Academic literature on the topic 'Steady-state dissociation constant'

The effect of concentration of acetohydroxamic acid (AHA) on inhibition of jack bean urease in phosphate buffer, pH 7.0, at 25 degrees C, was studied. The measurements were performed at urease concentration of 2.5 mg/100 cm3 for concentrations of urea and AHA ranging in the range of 2-50 mmol dm-3 and 0.25-10 mmol dm-3, respectively. The reactions were monitored by two techniques: analytical and enthalpimetric. For the analytical technique the growth of ammonia concentration in the course of the reaction was determined. From the recorded progress curves the following parameters were calculated for each inhibitor concentration: the initial reaction rate, the steady-state rate and the inversion constant. From these parameters the inhibition constants of the initial and steady-state stages of the reaction, Ki and Ki, were calculated. The former constant did not change whereas the latter one proved to decrease quickly with an increase in inhibitor concentration. This behaviour resulted from the fact that the inactive complex EI was not a product of internal inversion but was formed in the reaction: 2/3I + EI-->(EI.2/3I). The dissociation constant of this complex is equal to about 0.3 x 10(-3) (mol dm-3)2/3.

Abstract IL-12 is a 75-kDa heterodimeric cytokine composed of disulfide-bonded 35-kDa and 40-kDa subunits. Included among the biologic activities mediated by IL-12 is induction of proliferation of PHA-activated human PBL. The concentration of IL-12 required to stimulate maximum proliferation of PHA-activated lymphoblasts is 50 to 100 pM. In this study, highly purified 125I-labeled IL-12 (7 to 15 microCi/microgram; 50 to 100% bioactive) was used to characterize the receptor for IL-12 on 4-day PHA-activated lymphoblasts. The binding of 125I-labeled IL-12 to PHA-activated lymphoblasts was saturable and specific because the binding of radiolabeled ligand was only inhibited by IL-12 and not by other cytokines. The kinetics of [125I]IL-12 binding to PHA-activated lymphoblasts was rapid at both 4 degrees C and 22 degrees C; reaching equilibrium within 60 min. At 22 degrees C, the rate of dissociation of [125I]IL-12 was slow in the absence of competing IL-12 (t1/2 = 5.9 h) and more rapid in the presence of 25 nM competing IL-12 (t1/2 = 2.5 h). The kinetically derived equilibrium dissociation constant ranged from 10 to 83 pM. Analysis of steady state binding data by the method of Scatchard identified a single binding site with an apparent equilibrium dissociation constant of 100 to 600 pM and 1000 to 9000 sites/lymphoblast. The equilibrium dissociation constant for competing ligands and sites per cell calculated from unlabeled IL-12 competition experiments ranged from 164 to 315 pM and 1067 to 3336, respectively, which is in good agreement with the values determined from steady state binding. The variations in KD and sites per cell were dependent on the individual preparations of lymphoblasts. Although the steady state binding data were consistent with a single class of high affinity binding sites, the kinetic dissociation data indicates a cooperative interaction between receptors on PHA-activated lymphoblasts. Affinity cross-linking of surface bound [125I]IL-12 to PHA-activated lymphoblasts at 4 degrees C identified a major complex of approximately 210 to 280 kDa. Anti-IL-12 antibodies also immunoprecipitated a complex of approximately 210 to 280 kDa that was produced by cross-linking unlabeled IL-12 to 125I-labeled lymphoblast cell-surface proteins. Cleavage of this complex with reducing agent identified one radiolabeled protein of approximately 110 kDa. These data suggest that the IL-12 binding site on PHA-activated lymphoblasts may be composed of a single protein of approximately 110 kDa.(ABSTRACT TRUNCATED AT 400 WORDS)

Three models for the hepatic uptake of thyoxine (T4) from human plasma were considered: 1) uptake occurs exclusively via the pool of free T4 after spontaneous dissociation of T4-plasma-protein complexes, 2) uptake occurs primarily via the pool of bound T4 by the interaction of one or more binding proteins with the cell-surface membrane, and 3) uptake occurs primarily by "enhanced dissociation" of T4 from one or more of its binding proteins within the sinusoids. Each of these models was examined in relation to well-accepted unidirectional uptake and steady-state kinetics data that indicate that 1) between 4 and 24% of the T4 in normal human serum is taken up unidirectionally by the liver in a single pass, and 2) the in vivo disposal rate of T4 is unaffected by primary changes in the plasma concentration of thyroid hormone-binding globulin. Both analytical and numerical techniques were used. The first two models were found to be compatible with both the steady-state kinetics data and the unidirectional uptake data, given certain assumptions in each of the models. Although theoretically distinguishable on the basis of unidirectional uptake data, uncertainty over the true uptake (influx) rate constant for free T4 prevented resolution between these two models. In contrast, the third model, that of enhanced dissociation [W. M. Pardridge, Am. J. Physiol. 252 (Endocrinol. Metab. 15): E157-E164, 1987], was found, as currently formulated with respect to T4, to be incompatible with both the steady-state kinetics data and the unidirectional uptake data.(ABSTRACT TRUNCATED AT 250 WORDS)

The properties of the small fraction of tetrodotoxin (TTX)-sensitive Na channels that remain open in the steady state were studied in internally dialyzed voltage clamped squid giant axons. The observed Ussing flux ratio exponent (n′) of 0.97 ± 0.03 (calculated from simultaneous measurements of TTX-sensitive current and 22Na efflux) and nonindependent behavior of Na current at high internal [Na] are explained by a one-site (“1s”) permeation model characterized by a single effective binding site within the channel pore in equilibrium with internal Na ions (apparent equilibrium dissociation constant KNai(0) = 0.61 ± 0.08 M). Steady-state open probability of the TTX-sensitive channels can be modeled by the product pap∞, where pa represents voltage-dependent activation described by a Boltzmann distribution with midpoint Va = −7 mV and effective valence za = 3.2 (Vandenberg, C.A., and F. Bezanilla. 1991. Biophys. J. 60:1499–1510) coupled to voltage-independent inactivation by an equilibrium constant (Bezanilla, F., and C.M. Armstrong. 1977. J. Gen. Physiol. 70:549–566) Keq = 770. The factor p∞ represents voltage-dependent inactivation with empirical midpoint V∞= −83 ± 5 mV and effective valence z∞ = 0.55 ± 0.03. The composite pap∞1s model describes the steady-state voltage dependence of the persistent TTX-sensitive current well.