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PubMedDate not supplied

Measurement of urinary somatomedin C by a chemiluminescent method.

We present a method for the measurement of the total Somatomedin C (SmC) content in human early morning urine samples after dialysis, extraction, and concentration. We modified a chemiluminescence immunoassay, previously developed for SmC determination in serum, for analysis of SmC in urine. Appropriate sensitivity was obtained by the preparation of a new chemiluminescent tracer (AEEI-COOH-SmC) and the optimization of a competitive non-equilibrium immunoassay system which had a detection limit of 0.24 fmol SmC per tube.

Carrier Proteins↗
PubMed1978

Cobalt exchange in horse liver alcohol dehydrogenase.

The preparation of metal hybrid species of horse liver alcohol dehydrogenase is made possible by the development of carefully delineated systems of metal in equilibrium metal exchange employing equilibrium dialysis. The conditions which are optimal for the site-specific replacement of the catalytic and/or noncatalytic zinc atoms of the native enzyme by cobalt are not identical with those which are utilized for substitution with 65Zn. Thus, while certain 65Zn hybrids can be prepared by exploiting the differential effects of buffer anions, the cobalt hybrids are generated by critical adjustments in the pH of the dialysate. Factors which may determine the mechanism of metal replacement reactions include acid-assisted, ligand-assisted, and metal-assisted dechelation, steric restriction, and ligand denticity as well as physicochemical properties of the enzyme itself. The spectral characteristics of the catalytic and noncatalytic cobalt atoms reflect both the geometry of the coordination complexes and the nature of the ligands and serve as sensitive probes of these loci in the enzyme.

Alcohol Oxidoreductases↗
PubMedDate not supplied

Nonlinear gradient isotherm parameter estimation for proteins with consideration of salt competition and multiple forms.

Salt gradients in ion-exchange chromatography are routinely used to speed separation of proteins and to concentrate products, but systematic optimization of these gradients requires protein equilibrium data as a function of salt concentration. An understanding of conformational changes, aggregation, and salt effects, which include both competition and affinity modulation, is important for equilibrium isotherm parameter estimation. In this study, gradient elution of bovine serum albumin (BSA) in anion exchange was well predicted by a salt-modulated nonlinear isotherm which considers salt competition. The isotherm was able to predict BSA gradient elution from batch equilibrium data. The same isotherm was also able to predict elution for various gradient slopes when fitted to an intermediate slope gradient experiment. If multiple forms due to aggregation or denaturation exist, isotherm parameters are readily averaged in batch experiments because of the long equilibration times. Similarly, gradient experiments yield averaged parameters because the salt gradient tends to merge the closely eluting forms. However, in isocractic elution, if the reaction rate is not rapid enough to give a merged peak, the estimated isotherm parameters are only fair predictors of gradient behavior and vice versa. Slower flow rates in isocratic elution can help reduce the discrepancy by allowing forms to merge through interconversion. As an alternative to determining averaged parameters, consideration of two binding forms, using VERSE-LC, an advanced rate model, gave good agreement with experimental data over the entire range of salt gradient durations.

Animals↗
PubMed1987

A comparison of alfentanil pharmacokinetics in children and adults.

The pharmacokinetics of alfentanil have been studied in eight children aged between 4 and 8 yr and five adults during general anesthesia. All patients were given 20 micrograms/kg alfentanil as an intravenous bolus injection. Plasma concentrations were measured at intervals up to 6 h by radioimmunoassay. Plasma protein binding was measured by equilibrium dialysis using tritiated alfentanil. The optimal pharmacokinetic model for alfentanil was an open two-compartment model. Total apparent volume of distribution (Vdss) was 457 +/- 160 ml/kg in adults and 163 +/- 110 ml/kg in children (P less than 0.01). When recalculated by surface area Vdss was still decreased in children (P less than 0.01). Plasma clearance (Cl) was similar in the two groups. Terminal elimination half-life was significantly shorter in children (40 +/- 9 min) than in adults (97 +/- 22 min; P less than 0.01). The shorter elimination half-life could be due to the smaller total apparent volume of distribution in children. Plasma protein binding was comparable between children and adults and could not explain the smaller volume of distribution in children. It is suggested that the smaller volume of distribution of alfentanil in children is a result of the decreased percentage of fat tissue in children.

Adult↗
PubMed1976

Optimal conditions and specificity of interaction of a distinct class of nonhistone chromosomal proteins with DNA.

A subclass of nonhistone chromatin proteins with high DNA affinity has been isolated from rat liver. The interaction of the isolated proteins with DNA in vitro was characterized utilizing a nitrocellulose filter binding technique. The temperature, time, concentration, ionic strength, and pH dependence were characterized. Optimal interaction was observed at 0.19 M naCl, pH 7.5 with a protein to DNA ratio of 13 (w/w). Equilibrium and kinetic competition experiments indicated that these proteins interact optimally with A-T rich and single-stranded DNA. The data also suggest that these proteins might affect the helixcoil transiton of DNA.

Animals↗
PubMed1992

Binding of platelet-activating factor to oviductal membranes during early pregnancy in the rabbit.

The present study explores the ability of rabbit oviductal membranes to bind tritiated platelet-activating factor [3H]PAF on days 3 and 6 of pregnancy. Under optimal conditions (25 degrees C, 120 min) equilibrium saturation analysis revealed only one class of binding sites, characterized by Kd s(nM), 80.03 +/- 11.60 and 11.17 +/- 7.09 and Bmaxs, (pmol/mg protein), 5.25 +/- 2.23 and 1.08 +/- 0.22 (N = 3, mean +/- SEM) for ampullar membranes on days 3 and 6, respectively. The corresponding values for isthmic membranes were Kds, 86.56 +/- 12.01 and 52.43 +/- 30.49 and Bmaxs, 9.41 +/- 0.67 and 2.88 +/- 1.96 for days 3 and 6, respectively. Significant differences between days 3 and 6 were observed only in the binding affinities for the ampullar membranes and the binding capacities for the isthmic binding sites. [3H]PAF binding was inhibited in the following order of decreasing potency: lyso-PAF greater than PAF C18:0 greater than U66985 greater than PAF C16:0 for day 3 ampullar membranes; and lyso-PAF C16:0 greater than PAF C18:0 greater than U66985 greater than PAF C16:0 for day 6 ampullar membranes. These studies show the existence of specific oviductal membrane PAF binding sites, the binding parameters of which may be related to the stage of pregnancy, rather than to the spatial location along the oviduct. The relative proportion of endosalpinx to myosalpinx between the ampulla and isthmus may have masked inherent differences and account for the relatively low affinity binding. The physiological significance of oviductal membrane PAF binding is yet to be established.

Animals↗
PubMed1977

A pregnenolone-binding protein in soluble fraction of guinea pig adrenal cortex.

A pregnenolone-binding component has been detected in the soluble fraction of the guinea pig adrenal cortex. Enzymatic degradation studies revealed that the binding component was a protein. The binding was destroyed at 60 degrees but was not inhibited by sulfhydryl reactants. Pregnenolone was bound optimally at pH 7 to 7.5 The equilibrium association constant at 0 degrees was 10(7) M-1. The pregnenolone-binding protein had an apparent molecular weight of 58,000, as determined by gel filtration. With the exception of pregnenolone sulfate, structurally similar steroids did not interfere with pregnenolone binding. No such binding activity was detected in the guinea pig liver and kidney. Serum contained pregnenolone-binding activity which was distinguishable from the adrenal cytosol factor by a variet of physicochemical means. The physiological importance of this finding remains to be determined.

Adrenal Cortex↗
PubMedDate not supplied

A strategy for efficient characterization of macromolecular heteroassociations via measurement of sedimentation equilibrium.

A method is proposed for the selection of experimental conditions for sedimentation equilibrium experiments that will provide maximal information about the values of equilibrium association constants within a given scheme for heteroassociation of two solute components. A discriminator function is proposed that indicates the sensitivity of the experimentally observed gradient or gradients to alterations in the underlying association constants. The value of this function is plotted or tabulated as a function of the concentrations of the two components, over a broad range of solution compositions. It is suggested that experiments performed with loading compositions corresponding to large absolute values of the discriminator function will yield the most information with respect to determination of the underlying association constants. This method was tested by predicting optimal conditions for three different types of sedimentation equilibrium experiments: (i) measurement of total (natural) solute absorbance; (ii) measurement of individual component gradients via measurement of tracer absorbance; and (iii) global analysis of multiple experiments. Experimental data resulting from sedimentation equilibrium experiments carried out under the specified conditions were simulated by addition of realistic levels of random error to calculated equilibrium gradients. The simulated data were then analyzed exactly as real experimental data, i.e., without prior knowledge of the underlying association constants. It was found that the highest accuracy and precision in determination of heteroassociation constants are obtained by global analysis of multiple experiments performed using significantly different loading compositions, each of which is selected from 'sensitive' regions of the discriminator map.

Centrifugation, Density Gradient↗
PubMed1976

Hydrolysis-resynthesis equilibrium of the lysine-15--alanine-16 peptide bond in bovine trypsin inhibitor (Kunitz).

Catalytic amounts of bovine beta-trypsin, bovine alpha-chymotrypsin and porcine plasmin establish a true thermodynamic equilibrium between virgin (I) (reactive site Lys15-Ala16 peptide bond intact) and modified (I) (this bond hydrolyzed) bovine trypsin/kallikrein inhibitor (Kunitz). The very slow reaction rates for attaining equilibrium are pH-dependent and differ for different enzymes. Optimal rates are for beta-trypsin at pH 3.75, for alpha-chymotrypsin at pH 5.5, and for plasmin at pH 5.0. Under conditions of optimum pH the equilibrium is reached with the highest rate by plasmin. In 10(-5)M inhibitor solutions the equilibrium concentrations of virgin and modified inhibitor are established by plasmin after almost 300 days starting from either pure virgin or pure modified inhibitor. Thus, the hydrolysis constant KHyd = [I]/[I] is determined to be 0.33 at pH 5.0. In spite of many unsuccessful attempts, this demonstrates that the reactive site peptide bond Lys15-Ala16 in the bovine trypsin inhibitor (Kunitz) can be hydrolyzed by catalytic amounts of endopeptidase. It further confirms that the hydrolyzed Lys15-Ala16 peptide bond in modified inhibitor is subject to thermodynamic control resynthesis.

Alanine↗
PubMed1985

Synthesis of ATP catalyzed by the (Ca2+ + Mg2+)-ATPase from erythrocyte ghosts. Energy conservation in plasma membranes.

The (Ca2+ + Mg2+)-ATPase from erythrocyte ghosts catalyzed the hydrolysis of ATP together with the synthesis of ATP or ATP in equilibrium 'Pi exchange. The modulation of the ATPase reaction cycle was controlled by high- and low-affinity calcium-binding sites asymmetrically located on the enzyme. Calmodulin accelerated the reaction cycle in both directions, stimulating the overall turnover of the enzyme. Calcium transport was achieved utilizing optimal conditions for the expression of the ATP in equilibrium Pi exchange system.

Adenosine Triphosphate↗
PubMed1991

Physiological prediction of muscle forces--I. Theoretical formulation.

A physiological model for predicting muscle forces is described. Rigid-body mechanics and musculoskeletal physiology are used to describe the dynamics of the segment model and muscle model. Unknown muscle and joint contact forces outnumber the equilibrium equations resulting in an indeterminate problem. Mathematical optimization is utilized to resolve the indeterminacy. The modeling procedure relies entirely on established physiological principles. Data describing the muscle anatomy and body structures are included. A model defining the force-length-velocity-activation relationship of a muscle is adopted. The force a muscle produces is assumed to be proportional to its maximum stress, physiological cross-sectional area, activation, and its functional configurations including the muscle architecture, muscle length, contracting velocity, and passive tension. These factors are incorporated into inequality equations which limit the force for each muscle. Minimal muscular activation is forwarded as the optimization criterion for muscle force determination.

Biomechanical Phenomena↗
PubMed1991

Physiological prediction of muscle forces--II. Application to isokinetic exercise.

The successful application of a physiological model of the musculoskeletal system capable of accounting for nonequilibrium dynamic loading and predicting individual muscle forces in the knee is presented. The model incorporates rigid-body mechanics and musculoskeletal physiology. Unknown muscle and joint contact forces outnumber the equilibrium equations resulting in an indeterminant problem. Mathematical optimization is utilized to resolve the indeterminacy. The model is used to estimate individual muscle forces during isokinetic exercise. Five subjects were tested at speeds of 60 degrees/s and 180 degrees/s. A newly proposed optimal criterion, minimizing muscular activation, results in muscle force predictions which have significantly higher correlations with myoelectric activity than other linear and nonlinear optimal criteria. The results demonstrate that properly constrained linear programming methods do not limit the number of active muscles and allow for uniform recruitment of the active muscles.

Adult↗
PubMed1989

Computer simulation of the hydrostatic skeleton. The physical equivalent, mathematics and application to worm-like forms.

The functional principles of a hydrostatic skeleton were combined to obtain a physical model which includes geometry, number and length-tension relationships of the elastic elements in the body wall, internal volume and internal pressure. The model skeleton with pre-set internal volume assumes a certain shape and develops a specific internal pressure in order to minimize the potential energy stored in the elastic elements. This shape is calculated as equilibrium state by using finite element methods and optimization techniques. This model is flexible enough to accommodate different geometries and length-tension-relationships of the elastic elements. Presently, the model is implemented with linear length-tension relationships and certain geometrical restrictions, such as uniform width over the entire animal, and rectangular cross sections; the general case is outlined. First simulations with the "unit-worm" yield stable solutions, i.e. stable shapes for all combinations of parameters tested so far. They define the conditions for bringing all muscles to an optimal operating point. We detected a pressure maximum with increasing volume, assessed the contribution of circular muscles to bending, and determined the shapes of animals with different muscle activations in each body half (Chapman-matrix). We summarize our results by the volume rule and stabilization rule, two simple concepts which predict changes in shape as the result of muscle activation.

Animals↗
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↗
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↗