Cooperativity Research Articles

The "n' effect in molecular recognition.

The cooperativity which exists in crystal melting and many biological molecular recognition phenomena arises from extended arrays of weak interactions. We present a correlation between the melting temperature of a crystal and the intermolecular energy (which is evident only when compounds possessing several or many internal rotors are excluded). The correlation is used as the basis for a model of crystal melting which is capable of estimating the melting temperature of crystals. This model provides the basis for an understanding of the sharpness of the crystal melting transition for organic and inorganic substances. The cooperativity illustrated by the extended arrays of weak interactions, or the "n' effect, is extended to analogous order/disorder transitions in biological systems, such as the "melting' of DNA and RNA duplexes, providing insights into the physical properties of these structures.

Photolinker-polymer-mediated immobilization of monoclonal antibodies, F(ab')2 and F(ab') fragments.

Photolinker-polymer-mediated covalent immobilization of antibodies, F(ab') and F(ab')2 fragments has been achieved by light-dependent coupling procedures. Anti-alpha-foetoprotein (anti-AFP) monoclonal antibodies were covalently linked to microplates by layer-coating procedures, which entail antibody photoimmobilization to a photolinker-polymer-precoated surface. In this and the co-coating procedure described, diazirine-functionalized BSA (T-BSA) served as the multifunctional light-activatable linking agent (photolinker polymer). Prior to photo-activation, F(ab')2 or F(ab') fragments derived from anti-(prostate-specific antigen) monoclonal antibodies were mixed and co-coated with the photolinker polymer on to polystyrene microplates. The immunoreagents remained immunologically active after 350 nm irradiation (irradiance 0.7 mW.cm-2 for 20 min). Immuno-responses of photoimmobilized monoclonal anti-AFP antibodies were equivalent to signal intensities obtained with physically adsorbed antibodies. Photoimmobilization of anti-PSA F(ab') fragments in the presence of T-BSA revealed exponential binding characteristics indicating stabilizing molecular co-operativity of the BSA constituent. Co-coating procedures yielded 62 and 65% binding of applied 14C-labelled F(ab')2 and F(ab') fragments respectively. Covalency of antibody binding was inferred from: (i) the strict dependence of photoreagent availability; (ii) the light-dependence of the immobilization process; and (iii) the reversibility of immunocomplexation after acid treatment.

Studies on tryptophan residues of fructose-6-phosphate-2-kinase

The results of ultraviolet spectroscopy of NBS titrating PFK-2 indicate that the enzyme has four tryptophase residues. Among them two are essential. There is a strong emission fluorescence at the 335 nm region when chicken liver PFK-2 excited with UV light as 295 nm. This fluorescence can specifically reflect the states of tryptophane residues of the enzyme. Substrate Fru6P does not affect the fluorescence but ATP causes a quenching of the fluorescence. The maximum quenching is about 30%. The binding of ATP to PFK-2 has been monitored by fluorescence. It has been obtained that K-i is 0.033 mmol/L in the low ATP range, while it is 0.23 mmol/L in the high ATP concentration range. This illustrates that the binding of ATP to the enzyme possesses a negative cooperativity. Both Mg-2+ and inorganic phosphate are activators which reduce the K-i values. Fru6P and Fru2, 6P-2 have no effects on K-i. The binding at ATP induces a relatively large change in the conformation of the enzyme. It has been noted that in the presence of ATP the inactivation rate of the enzyme by NBS slightly increases. From the quenching of fluorescence, the Stern-Volmer constant (K-sv) is 4.0 either in the presence or in the absence of ATP as measured by using acrylamide to quench the tryptophane fluoresence of the enzyme.

Haemoglobin and cooperativity

Haemoglobin and cooperativity

PROPERTIES OF CHICKEN LIVER PHOSPHOFRUCTOKINASE-2

Phosphofructokinase-2 was purified to homogeneity from chicken livers by homogeniza-tion, polyethylene glycol fractionation and column chromatography on DEAE-Sephadex A-50 and Blue-Sepharose 4B. Some properties of the enzyme were as follows: (i) The saturation curve of the enzyme for fructose 6-phosphate showed hyperbolic and the Km of fructose 6-phosphate was affected by inorganic phosphate while Vmax was not; (ii) the binding of ATP to the enzyme was of negative cooperativity with a Hill coefficient of 0.56; (iii) the activity of the enzyme was completely lost in the presence of EDTA. The enzyme was activated by Mg2+ at low concentrations, but inhibited by Mg2+ at high concentrations; (iv) the enzyme was stable below 30℃ and easily lost its activity when the temperature was above 40℃; (v) the activity of the enzyme was stable at the range of pH 7-9, increased at pH 9.0-9.5 and decreased when pH was over 9.5; (vi) the enzyme was sensitive to trypsin and ATP protected the enzyme against the proteolysis of trypsin.

A New Paradigm From an Old Theory: Cooperativity in Evolution Examined and Taught Through Frank Herbert's Novel Hellstrom's Hive

A New Paradigm From an Old Theory: Cooperativity in Evolution Examined and Taught Through Frank Herbert's Novel Hellstrom's Hive

Purification and kinetics of mouse liver fructose 6-phosphate, 2-kinase.

The mouse liver fructose 6-phosphate, 2-kinase was purified by ultracentrifugation, polyethylene glycol precipitation, and subsequently by chromatography on DEAE-Sephadex, Blue-Sepharose and phasphocellulose columns. Gel filtration and SDS polyacrylamide electrophoresis showed that the enzyme has a molecular weight of 110,000 with two identical subunits. Mg2+ is essential for its activity. The activation of the enzyme by Mg2+ showed a positive cooperativity. The substrate saturation curve for fructose 6-phosphate was sigmoidal and for ATP was hyperbolic. The Km's for ATP increased with decrease in concentrations of fructose 6-phosphate indicating that the sequence for the substrates binding was in an ordered mechanism with respect to fructose 6-phosphate prior to ATP. An ionizable residue at the active site with pKa 9.5 was essential for the ATP binding and the pKa shifted to 9.8 after the binding of ATP.

Chemically crosslinked lactate dehydrogenase: stability and reconstitution after glutaraldehyde fixation.

Lactate dehydrogenase (LDH) from pig heart was stabilized in its tetrameric state by crosslinking with glutaraldehyde. The product (containing 82% tetramers, 9% dimers, 6% monomers, and 3% aggregates) retained up to approximately equal to 60% of its original activity without detectable changes in the spectral and catalytic properties of the enzyme. Fixation of the native quaternary structure enhances the stability of the enzyme: the equilibrium transitions of heat and guanidine deactivation are shifted from 60 to 65 degrees C and CG.HCl = 0.3 to 1.0 M, respectively. Deactivation and denaturation of the crosslinked enzyme do not coincide: the equilibrium transition of fluorescence emission at CG.HCl approximately equal to 3.5 M is paralleled by a significant decrease in cooperativity. The kinetics of thermal denaturation allow structural transitions within a partially crosslinked, native-like species (less than or equal to 10%) to be separated from the unfolding of the fully crosslinked enzyme. The corresponding first-order rate constants at CLDH = 10 micrograms/ml and 60 degrees C are 4.5 X 10(-4) and 2.7 X 10(-5)s-1, respectively. The yield of reconstitution of the crosslinked enzyme after acid denaturation does not exceed 30%; the rate of reactivation is fast due to the significant residual structure of the acid-denatured enzyme. Rate-determining association (which dominates the reconstitution of the unmodified enzyme) does not contribute to the kinetic mechanism. After complete randomization in 6 M guanidinium chloride, incorrect near-neighbor interactions between the crosslinked subunits essentially block the reconstitution of the native structure and function.

Cooperativity in Biochemistry: Cooperativity Theory in Biochemistry . Steady- State and Equilibrium Systems. Terrell L. Hill. Springer-Verlag, New York, 1985. xvi, 459 pp., illus. $120.

Cooperativity in Biochemistry: <i>Cooperativity Theory in Biochemistry</i> . Steady- State and Equilibrium Systems. Terrell L. Hill. Springer-Verlag, New York, 1985. xvi, 459 pp., illus. $120.

The cooperative behavior of yeast D-glyceraldehyde-3-phosphate. Dehydrogenase as studied by the formation of the fluorescent NAD derivative.

The ultraviolet irradiation of the yeast D-glyceraldehyde-3-phosphate dehydrogenase carboxymethylated at the active site Cys residues, as with the rabbit muscle enzyme, led to the formation of a fluorescent NAD derivative with an emission maximum at 410 nm. Similar results were obtained with the enzyme selectively carboxymethylated at only 2 of its 4 active site Cys residues. The binding of NAD+ to both the carboxymethylated enzymes is non-cooperative or only weakly negatively cooperative when determined by NAD+ quenching of the intrinsic protein fluorescence. However, determinations of the amount of fluorescent NAD derivative formed under different NAD+ concentrations show that both the carboxymethylated enzymes appeared to bind NAD+ with positive cooperativity as in the case of the binding of NAD+ to the native apoenzyme. This seems to suggest that the spatial positioning of the nicotinamide moiety at the active site of the irradiated enzyme resembles more closely that of the nicotinamide ring in the native holoenzyme as compared to the carboxymethylated enzymes.

Cooperativity in thermally induced intersystem crossing in solids: Fe(phen)2(NCR)2, R = BH3, BPh3, S, Se

Analyse de donnees sur les moments magnetiques en fonction de la temperature de ces complexes pour etudier les contributions cooperatives a la conversion de spin singulet/quintuplet, a l'aide d'une formulation thermodynamique generalisee simple des interactions intermoleculaires et de domaines

Cooperativity of Biopolymers

Cooperativity of Biopolymers

Cooperativity and composition of the linear amylose-iodine-iodide complex

Cooperativity and composition of the linear amylose-iodine-iodide complex