Evidence For Negative Cooperativity Research Articles

NAD(P)H quinone oxidoreductase (NQO1): an enzyme which needs just enough mobility, in just the right places.

NAD(P)H quinone oxidoreductase 1 (NQO1) catalyses the two electron reduction of quinones and a wide range of other organic compounds. Its physiological role is believed to be partly the reduction of free radical load in cells and the detoxification of xenobiotics. It also has non-enzymatic functions stabilising a number of cellular regulators including p53. Functionally, NQO1 is a homodimer with two active sites formed from residues from both polypeptide chains. Catalysis proceeds via a substituted enzyme mechanism involving a tightly bound FAD cofactor. Dicoumarol and some structurally related compounds act as competitive inhibitors of NQO1. There is some evidence for negative cooperativity in quinine oxidoreductases which is most likely to be mediated at least in part by alterations to the mobility of the protein. Human NQO1 is implicated in cancer. It is often over-expressed in cancer cells and as such is considered as a possible drug target. Interestingly, a common polymorphic form of human NQO1, p.P187S, is associated with an increased risk of several forms of cancer. This variant has much lower activity than the wild-type, primarily due to its substantially reduced affinity for FAD which results from lower stability. This lower stability results from inappropriate mobility of key parts of the protein. Thus, NQO1 relies on correct mobility for normal function, but inappropriate mobility results in dysfunction and may cause disease.

Direct Electrochemistry of Shewanella oneidensis Cytochrome c Nitrite Reductase

Shewanella oneidensis cytochrome c nitrite reductase (soNrfA) is a dimeric enzyme that plays a key role in bacterial nitrate respiration. The periplasmic reductase catalyzes the six‐electron reduction of nitrite to ammonia but can also catalyze the two‐electron reduction of hydroxylamine, a proposed intermediate in the nitrite reduction pathway. Here we use Protein Film Voltammetry to investigate the electrochemical and catalytic properties of soNrfA and compare its properties to the related Escherichia coli nitrite reductase (ecNrfA). Our findings highlight the similarities and differences between the two enzymes: most notably, all detected electron transfer steps are one‐electron in nature, contrary to what has been observed in ecNrfA. This suggests that for soNrfA the six‐electron reduction of nitrite is carried out in a series of one‐electron steps. Additionally we observe substrate inhibition during nitrite turnover and evidence of negative cooperativity during hydroxylamine turnover which suggests individual monomers within a ccNiR homodimer interact with one another during catalysis.

Thermodynamic and Voltammetric Characterization of the Metal Binding to the Prion Protein: Insights into pH Dependence and Redox Chemistry

The prion protein is a high-affinity copper binding protein that plays a role in the neurodegenerative prion diseases when it is converted into an altered isoform. The function of the protein remains controversial, but its relationship to its metallochemistry has prompted further investigation. While many researchers continue to use short peptide models for binding studies, the clear discrepancy between data obtained with such models when compared to those of full-length recombinant proteins requires clarification with this more appropriate model. Isothermal titration calorimetry was used to assess metal affinity for PrP. Using both full-length native and recombinant prion protein, we have demonstrated that the prion protein binds copper but has little affinity for other metals. Metal binding is highly pH sensitive, being optimal at pH 7.5 for copper, nickel, and zinc and at pH 5.5 for iron. Metal binding affinity for PrP was not altered by protein glycosylation. The use of suitable thermodynamic modeling reveals complex and cooperative copper binding, with evidence of negative cooperativity within the octarepeat region. Cyclic voltammetry was utilized to assess the electrochemistry of copper-charged prion protein, and we show that mPrP has a redox potential of 0.03 +/- 0.01 V versus the saturated calomel electrode at pH 7. The analysis also indicated that PrP is able to undergo reversible redox cycling with equal oxidative and reductive charges that are largely dependent on the copper bound to the octarepeat. The fifth site provides a small contribution to this redox activity, but only when the octarepeat is present. These results show conclusively that PrP can utilize copper for electron transfer, which would be expected for a radical detoxifying enzyme, and that the octarepeat region is the functional domain.

The Affinity of Copper Binding to the Prion Protein Octarepeat Domain: Evidence for Negative Cooperativity

The prion protein (PrP) binds Cu(2+) in its N-terminal octarepeat domain, composed of four or more tandem PHGGGWGQ segments. Previous work from our laboratory demonstrates that copper interacts with the octarepeat domain through three distinct coordination modes at pH 7.4, depending upon the precise ratio of Cu(2+) to protein. Here, we apply both electron paramagnetic resonance (EPR) and fluorescence quenching to determine the copper affinity for each of these modes. At low copper occupancy, which favors multiple His coordination, the octarepeat domain binds Cu(2+) with a dissociation constant of 0.10 (+/-0.08) nM. In contrast, high copper occupancy, involving coordination through deprotonated amide nitrogens, exhibits a weaker affinity characterized by dissociation constants in the range of 7.0-12.0 microM. Decomposition of the EPR spectra reveals the proportions of all coordination species throughout the copper concentration range and identifies significant populations of intermediates, consistent with negative cooperativity. At most copper concentrations, the Hill coefficient is less than 1.0 and approximately 0.7 at half copper occupancy. These findings demonstrate that the octarepeat domain is responsive to a remarkably wide copper concentration range covering approximately 5 orders of magnitude. Consideration of these findings, along with the demonstrated ability of the protein to quench copper redox activity at high occupancy, suggests that PrP may function to protect cells by scavenging excess copper.

Kinetic Characterization of Mutations Found in Propionic Acidemia and Methylcrotonylglycinuria: EVIDENCE FOR COOPERATIVITY IN BIOTIN CARBOXYLASE

Acetyl-CoA carboxylase catalyzes the committed step in fatty acid synthesis in all plants, animals, and bacteria. The Escherichia coli form is a multifunctional enzyme consisting of three separate proteins: biotin carboxylase, carboxyltransferase, and the biotin carboxyl carrier protein. The biotin carboxylase component, which catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the carboxylate source, has a homologous functionally identical subunit in the mammalian biotin-dependent enzymes propionyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase. In humans, mutations in either of these enzymes result in the metabolic deficiency propionic acidemia or methylcrotonylglycinuria. The lack of a system for structure-function studies of these two biotin-dependent carboxylases has prevented a detailed analysis of the disease-causing mutations. However, structural data are available for E. coli biotin carboxylase as is a system for its overexpression and purification. Thus, we have constructed three site-directed mutants of biotin carboxylase that are homologous to three missense mutations found in propionic acidemia or methylcrotonylglycinuria patients. The mutants M169K, R338Q, and R338S of E. coli biotin carboxylase were selected for study to mimic the disease-causing mutations M204K and R374Q of propionyl-CoA carboxylase and R385S of 3-methylcrotonyl-CoA carboxylase. These three mutants were subjected to a rigorous kinetic analysis to determine the function of the residues in the catalytic mechanism of biotin carboxylase as well as to establish a molecular basis for the two diseases. The results of the kinetic studies have revealed the first evidence for negative cooperativity with respect to bicarbonate and suggest that Arg-338 serves to orient the carboxyphosphate intermediate for optimal carboxylation of biotin.

Cofactor Binding to Escherichia colid-3-Phosphoglycerate Dehydrogenase Induces Multiple Conformations Which Alter Effector Binding

The inhibition of Escherichia coli d-3-phosphoglycerate dehydrogenase by l-serine is positively cooperative with a Hill coefficient of approximately 2, whereas the binding of the inhibitor, l-serine, to the apoenzyme displays positive cooperativity in the binding of the first two serine molecules and negative cooperativity in the binding of the last two serine molecules. An earlier report demonstrated that the presence of phosphate appeared to lessen the degree of both the positive and negative cooperativity, but the cause of this effect was unknown. This study demonstrates that the presence of intrinsically bound NADH was responsible to a substantial degree for this effect. In addition, this study also provides evidence for negative cooperativity in NADH binding and for at least two NADH-induced conformational forms of the enzyme that bind the inhibitor in the physiological range. Successive binding of NADH to the enzyme resulted in an increase in the affinity for the first inhibitor ligand bound and a lessening of both the positive and negative cooperativity of inhibitor binding as compared with that seen in the absence of NADH. This effect was specific for NADH and was not observed in the presence of NAD+ or the substrate alpha-ketoglutarate. Conversely, the binding of l-serine did not have a significant effect on the stoichiometry of NADH binding, consistent with it being a V-type allosteric system. Thus, cofactor-related conditions were found in equilibrium binding experiments that significantly altered the cooperativity of inhibitor binding. Since the result of inhibitor binding is a reduction in the catalytic activity, the binding of inhibitor to these NADH-induced conformers must also induce additional conformations that lead to differential inhibition of catalytic activity.

Evidence for negative cooperativity among human thyrotropin receptors overexpressed in mammalian cells

Evidence for negative cooperativity among human thyrotropin receptors overexpressed in mammalian cells

Evidence for negative cooperativity among human thyrotropin receptors overexpressed in mammalian cells.

The complementary DNA for the human TSH receptor (TSHR) translated region was amplified in the genome of stably transfected Chinese hamster ovary (CHO) cells using a dihydrofolate reductase minigene. Immunoprecipitation of TSHR in whole cells precursor-labeled with [35S]methionine and [35S]cysteine revealed an approximately 10-fold increase in TSHR expression in cells stabilized in 10,000 nM methotrexate (TSHR-10,000 cells) compared to cells with the same gene not subjected to amplification (TSHR-0 cells). Similarly, [125I]TSH cross-linking to the surface of intact CHO cells revealed a progressive increase in TSH-binding sites with dihydrofolate reductase minigene amplification, with a 12.8-fold increase in TSHR in TSHR-10,000 vs. TSHR-0 cells. Based on the known number of TSHR expressed by TSHR-0 cells, TSHR-10,000 express approximately 1.9 x 10(6) TSHR on their surface. Two ligand-TSHR complexes were evident under reducing conditions, representing the single chain holoreceptor of about 115 kDa and a dissociated A subunit of about 60 kDa. In the absence of TSH, basal cAMP levels in TSHR-10,000 cells were greater than those in TSHR-0 cells (6-fold in isotonic medium and 18.5-fold in hypotonic medium), indicating that the unliganded TSHR has significant constitutive activity. We assessed the kinetics of TSH binding to CHO cells overexpressing the TSHR using [125I]TSH in the presence of increasing concentrations of unlabeled TSH as well as by attempted saturation with labeled ligand. Surprisingly, in contrast to TSHR-0 cells (Kd = approximately 5 x 10(-10) M), we observed progressively lower affinities for TSH binding by TSHR-800 cells (Kd = approximately 10(-9) M) and TSHR-10,000 cells (Kd = approximately 2 x 10(-9) M). In summary, we report a high level of expression of TSHR in CHO cells and confirm the high constitutive activity of the TSHR in the absence of ligand as well as the binding of TSH to the single subunit, uncleaved TSHR. Moreover, we found that a high level of expression is associated with apparent negative cooperativity among the TSHR in terms of their affinity for ligand.

Sequence-dependent effects of spermine on the thermodynamics of the B-DNA to Z-DNA transition.

Spermine has been shown to bind to and stabilize a number of altered DNA conformations, including left-handed Z-DNA. Here, we have quantitatively studied the effects of spermine on the negative supercoil-induced transition from B- to Z-DNA. We have determined the intrinsic association constants for and the effective number of ligands that bind to both B- and Z-DNA. The intrinsic affinity of spermine for Z-DNA is approximately 10 times higher for d(CA/TG) (KZP = 1.2 x 10(8) M-1) than for d(CG) dinucleotides (KZP = 1.5 x 10(7) M-1), and both are greater than that for B-DNA (KBP = 1.4 x 10(5) M-1). This accounts for the stabilization of Z-DNA by spermine. The number of spermine accommodated by Z-DNA (nZ) is sequence-dependent [nZ = 0.6 spermine per 18 d(CA/TG) dinucleotides and 2.3 for 12 d(CG) dinucleotides]. The value of nZ of < 1 was interpreted as evidence for negative cooperativity in spermine binding to d(CA/TG) dinucleotides. Thus, although d(CA/TG) sequences saturate at lower spermine concentrations, the ligand has an overall greater effect on the stability of d(CG) dinucleotides as Z-DNA. B-DNA accommodates more spermines per base pair than either sequence as Z-DNA. At higher concentrations (> 10 microM), spermine destabilizes Z-DNA. Using these parameters in a model for competitive spermine binding to B-DNA and Z-DNA, we can make predictions for how potential Z-DNA sequences found in the human genome are affected by cellular levels of superhelical density and spermine.

Substrate binding to chloramphenicol acetyltransferase: evidence for negative cooperativity from equilibrium and kinetic constants for binary and ternary complexes

Chloramphenicol acetyltransferase (CAT) catalyzes the acetyl-CoA-dependent acetylation of chloramphenicol by a ternary complex mechanism with a rapid equilibrium and essentially random order of addition of substrates. Such a kinetic mechanism for a two-substrate reaction provides an opportunity to compare the affinity of enzyme for each substrate in the binary complexes (1/Kd) with corresponding values (1/Km) for affinities in the ternary complex where any effect of the other substrate should be manifest. The pursuit of such information for CAT involved the use of four independent methods to determine the dissociation constant (Kd) for chloramphenicol in the binary complex, techniques which included stopped-flow measurements of on and off rates, and a novel fluorometric titration method. The binary complex dissociation constant (Kd) for acetyl-CoA was measured by fluorescence enhancement and steady-state kinetic analysis. The ternary complex dissociation constant (Km) for each substrate (in the presence of the other) was determined by kinetic and fluorometric methods, using CoA or ethyl-CoA to form nonproductive ternary complexes. The results demonstrate an unequivocal decrease in affinity of CAT for each of its substrates on progression from the binary to the ternary complex, a phenomenon most economically described as negative cooperativity. The binary complex dissociation constants (Kd) for chloramphenicol and acetyl-CoA are 4 microM and 30 microM whereas the corresponding dissociation constants in the ternary complex (Km) are 12 microM and 90 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Distinct platelet thromboxane A2/prostaglandin H2 receptor subtypes. A radioligand binding study of human platelets.

Thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) may aggregate platelets via a common membrane receptor(s). To further characterize this receptor, binding of the radiolabeled TXA2/PGH2 mimetic [125I]BOP to washed human platelets (WP) was investigated. [125I]BOP was competitively displaced from its platelet binding site by stable TXA2/PGH2 analogues. Competition curves were shallow with Hill coefficients of -0.73 +/- 0.05 (P less than 0.001 different from unity) (90 +/- 1% specific binding). Scatchard plots were curvilinear and most consistent with two binding sites; a high-affinity site with Kd of 234 +/- 103 pM, Bmax of 0.7 +/- 0.3 pM/mg protein (180 +/- 87 sites/WP), and a lower affinity site with Kd of 2.31 +/- 0.86 nM, Bmax of 2.2 +/- 0.3 pM/mg protein (666 +/- 65 sites/WP). [125I]BOP association and dissociation kinetics gave a Kd of 157 pM without evidence of negative cooperativity. The EC50 for I-BOP-induced initial Ca2+ increase was 209 +/- 24 pM, shape change was 263 +/- 65 pM, and aggregation was 4.4 +/- 0.5 nM. Parallel binding studies using the TXA2/PGH2 receptor antagonist [125I]PTA-OH showed a single binding site. The rank order for TXA2/PGH2 analogues to displace [125I]PTA-OH was identical to that for [125I]BOP. These studies indicate that [125I]BOP binds to two distinct sites on human platelets that may represent platelet TXA2/PGH2 receptor subtypes. The close correlation of IC50 values for I-BOP-induced platelet shape change and aggregation with the two Kds for [125I]BOP binding suggests that these platelet responses may be independently mediated by the two putative receptors.

Interaction of human monomeric C3b with its receptor (complement receptor type 1, CR1) on neutrophils. Evidence for negative cooperativity.

The binding of highly purified monomeric 125I-C3b to its receptor (CR1) on resting human polymorphonuclear neutrophils (PMN) was analyzed under equilibrium conditions, at 4 degrees C and low ionic strength. Scatchard analysis of specific binding data yielded curvilinear concave upward plots, which resulted from the presence of site-site interactions of the negative type among PMN C3b-receptors (negative cooperativity), as shown by dissociation kinetic experiments. Indeed, the dissociation rate of 125I-C3b from PMN was markedly increased in the presence of an excess of unlabeled C3b in the dilution medium and was directly dependent on the degree of initial receptor occupancy with the radioligand. These interactions occurred when 2% of the receptors were occupied with 125I-C3b and resulted in a 4-fold decrease in CR1 affinity when the receptor went from its "empty" to its "filled" conformation. In a disease associated with a continuous production of C3b (factor I deficiency), CR1 on in vivo circulating PMN was found to be in a "low affinity" and "high dissociating" state similar to that of normal CR1 at high occupancy. Finally, negative cooperativity among CR1 sites disappeared after PMN activation with chemotactic peptides.

Oxygen equilibria of Octopus dofleini hemocyanin.

Oxygen binding by Octopus dofleini hemocyanin was examined under very nearly physiological conditions. The effects of pH, ionic composition, temperature, and aggregation were controlled so that the role each plays in modulating oxygen binding can be isolated. There is a very large effect of pH on affinity, the Bohr effect (delta log P50/delta pH = -1.7), which is the same at 10 and 20 degrees C. However, cooperativity is substantially altered over the same range of pHs at the two temperatures. The allosteric properties were examined by comparing the experimental data points to curves generated by use of the Monod-Wyman-Changeux model. A computer-fitting process was developed which allowed the individual allosteric parameters to be varied independently until the best fit could be determined. The relationship between kR and kT is responsible for the effect of pH on cooperativity. A change in the allosteric properties of the T form is primarily responsible for the differences due to temperature. Changing cation concentrations when the molecule is in the fully aggregated 51S form alters affinity without influencing cooperativity. The effect of Mg2+ is much greater than that of Na+. If the 51S decamer is dissociated to 11S monomers by removing divalent cations, oxygen binding is noncooperative. There is evidence for negative cooperativity, indicating heterogeneity of function within the subunit which contains seven oxygen binding domains. Association into decamers generates conformational change which results in a much wider range of allosteric function.

Nerve growth factor receptors: analysis of the interaction of ßNGF with membranes of chick embryo dorsal root ganglia

The binding of the beta subunit of Nerve Growth Factor (beta NGF) to membrane preparations of 8-day chick embryo dorsal root ganglia (DRG) has been investigated under conditions similar to those used to study the binding of beta NGF to intact single cell dissociates of DRG. The equilibrium binding data reveal heterogeneityy of binding that is more complex than that seen with intact cells. Binding is not saturable up to 125I beta NGF concentrations of 10(-8) M. Steady-state and kinetic binding data show two sites with dissociation constants similar to those found on DRG cells. In addition, displacement data reveal a binding component with lower affinity (Kd = 10(-6) M) which is not found on intact cells. As with intact cells, the difference in the affinities of the two high affinity sites has been shown to be due to different rate constants of dissociation. The kinetics of dissociation of NGF are slower with membranes than with cells, and dissociation characteristics of 125I beta NGF change with increasing time of exposure to membranes. Degradation of 125I beta NGF during incubation with membranes is minimal and does not complicate the analysis of steady-state binding. Insulin does not bind to either of the two high affinity sites. Heterogeneity of the 125I beta NGF preparation and cooperativity of binding as a cause for the heterogeneity of the binding of NGF has been ruled out. Although there was an apparent increase in the rate of dissociation of 125I beta NGF in the presence of unlabelled NGF, a finding previously interpreted as evidence for negative cooperativity, this was shown to be independent of receptor site occupancy by NGF, and in part due to isotopic dilution within a diffusion barrier around the membranes.

Evidence for negative cooperativity among β-adrenergic receptors in cardiac and lung tissue of guinea pig

Stereospecific binding sites for (−)-[ 3H]dihydroalprenolol, a β-adrenergic antagonist, have been identified in guinea-pig myocardial cell membranes and lung tissue preparations. A close correlation between the abilities of β-adrenergic antagonists to compete with labelled antagonist binding and to block the response of isoproterenol plus Gpp(NH)p† stimulated adenylate cyclase has been found. Equilibrium binding of (−)-[ 3H]dihydroalprenolol to binding sites of cardiac and lung tissue displays characteristics consistent with negative cooperativity among the DHA binding sites: Scatchard plots are curvilinear with upward concavity. The existence of site-site interactions of the negatively cooperative type was demonstrated directly by the ability of unlabelled (−)alprenolol to accelerate the dissociation of (−)-¦ 3H¦dihydroalprenolol under conditions where rebinding of the radioligand does not occur.

The interaction of caerulein with the rat pancreas. 1. Specific binding of [3H]caerulein on plasma membranes and evidence for negative cooperativity.

1. The binding of [3H]caerulein (a stable, biologically active labeled analog of cholecystokinin-pancreozymin) to semi-purified rat pancreatic plasma membranes was investigated. The binding was dependent on time and temperature, as well as saturable, specific and reversible. This process was pH-dependent and optimal at pH 7.0. Cysteine and serine residues in plasma membranes were of importance for binding. Mg2+ favored the binding. 2. The acceleration of the dissociation of [3H]caerulein in the presence of an excess of native caerulein suggests that binding was characterized by a negative cooperativity. The fast dissociation state evoked by a high degree of occupancy by caerulein was inhibited by lowering the temperature, by decreasing the pH, or by the presence of wheat germ agglutinin.

Insulin receptor: Interaction with nonreceptor glycoprotein from liver cell membranes

In crude receptor preparations (either particulate or soluble) of rat liver membranes, the insulin receptor exhibits complicated binding kinetics (two binding plateaus, half-saturated at approximately 60 pM and 700 pM insulin) and an apparent chromatographic heterogeneity, suggested by the presence of two detectable, soluble insulin-binding components with apparent Stokes radii of 72 A and 38 A. In contrast, the insulin receptor isolated by affinity chromatography exhibits a simple binding isotherm (half-maximal saturation of binding at 700 pM insulin) without evidence for negative cooperativity and behaves as a single component (apparent Stokes radius of 38 A) upon chromatography on Sepharose 6B. The apparent discrepancies between the properties of the unpurified insulin receptor and the affinity-purified receptor can be attributed to the presence in crude preparations of a nonreceptor constituent(s) having properties consistent with those of a membrane glycoprotein. A glycoprotein fraction from such crude soluble membrane preparations, freed from insulin receptor and subsequently partially purified using concanavalin-A-agarose, when combined with affinity-purified insulin receptor, causes both a reappearance of the complicated binding kinetics and an increase in the receptor's apparent Stokes radius from 38 A to 72 A. Similar results are observed for a glycoprotein fraction obtained from rat adipocyte membranes but are not observed for an identical fraction isolated from human erythrocyte membranes. We conclude that the insulin receptor in rat liver membranes can interact with another nonreceptor membrane glycoprotein that may represent either a nonrecognition moiety of the receptor oligomer or an effector molecule to the biological action of insulin.

The monocyte as a model for the study of insulin receptors in man.

We have characterized the cellular composition of preparations isolated from peripheral blood by Ficoll-Isopaque gradient centrifugation. 125I-insulin binding to every cell type was measured. A highly significantly positive correlation between specific cell binding fraction and the monocyte concentration of the heterogeneous cell suspension was demonstrated. Depletion of monocytes reduced the insulin binding approximately 80%, which confirms previous findings by other investigators. The granulocytes possessed the second highest binding ability, but only one fourteenth of that of monocytes. Compared to the lymphocyte the monocyte had about 25 times greater insulin binding. Also thrombocytes bound insulin and contamination with these meant that their contribution to the total specific cell binding was not negligible. A reduction in these contaminants is essential. We found that insulin binding to erythrocytes was insignificant. A method of calculating the specific insulin binding to monocytes alone is introduced. The monocyte-insulin-receptor possesses specificity. Only an insignificant degradation of receptor bound insulin could be shown. Evidence of negative cooperativity between receptors was found. Consequently monocytes are considered a useful model for insulin receptor studies in man.

Corrections - Evidence for Negative Cooperativity in the Adsorption of Phosphorylase b on Hydrophobic Agaroses

ADVERTISEMENT RETURN TO ISSUEPREVArticleCorrections - Evidence for Negative Cooperativity in the Adsorption of Phosphorylase b on Hydrophobic AgarosesHerbert JennissenCite this: Biochemistry 1977, 16, 10, 2312Publication Date (Print):May 17, 1977Publication History Published online12 November 2003Published inissue 17 May 1977https://doi.org/10.1021/bi00629a603RIGHTS & PERMISSIONSArticle Views4Altmetric-Citations2LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (82 KB) Get e-Alerts Get e-Alerts

Evidence for negative cooperativity in the adsorption of phosphorylase b on hydrophobic agaroses.

The concept of cooperativity appears to be the key to the understanding of the complex mechanisms underlying the adsorption of proteins to agaroses substituted with hydrophobic alpha-aminoalkanes. The adsorption of phosphorylase b occurs through the positive cooperative interaction of a critical number of approximately 3-5 butyl and a higher number of methyl residues with ocrresponding sites on the enzyme. The amount of adsorbed phosphorylase b per millileter of packed gel (methyl-, butyl-Sepharose) in the absence and presence of 1.1 M ammonium sulfate at temperatures between 0 and 34 degrees C is a power function of the free solute equilibrium concentration (Freundlich isotherm). In contrast, the adsorption of cyanmyoglobin to phosphocellulose is described by the Langmuir equation. The surface coverage dependent isosteric heats of adsorption for phosphorylase b indicate an endothermic reaction only on the butyl-Sepharose in the presence of high salt concentrations. Scatchard plots of the Freundlich isotherms of phosphorylase b are concave upwards, typical of negative cooperativity. Hill plots of these isotherms (5-70% saturation) yield coefficients between nH = 0.39 and 0.71. At high surface coverages, the Hill coefficients approach unity. Apparent association constants (K0.5) of 4-39 X 10(4) M-1 are calculated for the adsorption of phosphorylase b, as compared to 2-9 X 10(4) M-1 for the adsorption of cyanmyoblobin. In general, negative cooperativity of binding may be explained by changes in the affinity of the ligand for the matrix, due to the sequential multivalent adsorption, and competition of phosphorylase b molecules for the critical number of alkyl residues (nonindependence of binding) on one side and to variations in the configuration of binding and entropy on the other.