The affinity of human, rabbit and bovine thrombins for sepharose-lysine and other conjugates.

Human, rabbit and bovine thrombins are shown to possess marked affinities for Sepharose-lysine. Using either Xa-activated crude prothrombins (human, rabbit) or a commercial thrombin sample (bovine), the enzyme was isolated in a single chromatographic step by the affinity medium and preparations of high specific activity were obtained. The relevance of bound-lysine for the affinity of the thrombins was studied using other Sepharose conjugates with structures related to Sepharose-lysine. Using freshly activated prothrombins it was found that human and rabbit thrombin uptake required a conjugate with a spacer chain containing a minimum of four carbon atoms in length which supported a terminal amino group. As the thrombin activity aged, affinity for the terminal amino group decreased but the hydrophobic spacer chain became essential for enzyme binding. The active centre of thrombin was not involved in binding to Sepharose-lysine.     

High affinity binding of paclitaxel to human serum albumin.

Paclitaxel, a very potent antitumor agent is a hydrophobic molecule with low aqueous solubility. Its currently used formula (Taxol) contains the drug in a 1 : 1 (v/v) mixture of ethanol and Cremophor EL. To minimize vehicle-related toxicity, we developed a novel, water-soluble formulation in which paclitaxel is bound noncovalently to human serum albumin. For this purpose, studies of the paclitaxel-albumin binding equilibrium were performed. Paclitaxel dissolved in ethanol was added to the aqueous solution of human serum albumin. Precipitated paclitaxel was removed and unbound drug was separated by ultrafiltration. Paclitaxel concentration was measured by RP-HPLC. Binding data were evaluated based both on the Scatchard plot and the general binding equation describing binding equilibria with the stepwise stoichiometric binding constants. The Scatchard plot was found to be curvilinear with a slight positive slope of the final part. Parameters of high affinity specific binding were determined from the initial part of the curve (nsp = 1.3 and Ksp = 1.7 x 10(6) M(-1)). Stoichiometric binding constants were estimated by fitting the general binding equation to the experimental data (K1 = 2.4 x 10(6) M(-1) and K2 = 1.0 x 10(5) M(-1)). Saturation of the protein with paclitaxel, similarly to other ligands of albumin, could not be reached. The greatest observed value of r (number of paclitaxel molecules bound to one albumin molecule) was 6.6.     

High affinity divalent cation binding to actin. Effect of low affinity salt binding

Monomeric actin labeled with the fluorescent probe N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-I-AEDANS-actin) displays a fast fluorescence intensity increase immediately upon addition of salt and then a slow fluorescence intensity change concurrent with Ca2+/Mg2+ exchange at the high affinity divalent cation binding site on actin. The fast change appears to reflect competitive binding of K+ at low affinity (nonspecific) sites and of Mg2+ or Ca2+ at low and intermediate affinity sites. Binding of cation at the low affinity sites (but apparently not at the intermediate affinity sites) results in an increase in k-Ca and k-Mg and thus a decrease in affinity for divalent cations at the high affinity site. The effect of Mg2+ on k-Ca is twice that of K+ for equal fractional saturations of the low affinity binding, and the effect of K+ and Mg2+ together on k-Ca reflects competitive binding at the low affinity sites. Thus the affinity and kinetics of divalent cation binding at the high affinity site of actin are significantly affected by concurrent cation binding at low affinity sites.     

High affinity binding of human interleukin 4 to cell lines

Purified human recombinant interleukin 4 (IL-4) was radio iodinated to high specific radioactivity without loss of biological activity. 125I-IL-4 bound specifically to the Burkitt lymphoma Jijoye cells and other cell lines. Jijoye cells showed a high affinity for 125I-IL-4 (Kd approximately equal to 7 10(-11) M) and displayed 1200-1400 specific receptors per cell at 4 degrees C or 37 degrees C. The equilibrium dissociation constant (Kd) corresponds to the IL-4 concentration which induces 50% maximal expression of the low affinity IgE receptor (Fc epsilon RL/CD23) on Jijoye cells. At 4 degrees C the rate constant of association K1 is 1.7 x 10(6) M-1 s-1 and the rate contant of dissociation k -1 is 1.3 x 10(-4) s-1 (t 1/2 = 91 min.) No human recombinant lymphokines other than IL-4 were able to compete for the binding of 125I-IL-4 to its receptor.     

High affinity binding of VIP to human lung cancer cell lines

The binding of ¹²⁵I-VIP to human lung cancer cell lines was investigated. Radiolabeled VIP bound to adenocarcinoma, squamous cell carcinoma, large cell carcinoma and small cell lung cancer (SCLC) cell lines. As SCLC cell line NCI-N592 bound radiolabeled VIP well, its binding was further characterized. ¹²⁵I-VIP bound to membranes in a specific and time dependent manner. ¹²⁵I-VIP bound with high (Kd=0.8 nM) and moderate affinity (Kd=66 nM) to two classes of sites. Pharmacology studies indicated that the order of peptide potency was VIP PHI > secretin > VIP^10–28. Because VIP receptors are present on human lung cancer cells, VIP may function as a regulatory peptide in lung cancer.     

High affinity binding of immunoglobulin G fragments to human polymorphonuclear leukocytes

Human neutrophil elastase splits IgG into Fc, Fabc, and Fab fragments. The Fc and Fabc fragments bind with high affinity (KD 2.1 and 2.5 nM respectively) to a small number of binding sites (1175 and 1370 sites/cell respectively) on untreated human polymorphonuclear leukocytes. Molecular mass determination of the binding site by crosslinking of Fc fragments to the neutrophils followed by SDS electrophoresis yields one band corresponding to a molecular mass of 67 kDa for the binding site. Incubation of neutrophils with rIFN-gamma (50 ng/ml, 18 h, 37 degrees C) enhances the expression of binding sites by about 6 fold to about 14,500 sites/cell, while the binding affinity and the molecular mass of the ligand receptor complex remain constant. By comparison with known affinities of leukocyte Fc receptors it is concluded that IgG fragments bind to the high affinity FcRI receptor of human neutrophils.     

High affinity Concanavalin A binding to sterol-depleted L cells.

Binding of Concanavalin A to mouse L cells which were grown in serum free, chemically defined medium and depleted of their membrane sterol by blocking their de novo sterol synthesis was investigated. Kinetic analysis of binding data implied positive cooperativity, with two different affinities for Con A, in both experimental and control cultures. The amount of Con A bound to the cell surface at saturation was approximately 0.5 picomoles per mg cellular protein in controls and approximately 1.0 picomoles per mg cellular protein in 25-hydroxycholesterol treated cultures (which had a reduced sterol concentration of up to 50% in their plasma membranes). This phenomenon was reversed when cholesterol or mevalonate was added to the inhibited cultures to compensate for their inability to synthesize sterol. Our findings indicate that lectin binding to specific glycoprotein receptors is influenced by membrane lipid composition.     

Specific binding of human alpha interferon to a high affinity cell surface binding site on bovine kidney cells

Virus-induced human alpha interferon (HuIFN-alpha) derived from Namalwa cells and purified to a specific activity of 2 X 10(8) units/mg of protein was radiolabeled with 125I-labeled Bolton and Hunter reagent to a specific activity of 4-12 microCi/micrograms of protein. The binding of this 125I-IFN to bovine kidney cells was examined at 4 degrees C. Scatchard analysis of the binding data indicate the presence of 650 binding sites/cell and binding of the ligand with an apparent Kd of 6 X 10(-11) M. Trypsin or acid treatment of cells to which 125I-IFN was bound resulted in the release of greater than or equal to 77% of the radioactivity, indicating a majority of radiolabeled material was bound to the cell surface. Antibodies against human leukocyte IFN but not antibodies against human fibroblast IFN inhibited the binding of radiolabeled IFN to the cells. The binding of 125I-IFN was not inhibited by a 75-fold molar excess of mouse IFN but was inhibited 30% by a 200-fold molar excess of human beta (fibroblast) IFN. These data are compatible with the Lower biological activities of these IFNs on bovine kidney cells. Several Escherichia coli derived HuIFN-alpha s inhibited the binding of the radiolabeled IFN to the same extent as native HuIFN-alpha s, but four fragments of HuIFN-alpha 1, an E. coli-derived 86 amino acid NH2-terminal fragment as well as 3 different synthetic carboxy-terminal fragments of 140, 56, or 46 amino acids did not inhibit binding.     

High affinity progesterone binding sites of human uterine microsomal membranes

Microsomal membranes sedimented at 40 000 g were prepared from human myometrium samples. The progesterone binding properties of microsomal suspensions were determined by incubating microsomes and [3H]progesterone at 4 degrees C. Dextran-coated charcoal was used for the separation of bound and free steroids. Membrane-associated progesterone binding sites of high affinity were identified in microsomes prepared from pregnant and nonpregnant uteri. The binding was saturable (Kd approximately 4 X 10(-9) M, concentration of binding sites 400-900 fmol/mg microsomal protein) and specific for natural progesterone. Of 21 steroids tested only 21-hydroxy-4-pregnene-3,20-dione, 17 alpha-hydroxyprogesterone and testosterone showed moderate competition against progesterone with relative affinities between 7.0-20.0% (R.A. of progesterone 100%). 5 alpha-Dihydroprogesterone and 5 alpha-dihydrotestosterone showed weak cross reaction (relative affinities 2.5 and 2.0%, respectively). Corticosteroids, estrogens and the 5 synthetic progestins tested showed only weak competition with relative affinities lower than 1.0%. These microsomal progesterone binding sites of high affinity and limited capacity resemble steroid hormone receptors but they are different from the soluble cytosolic progesterone receptor of human uterus in terms of steroid specificity. The physiological function of this microsomal progesterone receptor is unknown.     

High affinity thyroxine binding to purified rat liver plasma membranes.

Two sets of high-affinity thyroxine binding sites (K_D 0.39 ± 0.06 nM and 23 ± 5 nM) were detected on purified rat liver plasma membranes. Thyroxine is bound with high stereospecificity regarding iodine substituents and alanine side chain modifications of the molecule. Thyroxine binding is inhibited by -SH blocking agents and proteases. The highest affinity thyroxine binding site is also affected by phospholipase A and is distinct from triiodothyronine binding sites present in the membrane preparations; arguments are given for its plasmalemma origin.     

4,4'-Bis[8-(phenylamino)naphthalene-1-sulfonate] binding to human thrombins: a sensitive exo site fluorescent affinity probe

The binding of the fluorescent probe 4,4'-bis[8-(phenylamino)naphthalene-1-sulfonate] (bis-ANS) to human alpha- and gamma-thrombins was investigated. Bis-ANS binds in a 1:1 complex to both forms of the enzyme, with Kd = 14.8 +/- 2.2 microM and 5.8 +/- 1.0 microM for alpha- and gamma-thrombin, respectively, at pH 7.0 [25 mM tris(hydroxymethyl)aminomethane, 0.15 M NaC1]. Fluorescence changes upon complexation included a considerable (approximately 30-nm) blue shift in the fluorescence emission maximum as well as a dramatic increase in the fluorescence emission intensity: a 70-fold enhancement was observed with alpha-thrombin vs. a approximately 220-fold enhancement with gamma-thrombin. Proflavin was not displaced upon bis-ANS binding. The unknown thrombin effectors ATP, Ca(II)ATP, Co(III)ATP, phosphate, and pyrophosphate bound with enhancement of the fluorescence of the bis-ANS-alpha-thrombin complex. The two inhibitors benzamidine and p-chlorobenzylamine as well as heparin caused decreases in bis-ANS-thrombin fluorescence: valerylamidine had no effect on the fluorescence of the bis-ANS-thrombin complex. Kinetic measurements with two chromogenic substrates, S-2238 and S-2160, indicated that bis-ANS acts as a partial noncompetitive inhibitor of thrombin amidase activity. The kinetic evidence combined with the ligand binding results suggests that bis-ANS does not overlap the catalytic site. The fluorophore ANS complexed with equal affinity to both alpha- and gamma-thrombins (Kd = 24 +/- 4 microM); however, the gamma-thrombin-ANS complex emission at 470 nm was enhanced 26% more than that for the alpha form.     

High affinity binding of thrombin to platelets. Inhibition by tetranitromethane and heparin

[¹²⁵I] iodo-α-thrombin has been modified at the macromolecular substrate binding site in order to study the importance of this region in the platelet-thrombin interaction. Modification was effected by the nitration of tyrosine residues with tetranitromethane. This chemical modification abolished the ability of the enzyme to bind with a high affinity to the platelet surface but did not significantly alter low affinity binding. The presence of heparin was also found to inhibit high affinity binding. These results indicate that the high affinity binding site interacts with the fibrinogen binding region of the thrombin molecule and suggests that there are two distinct classes of binding sites for thrombin on the platelet membrane.     

High affinity GnRH binding to testicular membrane homogenates

The binding of GnRH to membrane homogenates from collagenase-dissociated normal rat interstitial cells is shown to be of high affinity and specific. The dissociation constant for the radioligand used, [¹²⁵I-Tyr⁵, D-Ala⁶, N^αMeLeu⁷, Pro⁹-NEt]-GnRH is 0.26 ± 0.02 nM and the number of binding sites is 17 fmoles/mg membrane homogenate. There is one GnRH antagonist whose affinity is greater for the pituitary than for the testis. This antagonist has the same affinity for testis homogenates as for ovarian homogenates.     

High affinity L-triiodothyronine binding to right-side-out and inside-out vesicles from rat and human erythrocyte membrane

3,5,3'-Triiodo-L-thyronine (L-T3)-binding sites from rat and human red cells were characterized as to their distribution between the two surfaces of the membrane. Analysis of L-T3 binding to sealed right-side-out and inside-out vesicles from erythrocyte membrane revealed that high affinity L-T3-binding sites are located on the external side in rat erythrocytes and on the internal side in human red cells. These results were further confirmed by preincubation of intact red cells with p-chloromercuribenzoate, a slowly permeant reagent that interacts reversibly with SH groups of proteins. Following this treatment only the SH groups of L-T3 sites from rat erythrocytes were found to be blocked. Scatchard analysis of the binding data for rat right-side-out and human inside-out vesicles showed high affinity sites with Kd values of 0.2 x 10(-10) and 2 x 10(-10) M, respectively. The results suggest that the orientation of L-T3-binding sites in the erythrocyte membrane is species-dependent.     

High binding affinity of electronegative LDL to human aortic proteoglycans depends on its aggregation level

Electronegative LDL [LDL(-)] is an atherogenic subfraction of plasma LDL that has increased apolipoprotein E (apoE) and apoC-III content, high density, and increased susceptibility to aggregation. These characteristics suggest that LDL(-) could bind to proteoglycans (PGs); therefore, our aim was to evaluate its affinity to PGs. Binding of LDL(-) and native LDL [LDL(+)] to human aortic PGs was determined by precipitation of LDL-glycosaminoglycan complexes, LDL incubation in PG-coated microtiter wells, and affinity chromatography on PG column. All methods showed that LDL(-) had higher binding affinity to PGs than did LDL(+). PG capacity to bind LDL(-) was increased approximately 4-fold compared with LDL(+) in precipitation and microtiter assays. Chromatography on PG column showed LDL(-) to consist of two subpopulations, one with higher and one with lower PG binding affinity than LDL(+). Unexpectedly, the lower PG affinity subpopulation had increased apoE and apoC-III content. In contrast, the high PG affinity subpopulation presented phospholipase C (PLC)-like activity and increased aggregation. These results suggest that PLC-like activity could alter LDL lipid composition, thereby promoting particle aggregation and binding to PGs. This propensity of a subpopulation of LDL(-) to bind to PGs could facilitate its retention in the extracellular matrix of arterial intima and contribute to atherosclerosis progression.     

High affinity binding sites for somatostatin to rat pituitary

Binding sites for somatostatin (SS) are described in rat pituitary membranes using either [¹²⁵I-Tyr¹¹]-SS-14 or [Leu⁸, D-Trp²², ¹²⁵I-Tyr²⁵]-SS-28 as radioligands; in each case saturable and high affinity binding sites with K_D's for SS of 1.09 and 0.95 nM respectively have been characterized. The binding capacity is 100 f mols/mg protein. The potencies of various SS analogs measured in the radioreceptor assay are in agreement with the potencies in a bioassay measuring inhibition of growth hormone release; in particular, SS-28 is slightly less potent than SS-14. A comparison of these data with those describing SS binding in brain and pancreas suggests that some pharmacological differences may exist between pituitary, brain and pancreas binding sites for SS.     

High affinity streptococcal binding to human fibronectin requires specific recognition of sequential F1 modules

Fibronectin (Fn) binding by the Streptococcus pyogenes protein SfbI has been shown to trigger integrin-dependent internalization of this pathogen by human epithelial and endothelial cells. Here, using nuclear magnetic resonance spectroscopy and isothermal titration calorimetry in a dissection approach, the basis for the specificity and high affinity of the interaction between the N-terminal domain of Fn and SfbI is revealed. Each of the five Fn type 1 modules is directly involved in the interaction and is recognized by short consecutive motifs within the repeat region of SfbI. Crucially, these motifs must be combined in the correct order to form a high affinity ligand for the N-terminal domain of Fn.