Norepinephrine effectively reverses vanadate inhibition by reducing vanadate (+5) to oxovanadium (+4). This reduction by norepinephrine is sufficiently fast for these purposes--addition of vanadate after norepinephrine shows no inhibition of ATPase activity. The mathematical estimation procedure is generally useful for estimation of dissociation rates of other reversible inhibitors which can be quickly inactivated after dissociation from the enzyme. The rate of dissociation of vanadate from dynein with ATP and 2-N3ATP as substrates using this method was estimated to be in the ranges 0.0023-0.0042 and 0.0057-0.0075 s-1, respectively. These rates permit estimation of the rates of vanadate association with dynein by using the reported dissociation constant for vanadate. The results are consistent with the very fast and potent inhibition of dynein ATPase activity observed.

Adenosine Triphosphatases↗
PubMed1985

Factors affecting the dissociation and reconstitution of ribulose-1,5-bisphosphate carboxylase/oxygenase from Aphanothece halophytica.

Factors affecting the mutual interaction between the catalytic core [octamer of large subunit (A)] and the small subunit (B) comprising ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from the superhalophilic cyanobacterium, Aphanothece halophytica, were investigated. The enzyme molecule dissociated into the catalytic core highly depleted of subunit B and the monomeric form of subunit B during density gradient centrifugation (15 h, 4 degrees C) in a sucrose solution of low ionic strength ([I] less than or equal to 50 mM), whereas dissociation was effectively prevented in the presence of 0.3 M KCl. Under the latter condition, dissociation of the enzyme molecule was almost completely prevented by raising the temperature to 20 degrees C, suggesting hydrophobic interaction between catalytic core and subunit B. The addition of RuBP to the sucrose gradient was shown to effectively reduce the molecular dissociation, suggesting a close interaction between the catalytic site and the binding site of subunit B with the catalytic core directly or indirectly. The dissociation was accelerated at alkaline pH higher than 8.5. Reconstitution of the enzymatically active molecular form from the separated components, catalytic core highly depleted of subunit B and B1, was done under various conditions. Both carboxylase and oxygenase activities increased proportionately with the amount of subunit B and then became saturated. From the reconstitution kinetics of RuBP carboxylase, the binding constant of subunit B (KD) was estimated to be about 30 nM in the presence of bovine serum albumin under the usual assay conditions at pH 7.5 and 25 degrees C, but decreased to about 1 nM by the further addition of 0.3 M KCl. Alkaline pH (8.5 or 9) could increase KD by one order of magnitude. High KD was also observed as a result of lowering the temperature; however, the presence of 0.3 M KCl or 0.4 M sucrose or glycerol could effectively decrease the KD at low temperature from 900 nM to less than 50 nM. All these data indicate that the enzyme dissociation at low temperature can be prevented in vivo by cellular components such as salts, polyols, and substrate RuBP besides a factor of enzyme concentration.

Catalysis↗
PubMed1989

Dissociation of ferritins.

Apoferritins prepared from horse spleen and heart and rat heart and liver were dissociated by treatment with acetic acid (pH 1.3-3.0). Sedimentation velocity studies showed that apoferritins of spleen and liver (16-17 S) and heart (18-19 S) dissociated into material sedimenting near 3.2 S. Sedimentation equilibrium measurements determined that most of the material had a molecular weight of 38,000-43,000, corresponding to subunit dimers. Failure to dissociate into subunit monomers was confirmed by gel chromatography on Sephadex G-75 and G-150. With the exception of boiling in sodium dodecyl sulfate, further treatments with 0.1-0.4 M KCl, NaCl, 4-9 M urea, 0.01-0.5 M KSCN, 0.1-0.5% Triton X-100, 5-52% dimethylsulfoxide, 10% ethylene glycol, or 0.1% trifluoroacetic acid all failed to cause dissociation into individual subunits, as did exposure to 6 M guanidine-HCl or formic acid, or prior succinylation and/or nitration of the protein. Reassociation occurred between pH 4 and 7 but was not aided by the addition of Fe(II) or reducing agents. It is concluded that ferritins readily dissociate to subunit dimer units and that further dissociation does not occur without full denaturation of the protein.

Animals↗
PubMed1985

Dissociation kinetics of DNA-anthracycline and DNA-anthraquinone complexes determined by stopped-flow spectrophotometry.

The first order rate constants for the dissociation of daunorubicin, doxorubicin, and 1-; 1,4-; 1,5-; and 1,8-; N,N-diethylaminoethylamino-substituted anthraquinones from calf thymus DNA were determined using stopped-flow spectrophotometry. Sodium dodecyl sulphate was used to disrupt the equilibrium. In all cases there was an increase in the rate constant with temperature. The dissociation rate constants at 20 degrees, 25 degrees and 37 degrees, were in the order 1-; much greater than 1,8-; greater than 1,4-; greater than daunorubicin and doxorubicin greater than 1,5-disubstituted anthraquinone. The 1,5-disubstituted anthraquinone (VII) thus shows the slowest rate of dissociation from DNA; the DNA complex dissociating more slowly than the DNA complexes of the anthracyclines, daunorubicin and doxorubicin. The result is consistent with the data from computer graphics modelling studies [39] which show that DNA-breathing (transient base pair unstacking) has to occur to allow the docking of the 1,5-disubstituted anthraquinone (VII) into the receptor site. Hence once the 1,5-disubstituted anthraquinone molecule has intercalated into DNA, DNA-breathing is required before dissociation can take place. This is not necessary with the other compounds (though the 1,4-disubstituted anthraquinone (V) can bind in this manner as well). So the very slow dissociation of the DNA/1,5-disubstituted anthraquinone complex relative to that of the DNA complexes of the other compounds examined here, supports the proposed mode of binding [39].

Anthraquinones↗
PubMed1985

Dissociation of antibodies bound to surface-immobilized antigen.

The dissociation of antibodies bound to surface-immobilized antigen was investigated by the ELISA, using a hapten (TNP) as antigen. Antibody binding was found to be stable, and no half-time of dissociation could be defined within 69 h. The role of the bivalence of antibodies and the difference between a homogeneous and a heterogeneous reaction was investigated by comparing the dissociation rate of antigen-antibody complexes formed by monovalent Fab fragments from surface-immobilized antigen and the dissociation rate of TNP-lysine from antibodies in a homogeneous liquid phase. Fab fragments were found to dissociate with a half-time value of about 16 h, whereas the homogeneous binding of TNP-antibody dissociated with a half-time of less than 4 h, indicating that both the bivalence of antibodies and the solid phase contributed to the stability of surface-bound antigen-antibody complexes. Qualitative differences between antibodies produced by different clones in a polyclonal antibody response to TNP was investigated by a spot assay. The results indicated that a minority of the antibodies produced had the capacity of binding practically irreversibly to solid-phase-immobilized antigen. The impact of the results on the interpretation of data from solid-phase assays is discussed together with the biological importance of the findings.

Animals↗
PubMed1985

GABA, depressants and chloride ions affect the rate of dissociation of 35S-t-butylbicyclophosphorothionate binding.

The dissociation of 35S-TBPS was studied from binding sites of rat cerebral cortex. Monophasic dissociation plots became polyphasic and accelerated in the presence of micromolar concentrations of GABA suggesting the involvement of low (or super-low) affinity GABA receptors. The presence of the depressants etazolate, R(-)MPPB and ethanol resulted in similarly accelerated dissociation patterns. In contrast, the convulsants S(+)MPPB and pentamethylenetetrazol did not significantly affect the dissociation of TBPS. Dissociation initiated by dilution was not affected either by an excess of picrotoxin or by varying the equilibrium occupancy of the TBPS sites. These findings rule out the possibility of a kinetic cooperativity for the binding of convulsants. The removal of chloride ions also enhanced the rate of TBPS dissociation. Kinetic heterogeneity of the TBPS binding sites can be interpreted with allosteric interactions mediated by various sites at the GABA receptor complex coupled to different states of the chloride ionophore.

Animals↗
PubMed1980

The influence of epinephrine on prostacyclin (PGI2) induced dissociation of ADP aggregated plateletes.

Prostacyclin (PGI2) is a powerful inhibitor of platelet function and recent studies have indicated that it can dissociate aggregated platelets in vivo. The present investigation has evaluated the mechanism involved in the dissociation of ADP aggregated platelets by PGI2, the influence of epinephrine on the process of PGI2 mediated disaggregation, and effects of the catecholamine on the refractory state of PGI2 dispersed cells. Dissociation of ADP aggregates by PGI2 was concentration and time dependent, caused complete disappearance of sensitive platelet clumps and restored platelet discoid form. The dispersed platelets were refractory to further stimulation by ADP, thrombin, arachidonate or the calcium ionophore, A23187. Measurement of cAMP revealed no change in control levels after aggregation by ADP, but a sharp increase during and after dissociation by PGI2. Epinephrine added to ADP aggregates before or after PGI2 blocked disaggregation, or stopped dissociation if already in progress, and caused a rapid fall in the high levels of cAMP stimulated by PGI2. Agents to which PGI2 dissociated platelets were refractory, including ADP, arachidonate and A23187 caused no lowering of elevated cAMP levels. Reduction of high levels of cAMP in PGI2 dispersed platelets by epinephrine did not result in reaggregation. However, epinephrine addition to refractory platelets restored in large measure their sensitivity to aggregation by arachidonate, thrombin, ADP and A23187.

Adenosine Diphosphate↗
PubMed1987

Determination of the birth date and proliferative state of dissociated cells from fetal rat brain.

The proliferative status and time of origin of dissociated cells from cerebral cortex, basal ganglia, diencephalon, mesencephalon and rhombencephalon of the fetal rat brain have been analyzed. The distributions of cells in different phases of the cell cycle after dissociation and after 7 days in culture have been determined with flow cytofluorometry. Two separate DNA indicators, propidium iodide and Hoechst 33258, have been used. Almost all of the dissociated cells recovered from the 5 brain regions, between embryonic days 15 and 20, are in G1, or growth arrest phase of the cell cycle. In contrast, only about 55% of the liver cells (same fetal age) are in G1, or growth arrest phase. The times of the last in vivo mitoses of the dissociated cells have been determined by maternal injection of [3H]thymidine and autoradiography of cultures. The majority of the cells recovered on embryonic days 16 and 17 from the 5 brain regions appeared to have undergone DNA synthesis within a period of 24 h prior to dissociation. When the fetuses were sacrificed 96 h after the injection, less than 20% of the cells were labelled, and grain density was drastically reduced. Most labelled cells survive in the serum-free culture medium for more than 7 days. Dissociated cultures of synchronized and birth-dated cells from different brain regions of fetal rat should prove particularly useful for the study of cellular development and function.

Animals↗
PubMed1984

Dissociation of mitochondrial malate dehydrogenase.

The kinetics of the dissociation reaction under acidic conditions of the dimeric pig and chicken mitochondrial malate dehydrogenases (EC 1.1.1.37) have been studied. The dissociation of the pig enzyme is completely reversible. The pK for dissociation determined by light-scattering measurements agrees within experimental error with the pK value of 5.25 measured for a tyrosine-carboxylate pair. The rate constants for the dissociation reaction and for the protonation process of this tyrosine are in close agreement. Thus, the tyrosine-carboxylate pair can be used as indicator of the dissociation reaction. The dissociation of the chicken enzyme proceeds around pH 4.5 at a much lower rate. A true equilibrium between dimer and monomers is not found, since the monomer gradually unfolds at this pH. The monomers of both enzymes, pig and chicken mitochondrial malate dehydrogenase, show the same stability towards acid. The difference in stability of the dimeric forms, therefore, must be due to an altered subunit contact area.

Animals↗
PubMed1987

Glucagon receptor binding, dissociation and degradation in rat liver plasma membranes studied by a microperifusion method.

The association process of glucagon receptor binding in purified rat liver plasma membranes and prolonged incubation of the hormone-receptor complex at 30 degrees C did not result in degradation of bound labelled glucagon. In contrast, up to 95% of the non-membrane-bound labelled glucagon was degraded. The rate of spontaneous dissociation of the glucagon-receptor complex was slow, and amounted to about 0.1% per min of that bound. GTP greatly enhanced the rate of dissociation. Half the maximal dissociation of the complex was effected by 10(-5) mol/l of GTP under equilibrium binding conditions. At maximally effective concentrations of GTP, 80% of the glucagon-receptor complex was dissociated within 2 min. A microperifusion system for the perifusion of isolated plasma membranes was devised and used for the separation of labelled glucagon from the plasma membranes subsequent to a GTP-induced dissociation of the hormone-receptor complex. Rebinding of the dissociated peptide to fresh membranes showed that maximum binding ability was retained. The glucagon molecule was protected against degradation while bound to the receptor, indicating that the glucagon effector system is completely separate from the inactivating system(s) in isolated plasma membranes. Thus, the hormonal effect of glucagon could be exerted through the sequential interaction of each glucagon molecule with several receptors.

Animals↗
PubMed1989

Unexpected sudden death during acute myocardial infarction: role of primary electromechanical dissociation.

The causes of death during the acute phase of myocardial infarction were studied in 128 patients. Forty-three of these, who had no clear signs of cardiocirculatory failure, were considered to be cases of sudden and unexpected death. Thirty-two of these patients (74%) had electromechanical dissociation, defined as a sudden disappearance of an effective arterial pressure in the presence of adequate electrocardiographic complexes. Twenty-three patients who had been given this diagnosis were males and 9 females; 53% presented with anterior infarction, 31% with infero-posterior infarction, 3.5% with both anterior and infero-posterior and 12.5% with non-Q wave infarction. A previous episode of infarction was recorded in 31.2% of patients with electromechanical dissociation. Autopsy was performed in 84 patients, 23 of whom died with electromechanical dissociation. Half of the latter cases revealed cardiac rupture (secondary electromechanical dissociation), whereas in the other half death was due to primary electromechanical dissociation. The study stresses the relatively high incidence of this cause of death and the need to differentiate between the two different forms. Although at present the pathophysiology of primary electromechanical dissociation is not completely understood, we believe that recurrence of global or local ischemia may play a more important role than cardiovascular inhibitory reflexes.

Acute Disease↗
PubMed1982

Thermodynamics and mechanism of high-pressure deactivation and dissociation of porcine lactic dehydrogenase.

Lactic dehydrogenase (LDH) from pig heart and pig skeletal muscle can be reversibly dissociated into monomers at high hydrostatic pressure. The reaction can be quantitatively fitted by a reversible consecutive dissociation-unfolding mechanism according to N in equilibrium 4M in equilibrium 4M (where N is the native tetramer, and M and M two different conformations of the monomer) (K. Müller, et al., Biophys. Chem. 14 (1981) 101.). At p less than or equal to 1 kbar, the pressure deactivation of both isoenzymes (H4 and M4) is described by the two-state equilibrium N in equilibrium 4M. From the respective equilibrium constant and the temperature and pressure dependence of the change in free energy, the thermodynamic parameters of the dissociation/deactivation may be determined, e.g., for LDH-M4: delta GDiss = 110 kJ/mol, delta SDiss =-860 J/K per mol, delta HDiss= -124 kJ/mol (enzyme concentration 10 microgram/ml, in Tris-HCl buffer, pH 7.6, I = 0.16 M, 293 K, 0.8 kbar); the dissociation volume is found to be delta VDiss =-420 ml/mol (0.7 less than p less than 0.9 kbar). Measurements using 8-anilino-1-naphthalenesulfonic acid (ANS) as extrinsic fluorophore demonstrate that the occurrence of hydrophobic surface area upon dissociation parallels the decrease in reactivation yield after pressurization beyond 1 kbar. Within the range of reversible deactivation (p less than 1 kbar) no increase in ANS fluorescence is detectable, thus indicating compensatory effects in the process of subunit dissociation. 2H2O is found to stabilize the enzyme towards pressure dissociation, in accordance with the involvement of hydrophobic interactions in the subunit contact of both isoenzymes of LDH.

Anilino Naphthalenesulfonates↗
PubMed1985

Kinetics of dissociation of nogalamycin from DNA: comparison with other anthracycline antibiotics.

Stopped-flow spectrometry and simple mixing techniques have been employed to investigate the detergent-induced dissociation of anthracycline antibiotics from natural and synthetic DNAs. Both daunomycin and nogalamycin dissociate more slowly from poly(dG-dC) than from poly(dA-dT) but the difference is much more marked for nogalamycin. With an equimolar mixture of poly(dG-dC) and poly(dA-dT), or with poly(dA-dC).poly(dG-dT), dissociation of nogalamycin occurs very slowly. In all cases the release of antibiotic from a synthetic polynucleotide is a one-step process following a single exponential. Dissociation of daunomycin, adriamycin and iremycin from calf thymus DNA is a more complex reaction which requires a two-exponential fit, in contrast to earlier reports, but differences between the behaviour of the three antibiotics are minor. Dissociation of nogalamycin from natural DNA requires a three-exponential fit, is in general far slower, and depends upon the base composition, the level of binding and the time allowed for the complex to equilibrate. It is concluded that sequence selectivity is minimal or lacking for daunomycin, whereas nogalamycin binding is sequence dependent and probably involves migration of the antibiotic between DNA binding sites. There is an inverse correlation between dissociation rate constants and antibacterial potency in simple tests.

Animals↗
PubMed1988

Subunit dissociation and denaturation of Fasciolaria tulipa hemocyanin.

1. The hemocyanin from the marine snail, Fasciolaria tulipa has a molecular weight of 8.6 +/- 0.6 x 10(6) determined by light-scattering and a sedimentation constant of (105.9 +/- 1.1)S. 2. The dissociated subunits at pH 11 and in 8.0 M urea (pH 7.4) had molecular weights of 4.4 x 10(5) and 4.7 x 10(5), close to one-twentieth of the parent didecameric assembly. 3. The pH dependence of the molecular weight profile exhibited bell-shaped transitions in both the presence and absence of Ca2+ and Mg2+ ions. In the physiological pH range of about 7.5-8.2 in divalent ion-containing buffers neither the molecular weight behavior nor the sedimentation patterns suggest any significant dissociation. 4. Both the urea and the Hofmeister salt series were found to dissociate the didecameric hemocyanin assembly. The ureas exhibit increasing effectiveness as dissociating agents with the higher alkyl substituted members of the series, suggesting hydrophobic stabilization of the subunit assembly. 5. Denaturation of the hemocyanin subunits by the urea series follows the same trend in effectiveness as the dissociation reaction; the reagent concentrations required to cause unfolding of the globular domains of the hemocyanin chains were, however, much higher than those needed for dissociation.

Animals↗
PubMed1990

Rates of dissociation of steroid and thyroid hormones from human serum albumin.

A rapid filtration assay employing dextran-coated charcoal as acceptor particles for free hormone was used to measure rates of dissociation of steroid and thyroid hormones from human serum albumin. Modification of a previously described assay allowed measurements at 1-s intervals. Nevertheless, this still permitted only minimum estimates of the dissociation rate constants. The hormones studied were thyroxine, 3,5,3'-triiodothyronine, cortisol, corticosterone, testosterone, dihydrotestosterone, estradiol, progesterone, and aldosterone. The apparent dissociation rate constant of the thyroxine-albumin complex at 37 degrees C was 1.3 +/- 0.2 s-1 (t 1/2, 0.5 s). The apparent dissociation rate constants of the other hormone-albumin complexes at 37 degrees C generally exceeded 2 s-1 (t 1/2 less than 0.35 s). Apparent dissociation rate constants at 4 degrees C were only slightly lower. These findings indicate that steroid and thyroid hormones dissociate from albumin rapidly compared with the 1-s capillary transit times that characterize many tissues.

Aldosterone↗