Characterization of Kinetic Binding Properties of Unlabeled Ligands via a Preincubation Endpoint Binding Approach

The dissociation rates of unlabeled drugs have been well studied by kinetic binding analyses. Since kinetic assays are laborious, we developed a simple method to determine the kinetic binding parameters of unlabeled competitors by a preincubation endpoint assay. The probe binding after preincubation of a competitor can be described by a single equation as a function of time. Simulations using the equation revealed the degree of IC50 change induced by preincubation of a competitor depended on the dissociation rate koff of the competitor but not on the association rate kon. To validate the model, an in vitro binding assay was performed using a smoothened receptor (SMO) and [3H]TAK-441, a SMO antagonist. The equilibrium dissociation constants (KI) and koff of SMO antagonists determined by globally fitting the model to the concentration–response curves obtained with and without 24 h preincubation correlated well with those determined by other methods. This approach could be useful for early-stage optimization of drug candidates by enabling determination of binding kinetics in a high-throughput manner because it does not require kinetic measurements, an intermediate washout step during the reaction, or prior determination of competitors’ KI values.

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Head-to-tail polymerization of microtubules in vitro. Electron microscope analysis of seeded assembly.

The Journal of Cell Biology ◽

10.1083/jcb.84.1.141 ◽

1980 ◽

Vol 84 (1) ◽

pp. 141-150 ◽

Cited By ~ 172

Author(s):

L G Bergen ◽

G G Borisy

Keyword(s):

Electron Microscope ◽

Rate Constants ◽

Dissociation Constants ◽

Dissociation Rate ◽

Association Constants ◽

Directional Growth ◽

Microtubule Protein ◽

Electron Microscope Analysis ◽

Dissociation Rate Constants

Microtubules are polar structures, and this polarity is reflected in their biased directional growth. Following a convention established previously (G. G. Borisy, 1978, J. Mol. Biol. 124:565--570), we define the plus (+) and minus (-) ends of a microtubule as those equivalent in structural orientation to the distal and proximal ends, respectively, of the A subfiber of flagellar outer doublets. Rates of elongation were obtained for both ends using flagellar axonemes as seeds and porcine brain microtubule protein as subunits. Since the two ends of a flagellar seed are distinguishable morphologically, elongation of each end may be analyzed separately. By plotting rates of elongation at various concentrations of subunit protein, we have determined the association and dissociation rate constants for the plus and minus ends. Under our conditions at 30 degrees C, the association constants were 7.2 X 10(6) M-1 s-1 and 2.25 X 10(6) M-1 s-1 for the plus and minus ends, respectively, and the dissociation constants were 17 s-1 and 7 s-1. From these values and Wegner's equations (1976, J. Mol. Biol. 108:139--150), we identified the plus end of the microtubule as its head and calculated "s," the head-to-tail polymerization parameter. Surprisingly small values (s = 0.07 +/- 0.02) were found. The validity of models of mitosis based upon head-to-tail polymerization (Margolis et al., 1978, Nature (Lond.) 272:450--452) are discussed in light of a small value for s.

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Myosin: a noncovalent stabilizer of fibrin in the process of clot dissolution

Blood ◽

10.1182/blood-2002-10-3227 ◽

2003 ◽

Vol 101 (11) ◽

pp. 4380-4386 ◽

Cited By ~ 9

Author(s):

Krasimir Kolev ◽

Kiril Tenekedjiev ◽

Katalin Ajtai ◽

Ilona Kovalszky ◽

Judit Gombás ◽

...

Keyword(s):

Plasminogen Activator ◽

Rate Constant ◽

Dissociation Constants ◽

Dissociation Rate ◽

Covalent Modification ◽

Tissue Type ◽

Factor Xiiia ◽

Molar Ratio ◽

Fibrin Monomer

Abstract Myosin modulates the fibrinolytic process as a cofactor of the tissue plasminogen activator and as a substrate of plasmin. We report now that myosin is present in arterial thrombi and it forms reversible noncovalent complexes with fibrinogen and fibrin with equilibrium dissociation constants in the micromolar range (1.70 and 0.94 μM, respectively). Competition studies using a peptide inhibitor of fibrin polymerization (glycl-prolyl-arginyl-proline [GPRP]) indicate that myosin interacts with domains common in fibrinogen and fibrin and this interaction is independent of the GPRP-binding polymerization site in the fibrinogen molecule. An association rate constant of 1.81 × 102 M–1 · s–1 and a dissociation rate constant of 3.07 × 10–4 s–1 are determined for the fibrinogen-myosin interaction. Surface plasmon resonance studies indicate that fibrin serves as a matrix core for myosin aggregation. The fibrin clots equilibrated with myosin are stabilized against dissolution initiated by plasminogen and tissue-type plasminogen activator (tPA) or urokinase (at fibrin monomer-myosin molar ratio as high as 30) and by plasmin under static and flow conditions (at fibrin monomer-myosin molar ratio lower than 15). Myosin exerts similar effects on the tPA-induced dissolution of blood plasma clots. Covalent modification involving factor XIIIa does not contribute to this stabilizing effect; myosin is not covalently attached to the clot by the time of complete cross-linking of fibrin. Thus, our in vitro data suggest that myosin detected in arterial thrombi binds to the polymerized fibrin, in the bound form its tPA-cofactor properties are masked, and the myosinfibrin clot is relatively resistant to plasmin.

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CORRELATED STUDIES ON PLASMA FREE CORTICOSTERONE AND ON ADRENAL STEROID FORMATION RATE IN VITRO

Acta Endocrinologica ◽

10.1530/acta.0.xxxiv0513 ◽

1960 ◽

Vol XXXIV (IV) ◽

pp. 513-523 ◽

Cited By ~ 34

Author(s):

]. van der Vies ◽

R. F. M. Bakker ◽

D. de Wied

Keyword(s):

Sulfuric Acid ◽

Environmental Change ◽

Formation Rate ◽

Rat Plasma ◽

Rapid Decline ◽

Simple Method ◽

Adrenal Steroid ◽

Present Technique

ABSTRACT The effects of various stimuli on the rate of formation of adrenal cortical hormones in vitro and on the plasma free corticosterone were studied in rats. First, a simple method for the determination of corticosterone in rat plasma is described which is based on the fluorescence of this steroid in sulfuric acid. In contrast to procedures described by other workers the present technique does not require expensive spectro-fluorometers, but can be performed using a simple fluorometric unit consisting of components which are generally available. The results obtained with the two methods have shown that the steroid formation rate in vitro and the plasma free corticosterone increases after formalin stress, environmental change and injection of corticotrophin. Hypophysectomy leads to a marked and rapid decline of adrenal activity in vitro and of the plasma free corticosterone.

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A simple method for the in vitro determination of phagocytic activity of bovine polymorphonuclear leukocytes

Veterinary Immunology and Immunopathology ◽

10.1016/0165-2427(88)90056-6 ◽

1988 ◽

Vol 18 (2) ◽

pp. 139-147 ◽

Cited By ~ 1

Author(s):

A.M. Soler-Rodriguez ◽

J.M. Armas L.

Keyword(s):

Phagocytic Activity ◽

Polymorphonuclear Leukocytes ◽

Simple Method

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Rates and equilibria for a photoisomerizable antagonist at the acetylcholine receptor of Electrophorus electroplaques.

The Journal of General Physiology ◽

10.1085/jgp.86.2.235 ◽

1985 ◽

Vol 86 (2) ◽

pp. 235-256 ◽

Cited By ~ 15

Author(s):

M E Krouse ◽

H A Lester ◽

N H Wassermann ◽

B F Erlanger

Keyword(s):

Dissociation Constants ◽

Light Flash ◽

Dissociation Rate ◽

Dose Ratio ◽

Response Curves ◽

Competitive Antagonism ◽

Cis And Trans Isomers ◽

Order Of Magnitude ◽

Dissociation Rate Constants ◽

The Right

Voltage-jump and light-flash experiments have been performed on isolated Electrophorus electroplaques exposed simultaneously to nicotinic agonists and to the photoisomerizable compound 2,2'-bis-[alpha-(trimethylammonium)methyl]-azobenzene (2BQ). Dose-response curves are shifted to the right in a nearly parallel fashion by 2BQ, which suggests competitive antagonism; dose-ratio analyses show apparent dissociation constants of 0.3 and 1 microM for the cis and trans isomers, respectively. Flash-induced trans----cis concentration jumps produce the expected decrease in agonist-induced conductance; the time constant is several tens of milliseconds. From the concentration dependence of these rates, we conclude that the association and dissociation rate constants for the cis-2BQ-receptor binding are approximately 10(8) M-1 s-1 and 60 s-1 at 20 degrees C; the Q10 is 3. Flash-induced cis----trans photoisomerizations produce molecular rearrangements of the ligand-receptor complex, but the resulting relaxations probably reflect the kinetics of buffered diffusion rather than of the interaction between trans-2BQ and the receptor. Antagonists seem to bind about an order of magnitude more slowly than agonists at nicotinic receptors.

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277. Vitamin A and C in cow's milk, with a note on the synthesis of vitamin C in bovines

Journal of Dairy Research ◽

10.1017/s0022029900003460 ◽

1941 ◽

Vol 12 (2) ◽

pp. 109-118 ◽

Cited By ~ 6

Author(s):

S. N. Ray ◽

Karam Chand ◽

K. Govind Rau

Keyword(s):

Vitamin A ◽

Vitamin C ◽

Normal Growth ◽

Mammary Glands ◽

Carotene Content ◽

Simple Method ◽

Blood Constituents ◽

Sufficient Vitamin

1. A simple method is described for the determination of carotene and vitamin A in milk. Figures for the carotene content of milk were higher and those for vitamin A were lower than similar figures reported by Western workers.2. Figures for vitamin C in milk are similar to corresponding figures of English and American workers. Previous low values for vitamin C, reported by Indian workers, are due to destruction of the vitamin by light.3. The vitamin C concentration of milk is not subject to great individual variation, probably because cows cannot excrete in milk any vitamin C taken in with the food and because vitamin C of milk is produced by synthesis within the mammary glands from some simple blood constituents.4. To test the ability of bovines to synthesize vitamin C, young calves were kept for long periods on a strictly vitamin C-free diet. The concentration of vitamin in the blood and other tissues of these animals remained, however, at a normal level. Calves appear therefore to be able to synthesize sufficient vitamin C for their normal growth and activity.5. Unsuccessful attempts were made to produce vitamin C in vitro by growing bacteria isolated from various parts of the alimentary canal on media prepared from ingesta taken from the regions from which they were isolated.

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The modes of action of gallamine

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1981.0002 ◽

1981 ◽

Vol 211 (1183) ◽

pp. 181-203 ◽

Cited By ~ 28

Keyword(s):

Membrane Potential ◽

Equilibrium Constant ◽

Rate Constant ◽

Blocking Agent ◽

Dissociation Rate ◽

Neuromuscular Blocking ◽

Fluctuation Analysis ◽

Muscle Fibres ◽

Response Curves ◽

Kinetic Measurements

The action of gallamine, a classical competitive neuromuscular blocking agent, has been examined on voltage-clamped endplates of frog skeletal muscle fibres. Gallamine produces a parallel shift of the equilibrium log (concentration)–response curves in concentrations of up to about 40 μM. At a membrane potential of —70 mV the Schild plot of the dose ratios so measured has a gradient of slightly less than the theoretical value, for a competitive antagonist, of unity. The apparent equilibrium constant for ‘competitive’ block is about 2 μM, and is approximately independent of the membrane potential. Fluctuation analysis of the endplate current shows two components in the presence of gallamine. The results can be fitted, over the range tested, by a mechanism that involves block of open ion channels by gallamine in a manner similar to that by procaine or quaternary local anaesthetic analogues. The rate constants for this action are strongly dependent on the membrane potential. At — 100 mV the association rate constant is about 4 x 10 7 M -1 s -1 , the dissociation rate constant is about 600 s -1 , and the equilibrium constant about 15 μM. Other kinetic measurements (voltage-jump relaxation, and nerve-evoked endplate currents) give results consistent with this conclusion, but apparently these results are valid over a range of conditions narrower than that for fluctuation analysis.

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