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Results for “Equilibrium optimization”

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PubMed1988

The saturable high affinity association of factor X to ADP-stimulated monocytes defines a novel function of the Mac-1 receptor.

Initiation of the coagulation protease cascade as it assembles on cell surfaces requires limited proteolytic activation of the zymogen factor X. Not previously suspected to be the ligand of an organizing receptor on cell surfaces, we now describe that factor X specifically associates with cells of monocyte lineage and we identify the high affinity receptor for this zymogen. Following stimulation with ADP (10 microM), or with the ionophore ionomycin (1 microM), isolated human monocytes bind 125I-factor X in a saturable fashion with a dissociation constant (Kd) of 21.8-44.9 nM. Equilibrium binding analyses indicate that the reaction is optimal at room temperature, requires Ca2+ ions, and saturates at 128,500 +/- 21,300 molecules of 125I-factor X specifically associated with the cell surface. Molar excess of unlabeled factor X inhibits and reverses the binding, whereas the homologous gamma-carboxylated coagulation proteins factors II, VII, IX, IXa, and Xa are without effect. Similarly, chelation of divalent ions immediately dissociates bound 125I-factor X. The monoblast cell line U 937 and the monocytic cell line THP-1 when stimulated with ADP or ionomycin, bind 125I-factor X with characteristics similar to monocytes. Receptor identity was explored using antibodies to the leukocyte adhesive receptors Mac-1, LFA-1, and p150.95. Monoclonal antibodies specific for the alpha subunit of Mac-1 (M 1/70, LM 2/1) or for the common beta subunit (TS 1/18, 60.3) bound equally to resting and ADP- or ionomycin-stimulated cells and also completely blocked the binding of 125I-factor X to stimulated monocytes, U 937, or THP-1 cells. To distinguish between modulatory effects of the monoclonal antibodies and direct spatial hindrance binding of 125I-factor X to Mac-1 was analyzed directly. OKM10 anti-alpha subunit of Mac-1 monoclonal antibody immunoprecipitated 125I-factor X chemically cross-linked to its receptor on stimulated cells. In addition, the complement protein fragment C3bi, which is a recognized ligand for Mac-1, competitively inhibited the association of 125I-factor X. These findings indicate that human blood monocytes and less differentiated cells of this lineage possess an inducible receptor specific for factor X; and also support the conclusion that the heterodimeric leukocyte adhesive receptor Mac-1 functions as the specific receptor structure. We suggest that the novel properties of this receptor may be of importance in the organization and regulation of certain coagulation protease cascades on the monocyte surface.

Adenosine Diphosphate↗

PubMed1989

Perfection of a synaptic receptor: kinetics and energetics of the acetylcholine receptor.

The energetics and kinetics of activation of the acetylcholine receptor are evaluated in the context of optimizing rapid synaptic transmission. Physiological needs are used as the basis for estimating optimal values for the closed-to-open channel equilibrium constants of the liganded and unliganded receptor. An estimate is made of the maximum energy that can be derived from the binding of acetylcholine to a perfectly designed receptor binding site. Application of the principle of detailed balance shows that with only one ligand binding site the receptor will not be able to derive enough energy from acetylcholine binding to drive a sufficiently large change in the channel conformational equilibrium. This then provides a rationale for the existence of a second binding site, rather than the often invoked advantage of cooperativity. With two binding sites there is a considerable excess of binding energy and consequently considerable flexibility in how binding energy can be utilized. It is shown that the receptor must have at least one binding site that binds acetylcholine weakly when the channel is closed. This is essential to rapid response termination. However, making the other binding site bind more tightly can enhance and accelerate the activation of the receptor. To optimize both response activation and termination the best solution is to make the two binding sites different in their binding affinities. This qualitatively reproduces an experimental observation.

Calorimetry↗

PubMed1976

Glucocorticoid receptors: evidence for a second, non-glucocorticoid binding site.

Previous studies on cytoplasmic glucocorticoid receptors and enzyme induction led to the classification of steroids as inducers (optimal or sub-optimal), antagonists, or inactive steroids, with respect to their activity as glucocorticoids. The receptor was postulated to exist in allosteric equilibrium between two conformational states, one "active" and the other "inactive". Steroids behaved as inducers (optimal or sub-optimal), antagonists, or inactive steroids depending on their relative affinity for the active and inactive conformational state of the receptor. Another possible model would invoke multiple binding sites on a single receptor with interactions between the binding sites depending upon the particular steroid bound. To test this latter possibility, an experimental technique was developed to measure the rate of dissociation of tritiated dexamethasone ([3H]DM) or tritiated aldosterone ([3H]A) from the glucocorticoid receptor of rat liver or kidney cytosol. The dissociation of the [3H]DM-receptor at 25 C was not due to irreversible denaturation, and minimal recombination of the receptor with [3H]DM occurred. Progesterone and a number of other steroids consistently and significantly increased the dissociation rate of [3H]DM-receptor complexes in both liver and kidney cytosol. An identical effect was seen with hepatic glucocorticoid receptors labelled with [3H]A, like dexamethasone an optimal inducer. All steroids which enhanced glucocorticoid-receptor dissociation were either antagonists or sub-optimal inducers. Thus, it is postulated that glucocorticoid receptors have at least two classes of binding sites, and that occupation of the second site increases the dissociation rate of agonists from glucocorticoid receptors.

Aldosterone↗

PubMed1976

Metabolic control of respiratory neuronal activity and the accompanying changes in breathing movements of the rabbit. III. Phase shifts in respiratory neurons induced by inflation and collapse of the lung, hyperventilation, or metabolic modifiers.

Phase shifts between inspiratory-related and expiratory-related discharge patterns can be reversibly induced in respiratory neurons following volume changes of the lung, hypocapnic apnea as a result of hyperventilation, or superfusion with certain metabolic modifiers. Phase-spanning expiratory-inspiratory or inspiratory-expiratory discharges are frequently induced in those neurons which are activated either by pulmonary stretch receptors or collapse afferents. The same is true for regulatory effectors which activate key steps of the neuronal metabolism such as ADP, 3-phosphoglycerate, L-glutamine, fructose-6-phosphate and fructose-1,6-diphosphate. In contrast, inhibitory vagal inputs or superfusion with citrate, an inhibitory metabolic modifier, revert preexisting expiratory-inspiratory discharges into a phase-coupled inspiratory pattern. It is postulated that the respiratory neuronal networks represents a time-optimal control system which strives to adjust to a new equilibrium value in a minimum of time, following a given mechanical or chemical perturbation. Following the hypothesis advanced by Cohen (1974) that the phase-spanning units modulate the activity of the in-phase neurons, it is suggested that the additional recruitment of expiratory-inspiratory and inspiratory-expiratory units provides a measure of the quality of time-optimal control and hence a performance index of the system.

Animals↗

PubMedDate not supplied

Thyrotropin binding to porcine thyroid plasma membranes: kinetic and thermodynamic analyses.

Evaluation of TSH binding to plasma membranes of porcine thyroid revealed unique sensitivity to pH and temperature. Analysis of apparent equilibrium binding yielded a linear Scatchard plot at the optimal pH of 6.0, indicating one class of binding sites. At physiological pH 7.4 a curvilinear Scatchard plot was obtained, resolved by computer analysis into two classes of binding sites of different affinities and capacities. Treatment of membranes with phospholipase C resulted in a 20% decrease in the number of high affinity sites, but no change occurred in binding affinity. In contrast, low affinity sites were not altered. To evaluate the significance of the curvilinear Scatchard plot, the kinetics of association were examined. The intrinsic Kd (kd/ka) was 0.20 nM, a value essentially equivalent to that of the high affinity binding component. The 'negative cooperativity' model of hormone binding was evaluated by examining the effect of excess unlabeled TSH on dissociation rate. Dissociation of bound 125I-labeled TSH was biphasic, and was enhanced by unlabeled hormone, regardless of whether the membranes were prelabeled at pH 6.0 or 7.4. This effect was not correlated with curvilinear Scatchard plots, and therefore not proof of negative cooperativity. Binding sites for TSH were further distinguished by their sensitivity to temperature. A van't Hoff plot of temperature dependence of the apparent Kd of the high affinity site was linear from 4 to 37 degrees C. In contrast, the apparent Kd of low affinity binding did not vary with respect to temperature. These results demonstrate that there are at least two independent binding sites for TSH on porcine thyroid plasma membranes, distinguishable by their equilibrium binding properties.

Animals↗

PubMed1989

Evolutionary optimization of the catalytic effectiveness of an enzyme.

The kinetic and thermodynamic features of reactions catalyzed by present-day enzymes appear to be the consequence of the evolution of these proteins toward maximal catalytic effectiveness. These features are identified and analyzed (in detail for one substrate-one product enzymes) by using ideas that link the energetics of the reaction catalyzed by an enzyme to the maximization of its catalytic efficiency. A catalytically optimized enzyme will have a value for the "internal" equilibrium constant (Kint, the equilibrium constant between the substrates and the products of the enzyme when all are bound productively) that depends on how close to equilibrium the enzyme maintains its reaction in vivo. Two classes are apparent. For an enzyme that operates near equilibrium, the catalytic efficiency is sensitive to the value of Kint, and the optimum value of Kint is near unity. For an enzyme that operates far from equilibrium, the catalytic efficiency is less sensitive to the value of Kint, and Kint assumes a value that ensures that the rate of the chemical transformation is equal to the rate of product release. In each of these cases, the internal thermodynamics is "dynamically matched", where the concentrations of substrate- and product-containing complexes are equal at the steady state in vivo.

Biological Evolution↗

PubMed1985

Is ECT imaging with Tc(V)-99m dimercaptosuccinic acid useful to detect lung metastases of osteosarcoma?

ECT imaging, using Tc(V)-99m dimercaptosuccinic acid [Tc(V)-DMS] was performed in two patients with lung metastasis of osteosarcoma, and the results were compared with those of CT scan. Clear accumulation of Tc(V)-DMS was recognized in all cases in the same area that CT scans demonstrated. Tc(V)-DMS was labeled under optimal pH 8, had very low SnCl2 concentrations, an equilibrium between a stable form and a dissociated form of anion TcO4(3-) structurally similar to PO4(3-), and was postulated for tumor uptake. Considering this proposed mechanism for Tc(V)-DMS uptake by tumor cells, ECT imaging using this tracer could be of use in the early detection of lung metastasis of osteosarcoma.

Adolescent↗

PubMed1979

The question of adaptive sex ratio in outcrossed vertebrates.

Of various published theories of adaptive control of progeny sex ratio only two are plausible, a physiological theory by Trivers & Willard, and a demographic theory by Verner. The first applies to species in which sons and daughters impose different costs on parents, and in which only one or very few young are produced at once. They ought to show positive correlations in the sex of successive offspring and high sex-ratio variance among progenies. Verner's theory postulates a minimization of competition for mates in neighbourhoods subject to random fluctuation in sex ratio. Optimal progenies would exactly match the population's evolutionary equilibrium sex ratio. There would be little variance among progenies. Evidence from vertebrates is unfavourable to either theory and supports, instead, a non-adaptive model, the purely random (Mendelian) determination of sex. The apparent absence of parental control of progeny sex ratio is a serious theoretical difficulty.

Adaptation, Biological↗

PubMed1991

Characterization and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study.

A potent, synthetic cannabinoid was radiolabeled and used to characterize and precisely localize cannabinoid receptors in slide-mounted sections of rat brain and pituitary. Assay conditions for 3H-CP55,940 binding in Tris-HCl buffer with 5% BSA were optimized, association and dissociation rate constants determined, and the equilibrium dissociation constant (Kd) calculated (21 nM by liquid scintillation counting, 5.2 nM by quantitative autoradiography). The results of competition studies, using several synthetic cannabinoids, add to prior data showing enantioselectivity of binding and correlation of in vitro potencies with potencies in biological assays of cannabinoid actions. Inhibition of binding by guanine nucleotides was selective and profound: Nonhydrolyzable analogs of GTP and GDP inhibited binding by greater than 90%, and GMP and the nonhydrolyzable ATP analog showed no inhibition. Autoradiography showed great heterogeneity of binding in patterns of labeling that closely conform to cytoarchitectural and functional domains. Very dense 3H-CP55,940 binding is localized to the basal ganglia (lateral caudate-putamen, globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), cerebellar molecular layer, innermost layers of the olfactory bulb, and portions of the hippocampal formation (CA3 and dentate gyrus molecular layer). Moderately dense binding is found throughout the remaining forebrain. Sparse binding characterizes the brain stem and spinal cord. Densitometry confirmed the quantitative heterogeneity of cannabinoid receptors (10 nM 3H-CP55,940 binding ranged in density from 6.3 pmol/mg protein in the substantia nigra pars reticulata to 0.15 pmol/mg protein in the anterior lobe of the pituitary). The results suggest that the presently characterized cannabinoid receptor mediates physiological and behavioral effects of natural and synthetic cannabinoids, because it is strongly coupled to guanine nucleotide regulatory proteins and is discretely localized to cortical, basal ganglia, and cerebellar structures involved with cognition and movement.

Animals↗

PubMed1985

Space motion sickness and space vestibulology.

Given the relatively short period of the space flight missions and more inexperienced personnel expected to be aloft, if the time in space should be optimized, the critical processes which occur in the body equilibrium system during the period of initial exposure to space environment must be thoroughly investigated. It is most important that improved management of space motion sickness (including the prediction, prevention and countermeasures) is properly developed. The improvement will fully depend on advances in our research on the etiology and pathology of this debilitating condition.

Aerospace Medicine↗

PubMed1984

Adaptation of testicular follicle-stimulating hormone receptors to ambient temperatures in vertebrates: equilibrium analysis.

Gonadotrophin receptors of the rat, turtle, and newt testes had different optimal temperatures of affinity for rat FSH. The optimal temperature in the rat was around 37 degrees and that in the newt was between 15 and 20 degrees. The binding affinity in the turtle did not differ significantly at temperatures ranging between 0- and 40 degrees. These optimal temperatures coincided well with the temperatures at which gonadotrophin acts in each species under natural conditions.

Animals↗

PubMed1988

High pressure and anesthesia: pressure stimulates or inhibits bacterial bioluminescence depending upon temperature.

Although high pressure is often viewed as a nonspecific stimulus counteracting anesthesia, pressure can either excite or inhibit biological activity depending on the temperature at application. Temperature and pressure are two independent variables that determine equilibrium quantity, e.g., the state of organisms in terms of activity and anesthesia depth. We used the light intensity of luminous bacteria (Vibrio fischeri) as an activity parameter, and studied the effects of pressure and anesthetics on the bacteria's light intensity at various temperatures. The light intensity was greatest at about 30 degrees C at ambient pressure. When the system was pressurized up to 204 atm, the temperature for maximum light intensity was shifted to higher temperatures. Above the optimal temperature for the maximal light intensity, high pressure increased the light intensity. Below the optimal temperature, pressure decreased light intensity. Pressure only shifts the reaction equilibrium to the lower volume state (Le Chatelier's principle). When the volume of the excited state is larger than the resting state, high pressure inhibits excitation, and vice versa. Halothane 0.008 atm and isoflurane 0.021 atm inhibited the light intensity both above and below the optimal temperature. When pressurized, the light intensity increased in the high temperature range but decreased in the low temperature range, as in the control. Thus, high pressure seemingly potentiated the anesthetic action at low temperatures. When the ratio of the light intensity in bacteria exposed to anesthesia and those not exposed to anesthesia was plotted against the pressure, however, the value approached unity in proportion to the pressure increase.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthesia↗

PubMed1990

Contacts between the factor TUF and RPG sequences.

The yeast TUF factor binds specifically to RPG-like sequences involved in multiple functions at enhancers, silencers, and telomeres. We have characterized the interaction of TUF with its optimal binding sequence, rpg-1 (1-ACACCCATACATTT-14), using a gel DNA-binding assay in combination with methylation protection and mutagenesis experiments. As many as 10 base pairs appear to be engaged in factor binding. Analysis of a collection of 30 different RPG mutants demonstrated the importance of 8 base pairs at position 2, 3, 4, 5, 6, 7, 10, and 12 and the critical role of the central GC pair at position 5. Methylation protection data on four different natural sites confirmed a close contact at positions 4, 5, 6, and 10 and suggested additional contacts at base pairs 8, 12, and 13. The derived consensus sequence was RCAAYCCRYNCAYY. A quantitative band shift analysis was used to determine the equilibrium dissociation constant for the complex of TUF and its optimal binding site rpg-1. The specific dissociation constant (K8) was found to be 1.3 x 10(-11) M. The comparison of the K8 value with the dissociation constant obtained for nonspecific DNA sites (Kn8 = 8.7 x 10(-6) M) shows the high binding selectivity of TUF for its specific RPG target.

Base Sequence↗

PubMed1990

Equilibrium kinetics of the new experimental anti-tumour compound SK&F 104864-A in aqueous solution.

The equilibrium kinetics of lactone ring hydrolysis in the new experimental anti-tumour compound SK&F 104864-A. (S)-dimethylaminomethyl-10-hydroxycamptothecin hydrochloride, have been studied. Only one product is formed, SK&F 105992. A stability-indicating HPLC method has been optimized to perform the analysis. The pH is the main factor influencing equilibrium; at pH greater than or equal to 10 the lactone ring is quantitatively opened while at pH values less than or equal to 4 the lactone form is exclusively present. Other parameters, such as buffer ions and ionic strength, do not influence equilibrium. Complexation with dimethyl-beta-cyclodextrin stabilizes the lactone form. Other cyclodextrins do not show this stabilization.

Camptothecin↗

PubMedDate not supplied

Representation of phase equilibrium behavior of antibiotics.

The phase equilibrium behavior of biomolecules is important both in understanding the partition mechanisms and in the design and optimization of downstream recovery processes. Chen et al. (1989) proposed a molecular thermodynamic framework that successfully represents the liquid-solid equilibrium behavior of amino acids and small peptides as functions of temperature, ionic strength, solvent compositions, and pH. Based on this theoretical framework, this paper presents recent results in representing the liquid-solid equilibrium behavior (solubilities) and the liquid-liquid equilibrium behavior (phase partitioning) of beta-lactam antibiotics, which are amino acid derivatives and important chemotherapeutic agents.

Ammonium Chloride↗

PubMed1982

Thermodynamics of stacking and of self-association of the dinucleoside monophosphate m2(6)A-U from proton NMR chemical shifts: differential concentration temperature profile method.

Chemical shifts of base and sugar protons of the modified ribodinucleoside monophosphate N6-dimethyladenylyl(3'-5')uridine (m2(6)A-U) were measured at 100, 360 and 400 MHz in aqueous solution. Seven different samples were used with concentrations ranging from 0.28 mM to 32.7 mM. The temperature was varied from -5 degrees C to 105 degrees C. An internal temperature calibration was used. The effects of intermolecular self-association and of intramolecular stacking on the chemical shifts were quantitatively separated by means of a new approach: differential concentration/temperature profiles (DCTP). Several computational models were tested and the analysis allowed deeper insight into the behaviour of m2(6)A-U at the molecular level. The simple two-state approach for both self-association and stacking already afforded a significant improvement over models in which the association is entirely neglected. A computer least-squares analysis of the chemical shift behaviour of each individual proton yielded thermodynamic parameters for self-association and stacking. However, the two-state model did not suffice to reproduce accurately all of the observations. A satisfactory fit required two additional assumptions: (a) the aromatic protons experience different association shifts in stacked and in unstacked molecules: (b) a temperature-dependent conformational equilibrium exists between sets of unstacked microstates. The stacked state is taken to represent a single conformational species. The implementation of this extended model in the least-squares optimization allowed the reproduction of over one thousand chemical shift observations within experimental error. Thermodynamic equilibrium parameters deduced for intramolecular stacking are: delta H degrees x = -28.8 kJ mol-1, delta S degrees x = -93 J mol-1 K-1. These numbers agree well with those obtained earlier by us from circular dichroism spectra. The equilibrium enthalpy and entropy values deduced for the association process are: delta H degrees A = -35 kJ mol-1 and delta S degrees A = -95 J mol-1 K-1.

Adenosine Monophosphate↗

PubMed1981

L-Aspartate binding sites in rat cerebellum: a comparison of the binding of L-[3H]aspartate and L-[3H]glutamate to synaptic membranes.

The binding of L-[3H]aspartate to sonicated, extensively washed and preincubated cerebellar synaptic membranes was investigated. Binding was optimal under physiological conditions of pH and temperature, and attained equilibrium within 10 min. Binding was saturable, and Eadie-Hofstee analysis revealed interaction with a single population of binding sites (Kd = 874 nM and Bmax = 44 pmol/mg protein), which displayed no cooperativity (Hill coefficient approx. = 1). Specific [3H]aspartate was readily and reversibly displayed by unlabelled L-aspartate (the D-isomer being less than half as active) with a half-life of dissociation of 32 sec. Quisqualate, 4-fluoroglutamate and 2-amino-4-phosphonobutyrate, which are good displacers of [3H]glutamate binding, were only weakly active against the aspartate system. The excitatory amino acid antagonists, DL-alpha-aminoadipate, DL-alpha-aminosuberate and HA-966 were effective displacers, but the proposed aspartate receptor-preferring agonist, N-methyl-D-aspartate was inactive. Kainic acid exhibited negligible affinity for the aspartate binding site, in common with that for glutamate. While freezing or cold storage of membranes resulted in diminished [3H]-aspartate binding, lyophilization was not only able to confer substantial stability, but induced a marked increase in affinity of the binding site. Differential effects of various cations on [3H]aspartate binding were observed--monovalent cations reduced, while divalent cations enhanced L-[3H]aspartate binding.

Animals↗

PubMed1995

Optical detection of macromolecular heparin via selective coextraction into thin polymeric films.

Thin plasticized polymer films, poly(vinyl chloride) doped with a specific ion pairing quaternary ammonium compound, tridodecylmethylammonium chloride, and a lipophilic pH indicator, 3-hydroxy-4-(4-nitrophenylazo)phenyl octadeconate, are shown to exhibit significant and analytically useful optical response toward macromolecular heparin. The response mechanism is based on favorable extraction of heparin into the bulk organic film, owing to the specific ion-pairing complexation reaction between the quaternary ammonium species and the polyanion. A simultaneous coextraction of hydrogen ions results in protonation of the pH chromophore and hence a change in the optical absorbance of the polymeric film. When used in a limited volume/fixed exposure (10 min) detection mode, film absorbances change as a function of the initial heparin concentration in the range of 0.2-3.0 units/mL (1.2-18 micrograms/mL). The practical measurement response time is controlled by heparin diffusion through the stagnant diffusion layer adjacent to the surface of the film as well as within the bulk of the polymer film and is shown to increase with the molecular weight of the heparin species tested. No optical response to heparin is observed when a strong heparin complexing agent (e.g., protamine) is present in the test solution, suggesting that the polymer film can be used to conveniently monitor heparin-protamine (or other antagonist) titrations. The theory relating to the operation of the sensing film in either the equilibrium or the kinetic mode and the selectivity of the optimized film to heparin relative to small anions are presented.

Anions↗