Showing papers on "Cooperative binding published in 1970"

Cooperative binding to linear biopolymers. 2. Thermodynamic analysis of the proflavine-poly(L-glutamic acid) system.

Abstract: The binding of acridine orange to poly(α-l-glutamic acid) at pH 7.5 has been investigated by means of absorbance measurements at 492 nm. The equilibrium as well as chemical relaxation behavior is interpreted in the light of a basic model theory of cooperative binding. Binding and cooperativity are found to be stronger than in the previously studied case of proflavine. In principle, the static properties are analogous in both cases. The only distinct contrast is given by the fact that the degree of cooperativity is considerably affected by ionic strength and polymer to dye ratio. This is attributed to the effect of unfavorable electrostatic interactions which tend to prevent long aggregates of bound dye. Temperature jump measurements resulted in relaxation curves which agreed very well with the predictions of the theory. Mean relaxation times of cooperative binding could be determined and evaluated in terms of an effective rate constant for recombination of free dye with bound aggregates of dye. The effect of varying the concentration of added KCl can be attributed to blocking of reaction sites by bound K+ ions. Extrapolation to zero KCl concentration yields a diffusion controlled rate constant for cooperative binding of monomer dye (kRo is approx. 1.5 × 109 M–1 sec–1). The life time of terminal elements of bound aggregates of dye turns out to be about 400 μsec.

Cooperative Binding of Manganese (II) to Transfer RNA

Abstract: We have measured the binding of manganse ions to tRNA in the concentration range of 1 to 28 bound Mn2+ per tRNA at 23°. The free Mn2+ concentration was monitored by electron spin resonance. The Scatchard plot shows that the first four Mn2+ bind cooperatively, at a free Mn2+ concentration of 3 μM. The cooperativity presumably reflects a conformational change of tRNA. A resulting structure with 6–10 strong binding sites (ks= 1.8 × 105M−1), and 21–25 weak binding sites (kw= 5 × 103 M−1) is suggested. From the bound Mn2+ electron spin resonance we estimate a minimal distance between the strong sites of about 1.5 nm; this shows that the binding sites are spread on the molecule and not clustered together.

On the Prevalence of „Nonspecific” Binding at the Specific Binding Sites of Globular Proteins

Abstract: Strong binding of dyes to simple globular proteins takes place predominantly in areas overlapping the binding sites for substrates, coenzymes and prosthetic groups, in preference to other regions of the protein surface. The structure of the dyes bears no obvious relationship to that of the normal ligands. It is proposed that this phenomenon is a reflection of the special stereochemical features of such sites, their hydrophobicity relative to other portions of the protein surface, and, possibly, greater flexibility in these regions of the protein molecule. The binding properties of antibodies and bovine serum albumin are discussed in relation to this apparent versatility of protein binding sites towards structurally unrelated organic ligands.

Hybrid molecules of yeast and rabbit GPD containing native and modified subunits.

Abstract: CONVINCING evidence has been presented that molecules of glyceraldehyde-3-phosphate dehydrogenase (GPD) from yeast1 and from rabbit skeletal muscle2 each comprise four identical polypeptide chains and bind a maximum of four molecules of NAD per molecule of enzyme3–5. But whereas the binding of NAD to the yeast enzyme (Y4) at 40° C is considered3,4 to conform to the Monod, Wyman and Changeux model6 for allosteric interactions, which requires highly cooperative structural transitions of the subunits within a given enzyme molecule, the binding of NAD to rabbit GPD (R4) shows a negative cooperativity in that binding of successive molecules of NAD to a given GPD molecule diminishes the affinity for NAD of the remaining unoccupied sites5. As such the rabbit enzyme conforms to the “stepwise” model for ligand binding proposed by Koshland, Nemethy and Filmer7.

Cooperative binding of adenosine by polyuridylic acid: a further analysis.

Abstract: The dialysis data of Huang and Ts'o for the cooperative binding of adensine to polyuridylic acid are analyzed here using a grand-partition function Ising model method similar to that originally employed for polyelectrolytes by Rice and Nagasawa. An appropriate modification permitting the treatment of the sliding degeneracy of the two polyuridylic acid strands is also included. In addition to the previously estimated stacking energy of about −6 kcal/mole one also obtains the free energy change F for the transfer of a single adenosine molecule from a fixed site in solution to a fixed site on the polyuridylic acid. This binding energy falls in the range F = −140 to +620 cal/mole, indicating that binding in the 1:2 (purine: pyrimidine) complex is either very weak or repulsive. The absence of any comparable cooperative stacking of adenosines in solution at the same concentration together with the likely repulsive character of the binding implies that the stacking energy must contain a significant contribution from other processes than simple stacking of adenosines. A generalization of the theory to treat multicomponent binding and longer-range interactions is effected, and the form applicable to simultaneous binding of both adenosine and guanosine by polyuridylic acid is presented.

A Factor for the Binding of Aminoacyl Transfer RNA to Mammalian 40S Ribosomal Subunits

Abstract: A factor present in rat liver supernatant catalyzes binding of Phe-tRNA to 40S ribosomal subunits from rat skeletal muscle. This factor could be distinguished from aminoacyltransferase I by a number of criteria: (1) at lower concentrations of magnesium (5 mM) the 40S binding factor was approximately seven times as effective as T-I in catalyzing binding of Phe-tRNA to 40S subunits; (2) the kinetics of the binding reaction were different when catalyzed by the 40S binding factor, in particular the initial rate was greater than in the presence of T-I—indeed, the kinetics of the T-I catalyzed reaction resembled nonenzymic binding; (3) GTP was required for maximal binding of Phe-tRNA to 40S subunits in the presence of the 40S binding factor, but not for the T-I catalyzed reaction; (4) the 40S binding factor was inactivated by N-ethylmaleimide whereas T-I was not; (5) finally, the 40S binding factor was more susceptible to heat inactivation. Binding of aminoacyl-tRNA to 40S ribosomal subunits may be a paradigm for the initiation of protein synthesis, and the 40S binding factor may play a role in the process.

Studies on the Formation of Transfer Ribonucleic Acid-Ribosome Complexes, XII. Phenylalanyl-Oligonucleotide Binding to E. coli Ribosomes: Necessity for a Free Amino Group

Abstract: The binding of phenylalanyl-oligonucleotide, C-A-C-C-A-(Phe), to ribosomes requires the presence of a free amino group on the amino acid. Acetylation of the amino acid reduces the binding to ribosomes to about 1/20 of the binding observed with the intact Phe-oligonucleotide. Deacylation of the phenylalanyl-oligonucleotide eliminates binding of the free amino acid and markedly reduces the binding of the oligonucleotide (C-A-C-C-A). Neither 30S nor 50S subunits alone are sufficient for binding of the phenylalanyl-oligonucleotide; the presence of both subunits is necessary. The data suggest that phenylalanyl-oligonucleotide binding to ribosomes represents the binding of the aminoacyl-terminus of phenylalanyl-tRNA and that the presence of an amino acid with an unsubstituted amino group attached to the oligonucleotide (C-A-C-C-A) is required for binding to this potassium-dependent site. Furthermore, the ribosome itself has the capability of distinguishing between aminoacyl-oligonucleotides with N-substituted and unsubstituted amino acids-

The Influence of the 2′OH Group upon the Binding of Nucleotides by Basic Polyamino Acids

Abstract: The position of the phosphate group on the ribose of nucleoside monophosphates markedly influences their binding behavior with basic polyamino acids, such as poly-l-lysine and poly-l-arginine. Equilibrium dialysis of adenine and guanine derivatives has revealed that the binding of 5′- and 3′-ribonucleotides (group I, possessing a free 2′OH group) to basic polyamino acids is clearly different from that of 2′-ribo and 5′-deoxyribonucleotides (group II, the 2′OH group is blocked and absent, respect.). At low nucleotide to poly-l-lysine ratios, where the binding corresponds to the Langmuir isotherm, the binding constants of group I are smaller than those of group II. At higher nucleotide to polypeptide ratios the binding is cooperative and accompanied by a phase transition: guanine derivatives of group I show precipitation with poly-l-lysine at 0′, whereas with group II a gel-like phase is separated; with poly-l-arginine only precipitation occurs. Analysis of the cooperative binding with guanine nucleotides and poly-l-arginine revealed that group I possesses considerably higher nearest neighbour interaction energies than group II. The stacking coefficients decrease in the order 3′-GMP > 5′-GMP > 5′-dGMP > 2′-GMP. The implications of these results are discussed.

Conformational Effects of NAD+ on Yeast Glyceraldehyde-3-Phosphate Dehydrogenase

Abstract: As shown by hydrodynamic and spectroscopic techniques the enzyme-coenzyme interaction of glyceraldehyde-3-phosphate dehydrogenase with NAD is accompanied by structural changes. The tetrameric structure of the holoenzyme persists under all but strong dissociating conditions, proving that there is no change in the quaternary structure on binding. On the other hand, binding is accompanied by an increase in the sedimentation and diffusion coefficients which in the case of S 20,w 0 is too large to be explicable in terms of the increase in particle weight by bound ligand. Optical rotation, optical rotatory dispersion, and circular dichroism show parallel changes wich are shown to be conformational in origin, rather than a consequence of NAD attachment per se. Increasing temperature in the range 20–40°C leads to a transition from non-cooperative to cooperative binding of NAD+ to the apoenzyme. The hyperbolic profile of the structural parameters at 20° closely parallels the binding curve, while at 40° the sigmoidal binding curve and the data representing the structural changes deviate in a characteristic way consistent with a two-state “concerted” model for ligand binding.

Subunit Interactions in Glyceraldehyde-3-Phosphate Dehydrogenase: A Fluorometric and Calorimetric Analysis of DPN Binding as a Function of Temperature

Abstract: Some rather large effects of temperature on the kinetics of reactions catalyzed by the rabbit muscle and liver glyceraldehyde-3-phosphate dehydrogenases (GPD) and the results of Kirschner and coworkers (1) on the temperature perturbation of DPN binding by the enzyme from yeast prompted us to examine the temperature dependence of DPN and DPNH binding by the mammalian enzyme and to determine the thermodynamic parameters. In order to cope with the very strong coenzyme binding of the mammalian GPD, we employed the quenching of protein fluorescence as an indicator of complex formation (2) after establishing that the quenching was colinear with binding at the three high affinity sites of the protein. The fourth and weakest binding site does not give an optical signal for ligand addition and must be examined by separat ion methods (3, 4). A set of fluorescence quenching titrations at a series of temperatures is shown in Fig.1. The results at 2.5° are in approximate accord with estimates made by Conway and Koshland (3) at 4° and at much higher protein concentrations by the stepwise removal of bound DPN in a series of prolonged dialyses. The effect of increasing the temperature is to weaken the bind ing and to make the curves progressive I y steeper at their midpoints. Whereas three constants are required to describe the curve at 2.5° , the curve at 36° may be described by a sing le intrinsic binding constant. A similar but slightly less extreme set of curves was obtained in the titration of apo-enzyme with DPNH. The convergence of the three K’s with in-creasing temperature suggest that at still higher temperatures a positive cooperativity might develop, analo-gous to that observed with the yeast enzyme at 40°. However, stability restrictions have prevented us from going much above 36° so that this possibility could not be tested.