Exclusive Binding Sites Research Articles

A grazing incidence surface X-ray absorption fine structure (GIXAFS) study of alkanethiols adsorbed on Au, Ag, and Cu

Self-assembled monolayers of n-alkanethiols, CH 3–(CH 2) x –SH, on Au, Ag, and Cu have been studied with GIXAFS at the sulfur K-edge. For both pentanethiol and decanethiol monolayers on Ag and Cu, the three-fold hollow site is found to be the most probable sulfur binding site. However, observations for octadecanethiol indicate that the three-fold hollow site is not the exclusive binding site. In addition, the possible existence of disulfide bonds on the metal surface (adsorbed dialkyldisulfides) is not supported by the data. Preliminary results from monolayers on Au are also reported.

Potentiometric and spectroscopic studies of transition metal complexes of bis(imidazolyl) and bis(pyridyl) derivatives of amino acids

Copper(II) and zinc(II) complexes of H-Gly-BIMA (glycinamido-bis(imidazol-2-yl)methane) and copper(II), nickel(II), cobalt(II) and zinc(II) complexes of Prodipa (prolineamido-bis(pyridin-2-yl)methane) were studied by potentiometry, electron paramagnetic resonance and visible spectroscopy. The data revealed that both ligands are ambidentate. Imidazole and pyridine nitrogens of H-Gly-BIMA and Prodipa, respectively, are the exclusive binding sites in acidic media, but the metal binding ability of the bis(imidazolyl) residue is much higher than that of the bis(pyridyl) ligand. The coordination of the amino, amide and imidazole/pyridine nitrogens was concluded around physiological pH, which resulted in the formation of an imidazole bridged dimeric species in the case of H-Gly-BIMA.

Unusual potentiation by vinca alkaloids of the cytostatic and cytocidal action of methyl-3,5-diiodo-4-(4'-methoxyphenoxy) benzoate (DIME) and its nonhydrolyzable ethanone analog (DIPE) on MDA-MB-231 human mammary cancer cells.

Drug interaction between DIME or DIPE ¿1-[3, 5-diiodo-4-(4'-methoxyphenoxy)-phenyl]-ethanone¿ with vincristine and vinblastine on the growth rate of MDA-MB-231 human mammary cancer cells was determined by the median effect kinetic method. Mutually exclusive cellular binding sites were identified kinetically and isobologram analyses showed potentiation. The combind effect of 0.75 MICROM DIME and 2 nM vincristine demonstrated a nearly type of mutual activation. It was shown that the nonhydrolyzable DIME derivative DIPE is equivalent to DIME, but because of its biological stability is a preferred drug candidate. Vinblastine-DIME cooperative action is similar to that of vincristine-DIME (or DIPE). Activation of caspase 3 by both DIME and vincristine is greatly potentiated when both drugs are added simultaneously in a given proportion. We propose that following a primary binding of DIME and vinca alkaloids to microtubules, an as yet unrecognized mutual activation of caspase 3 apoptotic path is initiated, explaining DNA fragmentation and cell death. A subpopulation of cancer cells, capable of slow growth at 1.5 microM DIME was identified. This cell type was also killed by the DIME-vincristine drug combination.

Effects of surface imperfections on the binding of CH 3OH and H 2O on FeS 2(100): using adsorbed Xe as a probe of mineral surface structure

Studies are presented that investigate the adsorption and binding of CH 3OH and H 2O on the atomically clean (100) crystallographic plane of pyrite, FeS 2. Temperature programmed desorption suggests that both reactants adsorb molecularly at 90 K and desorb thermally between 170 and 400 K depending on the surface coverage. Photoemission of adsorbed xenon (PAX) suggests that the surface of pyrite is heterogeneous and contains a significant fraction of defect sites that are believed to be, at least in part, anion vacancy or sulfur-deficient sites. An upper limit of 0.2 is proposed for the fraction of surface sites that are defects on FeS 2(100). PAX indicates that these defect sites at low adsorbate coverage serve as the exclusive binding sites for H 2O and CH 3OH adsorbate. We speculate, on the basis of our ability to interpret PAX data for pyrite, that PAX may be of use for understanding the effect of short range order on adsorbate binding on other complex mineral surfaces. On the basis of high resolution electron energy loss spectroscopy, it is found that some dissociation of the adsorbate occurs on the pyrite. Vibrational data obtained with this technique suggests that FeO species result from the adsorbate decomposition. After saturation of the defect sites, further molecular adsorption is accommodated on the less reactive surface that we postulate is largely disulfide, the characteristic structural group of pyrite.

Binding of nickel(II) and copper(II) to the N-terminal sequence of human protamine HP2.

A potentiometric and spectroscopic (UV/vis and CD) study of Cu(II) and Ni(II) binding to the N-terminal pentadecapeptide of human protamine HP2 (HP2(1-15)) was performed. The results indicate that the N-terminal tripeptide motif Arg-Thr-His is the exclusive binding site for both metal ions at a metal to HP2(1-15) molar ratio not higher than 1. The very high value of protonation-corrected stability constant (log *K) for Ni(II)-HP2(1-15) complex, -19.29, indicates that HP2 has the potential to sequester Ni(II) from other peptide and protein carriers, including albumin. The same is likely for Cu(II) (log *K = -13.13). The CD spectra of Cu(II) and Ni(II) complexes of HP2(1-15) indicate that the N-terminal metal binding affects the overall conformation of the peptide that, in turn, may alter interaction of HP2 with DNA. These results imply HP2 as a likely target for the toxic metals Ni(II) and Cu(II).

Novel active site in Escherichia coli fructose 1,6-bisphosphate aldolase.

The molecular architecture of the Class II E. coli fructose 1,6-bisphosphate aldolase dimer was determined to 1.6 A resolution. The subunit fold corresponds to a singly wound alpha/beta-barrel with an active site located on the beta-barrel carboxyl side of each subunit. In each subunit there are two mutually exclusive zinc metal ion binding sites, 3.2 A apart; the exclusivity is mediated by a conformational transition involving side-chain rotations by chelating histidine residues. A binding site for K+ and NH4+ activators was found near the beta-barrel centre. Although Class I and Class II aldolases catalyse identical reactions, their active sites do not share common amino acid residues, are structurally dissimilar, and from sequence comparisons appear to be evolutionary distinct.

Molecular characterization of protein Sir, a streptococcal cell surface protein that binds both immunoglobulin A and immunoglobulin G.

Cell surface proteins that bind to the Fc part of immunoglobulin (Ig) A and/or IgG are expressed by many strains of the group A Streptococcus, an important human pathogen. Two extensively characterized proteins in this group of molecules are protein Arp that preferentially binds IgA and protein H that binds IgG. In addition, recent work has shown that many group A streptococcal strains express a novel type of Fc-binding protein, designated protein Sir, that binds both IgA and IgG. Protein Sir22, the molecule expressed by a strain of serotype M22, has now been purified and characterized after expression of the cloned gene in Escherichia coli. Dot-blot analysis with a large number of purified monoclonal Igs showed that protein Sir22 reacted with 19 out of 20 IgA proteins and with 19 out of 24 IgG proteins. The affinity constants for the reactions between protein Sir22 and Ig were determined to be 7.0 x 10(8) M-1 for serum IgA, 2.4 x 10(8 M-1 for secretory IgA, and 7.8 x 10(8) M-1 for IgG. Inhibition experiments showed that the bindings of IgA and IgG to protein Sir22 were mutually exclusive, indicating shared or contiguous binding sites. Analysis of the sequence of the sir22 gene indicated a gene product with 365 amino acid residues, including a 41-residue signal peptide. The processed form of the protein, 324 residues, has a calculated M(r) of 37,168. Deletion analysis of the sir22 gene showed that a 156-residue NH2-terminal fragment of protein Sir22 retained the ability to bind both IgA and IgG. The overall organization of protein Sir22 is similar to that of the IgA-binding protein Arp and the IgG-binding protein H. All three of these proteins are members of the M protein family and have a central repeat region of the C type.

Activity of the rat liver-specific aldolase B promoter is restrained by HNF3.

Although it contains binding sites for HNF1, NFY and C/EBP/DBP, the proximal promoter of the aldolase B gene is surprisingly weak when tested by transient transfection in differentiated hepatoma cells. This low activity could be due to overlapping between HNF1 and HNF3 binding sites in element PAB, from -127 to -103 bp with respect to the cap site. Replacement of the PAB region by a consensus HNF1 binding site unable to bind HNF3, results in a 30 fold activation of the promoter, in accordance with the hypothesis that activity of the wild-type promoter is normally restrained by HNF3 binding to PAB competitively with HNF1. Consistently, transactivation of the wild-type promoter by excess HNF1 is very high, most likely due to the displacement of HNF3, while the construct with the exclusive HNF1 binding site is weakly transactivated by HNF1. The inhibitory effect of HNF3 on HNF1-dependent transactivation is clearly due to competition between these two factors for binding to mutually exclusive, overlapping sites; indeed, when HNF1 and HNF3 sites are contiguous and not overlapping, the resulting promoter is as active as the one containing an exclusive HNF1 binding site. A construct in which PAB has been replaced by an exclusive HNF3 binding site is weakly expressed and is insensitive to HNF3 hyperexpression. DBP-dependent transactivation, finally, is independent of the nature of the element present in the PAB region.

A new class of potent thrombin inhibitors that incorporates a scissile pseudopeptide bond

A synthetic hirudin peptide analog corresponding to N′-acetyl [D-Phe 45, ArgΨ(COCH 2) 47, Gly 48]desulfo hirudin 55–65 (P79) was synthesized. Comparative kinetic studies showed that while recombinant hirudin (HV2) is a slow-tight binding inhibitor, P79 behaves as a classical competitive inhibitor of human α-thrombin ( K i=3.7±0.3 × 10 −10 M) and bovine α-thrombin (1.8±0.7 × 10 −9 M). P79 showed saturable inhibition of plasma APTT. The P 1 subsite of P79 is isosteric with the glycine residue of the natural thrombin substrate fibrinogen, but is proteolytically stable due to the incorporation of a ketomethylene pseudopeptide bond. The model active site-directed tripeptide [D-Phe-Pro-ArgΨ(COCH 2)CH 3COOCH 3, P79L] corresponding to the amino terminal of P79 also binds competitively to the active site of α-thrombin and inhibited the proteolysis of a tripeptidyl substrate with a K 1=17.9±2.1 μM (human) and 10.3±3.6 μM (bovine) α-thrombin. NMR experiments indicated that P79L and the corresponding amino terminal residues of P79 occcupy a mutually exclusive binding site on bovine α-thrombin while the carboxyl terminal tail of the latter adopts a similar bound conformation as the fragment hirudin??? which is known to interact with the ‘anion’ exosite. Taken together these results provide conclusive evidence that the high antithrombin activity of N???-acetyl[D-Phe 45, ArgΨ(COCH 2) 47, Gly 48]desulfo hirudin 45–65 stems from the concurrent interaction with the catalytic site and the putative ‘anion’ exosite through its respective NH 2- and COOH-terminal recognition sites.

Anion binding to the chloride pump, halorhodopsin, and its implications for the transport mechanism

The light-driven chloride pump, halorhodopsin, binds and transports chloride across the membrane, and to a lesser extent nitrate. Binding and transport kinetics, and resonance Raman spectra of the retinal Schiff base, with these anions suggest the existence of two mutually exclusive binding sites. One of these may be the uptake site, and the other the release site during the transport. Plausible locations can be suggested for these sites, because halorhodopsin is a small protein with few buried positively charged residues, and the primary structure of a second pigment with similar function has recently become available for comparison.

INTERACTION OF MONOSACCHARIDES AND RELATED COMPOUNDS WIT OXOCATIONS OF MO(VI), W(VI) AND U(VI)STUDIED BY NMR SPECTROSCOPY

Abstract Proton, 13C and 31P nuclear magnetic resonance spectroscopy has been used to study the complexation of M(VI), W(VI) and U(VI) oxocations with various aldoses, cyclic polyols and ribose-5-phosphate in aqueous solution. The aldoses D-mannose, D-lyxose and D-ribose from tridentate complexes with Mo(VI) and W(VI) at pH ≃ 5, via the 1,2,3-hydroxyl groups, which are cis to each other in these sugars. Other aldoe, like D-arabinose, D-glucos, D-xylose and D-galactose from weaker bidentate complexs with those ions because they can onl use the 1 and 3-cis hydroxyl groups in metal binding. These bidentate interactions also take place in the binding of U(VI) to D-ribose, at pH ≃ 110. However, sugars having 1,3,5-hydroxyl groups in the cis position do not from stable chelates with these oxocations, possibly due to steric crowding. In the case of ribose-5-phosphate, the phosphate group is the exclusive binding site for the three oxocations. except for U(VI) at ver basic pH(pH > 10), where the hdroxyl groups al...

C-reactive protein antigenicity on the surface of human lymphocytes.

We reported previously that C-reactive protein (CRP), when complexed to a multivalent binding specificity, can bind to a subset of lymphocytes that bear the IgG Fc receptor. Recently we showed that anti-CRP plus complement depletes natural killer function. We now show the detection of CRP on the surface (S-CRP) of a small percentage of PBL and on a minor population of phagocytic mononuclear cells. S-CRP is not detectable by direct immunofluorescence, but is readily discernible by using more sensitive techniques such as biotinylated anti-CRP with fluorescent avidin or indirect immunofluorescence with monoclonal anti-CRP. S-CRP is expressed in the absence of calcium, whereas the binding of complexed CRP is calcium dependent. S-CRP does not appear to be the exclusive binding site for CRP-CPS because anti-CRP does not block the binding of complexed CRP. Both the binding site for complexed CRP and S-CRP can be capped off; however, the kinetics of capping of these two surface molecules is different. Cells binding complexed CRP are OKT11-, whereas a significant number of PBL bearing S-CRP are OKT11+. Therefore cells bearing S-CRP appear to be a separate population from those that bind complexed CRP. These studies establish the presence of S-CRP on a subpopulation of lymphocytes, define conditions required for its detection, and speak to certain of the relationships between S-CRP and the binding site of CRP-CPS complexes.

Biochemical characterization of the amiloride-sensitive Na+/H+ antiport in Chinese hamster lung fibroblasts.

Net H+ fluxes across the plasma membrane of Chinese hamster lung fibroblasts (CC139) were monitored by pH-stat titration. Na+-depleted cells release H+ upon addition of Na+. Conversely Na+- or Li+-loaded cells take up H+ from the medium when shifted to a Na+,Li+-free medium. This reversible Na+ (or Li+)-dependent H+ flux is inhibited by amiloride and does not occur in digitonin-permeabilized cells. A similar Na+/H+ exchanger was identified in vascular smooth muscle cells, corneal and aortic endothelial cells, lens epithelial cells of bovine origin, and human platelets. Kinetic studies carried out with CC139 cells indicate the following properties: 1) half-saturation of the system is observed at pH = 7.8, in the absence of Na+; 2) external Na+ stimulates H+ release and inhibits H+ uptake in a competitive manner (Ki = 2-3 mM); 3) amiloride is a competitive inhibitor for Na+ (Ki congruent to 1 microM) and a noncompetitive inhibitor for H+; 4) a coupling ratio of 1.3 +/- 0.3 for the H+/Li+ exchange suggests a stoichiometry of 1:1. We conclude that CC139 cells possess in their plasma membrane a reversible, electroneutral, and amiloride-sensitive Na+/H+ antiporter, with two distinct and mutually exclusive binding sites for Na+ and H+. The rapid stimulation of the Na+/H+ antiporter in G0/G1-arrested CC139 cells upon addition of growth factors, together with the fact that intracellular H+ concentration is, under physiological conditions, around the apparent K0.5 of the system, strongly suggests a key role of this antiport in pHi regulation and mitogen action.

Urinary-proteinase inhibitor in premature infants and its comparison with amniotic-fluid acid-stable proteinase inhibitor

Urinary-antitryptic activity was found to be higher in neonates (2.25 ± 0.75 units, mean ± SD) than in infants (0.65 ± 0.50, 2–12 months old) or in children of 1–10 years old (0.63 ± 0.53). It was indicated in follow-up studies that a gradual decline of activity occurred in the first month of life, and stabilized by 30 days. The antitryptic activity in urine was higher in premature infants (5.97 ± 0.98) than in fullterm ones. The inhibitor was purified to homogeneity from the urine of premature infants by chromatography on DEAE-cellulose, concanavalin A-Sepharose 4B, and Sephadex G-100. The inhibitor which is a glycoprotein was found to have a molecular weight of 33,000 based upon SDS electrophoresis. The purified inhibitor appears to be made up of two identical polypeptide chains as suggested by electrophoresis in the presence of mercaptoethanol. The inhibitor was stable to exposure of a wide range of pH (1.0–11.8). Modification of the guanidino groups resulted in the loss of the antitryptic activity but not the antichymotryptic activity. The inhibitor was found to be similar to the proteinase inhibitor isolated from amniotic fluid (5) in terms of its molecular size, dimeric nature, carbohydrate content, and heat and acid stability and lability to pepsin treatment. However, the urinary inhibitor did not have mutually exclusive binding sites for trypsin and chymotrypsin, unlike the factor from amniotic fluid; the former inhibitor was more active on trypsin than on chymotrypsin.

A comparison of the interactions of the mitogenic and nonmitogenic lima bean lectins with human lymphocytes.

Binding and mitogenic characteristics of the 2 Phaseolus lunatus (lima bean) lectins have been examined with human peripheral blood lymphocytes. Chemical cross-linking of the nonmitogenic lima bean lectin produced a species that stimulated human lymphocytes as well as or better than the mitogenic lima bean lectin, which is a T lymphocyte mitogen with requirement for monocyte participation. The maximal stimulation and the dose response to the cross-linked lima bean lectin did not significantly differ from that observed with the mitogenic lectin. We have used fluorescein-labeled lima bean lectins to show that both lectins share mutually exclusive binding sites on lymphocytes. Our results strongly support the concept that multiple valence of lectins is important in inducing mitogenesis. Both the mitogenic and nonmitogenic lectins demonstrated selective binding by labeling only 70% od human peripheral blood lymphocytes. The fraction not binding lectin is a population of T lymphocytes. Competitive binding studies with the lima bean lectins and other N-acetyl-D-galactosamine-specific lectins suggest that the cell surface receptors for these various lectins may be quite different. We have also studied the response of human lymphocytes to the lima bean lectins and soybean agglutinin after neuraminidase treatment. As previously demonstrated (22), neuraminidase treatment of the cells drastically altered the binding and mitogenic response to soybean agglutinin. Lima bean lectin binding to the treated cells was also markedly increased, but the mitogenic response was essentially unaffected.

Zinc, cadmium, and iron interactions during intestinal absorption in iron-deficient mice.

Zinc absorption from a test dose of (65Zn) zinc chloride was increased in mice with a high capacity to absorb iron induced by a low-iron diet. When radiolabelled zinc chloride in concentrations varying from 0.025 to 0.30 mM was perfused through open-ended duodenal loops of mice fed this diet, the proportion of zinc taken up from the lumen and transferred to the body was greater from lower than from higher doses. The addition of iron to the perfusate inhibited zinc uptake and transfer, and zinc had a similar effect on iron absorption. Cadmium, a potent inhibitor of iron uptake in mice fed a low-iron diet, impaired zinc uptake under these dietary conditions. These results suggest that in dietary-induced iron deficiency there are analogous mucosal binding sites for the uptake of iron and zinc. There also appear to be mutually exclusive binding sites for the absorption of these metals: radiolabelled iron absorption from an intragastric test dose was enhanced in mice with a high capacity to absorb iron produced by bleeding, whereas the absorption of zinc was not increased.