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Showing papers on "Cooperativity published in 1978"


Journal ArticleDOI
TL;DR: The results indicate that the binding of trifluoperazine to the calcium-dependent activator of phosphodiesterase is selective and suggest that this binding may explain some of the biochemical and pharmacological actions of this antipsychotic agent.

191 citations


Journal ArticleDOI
TL;DR: In vitro polymerization of pig brain tubulin, highly purified and deprived of microtubule-associated proteins, was followed by turbidimetry, which yielded a kinetic analysis of the polymerization process, which consisted of a slow spontaneous nucleation followed by the growth process.
Abstract: In vitro polymerization of pig brain tubulin, highly purified and deprived of microtubule-associated proteins, was followed by turbidimetry. Treatment of the data yielded the relation existing between the observed turbidity and the amount of polymer formed. This allowed a kinetic analysis, according to Oosawa's theories, of the polymerization process, which consisted of a slow spontaneous nucleation followed by the growth process. The apparent elongation rate constant was closely related to the nucleation process and exhibited a highly cooperative variation with tubulin concentration. The cooperativity was indicative of the size of the nucleus which appears to remain the same whether sheets or microtubules are formed. Magnesium ions appear to play a role in the polymorphism of tubulin polymers, the proportion of microtubules to sheets increasing with magnesium ion concentration. From kinetic experiments evidence was provided for GDP binding in competition with GTP, with a sixfold lower affinity. The tubulin-GDP complex could participate in microtubules elongation, but was not able to form nuclei. The critical concentration of tubulin in the presence of GDP was roughly twice as high as in the presence of GTP.

159 citations


Journal ArticleDOI
TL;DR: The thermodynamic constants of oxygen binding to cobalt "picket fence" porphyrin complexes, meso-tetra(a,o, a,o- o-pivalamidophenyl)porphyrinatocobalt(II)- 1 -methylimidazole and 1,2-dimethylimodazole, are reported and appear to be qualitatively consistent with the lowered AGil of CoHb.
Abstract: The thermodynamic constants of oxygen binding to cobalt "picket fence" porphyrin complexes, meso-tetra(a,o,a,o- o-pivalamidophenyl)porphyrinatocobalt(II)- 1 -methylimidazole and 1,2-dimethylimidazole, are reported. In contrast to pre- viously studied cobalt porphyrins, these complexes bind oxygen with the same affinity as cobalt substituted myoglobin and he- moglobin. Solvation effects are discussed as the source of this difference. The use of sterically hindered axial bases as models of T state hemoglobin is discussed. In studies of myoglobin (Mb) and hemoglobin (Hb), the replacement of the neutral iron porphyrin prosthetic group with different metalloporphyrins has proved to be a useful tech- nique.2 Artificial hemoglobins containing ~inc,~.~ manga- ne~e,~-* ~opper,~ and nickel9 have been reconstituted, and their properties compared with those of the native iron proteins. These artificial systems, however, are incapable of reversible oxygenation. In contrast, cobalt substituted hemoglobin and myoglobin (CoHb and CoMb)l0 are functional,I1-l3 although their oxygen affinities are 10-100 times less than those of native Hb and Mb. CoHb exhibits cooperativity in oxygen binding, though to a lesser degree than Hb. The extent of this cooperativity is conveniently expressed as AG;, , the free energy difference between the intrinsic binding of the first and the fourth 02 to Hb.13b,c For CoHb, AG:l is roughly one-third that of Hb under comparable condition^.'^^ Because of the different stereo- chemical and electronic factors involved in binding oxygen to a cobalt porphyrin, the observation of cooperativity in CoHb has been variously used either to que~tion~9~~J~ or s~pport'~~,'~ the elegant proposal of Perutz concerning the molecular mechanism of cooperativity in natural Hb.16 At the heart of this proposal is the assumption, based on earlier ideas of Hoard" and Williams,18 that the high-spin iron in the unli- gated, low 02 affinity form of Hb (T state) lies out of the porphyrin plane, and that on binding 02, the iron becomes low spin and moves into the plane. The resulting motion of the proximal imidazole (0.6 A) then causes conformational changes in the protein which produce a higher 02 affinity quaternary form of the protein (R state). In the deoxy form of coboglobin, cobalt is low spin rather than high spin and the best estimates from simple cobalt model systems indicate that the proximal imidazole in CoHb moves -0.4 A upon oxygen- ation'4,'9,20 as opposed to the 0.6 8, for Hb. The resulting motion of the proximal histidine upon binding 02 will therefore only be two-thirds as great. This seems to be qualitatively consistent with the lowered AGil of CoHb.

150 citations


Journal ArticleDOI
TL;DR: The enhancement of the GABA effect produced by sodium chloride showed sigmoidal kinetics indicating cooperativity, but no evidence could be found for the involvement of sodium-dependent GABA binding in this interaction.

148 citations


Journal ArticleDOI
TL;DR: It is shown that the qualitative differences in the kinetics of NO, O2, CO, and bulky isocyanides can be understood in structural terms in a framework based on transition state theory.
Abstract: Experimental evidence indicates that the way cooperativity in hemoglobin is manifested kinetically depends on the nature of the ligand. It is shown that the qualitative differences in the kinetics of NO, O2, CO, and bulky isocyanides can be understood in structural terms in a framework based on transition state theory.

88 citations


Journal ArticleDOI
TL;DR: Isotopic probes promise to be a powerful tool in the study of alternating site cooperativity for those enzymes where suitable isotopic exchange reactions exist and at low ADP or Pi concentrations extensive reversal of bound ATP formation occurs before the bound ATP is released.

85 citations


Journal ArticleDOI
TL;DR: Data are presented which suggest that ATP promotes the binding of efrapeptin to the enzyme, andications that efrapptin is a catalytic site inhibitor make these results difficult to reconcile with a simple mechanistic scheme involving a single independnet catalytic sites for ATP synthesis and hydrolysis.

77 citations


Book
01 Jun 1978
TL;DR: This chapter discusses molecular models for Cooperativity and Allosteric Interactions in Multisubunit Proteins, as well as special types of Cooperative Systems.
Abstract: 1 Basic Concepts of Allosteric Control.- 2 The Structure of Multisubunit Proteins.- I. General Principles.- II. Other Types of Protein Assemblies.- 3 Cooperativity in Multisubunit Proteins - The Basic Concepts.- I. The Hill Equation.- II. The General Adair Equation.- III. The Statistical Correction.- IV. The Hill Coefficient in Terms of Intrinsic Ligand Affinities.- V. The Hill Coefficient at 50% Ligand Saturation.- VI. The Maximal Hill Coefficient.- VII. The Limiting Values of the Hill Slope.- VIII. The Allosteric Dimer.- IX. The Multi-Dimer Case.- X. The Allosteric Tetramer.- XI. The General Tetrameric Case.- 4 The Energy of Subunit Interactions.- I. Determination of Intersubunit Interaction Energy.- II. The Hill Coefficient and the Intersubunit Interaction Energy.- III. The Meaning of Intersubunit Energy of Interaction.- 5 Molecular Models for Cooperativity and Allosteric Interactions.- I. Introduction.- II. The Monod-Wyman-Changeux (MWC) Concerted Model.- 1. Basic Assumptions of the Concerted Model.- 2. The Allosteric Dimer Analyzed by the MWC Model.- 3. Allosteric Inhibition and Allosteric Activation in the MWC Model.- 4. The General Case.- 5. Phenomena Explained by the Concerted Model.- 6. Cooperativity in the Monod-Wyman-Changeux Model.- III. The Koshland-Nemethy-Filmer (KNF) Sequential Model.- 1. Basic Assumptions of the Sequential Model.- 2. The Allosteric Dimer Analyzed by the KNF Model.- 3. Allosteric Activation and Allosteric Inhibition in the KNF Model - the Dimer Case.- 4. The KNF Model - the Tetramer Case.- 5. The Influence of the Intersubunit Binding Domains on the Nature of Subunit Interactions.- IV. The Conformational State of the Protein.- 1. Exclusive Binding in the MWC Model.- 2. The Nonexclusive Binding in the MWC Model.- 3. The Simple Sequential KNF Model.- 4. The General Sequential KNF Model.- 5. Measuring R?.- V. Comparison Between the KNF Model and the MWC Model.- 6 Special Types of Cooperative Systems.- I. Cooperativity Resulting from Ligand-Coupled Protein Association or Dissociation.- 1. Ligand-Coupled Monomer-Dimer Equilibrium.- 2. The General Case.- II, Negative Cooperativity.- III. Protein Association and Dissociation Coupled to Ligand Binding.- 1, Dimerization Coupled to Ligand Binding.- 2. Monomer Multimer Equilibrium Coupled to Ligand Binding.- References.

74 citations


Journal ArticleDOI
TL;DR: In this paper, a fluorimetric-polarographic method for recording oxygen equilibrium curves was developed: with a favorable geometrical arrangement and low hemocyanin concentration, the error induced by reabsorption of emitted light was minimal (working range 0.02-0.05 O.D).
Abstract: 1. Fluorescence (F) of the hemocyanin ofEurypelma californicum is strongly dependent on the degree of oxygenation (Fig. 2). Maximum excitation is found at 292 to 294 nm. There is only a small shift of maximum emission from 345 nm in oxygenated to 350 nm in deoxygenated hemocyanin, indicating that mainly tryptophan is responsible for oxygenation-dependent fluorescence (Fig. 3). Fluorescence enhancement depends linearly on the degree of deoxygenation (Fdeoxy/Foxy is about 16 at pH 7.4; Fig. 4). 2. Based on fluorescence quenching upon oxygenation, a fluorimetric-polarographic method for recording oxygen equilibrium curves was developed: With a favourable geometrical arrangement and low hemocyanin concentration, the error induced by reabsorption of emitted light is minimal (working range 0.02–0.2 mg/ml, corresponding to ca. 0.005–0.05 O.D. at 340 nm; Fig. 5). Data obtained by this method are in excellent agreement with data obtained by photometry (Figs. 6 and 7). 3. Oxygen affinity and cooperativity between oxygen binding sites ofEurypelma hemocyanin are strongly modified by protons: There is a very pronounced Bohr effect with a maximum between pH 8.0 and 8.4 (ΔlogP50/ΔpH=−1.2; Fig. 7). Cooperativity is maximal at about pH 8.0 (n50=7) and decreases towards low and high pH (Fig. 7). Oxygen affinity is independent of hemocyanin concentration, cooperativity, however, is slightly increased at high hemocyanin concentration. 4. Modification of oxygen affinity and cooperativity is interpreted in the framework of the Monod, Wyman and Changeux (1965) model. SinceK Tass andK Rass could not be estimated directly from the Hill plots, the intrinsic association constants of the first and the last oxygenation step,K1 andK24, were determined by means of a modified Scatchard plot (Edsall et al., 1954);K1=0.0036 mm Hg−1=0.0022×106 M−1;K24=2.69 mm Hg−1=1.636×106 M−1. With [T0]≫[R0],K1 representsK Tass , whereasK24 ([T0]≪[R0]) is equal toK Rass . From these constants, the MWC parameterc was calculated to be 0.00133 (c=K1/K24). The total free energy of interaction, ΔF1, is 3.9 kcal/site (25°C).

70 citations


Journal ArticleDOI
TL;DR: K1 is equal to KT, the oxygen affinity of the T state of hemoglobin, for all these hemoglobins and was increased in all of them when compared to normal hemoglobin at pH 7.9, which shows that the breakage of the Bohr group salt bridges by increasing pH or specific modification changes KT.

67 citations


Journal ArticleDOI
TL;DR: 8-Azido-adenosine 5'-triphosphate (n83ATP) is a suitable photoaffinity label for F1 ATPase from Micrococcus luteus and n83AMP, a cooperativity of the beta subunits carrying nucleotide binding sites is suggested.
Abstract: 1. 8-Azido-adenosine 5'-triphosphate (n83ATP) is a suitable photoaffinity label for F1 ATPase from Micrococcus luteus. The nucleotide is a substrate in the presence of bivalent cations and inhibits the enzyme irreversibly upon irradiation with ultraviolet light above 300 nm. 2. More than 80% of the label is covalently bound to the beta subunits in the presence of bivalent cations. Labeling and inactivation is decreased by protection with ADP, ATP or adenyl-5'-yl imidodiphosphate. To a much smaller degree the alpha subunits also become labeled. 3. n83AMP does not specifically bind to the beta subunits upon irradiation. Like n83ATP and n83ADP, it also labels the alpha subunits to a small extent. 4. The F1 ATPase is inactivated after a single beta subunit per F1 complex has become labeled. A cooperativity of the beta subunits carrying nucleotide binding sites is suggested.

Journal ArticleDOI
TL;DR: 1,2-Dimethylimidazole has been successfully used to mimic the presumed restraint of T state hemoglobin and model cobalt porphyrins show a smaller decrease in O2 affinity than the analogous iron p Morphyrins when the axial base is hindered.
Abstract: O2 binding to a series of ferrous and cobaltous "picket fence" porphyrins is reported N-Methylimidazole and covalently attached imidazoles gives O2 binding to ferrous porphyrins with deltaH degrees =-162 kcal/mol (-677 kJ/mol) and deltaS degrees =-40 eu (standard state, 1 atmosphere O2) Similar studies with cobaltous porphyrins yield deltaH degrees =- 128 kcal/mol (-535 kJ/mol) and deltaS degrees =- 39 eu These values match well those of myoglobin and isolated subunits of hemoglobin and their cobalt reconstituted analogues 1,2-Dimethylimidazole has been successfully used to mimic the presumed restraint of T state hemoglobin In direct analogy to the decreased cooperativity shown by cobalt-substituted hemoglobin, model cobalt porphyrins show a smaller decrease in O2 affinity than the analogous iron porphyrins when the axial base is hindered Thermodynamic data are presented The molecular mechanism of cooperativity in hemoglobin is discussed

Journal ArticleDOI
TL;DR: These findings support the assumption that clustering of receptors and conformational changes in membrane structure are prerequisites for mitogenic stimulation.

Journal ArticleDOI
TL;DR: lipids are investigated as a bidimensional medium required for the movement of Coenzyme Q, a lipid-soluble cofactor of the mitochondrial respiratory chain, and as a hydrophobic environment necessary to impose the proper conformation to membrane-bound enzymic proteins to postulate a working hypothesis for the mechanism of action of general anesthetics.
Abstract: The phospholipid requirement of membrane-bound enzymes may depend on several reasons. In our laboratory we have investigated lipids (1) as a bidimensional medium required for the movement of Coenzyme Q, a lipid-soluble cofactor of the mitochondrial respiratory chain, and (2) as a hydrophobic environment necessary to impose the proper conformation to membrane-bound enzymic proteins. We have found that Coenzyme Q, once reduced by NADH dehydrogenase, must cross the inner mitochondrial membrane; only quinones having long isoprenoid side chains can easily cross phospholipid bilayers, and this is the reason why a short chain quinone such as CoQ-3 inhibits NADH oxidation. The incapability of short quinones to cross lipid bilayers is due to their disposition in the lipid bilayer, stacked within the phospholipids. The conformational role of lipids has been investigated indirectly observing the kinetics of membrane-bound enzymes, e.g. the mitochondrial ATPase, and directly by circular dichroism. Lipid removal or lipid perturbation with organic solvents induce a decrease of α-helical content in mitochondrial proteins, and give rise to a series of kinetic changes in ATPase, including uncompetitive inhibition, increased activation energy, and loss of cooperativity in oligomycin inhibition. The recognition of a conformational role of lipids has allowed us to postulate a working hypothesis for the mechanism of action of general anesthetics. Such drugs have been found by us, by means of spin labels and fluorescent probes, to disrupt lipid protein interactions in several membranes, including synaptic membranes. The loosening of such interactions is believed to induce conformational changes, which will alter ion transport systems necessary to the propagation of neural impulses. Conformational changes induced by anesthetics have been found by us both directly by circular dichroism and indirectly by enzyme kinetics. The conformational effect of anesthetics is not directly exerted on the porteins but is mediated through the lipids. In agreement with this hypothesis we have found that membrane-bound acetylcholinesterase is inhibited by anesthetics, whereas the solubilized enzyme is not inhibited. However, binding of the solubilized enzyme to phospholipids restores anesthetic inhibition.

Journal ArticleDOI
TL;DR: It was concluded that the binding sites for L5 and L18 in the 5s RNA are functionally related to each other, but distinct from that for protein L25.
Abstract: Interactions of 5s RNA from Escherichia coli with 50s ribosomal subunit proteins L5, L18, and L25 have been evaluated by a number of criteria. From the dependence of complex formation on protein and RNA concentration in TMK buffer (50 mM Tris-HCI (pH 7.6)-20 mM MgC12-300 mM KCI), it was inferred that the three proteins differ substantially in their affinity for the nucleic acid. Measurement of the stoichiometry of association in the presence of excess protein revealed that molar protein:RNA binding ratios for L5, L18, and L25 at saturation were 0.6:1, l. l : l, and 0.7:1, respectively. The RNA molecule therefore contains no more than one specific site of attachment for each of the proteins. Solution conditions were varied to assess the effects of pH, Mg2+ concentration, and K+ concentration on the stability of the interactions. Optimal binding was observed for the L5-5s RNA complex at pH 6.5-9, [Mg2+] of 10-20 mM and [K'] of 300 to 400 mM; for the L18-5s RNA complex at pH 7.5-9. [Mg2+] of 10-20 mM and [K+] of 100-200 mM; and for the L25-5s RNA complex at pH 7.5-9, [Mg2+] of 0.3-20 mM, and [K+] of 200-300 mM. In a separate series of experiments, the association of L5 in TMK buffer was found to be cooperatively stimulated by L18 at component concentrations roughly tenfold less than were required for the association of L5 alone. The mutual influence of these two proteins upon one another was also clearly manifested in assays involving variation of pH and ionic environment. From the pattern of cooperativity, it was concluded that the binding sites for L5 and L18 in the 5s RNA are functionally related to each other, but distinct from that for protein L25.

Journal ArticleDOI
TL;DR: Findings indicate the presence of a cooperative (allosteric) interaction between warfarin and the oleate ion for albumin binding and suggest a conformational transition in the albumin molecule as a result of interactions.

Journal ArticleDOI
TL;DR: The solid-gas O2 binding equilibrium has been studied for ferrous "picket fence" porphyrinates with sterically hindered axial imidazoles, and direct analogies are drawn to the cooperativity shown in O2binding by hemoglobin.
Abstract: The solid-gas O2 binding equilibrium has been studied for for ferrous "picket fence" porphyrinates with sterically hindered axial imidazoles. Such systems show significant cooperativity in their binding of O2: at low O2 pressures a low O2 affinity form exists, and at high O2 pressures a higher O2 affinity form develops. Direct analogies are drawn to the cooperativity shown in O2 binding by hemoglobin. These model systems mimic hemoglobin quantitatively.


Journal ArticleDOI
01 Sep 1978-Science
TL;DR: The results suggest that no transient constraint of the heme group by the globin structure occurs on this time scale, and thus establish a temporal sequence for the early events that may participate in the stereochemical trigger mechanism of hemoglobin cooperativity.
Abstract: Resonance Raman spectra of oxyhemoglobin, deoxyhemoglobin, carboxyhemoglobin, and the corresponding myoglobin derivatives have been obtained with 7-nanosecond laser pulses at 531.8 nanometers. The results suggest that no transient constraint of the heme group by the globin structure occurs on this time scale, and thus establish a temporal sequence for the early events that may participate in the stereochemical trigger mechanism of hemoglobin cooperativity.


Journal ArticleDOI
TL;DR: A comparison of the oxygen binding properties of β-and α-hemocyanin shows that these proteins are complementary in their behaviour, with respect to the responses to changes in H+ anmd Ca2+ concentrations.
Abstract: The dissociation behaviour of Helix pomatiaβ- hemocyanin as influenced by pH, calcium-iopn concentraion and ionic strength, and the oxygen vinging properiotes were investigated. The dissociation behaviour of β-hemocyuanin and α-hemocyanin differs distinctly in a number of properties: (a) they show a different response to high salt concentration, (b) the stabilizing influence of calcium ions on the whole molecules of β-hemocyanin is smaller than that on α-hemocyanin, (c) half molecules of β-hemocyanina are stablized to a large extent in teh presence of calcium ions and (d) at low ionic strength no peak separation between one-tenth and one-twentieth molecules of β-hemocyanin is observed in the ultracentrifuge, which indicates an equilibrium between the species. The oxygen binding behaviour of β-hemocyanin shows the typical features of proteins with a large hnumber of binding sites. The Hill plot is characterized by a high value of the Hill coefficient (maxm nH=7), coupled to a low value for the interatrion free energy per site. The oxygen binding behaviour can bacisally be described by a mocdified tewo-state allosteric model with the variation, imposed by teh expeimental data, taht KT is pH-dependent (negative Bohr effect), while KR is pH-indepenmdent. Regulation of the cooperativity by teh alloosteric effector H+ is mainly due to changes in the allosteric equilibrium constant. In order to describe the cooperativity as a function of pH, the minimum number of interacting binding sites has to be varied, indicating that the size of the ‘functional constellation’ is variavle. This finding may have important implications on the relationship between the so-called ‘structural’ and ‘functional constellation’. A comparison fo the oxygen binding properties of β-and α-hemocyanin shows that these proteins are complementary in their behaviour, with respect to the responses to changes in H+ anmd Ca2+ concentrations. This may be of importance for the animal in order ito meet specific physiological and enviromental requirements.

Journal ArticleDOI
TL;DR: Folate binding was virtually insensitive to changes in ionic strength and temperature and Cooperativity as well as dependence of affinity on the folate binding protein concentration did moreover disappear.

Journal ArticleDOI
TL;DR: The formation of the ternary complex composed of actin, 5'-adenylyl imidodiphosphate [AMP-P(NH)P], and myosin subfragment 1 (S-1) was studied using the analytical ultracentrifuge with UV optics, which enabled the direct determination of the extent of dissociation of actIn.
Abstract: The formation of the ternary complex composed of actin, 5′-adenylyl imidodiphosphate [AMP-P(NH)P], and myosin subfragment 1 (S-1) was studied using the analytical ultracentrifuge with UV optics, which enabled the direct determination of the extent of dissociation of actin·S-1 (acto·S-1) by AMP-P(NH)P. In contrast to the reaction with ATP, at saturating levels of AMP-P(NH)P (1.5 mM), extensive formation of the ternary acto·S-1·AMP-P(NH)P complex occurs at 22°. With 40 μM actin present, AMP-P(NH)P causes almost no dissociation of the acto·S-1 complex at 0.04 M ionic strength, while even at 0.22 M ionic strength one-third of the S-1 remains associated with actin and AMP-P(NH)P in a ternary complex. A detailed study of the binding of S-1·AMP-P(NH)P to actin using the Scatchard plot analysis shows that, at saturation, 1 mol of S-1·AMP-P(NH)P binds per mol of actin monomer. There appears to be no cooperativity occurring as the S-1·AMP-P(NH)P binds along the actin filament, with the possible exception of a slight positive cooperativity when most of the sites on the actin filament are saturated. The turbidity of the ternary complex is identical to the turbidity of acto·S-1 alone. Preliminary experiments with the two-headed subfragment of myosin, heavy meromyosin (HMM), show that the binding of HMM·[AMP-P(NH)P]2 to actin is only about twice as strong as the binding of S-1·AMP-P(NH)P to actin, indicating that the second head contributes very little to the free energy of binding.

Journal ArticleDOI
TL;DR: This paper showed that 3H-dihydroergocryptine (3H-DHE) appears to bind to two classes of sites with limited capacity, differing in their specificities and affinities.
Abstract: In crude rat cardiac membrane preparations, [3H]dihydroergocryptine (3H-DHE) appears to bind to two classes of sites with limited capacity, differing in their specificities and their affinities. The first class of binding sites interacts preferentially with the postsynaptic alpha-adrenoceptor blocker ARC239, as can be expected for postsynaptic alpha-adrenoceptors. The binding of 3H-DHE to these receptors follows the law of mass action, with a high affinity for 3H-DHE (Kd 25 degrees C = 1.67 +/- 0.37 nM). Postsynaptic saturating levels of 3H-DHE are necessary to occupy the second class of binding sites. These sites exhibit a preferential affinity for presynaptic ligands such as clonidine and yohimbine, as would be expected for presynaptic alpha-adrenergic receptors. This presynaptic binding shows a markedly positive homotropic cooperativity (Hill n = 2.88) with initial and final apparent Kds of 23 and 0.83 nM, respectively. Free energy of interaction between sites is of the order of 2 kcal (8.36 kJ)/mol of sites. These characteristics provide a rational molecular basis for the functional role of presynaptic alpha-adrenoceptors that mediate the inhibition of norepinephrine release from nerve endings.

Journal ArticleDOI
TL;DR: Bifunctional affinity reagents of this type could be especially useful for placing the p-nitrophenyl chromophore adjacent to a binding site without blocking it, and could be photocrosslinkers.
Abstract: 4-Nitrophenyl ethers are proposed as new high-yield photoreagents for protein crosslinking and affinity labeling. These are totally unreactive in the dark under biological conditions, but react quantitatively with amines at pH 8 upon irradiation with 366-nm light. The reaction of monoalkoxy-p-nitrobenzenes with an amine yields the corresponding free alcohol and substituted nitrophenylamine. In essence, the nitrophenyl group is transferred from the alcohol to the amine. Bifunctional affinity reagents of this type could be especially useful for placing the p-nitrophenyl chromophore adjacent to a binding site without blocking it. The corresponding 2-methoxy-4-nitrophenyl ethers react with amines by displacement of the methoxyl group. Thus, bifunctional reagents of this class could be photocrosslinkers. A maleimide-containing 2-methoxy-4-nitrophenyl ether was attached to human fetal hemoglobin at γ-cysteine F9 stoichiometrically. Subsequent ultraviolet irradiation yielded a γ-γ crosslinked hemoglobin in 80% yield. The oxygenation properties of the derivative indicate that it is locked in a high affinity conformation and that all cooperativity is lost.


Journal ArticleDOI
TL;DR: The two-electron accepting behavior of the 330-nm chromophore is the exception rather than the rule, and the distribution of electron equivalents among the redox sites was found to be reductant dependent.
Abstract: Rhus laccase (monophenol monooxygenase, monophenol,dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) an O2/H2O oxidoreductase containing four copper ions bound to three redox sites (type 1, type 2, and type 3 Cu pair), was titrated anaerobically with several reductants having various chemical and thermodynamic properties. The distribution of electron equivalents among the redox sites was found to be reductant dependent. When the data for titration by various reductants of the type 3 site were plotted against those of the type 1 site according to the Nernst formalism, the slope n varied from 2.0 to 1.0. The redox potential of the reductant's first oxidation step is qualitatively correlated with the value of n and is suggested as the factor that modulates the electron distribution. Such a behavior implies a nonequilibrium situation. A very good simulation of the data was provided by an analysis assuming a formally variable cooperativity between the two type 3 copper ions. This apparent variability is suggested to result from a process whereby sufficiently strong reductants induce a transition of the type 3 site from a cooperative two-electron acceptor to a pair of independent one-electron acceptors. This uncoupled state of the type 3 site is considered metastable. Other possible models were also investigated. Summarizing the available data, we conclude that the two-electron accepting behavior of the 330-nm chromophore is the exception rather than the rule.


Journal ArticleDOI
TL;DR: The interaction parameters between the enzyme and its effectors at the Mg2+ site have been determined and the pH dependence of the apparent affinity studied and heterotropic interactions between K+ and Na+ sites have been studied.
Abstract: 1 Specificity at the Mg2+ site has been investigated with P-nitrophenylphosphate and dinitrophenylphosphate as substrates. The interaction parameters between the enzyme and its effectors at the Mg2+ site have been determined. Ni2+ and Cd2+ can be added to the series of known Mg2+ analogues. Tb3+ also inhibits the enzymatic activity and appears as a promising tool for further studies since it interacts with the (Na+,K+)-ATPase in a relatively high affinity process. 2 Interactions between the Mg2+ and Na+ sites have been studied by the P-nitrophenylphosphatase activity. Na+ decreases the apparent affinity of the enzyme for Mg2+ without strong modification of the cooperativity index. 3 Kinetic parameters for ATPase, P-nitrophenylphosphatase and dinitrophenylphosphatase activations by K+ analogues have been determined and the pH dependence of the apparent affinity studied. 4 Heterotropic interactions between K+ and Na+ sites have been studied with K+, Tl+ and NH+4. Three substrates have been used. Firstly, with dinitrophenylphosphate, increasing Na+ concentration changes the positive cooperativity for K+ to a negative one, then back to a positive cooperativity. Simultaneously, the apparent affinity increases then decreases. Secondly, with ATP, an increase of the Na+ concentration decreases the apparent affinity for K+ and changes the cooperativity from a negative to a positive one. Thirdly, with P-nitrophenylphosphate, interactions resemble those obtained with dinitrophenylphosphate. When added at concentrations which stimulate the activity, ATP imposes its type of heterotropic interactions between Na+ and K+ sites.

Book ChapterDOI
01 Jan 1978
TL;DR: It appears that in normal human hemoglobin the binding of anionic cofactors directly influences the oxygen affinity by neutralizing the charged groups of the diphosphoglycerate binding site and thereby stabilizing the deoxy conformation.
Abstract: Hemoglobin is in a state of dynamic equilibrium between high and low affinity forms As in the case of many other enzymes, the equilibrium can be shifted by binding effector molecules at sites remote from the protein's active site We do not yet know the rules by which nature has chosen anionic or cationic effectors as control molecules for specific enzyme forms, but it appears clear that in the case of human hemoglobin the oxygen affinity is primarily modulated by the concentration of anions in the external medium In many laboratories around the world the question of how this anionic control is achieved is under investigation This investigation began many years ago and many of the questions that faced early investigators of hemoglobin function are still facing researchers today Detailed studies of the structural and functional properties of human hemoglobin variants have provided greater insight into the mechanism by which cofactor binding is linked to oxygen binding Of particular interest in this regard are the substitutions which involve the eight positively charged residues of the 2,3-diphosphoglycerate binding site The characteristics of these hemoglobin mutants and their response to organic and inorganic anions suggest that the equilibrium between high and low affinity conformations of hemoglobin is strongly affected by the net positive charge in the central cavity Thus it appears that in normal human hemoglobin the binding of anionic cofactors directly influences the oxygen affinity by neutralizing the charged groups of the diphosphoglycerate binding site and thereby stabilizing the deoxy conformation