Total Number Of Binding Sites Research Articles

Insights into the role of extracellular DNA in heavy metal adsorption

Extracellular polymeric substances (EPS) participate in heavy metal adsorption in the aquatic environments. Extracellular DNA (eDNA) is an essential component of EPS, but its involvement in metal binding remains ambiguous. Herein, the role of eDNA in Cd(II) and Ni(II) adsorption was described using a combination of semi-quantitative and qualitative approaches. EPS were extracted from Burkholderia sp. MBR-1 and eDNA accounted for 6.9% of the total mass of EPS. The eDNA in the extracted EPS was digested using the DNase II to prepare an eDNA-free EPS sample. Potentiometric titration unveiled that the number of total binding sites of the eDNA-free EPS was 19% lower than the untreated EPS. The Cd(II) and Ni(II) adsorption capacity of the eDNA-free EPS was lower than the untreated EPS at the pH range of 4–7. At pH 7, the results of batch adsorption experiments showed that removing eDNA from EPS resulted in declines of 12.6% and 15.7% in the adsorption capacities for Cd(II) and Ni(II), respectively. Furthermore, Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy unraveled that the phosphoryl groups and purines of eDNA are responsible for Cd(II) and Ni(II) complexation. The results demonstrated that eDNA plays an essential role in heavy metal adsorption.

Exocytic Trafficking Is Required for Nicotine-induced Up-regulation of α4β2 Nicotinic Acetylcholine Receptors

The primary target for nicotine in the brain is the neuronal nicotinic acetylcholine receptor (nAChR). It has been well documented that nAChRs respond to chronic nicotine exposure by up-regulation of receptor numbers, which may underlie some aspects of nicotine addiction. In order to investigate the mechanism of nicotine-induced nAChR up-regulation, we have developed a cell culture system to assess membrane trafficking and nicotine-induced up-regulation of surface-expressed alpha(4)beta(2) nAChRs. Previous reports have implicated stabilization of the nAChRs at the plasma membrane as the potential mechanism of up-regulation. We have found that whereas nicotine exposure results in up-regulation of surface receptors in our system, it does not alter surface receptor internalization from the plasma membrane, postendocytic trafficking, or lysosomal degradation. Instead, we find that transport of nAChRs through the secretory pathway to the plasma membrane is required for nicotine-induced up-regulation of surface receptors. Therefore, nicotine appears to regulate surface receptor levels at a step prior to initial insertion in the plasma membrane rather than by altering their endocytic trafficking or degradation rates as had been previously suggested.

Melanin counter act puromycin-induced inhibition of collagen and DNA biosynthesis in human skin fibroblasts

Puromycin is an experimental anti-tumor antibiotic acting through inhibition of protein synthesis. Because of its untoward side effects (as inner ear and renal lesions) the antibiotic was not approved for clinical trials. The mechanism underlying the organ specificity of the side effect is not understood. In view of the fact that a number of drugs form with melanin complexes that affect their pharmacological activity, we determined whether puromycin interacts with melanin and how this process affects biosynthesis of collagen in cultured human skin fibroblasts. Our results indicate that puromycin forms complexes with melanin. The amount of puromycin bound to melanin increases with increase of initial drug concentration. The Scatchard plot analysis of the drug binding to melanin has shown that at least two classes of independent binding sites are implicated in the puromycin-melanin complex formation: one class of strong binding sites with the association constant K 1 = 1.84 × 10 6 M −1, and the second class of weak binding sites with the association constant K 2 = 5.26 × 10 3 M −1. The number of total binding sites were n 1 = 0.1260 and n 2 = 0.2861 μmol puromycin per 1 mg melanin. We found that puromycin induced inhibition of collagen and DNA biosynthesis (IC 50 ∼ 2 μM). Melanin at 100 μg/ml produced about 20% inhibition of DNA synthesis, but it had no effect on collagen biosynthesis in cultured fibroblasts. However, the addition of melanin (100 μg/ml) to puromycin - treated cells (2 μM) abolished the inhibitory action of puromycin on collagen and DNA biosynthesis. We have suggested that IGF-I receptor expression, involved in collagen metabolism, may be one of the targets for puromycin - induced inhibition of collagen biosynthesis. It was found that melanin abolished puromycin induced decrease in the expression of IGF-I receptor as well MAP kinases expression: ERK1 and ERK2 as shown by Western immunoblot analysis. These data suggest that tissue specific pharmacological activity of puromycin may depend on the melanin abundance in tissues.

Melanin potentiates gentamicin-induced inhibition of collagen biosynthesis in human skin fibroblasts

One of the recognized side effects of gentamicin is ototoxicity. The mechanism underlying the organ specificity of this side effect of gentamicin has not been fully established. In view of the fact that a number of pharmacologic agents are known to form complexes with melanin and melanin is an abundant constituent of the inner ear tissues, we determined whether gentamicin interacts with melanin and how this process affects the biosynthesis of collagen in cultured human skin fibroblasts. Our results indicate that gentamicin forms complexes with melanin. The amount of gentamicin bound to melanin increases with increasing of initial drug concentration. The Scatchard plot analysis of drug binding to melanin showed that at least two classes of independent binding sites are implicated in gentamicin–melanin complex formation: one class with an association constant K 1∼4×10 3 M −1, and the second class with an association constant K 2∼3×10 2 M −1. The number of total binding sites ( n 1+ n 2) was calculated as about 1.36 μmol gentamicin per 1 mg melanin. We have suggested that prolidase, an enzyme involved in collagen metabolism, may be one of the targets for gentamicin-induced inhibition of collagen biosynthesis. We found that gentamicin-induced inhibition of prolidase activity (IC 50∼100 μM) and collagen biosynthesis (IC 50∼100 μM). At this concentration of gentamicin, DNA biosynthesis in human skin fibroblasts was inhibited only by about 30%. Melanin at 100 μg/ml produced about 25% inhibition of DNA synthesis and about 30% inhibition of prolidase activity, but it had no effect on collagen biosynthesis in cultured fibroblasts. However, the addition of melanin (100 μg/ml) to gentamicin-treated cells (100 μM) augmented the inhibitory action of gentamicin on collagen and DNA biosynthesis and partially reversed its inhibitory effect on prolidase activity. A melanin-induced augmentation of the inhibitory effects of gentamicin on collagen and DNA biosynthesis may explain the mechanism for the organ specificity of gentamicin-induced hearing loss in patients administered this drug.

Selective alterations in binding kinetic parameters and allosteric regulation of N-methyl-D-aspartate receptors after prolonged seizures in the developing rat brain.

Among glutamate receptor subtypes, the N-methyl-D-aspartate (NMDA) receptor plays a key role in brain development and cognitive processes, and mediates excitotoxic injury. To test the hypothesis that prolonged seizures may affect NMDA receptor characteristics in the developing brain, a 30-min episode of generalized seizures was induced in rats at 5, 10, 15 and 25 d of age by i.p. administrations of bicuculline, NMDA receptors were analyzed using specific binding of [3H]-labeled (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]-cycloheptene-5,10-imin e maleate (MK-801) in brain membrane preparations, and allosteric regulation was studied by addition of glutamate (10 microM) and glycine (10 microM). In control pups, total number of binding sites increased between 5 and 25 d, Bmax values varying from 1032 +/- 93 to 2311 +/- 449 fmol/mg protein, whereas receptor affinity decreased with age, the affinity constant (Kd) changing from 20.9 +/- 2.0 to 29.1 +/- 2.0 nM. Activation of NMDA receptors by glutamate and glycine led to age-dependent decreases in Kd values, from 30% at 5 d to 72% at 25 d. Seizures altered receptor density only at 5 d (by 40%). Receptor affinity was increased after seizures at 5, 15 and 25 d (from 12 to 60%). The capacity of receptor activation by glutamate and glycine was significantly reduced by seizures at 5 d. There was no change either in density nor affinity of receptors at 10 d. Therefore, as previously shown for central adenosine and benzodiazepine receptors, sustained seizures are able to alter the characteristics of NMDA receptors in a specific way depending on the maturational stage, suggesting developmental changes in the mechanisms of brain response to seizures.

Effects of aging on pituitary growth hormone-releasing factor receptor binding sites: in vitro mimicry by guanyl nucleotides and reducing agents

We have investigated the effect of 5′-guanylylimidodiphosphate (Gpp(NH)p) and two disulfide bond reducing agents, reduced glutathione (GSH) and dithiothreitol (DTT), on the modulation of [ 125I-Tyr 10]hGRF(1–44)NH 2 binding to GRF receptor binding sites, in pituitaries of young and aging rats. In pituitaries from 2-month-old rats, Gpp(NH)p (0.1–1.0 mM), GSH and DTT (1–50 mM) exhibited a partial but concentration-dependent inhibitory effect on GRF specific binding. These effects were associated with a conversion of the high affinity GRF binding sites to lower affinity sites and to a reduction of the apparent number of total binding sites (high and low). No potentiation of these effects was observed when Gpp(NH)p (1 mM) and DTT (1 mM) were combined. In pituitaries from 14-month-old rats, Gpp(NH)p (1 mM) was capable of modulating GRF binding parameters in a similar fashion to that in pituitaries from 2-month-old rats. In pituitaries from 18-month-old rats, the high affinity GRF binding sites were already blunted and neither Gpp(NH)p nor Gpp(NH)p plus DTT significantly altered GRF binding parameters. In addition, in 20-month-old rats, the affinity of hGRF(1–29)NH 2 and that of the full antagonist N α-Ac-[ d-Arg 2, Ala 15]rGRF(1–29)NH 2 were respectively decreased 9.3- and 9.9-fold. Our results suggest that in aging, alterations of GRF receptor binding sites could involve disulfide bond reduction or other structural modifications leading to conformational changes, similar to those induced by GSH or DTT. Such structural changes may prevent an efficient coupling of the GRF receptor with its ligands and G-protein, leading to a loss of somatotroph responsiveness.

Selective loss of binding sites for the iodinated alpha-neurotoxin I from Naja mossambica mossambica venom upon enzymatic deglycosylation of Torpedo electric organ membranes.

Removal of asparagine-linked carbohydrate chains from Torpedo marmorata electric organ membranes was found to inhibit the binding of the iodinated alpha-neurotoxin I from Naja mossambica mossambica snake venom to its receptor. Optimal deglycosylation of membranes by endoglycosidase F resulted in a 55% inhibition of alpha-neurotoxin-I-saturable binding. Under these conditions, up to 70% of concanavalin A binding was also lost, indicating an efficient removal of mannose-rich carbohydrate chains. Saturation binding experiments at equilibrium on membranes incubated in the absence of endoglycosidase F indicated, when analyzed by Scatchard plots, the presence of two classes of high-affinity binding sites for alpha-neurotoxin I (kd = 9 pM and 68 pM respectively) with capacities of 24 and 14 pmol/mg membrane proteins, respectively. After endoglycosidase F treatment, only the former class of binding sites (Kd = 11 pM) was recovered together with a 45% reduction in the number of total binding sites. Dissociation experiments further confirmed the presence of two types of toxin-receptor complexes in control membranes and the selective loss of the rapidly dissociating component upon deglycosylation. The binding of alpha-neurotoxin I to its receptor, deglycosylated or not, was totally inhibited by carbamoylcholine, d-tubocurarine or alpha-bungarotoxin. These findings show that the neurotoxin binding sites present on the acetylcholine receptor can be discriminated on the basis of their differential susceptibility to the removal of asparagine-linked carbohydrate chains.

Calcium binding by spinach stromal proteins

Calcium binding to spinach (Spinacia oleracea L.) stromal proteins was examined by dual-wavelength spectrophotometry using the metallochromic indicator tetramethylmurexide. The data are consistent with the existence of at least two, probably independent, classes of binding sites. The total number of binding sites varied between 90-155 nmol·mg(-1) protein with "average" binding constants of 1.1-2.7·mM(-1). Both Mg(2+) and La(3+) inhibited calcium binding competitively, with "average" inhibitor constants of 0.26·mM(-1) and 39.4·mM(-1), respectively; an increase in the potassium concentration up to 50 mM had no effect. In a typical experiment a decrease in pH (7.8 to 7.1) resulted in a decrease in the total number of calcium binding sites from 90 to 59 nmol·mg(-1) protein, but in an increase of the "average" affinity from 2.7 to 4.5·mM(-1). Calculations, using these data and those of Gross and Hess (1974, Biochim. Biophys. Acta 339, 334-346) for binding site I of washed thylakoid membranes, showed that the free-Ca(2+) concentration in the stroma under dark conditions, pH 7.1, is higher than under light conditions, pH 7.8. The physiological relevance of the observed calcium binding by stromal proteins is discussed.

Binding of 125I-insulin to the human histiocytic lymphoma cell line U-937: Effect of differentiation with retinoic acid

The human histiocytic lymphoma line U-937 consists of cells having characters of immature monocytes. We have demonstrated that these cells posses highly specific insulin receptors with binding properties similar to that found for mature human blood monocytes. 125I-insulin binding increased progressively with time to reach a maximum at 90 min at 22°C and was proportional to the number of cells in the incubation medium. Insulin degradation as assessed by TCA precipitation was negligible. Scatchard analysis of the binding data was curvilinear and the total number of insulin binding sites was around 13,500. The average affinity profile gave an ‘unoccupied site’ affinity constant of 1.34 nM −1. When the U-937 cells were induced to differentiate into morphologically and functionally monocyte-like cells, after incubation with retinoic acid, the total number of binding sites decreased significantly with no change in the affinity of the hormone for its receptor.

Insulin receptors in AH-66 ascites hepatoma cells and liver of tumor-bearing rats.

Properties of insulin receptor on plasma membranes isolated from AH-66 ascites hepatoma cells and liver of tumor-bearing rats were studied. Specific binding (total binding minus nonspecific binding) of 125-I-labeled insulin to plasma membranes from AH-66 tumor cells and liver of normal and tumor-bearing rats were 33, 31, and 16% of a fixed amount of labeled insulin (1 x 10(5) cpm) added to each membrane preparation, respectively. Using Nisonoff plot, these membranes were found to possess at least two types of insulin receptors with a high affinity-low capacity and a low affinity-high capacity, as has been shown in normal liver. Total number of binding sites (high affinity plus low affinity sites, 8.4 x 10(-12) mol/mg protein) in hepatoma cells was more than that (7.0 x 10(-12) mol/mg protein) in normal rat liver. However, kinetic constants of binding in receptors of two types on tumor cells and tumor-bearing rat liver were similar to those of membrane receptors from normal rat liver. Insulin receptors of the hepatoma cells were considered to be highly specific for insulin from the results of competition with other peptide hormones. Inhibition of insulin binding with the tumor cell membranes by concanavalin-A, a competitive inhibitor for insulin receptor sites, did not differ very greatly from that of normal liver.

Interaction of a fluorescent reagent, fluorescein mercuric acetate, with nucleic acids

Fluorescein mercuric acetate (fluorescein Hg Ac), which is a fluorescent thiol reagent, was shown to bind to various nucleic acids by measuring the changes in its absorption and fluorescence properties. Up to a critical concentration of free fluorescein Hg Ac (1 · 10 −7 M for calf thymus DNA, with 42% GC, and 2 · 10 −7 M for Micrococcus lysodeikticus DNA, with 72% GC) this reagent appears to bind selectively to single-stranded sections in DNA. Above this critical concentration, cooperative binding to double helical DNA occurs, and denatured DNA is obtained after removal of bound fluorescein Hg Ac by dialysis against 1 M KCl. These facts indicate that fluorescein Hg Ac causes the denaturation of double helical DNA prior to binding as has been shown in the case of methylmercuric hydroxide. The binding of fluorescein Hg Ac to DNA is much stronger than that of methylmercuric hydroxide. The number of total binding sites for fluorescein Hg Ac is close to the number of base pairs for both calf thymus DNA and M. lysodeikticus DNA. Furthermore, it was shown that fluorescein Hg Ac binds to thymidine, deoxyguanosine, poly(U) and poly(G). Since fluorescence quenching of fluorescein Hg Ac accompanies its complex formation with DNA and the affinity is markedly high as indicated by the association constant of 6.8 · 10 7 M −1 for single-stranded calf thymus DNA, fluorescein Hg Ac can be used for the structural studies of small amounts of nucleic acids.

Estradiol receptors in the pituitary and anterior hypothalamus of the rat: Measurement by agar gel electrophoresis

Agar gel electrophoresis has been applied to the measurement of the high-affinity saturable estrogen binding component in the cytosol of rat pituitary and anterior hypothalamus. Under the conditions employed, the number of available binding sites can be determined in small samples with good precision and accuracy. The total number of estrogen binding sites in the anterior hypothalamus of 28-day-old females was measurably (⋍15%) greater than that in males. A significant difference in the pituitary could not be demonstrated. The total number of binding sites in the pituitary was twice that in the anterior hypothalamus in both males and females.

Spectrophotometric Investigation of the Interactions between Disperse Dyes and Sodium Salt of 2-Naphthalenesulfonic Acid-Formaldehyde Condensates

The absorption spectra of disperse dye in aqueous solution were changed by addition of the sodium salt of 2-naphthalenesulfonic acid-formaldehyde condensates (abbreviatted as 2-NSF). If all of the binding sites in polymer to a dye are assumed to be equivalent and independent of each other, and if the number of total binding sites is much larger than the number of occupaied sites, the apparent molar absorptivity (e. pp) is given by eap, =(sf-e. pp)/Pel/nK+Eb where ef and eb are the molar absorptivities of the free and bound dye respectively, P is concentration of the polymer, K is the intrinsic binding constant, and n is the number of the binding sites per one polymer molecule.The binding constant (nK) were obtained from the linear plots of e, versus (ef-e )/P, and the thermodynamic parameters were calculated. All values of the changes in enthalpy were negative and the contribution of enthalpy was generally greater than that of entropy. The binding character of dye with 2-NSF was discussed,