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PubMed1988

Conformational restrictions of the sheep testicular receptor discriminates pituitary lutropin and placental gonadotropins.

A membrane preparation from the testis of maturing Dorset-Leicester-Suffolk sheep, capable of discriminating pituitary LH (lutropin) from placental gonadotropins human choriogonadotropin (hCG) and equine choriogonadotropin is described. Maximum binding of 125I-oLH (ovine lutropin) to the testicular receptors occurred at 4 degrees C in a rapid manner, attaining equilibrium in 12-16 h. Under such optimal conditions, only unlabeled ovine LH or the structurally identical bovine LH effectively competed for receptor occupation. Other highly purified pituitary LH preparations from rat and human pituitaries were weakly (4-10%) active in displacement assays. Purified hCG or equine choriogonadotropin, which were highly potent in rat testicular LH receptor assays, could not compete with 125I-oLH for binding to the sheep LH receptor at 4 degrees C. Thus, the sheep testicular LH receptor was highly specific in recognizing pituitary LH conformation. The presence of an ovine/bovine LH alpha- or beta-subunit in recombinants with hCG subunit counterparts was required to generate an effective conformation capable of receptor recognition. Chemically deglycosylated hCG, containing 75% less carbohydrate and which showed greater binding to other LH receptors, failed to recognize sheep LH receptor, suggesting that excess carbohydrate in hCG was not a factor in hindering binding of the native placental hormone. Scatchard analysis using 125I-hCG/125I-oLH revealed that there were separate sites with similar affinities but vastly different capacities. The hCG binding sites, which could also be effectively occupied by oLH, were less than 10% of oLH binding sites. Thus, the Dorset-Leicester-Suffolk sheep testicular receptor provides an important and unique in vitro test system to distinguish pituitary LH from placental LH-like hormones. We infer that temperature-dependent conformational restrictions of the sheep testicular LH receptor are involved in recognizing differences in these highly similar and structurally homologous hormones.

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↗

PubMed1974

Purification and properties of malyl-coenzyme A lyase from Pseudomonas AM1.

1. Malyl-CoA lyase was purified 20-fold from extracts of methanol-grown Pseudomonas AM1. 2. Preparations of the enzyme were essentially homogeneous by electrophoretic and ultracentrifugal criteria. 3. Malyl-CoA lyase has a molecular weight of 190000 determined from sedimentation-equilibrium data. 4. Within the range of compounds tested, malyl-CoA lyase is specific for (2S)-4-malyl-CoA or glyoxylate and acetyl-CoA or propionyl-CoA. 5. A bivalent cation is essential for activity, Mg(2+) or Co(2+) being most effective. 6. Malyl-CoA lyase is inhibited by (2R)-4-malyl-CoA and by some buffers, but thiol-group inhibitors are without effect. 7. Optimal activity was recorded at pH7.8. 8. An equilibrium constant of 4.7x10(-4)m was determined for the malyl-CoA cleavage reaction. 9. The Michaelis constants for the enzyme are: 4-malyl-CoA, 6.6x10(-5)m; acetyl-CoA, 1.5x10(-5)m; glyoxylate, 1.7x10(-3)m; Mg(2+), 1.2x10(-3)m.

Centrifugation, Density Gradient↗

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↗

PubMed1988

DESIGN: computerized optimization of experimental design for estimating Kd and Bmax in ligand binding experiments. I. Homologous and heterologous binding to one or two classes of sites.

We have developed a versatile computer program for optimization of ligand binding experiments (e.g., radioreceptor assay system for hormones, drugs, etc.). This optimization algorithm is based on an overall measure of precision of the parameter estimates (D-optimality). The program DESIGN uses an exact mathematical model of the equilibrium ligand binding system with up to two ligands binding to any number of classes of binding sites. The program produces a minimal list of the optimal ligand concentrations for use in the binding experiment. This potentially reduces the time and cost necessary to perform a binding experiment. The program allows comparison of any proposed experimental design with the D-optimal design or with assay protocols in current use. The level of nonspecific binding is regarded as an unknown parameter of the system, along with the affinity constant (Kd) and binding capacity (Bmax). Selected parameters can be fixed at constant values and thereby excluded from the optimization algorithm. Emphasis may be placed on improving the precision of a single parameter or on improving the precision of all the parameters simultaneously. We present optimal designs for several of the more commonly used assay protocols (saturation binding with a single labeled ligand, competition or displacement curve, one or two classes of binding sites), and evaluate the robustness of these designs to changes in parameter values of the underlying models. We also derive the theoretical D-optimal design for the saturation binding experiment with a homogeneous receptor class.

Binding Sites↗

PubMed1995

Comparison of muscle forces and joint load from an optimization and EMG assisted lumbar spine model: towards development of a hybrid approach.

The purpose of this study was to determine whether the same estimates of individual muscle and L4/L5 lumbar joint compressive forces result from an optimization (OPT) compared to an electromyography (EMG) assisted approach for solving the inderminate moment equilibrium equations in the same anatomical model. Four male subjects performed near maximum, isometric, ramp efforts in trunk flexion, extension and lateral bending in a testing apparatus. The EMG approach was sensitive to subject and trial differences in the magnitudes of individual muscle forces needed to produce the same reaction moment. In contrast, the OPT method converged on a similar estimate of muscle forces for all subjects and trials producing the same moment. The OPT method predicted lower L4/L5 joint compression values, on average, by 32, 43 and 23% in trunk extension, flexion and lateral bending, respectively, because, unlike the EMG method, it could not predict co-contraction of anatomically antagonistic muscles. We incorporated the OPT method's advantage of forcing an equilibrium in the reaction moments into the EMG method in a new approach we have called 'EMG assisted optimization' (EMGAO). Muscle force estimates from the EMG and EMGAO methods differed from those from the OPT method, on average, by 123% (RMS) for flexion and extension and by 218% for lateral bends. Data from the two approaches result in different conclusions about spine mechanics. We have more confidence in the EMG assisted methods because they respond to variation in muscle synergy and co-contraction patterns commonly observed in different trials and subjects for the same reaction moments.

Adult↗

PubMed1981

The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. III. A minimal model.

A steady-state kinetic investigation of the effect of K+ on the Na+-enzyme activity of the (Na+ + K+)-ATPase in broken membrane preparations is reported. Analysis of the kinetic patterns obtained, together with the results reported in the first two articles of this series permit the following conclusions. 1. K+ inhibits the Na+-enzyme (the enzyme activity measured at micromolar substrate concentrations in the presence of Na+). The inhibition of non-competitive at low and competitive at higher K+ concentrations and is enhanced by free Mg2+. 2. The results indicate that the Na+-enzyme at steady-state tends to be accumulated in an enzyme-potassium complex when K+ is added. 3. The enzyme-potassium complex, in turn, binds Mg2+ in a dead-end fashion. The dissociation constant for the enzyme-K-Mg complex, estimated from the data, is 7.2 mM. The same value was obtained earlier for the Mg2+ inhibition constant of the substrate-free form of the (Na+ + K+)-enzyme (the enzyme activity measured with Na+ and K+ and at millimolar substrate concentrations) suggesting that the two constants describe the same equilibrium. 4. On the basis of the known (optimal) activity of the (Na+ + K+)-ATPase, relative to that of the Na+-ATPase, a rate constant condition is found which must be met if the Post-Albers kinetic scheme is to satisfy the data. Kinetic data for the phosphoenzyme indicate that this condition is not satisfied. 5. On the basis of the kinetic results a model for the hydrolytic action of (Na+ + K+)-ATPase is proposed. This model encompasses the Post-Albers scheme but contains two distinctive hydrolysis cycles (an 'Na+-enzyme cycle' and a '(Na+ + K+)-enzyme cycle') with widely different affinities for the substrates. Only one of the cycles (the Na+-enzyme cycle) involves acid-stable phosphorylated enzyme intermediates at discernible steady-state concentrations. Which of the two main cycles is predominant in any particular system is determined by the concentration of ligands and substrates. 6. According to this scheme, an enzyme preparation may exhibit both a high (Na+-enzyme) and a low ((Na+ + K+)-enzyme) substrate affinity, without the necessity of assigning more than one substrate site to a particular enzyme unit at any one time.

Adenosine Triphosphate↗

PubMed1984

Identification and characterisation of L-[3H]aspartate binding sites on rat spinal cord synaptic membranes.

The binding of L-[3H] aspartate to extensively-washed rat spinal cord synaptic membranes was investigated. Specific binding was enriched in synaptic membranes and was optimal under physiological conditions of temperature and pH. Equilibrium binding was established relatively slowly over a period of 30 min, and was totally reversible within 40 min. Saturation analysis revealed complex binding patterns. Two sites were clearly demonstrable, only one of which was shown to be saturable over the ligand concentration range employed in the study (0.1-10 microM). There was also some indication of the presence of a higher affinity site, although this was not investigated in any detail. Saturable binding demonstrated a KD = 1.4 microM and Bmax = 105 pmole/mg protein. Structure-activity studies with a range of amino acid analogues indicated that binding was stereospecific and was inhibited by a very restricted range of compounds. The most potent inhibitors of binding were L-glutamate and L-aspartate. There was no evidence for the involvement of NMDA receptors. Effects of possible endogenous modulators, including ions and guanosine nucleotides were investigated, and the chemical nature of the binding site probed with a number of protein-modifying agents.

Animals↗

PubMed1995

Method for measuring the activities of cholesteryl ester transfer protein (lipid transfer protein).

A continuous recording fluorescence assay was developed for cholesteryl ester transfer protein (CETP). The assay measures the increase in fluorescence accompanying the relocation of fluorescent lipids, cholesteryl esters and triglycerides, from a donor emulsion to an acceptor emulsion. In the absence of CETP, the quantum yields of the fluorescent lipids is low because their high concentrations in the donor emulsions result in self-quenching. CETP catalyzes the redistribution of the fluorescent lipids from the donor to the acceptor emulsions and fluorescence increases substantially. Efficient sonication and incorporation of apolipoproteins from human HDL into the emulsions significantly increased the transfer rates. Under optimal conditions, the redistribution of fluorescent compounds reaches equilibrium within < 30 min and the kinetics of this process are consistent with a simple, first-order reaction pathway. The redistribution kinetics support a mechanism of adsorption --> exchange --> desorption --> diffusion.

Boron Compounds↗

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↗

PubMed1994

Localization of nicotinic cholinergic receptors in rat brain: autoradiographic studies with [3H]cytisine.

There is a great deal of interest in the role of nicotinic acetylcholine receptors in the central nervous system, although their function is not well understood at present. Currently, central nicotinic receptors can be classified broadly as either alpha-bungarotoxin binding sites with low affinity for acetylcholine agonists, or as high-affinity agonist binding sites with low affinity for alpha-bungarotoxin. Neuronal nicotinic receptors with a high affinity for agonists are distributed widely in the central nervous system. Evidence from molecular biology and electrophysiology suggests that multiple nicotinic receptor types exist in the brain. In this study we have used the agonist [3H]cytisine as a ligand for autoradiography to generate a detailed quantitative map of the high-affinity agonist binding nicotinic receptor in the rat brain. Optimized binding conditions, characterization of the kinetic and equilibrium binding properties, and demonstration of the nicotinic pharmacology of this binding site in tissue sections confirm the usefulness of [3H]Cytisine as a ligand for nicotinic receptor autoradiography. [3H]Cytisine autoradiography provides excellent anatomic resolution with very low non-specific binding. This property has allowed us to describe variations in receptor density within subnuclei and gradients of receptor density in larger brain regions. Data from several studies suggest that the predominant high-affinity agonist binding nicotine receptor in the central nervous system is composed of the alpha 4 and beta 2 subunits. The data in the current study are consistent with the suggestion that [3H]cytisine labels only the alpha 4 beta 2 nicotinic receptor with high affinity, offering the possibility of localizing a specific nicotinic receptor subtype in the central nervous system. In summary, we characterize the optimum experimental conditions for the use of [3H]cytisine in tissue section autoradiography. [3H]Cytisine proves to be an excellent marker for nicotinic cholinergic receptors with a very high affinity and very low background. We provide a detailed quantitative characterization of nicotinic receptor density in the rat central nervous system and we find there are significant variations and gradients in receptor density within specific brain regions, including subregions previously thought to be homogeneous.

Alkaloids↗

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↗

PubMedDate not supplied

The role of lumbar spinal elements in flexion.

The forces induced in the disc and ligaments of a lumbar motion segment in resisting a quasi-static external load, using a semi-experimental approach, are presented. The lines of action of ligaments (direction cosines) and disc center for the initial position of the specimen were determined using a morphometer. The changes in these lines of action for a known external load were computed by using the three-dimensional load-deformation characteristics of an intact motion segment. The load-deformation behavior were obtained by applying a known load to the motion segment's superior vertebra and recording the motion produced. A seven dial gauge motion measuring system was used for this purpose. The six equations of equilibrium yielded a statically indeterminate model. A linear optimization technique in conjunction with a cost function enabled the computation of forces in the ligaments as well as forces and moments in the disc. This approach made it possible to determine the component forces without a priori knowledge of the structural properties of ligaments. Typically for an external flexion moment of 6.9 Nm the supraspinous ligament experienced the most force (60 N), followed by capsular ligaments (25 N), and transverse ligaments (15 N). A compressive force of 100 N within the disc was predicted. The load-deformation curve, obtained from this study, for the supraspinous ligament was nonlinear and is in agreement with published experimental results.

Aged↗

PubMedDate not supplied

Kinetic characterization of [125I] iodo-prolactin in binding to primary monolayer cultures of rabbit mammary epithelium.

Monocellular suspensions of epithelial cells from mammary glands of rabbits at 20-22 days of pregnancy were prepared by sequential dissociation with collagenase-hyaluronidase followed by Pronase. Maintenance in D-valine-substituted minimum essential medium (D-valine-MEM) supplemented with 10% dialyzed calf serum yielded monolayers enriched for rabbit mammary epithelial cells (RMEC). RMEC specifically and reversibly bound bovine PRL with Ka = 1.41-1.85 x 10(9)M-1. Association of lactogen with RMEC receptor followed bimolecular reaction kinetics with rate of 5.17 (+/- 0.75) x 10(5)M-1 sec-1 at 24 C, and 1.03 (+/- 0.11) x 10(6)M-1 sec-1 at 37 C. Dissociation was first order (K-1 = 5.97 (+/- 0.70) x 10(-5) sec-1) and was unaffected by the presence of lactogen. Specific binding determined with an excess of unlabelled bPRL was 66-77% of the total binding, and was optimal at pH 7.4. The binding reaction reached equilibrium in 2 h at 37 C, in 3 h at 24 C, and after 24 h at 4 C. Studies of binding capacity revealed the presence of 4.6-6.3 x 10(3) sites per cell, competition for which was limited to hormones demonstrating lactogenic activity. Recovered lactogen was not degraded by incubation with or dissociation from RMEC. Approximately 25% of the radioactivity remained associated with the cells even upon prolonged incubation. These studies demonstrated several advantages of RMEC for the investigation of hormone-receptor interaction and receptor regulation.

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↗