Showing papers on "Cooperativity published in 1984"
TL;DR: It is suggested that proteins and other biological macromolecules may have evolved to take functional advantage not only of mean conformational states but also of the inevitable thermal fluctuations about the mean.
Abstract: A general model is presented whereby lignand-induced changes in protein dynamics could produce allosteric communication between distinct binding sites, even in the absence of a macromolecular conformational change. Theoretical analysis, based on the statistical thermodynamics of ligand binding, shows that cooperative interaction free energies amounting to several kJ · mol-1 may be generated by this means. The effect arises out of the possible changes in frequencies and amplitudes of macromolecular thermal fluctuations in response to ligand attachment, and can involve all forms of dynamic behaviour, ranging from highly correlated, low-frequency normal mode vibrations to random local anharmonic motions of individual atoms or groups. Dynamic allostery of this form is primarily an entropy effect, and we derive approximate expressions which might allow the magnitude of the interaction in real systems to be calculated directly from experimental observations such as changes in normal mode frequencies and mean-square atomic displacements. Long-range influence of kinetic processes at different sites might also be mediated by a similar mechanism. We suggest that proteins and other biological macromolecules may have evolved to take functional advantage not only of mean conformational states but also of the inevitable thermal fluctuations about the mean.
642 citations
TL;DR: The initially simple model of reversible, bimolecular, and noncooperative interaction between receptor and insulin has been revised to include the existence of at least three affinity states that may be linked to modulation of the biological response induced by the insulin-receptor complex.
Abstract: During the last decade, earlier suggestions that insulin acts at the plasma membrane level via combination with receptors have been amply confirmed in studies of 125I-labeled insulin binding kinetics. Efforts have been devoted to the development of homogeneous, stable, and bioactive tracers, and a preparation of monoiodo[TyrA14]insulin showed 100-125% biological activity. The initially simple model of reversible, bimolecular, and noncooperative interaction between receptor and insulin has been revised to include the existence of at least three affinity states that may be linked to modulation of the biological response induced by the insulin-receptor complex. Thus negative cooperativity seems important in reducing oscillations of insulin action with variations in plasma insulin concentration, and formation of a high-affinity state or positive cooperativity may lead to desensitization of receptors. The kinetic phenomena suggest that receptor-binding affinity and function are actively regulated by insulin itself. At present the receptor model is purely functional and does not imply molecular mechanisms. However, recent advances in the analysis of receptor structure and biochemistry promise that the molecular equivalents of the kinetic phenomena may be elucidated in the near future. Furthermore the reaction between receptor and insulin is irreversible because of degradation of receptor-bound insulin, which may result in termination of the metabolic activation. Morphological and biochemical work suggests that internalization of the receptor-insulin complex from the plasma membrane transfers insulin to intracellular organelles like the lysosomes, the Golgi apparatus, or nucleus, where degradation by insulin protease takes place, whereas the receptor is recycled back to the membrane. Recent advances in the studies of biosynthesis and cellular dynamics of receptors indicate that intracellular processing and redistribution of binding sites may play a role in the mechanism of insulin action. Insulin receptors are widely distributed in all cell types, but evidence has accumulated that receptors show tissue and species variations in their functional properties regarding binding affinity, insulin specificity, cooperativity, and insulin degradation and in structural properties such as antigenic determinants and glycosidic composition. Perhaps these differences reflect cellular adaptations and variations in the physiological role of insulin.(ABSTRACT TRUNCATED AT 400 WORDS)
197 citations
TL;DR: Two polypeptides of Mr approximately 29,000 and 27,000 have been identified in human erythrocyte membranes that cross-react specifically with affinity purified antibodies to chicken gizzard tropomyosin, indicating that they are membrane-bound proteins under physiological conditions.
176 citations
TL;DR: The notion that the proton motive force generated by respiration is required for conformational changes of the F1 sector of the H+-ATPase complex has gained acceptance and is supported by kinetic data and by the demonstration of partial site reactivity in inactivation experiments performed with selective chemical modifiers.
Abstract: This review concerns the catalytic sector of F1 factor of the H+-dependent ATPases in mitochondria (MF1), bacteria (BF1) and chloroplasts (CF1). The three types of Ft have many similarities with respect to the structural parameters, subunit composition and catalytic mechanism. An α3β3γ2δ2e2 stoichiometry is now accepted for MF1 and BF1; the α2β2γ2δ2ɛ2 stoichiometry for CFI remains as matter of debate. The major subunits α, β and γ are equivalent in MF1, BF1 and CF1; this is not the case for the minor subunits δ and e. The δ subunit of MFI corresponds to the e subunit of BF1 and CF1, whereas the mitochondria) subunit equivalent to the δ subunit of BF1 and CF1 is probably the oligomycin sensitivity conferring protein (OSCP). The a β γ assembly is endowed with ATPase activity, β being considered as the catalytic subunit and y as a proton gate. On the other hand, the 6 and E subunits of BFI and CFI most probably act as links between the F1 and F0 sectors of the ATPase complex. The natural mitochondria) ATPase inhibitor, which is a separate protein loosely attached to MF1, could have its counterpart in the E subunit of BF1 and CF1. The generally accepted view that the catalytic subunit in the different F1 species is β comes from a number of approaches, including chemical modification, specific photolabeling and, in the case of BF1, use of mutants. The a subunit also plays a central role in catalysis, since structural alteration of a by chemical modification or mutation results in loss of activity of the whole molecule of F1. The notion that the proton motive force generated by respiration is required for conformational changes of the F1 sector of the H+-ATPase complex has gained acceptance. During the course of ATP synthesis, conversion of bound ADP and Pi into bound ATP probably requires little energy input; only the release of the F1-bound ATP would consume energy. ADP and Pi most likely bind at one catalytic site of F1, while ATP is released at another site. This mechanism, which underlines the alternating cooperativity of subunits in F1, is supported by kinetic data and also by the demonstration of partial site reactivity in inactivation experiments performed with selective chemical modifiers. One obvious advantage of the alternating site mechanism is that the released ATP cannot bind to its original site. The chemistry of the condensation reaction of ADP and Pi to form ATP has not yet been elucidated. Although implicitly admitted, definite evidence that the condensation reaction does not involve a phosphorylated intermediate has been acquired recently by analysis of the stereochemical course of the phosphoric residue transfer in ATP synthesis or hydrolysis. Whereas the catalytic events of ATP synthesis are well understood, the regulatory mechanism, and particularly the role of the so-called inhibitory peptides, remain enigmatic.
119 citations
TL;DR: Results indicated the cooperativity of Cd binding to double-stranded DNA and competition by Ca, Mg, and Zn for Cd-binding sites in DNA and their in vivo capacity to antagonize C d-induced carcinogenesis parallels that of these physiological essential metals.
115 citations
TL;DR: The initial velocity patterns for the amination reaction were obtained under conditions of hyperbolic kinetics produced by GlcNAc6 P; the Km values for the allosteric substrates were determined under the same conditions, and their dependence upon pH was studied.
92 citations
TL;DR: Possible allosteric regulation of fructose-6-P, 2-kinase and fructose 2,6-bisphosphatase in a reciprocal manner in vivo is discussed.
79 citations
TL;DR: The thermodynamics of ethidium ion binding to the double strands formed by the ribooligonucleotides rCA5G + rCU5G and the analogous deoxyribo‐oligonucleotide were determined by monitoring the absorbance versus temperature at 260 and 283 nm at several concentrations of oligonucleotide and ethidium bromide.
Abstract: The thermodynamics of ethidium ion binding to the double strands formed by the ribooligonucleotides rCA5G + rCU5G and the analogous deoxyribo-oligonucleotides dCA5G + dCT5G were determined by monitoring the absorbance versus temperature at 260 and 283 nm at several concentrations of oligonucleotides and ethidium bromide. A maximum of three ethidium ions bind to the oligonucleotides, which is consistent with intercalation and nearest-neighbor exclusion. For the ribo-oligonucleotide the binding mechanism is complex. Either two sites (assumed to be the intercalation sites at the two ends of the oligonucleotide) bind more strongly by a factor of 140 than the third site, or all sites are identical, but there is strong anticooperativity on binding (cooperativity parameter, 0.1). In sharp contrast, the binding to the same sequence (with thymine substituted for uracil) in the deoxyribo-oligonucleotide showed all sites equivalent and no cooperativity. For the ribo-oligonucleotides the enthalpy for ethidium binding is −14 kcal/mol. The equilibrium constants at 25°C depend on the model; either K = 6 × 105M−1 for the two strong sites (4 × 103M−1 for the weak site) or K = 2.5 × 105M−1 for the intrinsic constant of the anticooperative model. For the equivalent deoxyribo-oligonucleotide the enthalpy of binding is -9 kcal/mol and the equilibrium constant at 25°C is a factor of 10 smaller (K = 2.5 × 104M−1).
72 citations
TL;DR: The rate-determining step in the transition is found to occur in a mode which is kept local by distortion of nearby parts of the molecule, one especially important type of cooperativity, crank-like counterrotation of second-neighbor bonds, is identified.
Abstract: Conformational transitions in polymers involve large angle rotations about bonds The process must proceed in a way that does not require gross movements of the macromolecules The dynamics have been investigated by computer simulation and kinetic theory The rate-determining step in the transition is found to occur in a mode which is kept local by distortion of nearby parts of the molecule One especially important type of cooperativity, crank-like counterrotation of second-neighbor bonds, is identified Experiments which provide evidence about the dynamics of conformational transitions are discussed
69 citations
TL;DR: A slow transition model assumes that hexokinase D exists mainly in one conformation state (E1) in the absence of ligands and that the binding of glucose (or mannose) induces a conformational transition to EII, which would have a higher affinity for the sugar substrates and a higher catalytic activity than EI.
Abstract: Hexokinase D ('glucokinase') displays positive cooperativity with mannose with the same h values (1.5-1.6) as with glucose but with higher K0.5 values (8 mM at pH 8.0 and 12 mM at pH 7.5). In contrast, fructose and 2-deoxyglucose exhibit Michaelian kinetics [Cardenas, M. L., Rabajille, E., and Niemeyer, H. (1979) Arch. Biol. Med. Exp. 12, 571-580; Cardenas, M. L., Rabajille, E., and Niemeyer, H. (1984) Biochem. J. 222, 363-370]. Mannose, fructose, 2-deoxyglucose and N-acetylglucosamine acted as competitive inhibitors of glucose phosphorylation and decreased the cooperativity with glucose. Their relative efficiency for reducing the value of h to 1.0 was: fructose greater than mannose greater than 2-deoxyglucose greater than N-acetylglucosamine. Galactose, which is not a substrate nor an inhibitor, was unable to change the cooperativity. The competitive inhibition of glucose phosphorylation by N-acetylglucosamine or mannose was cooperative at very low glucose concentrations (less than 0.5 K0.5), suggesting the interaction of the inhibitors with more than one enzyme form. These and previously reported results are discussed on the basis of a slow transition model, which assumes that hexokinase D exists mainly in one conformation state (E1) in the absence of ligands and that the binding of glucose (or mannose) induces a conformational transition to EII. This new conformation would have a higher affinity for the sugar substrates and a higher catalytic activity than EI. Cooperativity would emerge from shifts of the steady-state distribution between the two enzyme forms as the sugar concentration increase. The inhibitors would suppress cooperativity with glucose by inducing or trapping the EII conformation. In addition, the model postulates that the different kinetic behaviour of hexokinase D with the different sugar substrates, cooperative with glucose and mannose and Michaelian with 2-deoxyglucose and fructose, is the consequence of differences in the velocities of the conformational transitions induced by the sugar substrates.
64 citations
TL;DR: It is demonstrated that the monomeric and dimeric structures of the nicotinic acetylcholine receptor of Torpedo californica electric tissue, reconstituted in planar lipid bilayers, are functionally different.
Abstract: It is demonstrated that the monomeric and dimeric structures of the nicotinic acetylcholine receptor of Torpedo californica electric tissue, reconstituted in planar lipid bilayers, are functionally different. The native dimer D of Mr 500,000 (heavy-form) exhibits a "single" channel conductance about twice as large as that of the monomer M of Mr 250,000 (light form). Under conditions where monomers aggregate, the conductance changes from the level of the monomer M to that of dimers M2. The dimer conductances (D and M2) seem to result from synchronous opening and closing of the two channels in the dimer, giving the impression of "single channel" activity. This channel cooperativity is apparently mediated by noncovalent interactions between the two monomers, since it requires no disulfide linkage between monomers. Both the monomers M and the dimers D and M2 show at least one substate of lower conductivity. The relative population of the two conductance levels depends on the ion type (Na+ and K+), indicating ion-specific channel states. Since the channel conductance of isolated dimers resembles those obtained from unextracted microsacs, the dimer with two synchronized channels appears to be the in vivo predominant gating unit. In the linear association of dimers, observed in the native membrane, channel synchronization may extend to more than two channels as suggested by oligomeric channel cooperativity in associations of monomers and dimers.
TL;DR: The complex of Lactobacillus casei dihydrofolate reductase with trimethoprim and NADP+ exists in solution as a mixture of approximately equal amounts of two slowly interconverting conformational states, and a partial structural model has been proposed.
Abstract: The complex of Lactobacillus casei dihydrofolate reductase with trimethoprim and NADP+ exists in solution as a mixture of approximately equal amounts of two slowly interconverting conformational states [Gronenborn, A., Birdsall, B., Hyde, E. I., Roberts, G. C. K., Feeney, J., & Burgen, A. S. V. (1981) Mol. Pharmacol. 20, 145]. These have now been further characterized by multinuclear NMR experiments, and a partial structural model has been proposed. 1H NMR spectra at 500 MHz show that the environments of six of the seven histidine residues differ between the two conformations. The characteristic 1H and 31P chemical shifts of nuclei of the coenzyme in the two conformations of the complex are identical in analogous complexes formed with a number of trimethoprim analogues, indicating that the nature of the two conformations is the same in each case. The pyrophosphate 31P resonances have been assigned to the two conformations, and integration of the 31P spectrum shows that the ratio of conformation I to conformation II varies from 0.4 to 2.3 in the complexes with the various trimethoprim analogues, the ratio for the trimethoprim complex itself being 1.2. Transferred NOE experiments, together with the 1H and 13C chemical shifts, indicate that in conformation II of the complex the nicotinamide ring of the coenzyme has swung away from the enzyme surface into solution; this is made possible by changes in the conformation of the pyrophosphate moiety. In conformation I, by contrast, the nicotinamide ring remains bound to the enzyme. 13C and 15N experiments show that trimethoprim is protonated on N1 in both conformations of the ternary complex. Analysis of the 1H chemical shifts of trimethoprim in terms of ring current effects shows that in conformation I of the ternary complex trimethoprim retains the same conformation as in its binary complex, but 13C, 15N, and 19F [using 2,4-diamino-5-(3,5-dimethoxy-4-fluoro-benzyl)pyrimidine] experiments show that the environment of both the pyrimidine ring and benzyl ring is affected by the proximity of the coenzyme. Less information is available about the conformation of the inhibitor in conformation II of the complex, but its environment is similar to that in the binary enzyme-inhibitor complex. The implications of the existence of these two conformations of the enzyme for understanding cooperativity in binding between NADP+ and trimethoprim are briefly discussed.
TL;DR: It is concluded that the acid groups ionize and hydrate rapidly and noncooperatively in both proteins, consistent with the known (lysozyme) and postulated (α‐lactalbumin) surface chemistry.
Abstract: Direct difference ir spectra are presented as a function of hydration for lysozyme and α-lactalbumin, and detailed sequential hydration molecular events identified. Despite the strong sequence homology between the two proteins, and their expected conformational similarity, the hydration behaviour of the polar groups is different for the two proteins. Using a Hill-type analysis, we conclude that the acid groups ionize and hydrate rapidly and noncooperatively in both proteins, consistent with the known (lysozyme) and postulated (α-lactalbumin) surface chemistry. The polar group hydration shows a clear cooperativity, which is quantitatively different in the two proteins. Complementary work suggests this cooperativity relates to a hydration-induced “loosening up” of the lysozyme conformation at about 55 mol water/mol protein. α-Lactalbumin appears to “open up” more easily for hydration than does lysozyme, consistent with its lower stability against thermal and acid denaturation.
TL;DR: The 1H NMR spectrum of calsequestrin in the calcium-free form is presented and is characteristic of a protein largely in the random coil configuration and a number of peaks in the aromatic region have been assigned based on their chemical shifts and sensitivity to pH.
TL;DR: The results showed that PK remains tetrameric both in the presence and in the absence of Phe; however, Phe induces a small decrease in the sedimentation coefficient of the enzyme; hence, it suggests a loosening of the protein structure.
Abstract: The mechanism of allosteric regulation of rabbit muscle pyruvate kinase (PK) was examined in the presence of the allosteric inhibitor phenylalanine (Phe). Steady-state kinetic, equilibrium binding, and structural studies were conducted to provide a broad data base to establish a reasonable model for the interactions. Phe was shown to induce apparent cooperativity in the steady-state kinetic measurements at pH 7.5 and 23 degrees C. The apparent Km for phosphoenolpyruvate was shown to increase with increasing Phe concentrations. These results imply that Phe reduces the affinity of PK for phosphoenolpyruvate. This conclusion was substantiated by equilibrium binding studies which yielded association constants of phosphoenolpyruvate as a function of Phe concentration. The binding constant of Phe was also determined at pH 7.0 and 23 degrees C. The effect of ligands on the hydrodynamic properties of PK was monitored by difference sedimentation velocity, sedimentation velocity, and equilibrium experiments. The results showed that PK remains tetrameric both in the presence and in the absence of Phe. However, Phe induces a small decrease in the sedimentation coefficient of the enzyme; hence, it suggests a loosening of the protein structure. The accessibility of the sulfhydryl residues of the enzyme also increases in the presence of Phe. Furthermore, the Phe-induced conformational change was approximately 90% complete when only 25% of the binding sites were saturated. This result suggested that the regulatory behavior of PK might satisfactorily be described by the two-state model of Monod-Wyman-Changeux [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118].
TL;DR: It is shown that in liganded hemoglobin the changes in structure that occur at the subunit interface upon the quaternary structure transition are accommodated away from the heme by relatively weak bonds in the protein.
Abstract: Using resonance Raman and infrared absorption spectroscopies, we show that there are no energetically significant structural changes at the heme upon the quaternary structure transition in six-coordinate hemoglobins. These observations are at variance with the presently accepted mechanism for cooperativity, which postulates severe strain in the T quaternary structure of liganded hemoglobin. By consideration of the present results, and studies on deoxyhemoglobins and photodissociated hemoglobins, a view of the distribution of the free energy of cooperativity emerges. In five-coordinate deoxyhemoglobins the iron-histidine bond is able to respond to the protein structure, thereby accounting for a wide variation (40 cm(1] in its frequency. In contrast, when a sixth ligand is present and the iron is pulled into plane, the histidine-heme-ligand complex becomes structurally rigid, thereby preventing protein-induced changes at the heme. Instead, in liganded hemoglobin the changes in structure that occur at the subunit interface upon the quaternary structure transition are accommodated away from the heme by relatively weak bonds in the protein.
TL;DR: Preliminary data in vivo suggest that estrogen administration to the animal can influence the relative titer of the low affinity sites and no cooperativity between the two sites has been detected in analysis using either rate kinetics or equilibrium methods.
TL;DR: Analysis of the kinetics of the reaction with oxygen and carbon monoxide of the homodimeric hemoglobin from the bivalve mollusc Scapharca inaequivalvis shows unequivocally that the presence of two different types of chain is not an absolute prerequisite for cooperativity in hemoglobins, contrary to currently accepted ideas.
01 Jan 1984
TL;DR: Garner et al. as discussed by the authors used the H,02-modified uncoupled (Na,K)-ATPase isolated from cultured bovine lenses to study the kinetics of ATP and p-nitrophenylphosphate and the action of the allosteric effectors, Na+ and K+, upon the hydrolysis of these substrates.
Abstract: The kinetics of hydrolysis of ATP and p-nitrophenylphosphate and the action of the allosteric effectors, Na+ and K+, upon the hydrolysis of these substrates were used to study the H,02-modified, uncoupled (Na,K)-ATPase isolated from cultured bovine lenses (Garner, W. H., Garner, M. H., and Spector, A. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2044-2048). Pure bovine renal (Na,K)-ATPase was modified by Hz02 in 150 mM KC1 and 20 mM MgClz to yield an enzyme with kinetic properties similar to the enzyme isolated from the HzOz-treated, cultured bovine lens. Hz02 modification changes the interaction of the ATP hydrolysis site from negative to positive kinetic cooperativity. HzOz modification dramatically alters Na+ stimulation of ATP hydrolysis and Na+ inhibition of p-nitrophenylphosphate hydrolysis while having little effect upon K+ control of the hydrolysis of these two substrates.
TL;DR: The spectral and thermodynamic data provide evidence that AO (and other intercalating agents) induces denaturation of dsucleic acids; the driving force of the denaturation is high affinity and cooperativity of binding of these ligands to ss nucleic acids.
Abstract: At high binding denstities acridine orange (AO) forms complexes with ds DNA which are insoluble in aqueous media. These complexes are characterized by high red- and minimal green-luminescence, 1:1 (dye/P) stoichiometry and resemble complexes of AO with ss nucleic acids. Formation of these complexes can be conveniently monitored by light scatter measurements. Light scattering properties of these complexes are believed to result from the condensation of nucleic acids induced by the cationic, intercalating ligands. The spectral and thermodynamic data provide evidence that AO (and other intercalating agents) induces denaturation of ds nucleic acids; the driving force of the denaturation is high affinity and cooperativity of binding of these ligands to ss nucleic acids. The denaturing effects of AO, adriamycin and ellipticine were confirmed by biochemical studies on accessibility of DNA bases (in complexes with these ligands) to the external probes. The denaturing properties of AO vary depending on th...
TL;DR: It was concluded that glycerate-2,3-P2 could function both as an activator and an inhibitor of casein kinase II in the erythroid cell by binding at the substrate binding site.
TL;DR: Results are extended to include ligands of any shape and cooperative interactions, and data on the cooperative binding of polymyxin to charged lipid bilayers are reevaluated.
TL;DR: Pure bovine renal (Na,K)-ATPase was modified by H2O2 in 150 mM KCl and 20 mM MgCl2 to yield an enzyme with kinetic properties similar to the enzyme isolated from the H 2O2-treated, cultured bovines lens.
TL;DR: Monomethoxypolyoxyethylene was covalently linked to human hemoglobin via an amide bond formed between amino groups of the protein and a carboxylic group introduced onto the polymer, and the resulting conjugates exhibited oxygen-binding characteristics quite similar to those of native hemoglobin.
TL;DR: A concerted, allosteric model is developed, and equations are derived for quantitative interpretation of the kinetic and equilibrium binding data of rabbit muscle pyruvate kinase at pH 7.5 and 23 degrees C to estimate the parameters which best predict the total body of experimental results.
Abstract: A concerted, allosteric model is developed, and equations are derivsd for quantitative interpretation of the kinetic and equilibrium binding data of rabbit muscle pyruvate kinase at pH 7.5 and 23 OC. The simplest model which seems likely to rationalize the experimental data involves two con- formational states. In this model, two simplifying assumptions are made. First, the affinities of pyruvate kinase for both substrate and inhibitor are assumed to depend only upon the conformational state of the tetrameric enzyme. Second, the rate of product formation is also assumed to depend only upon In the preceding paper (Oberfelder et al., 1984), it was shown that rabbit muscle pyruvate kinase (PK)' exhibits simple hyperbolic kinetics in the absence of Phe. The presence of Phe, however, induces sigmoidicity in the relation between activity and PEP concentration. This suggests a degree of cooperativity in PEP binding to PK which increases with Phe concentration. Results from equilibrium binding studies substantiate the kinetic observations in that the apparent affinity of PEP for PK is reduced in the presence of Phe which acts as a heter- otropic effector. Difference sedimentation velocity and sulf- hydryl titration experiments demonstrate unequivocally that Phe induces a gross structural change in PK and that com- pletion of the protein structural change precedes the saturation of the binding sites in PK by Phe. Thus, it was proposed that the regulation of rabbit muscle PK by Phe can be best rep- resented by the two-state model of Monod, Wyman, and Changeux (Monod et al., 1965). In this paper, methods are derived for quantitative interpretation of the kinetic and equilibrium binding data described in the preceding paper (Oberfelder et al., 1984). The results of such an analysis are presented, and their implications concerning the allosteric regulation of PK are discussed.
TL;DR: The results in the present study indicate that specific 3H-SP binding reaches a plateau only after 20 minutes of incubation, and the binding sites are saturable at a relatively low concentration of 3 H-SP.
TL;DR: The hemoglobin of the bullfrog, Rana catesbeiana, forms aggregates larger than tetramers in two ways, which results from the association of the deoxygenated forms of the two major components to form reversibly an aggregate which is believed to be a trimer, BC2.
TL;DR: Thepositive cooperativity of the unidirectional sulfate and phosphate fluxes under selfexchange conditions and the disappearance of the positive cooperativity under homoex exchange conditions indicate a cooperativity in the translocation process.
Abstract: The concentration dependence of the sulfate and the phosphate selfexchange and homoexchange fluxes was studied in resealed red cell ghosts (25° C, pH 7.3). The selfexchange fluxes were calculated from the rate constant of the tracer back-exchange and from the intracellular substrate anion content. The homoexchange fluxes were determined from the initial cis-to-trans tracer fluxes and the initial specific substrate anion activities at the cis-membrane side. Sulfate and phosphate concentrations ranging from approx. 2–100 mM were employed. The selfexchange fluxes of sulfate and of phosphate exhibit sigmoidal flux/concentration curves. The apparent Hill coefficients were in the range of 1.2–1.4 indicating a type of positive cooperativity. Under homoexchange conditions the positive cooperativity of the flux/concentration curves disappears. The outward homoexchange fluxes of sulfate and phosphate display a saturation kinetics with Hill coefficients close to 1.0. The inward homoexchange fluxes exhibit a negative type of cooperativity with Hill coefficients smaller than 1.0. The sulfate and the phosphate half-saturation concentrations for the outer and the inner membrane surface are equal in size and amount to approx. 35 mM for sulfate and to approx. 110 mM for phosphate, respectively. The positive cooperativity of the unidirectional sulfate and phosphate fluxes under selfexchange conditions and the disappearance of the positive cooperativity under homoexchange conditions indicate a cooperativity of the translocation process. The saturation of the outward homoexchange flux and the negative cooperativity of the inward homoexchange flux suggest a substrate anion binding according to the law of mass action at the inner and a negative cooperativity of substrate anion binding at the outer membrane surface.
TL;DR: Several hybridoma cell lines secreting antibodies specific to the membrane (Na+,K+)-dependent ATPase from lamb kidney medulla have been isolated by using the methods developed by Kohler and Milstein and one of these antibodies has been shown to be directed against a functional epitope or antigenic site of the catalytic (alpha) subunit of the enzyme.
Abstract: Several hybridoma cell lines secreting antibodies specific to the membrane (Na+,K+)-dependent ATPase from lamb kidney medulla have been isolated by using the methods developed by Kohler and Milstein. One of these antibodies (designated M7-PB- E9 ) has been shown to be directed against a functional epitope or antigenic site of the catalytic (alpha) subunit of the enzyme. Although this antibody was raised to the "native" holoenzyme, it has a higher apparent affinity toward the isolated, delipidated, and inactive alpha subunit than toward the holoenzyme. This antibody shows a 10-fold faster initial rate of binding to the alpha subunit than to the holoenzyme. The antibody dissociation rates from both isolated alpha subunit and holoenzyme are similarly slow, and the binding can be considered a pseudoirreversible reaction. By binding at this site, the antibody, however, acts like a "partial competitive inhibitor" with respect to ATP and acts as an uncompetitive or mixed competitive inhibitor with respect to the Na+ and K+ dependence of ATPase hydrolysis. This antibody also does not alter the cooperativity at either the Na+ or the K+ sites. The antibody causes a partial inhibition of the Na+- and MgATP-dependent phosphoenzyme intermediate formation but has no effect on either ADP in equilibrium ATP exchange or the K+-stimulated dephosphorylation step. In addition, the K+-dependent p-nitrophenylphosphatase activity of the enzyme was not affected. In the presence of Mg2+, the antibody stimulates the rate of cardiac glycoside binding [( 3H]ouabain) to the (Na+,K+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: Through studies of CO on- and off-rates, the [Mn(III),Fe(II)] hybrids are used to obtain the pH dependence of the association constants for binding the fourth CO to the individual Hb chains, and their pH dependence provides estimates of the proton release at each step.
Abstract: In mixed-metal [Mn,Fe] hybrid hemoglobins (Hb), the two chains of a single type, alpha or beta, are substituted with manganese protoporphyrin IX, which does not bind CO in either the Mn(II) or Mn(III) valency states Thus, CO binding by the two ferrous subunits of a hybrid with Mn of either valency represents a simplified two-step Hb ligation process in which ligands bind to a single-chain type Considering the [Mn(II),Fe(II)] hybrids, which are deoxy T-state analogues, at pH 66 both types bind CO with low affinity (alpha-Fe, 038 mmHg; beta-Fe, 071 mmHg) and noncooperatively (Hill coefficient n = 1) At elevated pH, both exhibit an increase in affinity (Bohr effect) and strong cooperativity, with the alpha-Fe hybrid having a higher degree of cooperativity (n approximately equal to 16) than beta-Fe (approximately equal to 13) at pH 90 The CO association constants for the Hb ligation routes in which the first two ligands bind to the same chain type are obtained from these measurements, and their pH dependence provides estimates of the proton release at each step Through studies of CO on- and off-rates, the [Mn(III),Fe(II)] hybrids are used to obtain the pH dependence of the association constants for binding the fourth CO to the individual Hb chains(ABSTRACT TRUNCATED AT 250 WORDS)