Independent sites

To understand the Na,K-pump mechanism it is obviously important to identify the cation pathway and the sites for binding and occlusion of Na and K relative to the intramembrane portion of the protein. The groups coordinating the cations should be identified and it should be known if the pump has independent sites for Na" and K" " or if the cations bind alternately to the same set of sites. With a stoichiometry of 3Na /2K per ATP split this would mean that two sites bind Na and alternately, while one site only binds Na". ... 

The interpretation of much of the binding data given so far is based upon the assumption that the high affinity binding sites represent a population of independent sites. In the unphosphorylated II" " these sites would open up either to the periplas-mic or cytoplasmic side of the membrane independently of each other. The assumption ignores the evidence that the enzyme is, in fact, multimeric and that the data... 

Guyenet, P. G. Aghajanian, G. K. (1979). ACh, substance P and met-enkephalin in the locus coeruleus pharmacological evidence for independent sites of action. 

The above procedures imply that (1) there is only a single type of site (2) binding occurs only to the transporter site (usually not the case for trace metals), and (3) the internalisation flux is negligible for the equilibration times that are employed [197,198], These conditions are rarely fulfilled for metal transporters. The interpretation of Scatchard plots is especially ambiguous in the presence of several independent sites. On the other hand, in the biomedical literature, where nonspecific adsorption is generally not a problem, values of 104 to 106 carriers per cell (ca. 10-13 to 10 11 carriers cm-2 of cell surface area), with even lower numbers determined for some receptors (e.g. haematopoetic growth factor [199]), are typically reported. 

Crystal interactions may lead to a number of readily observable consequences. The molecular geometries may be different in different polymorphic forms of a given compound or, as in the case above, in symmetry-independent sites in a given crystal a change of molecular substituents remote from the torsionally flexible bonds may change the molecular conformation, as a result of modification... 

An example of the second effect is provided by mono-sec-butyl phthalamide (17a) (56). In the crystal the two enantiomers of this molecule are miscible in all proportions. The racemate crystallizes in space group PI (two general positions in the unit cell) with four molecules per unit cell. Thus there are two molecules in the asymmetric unit. The sec-butyl moieties adopt the anti conformation (the two geometries are shown schematically in 17b) and exhibit conformational disorder to different extents at the two symmetry-independent sites. 

When the two wells are of similar energies, and the crystal structure allows, the above will no longer be the situation. We may then expect a number of consequences There may be a measurable displacement of the hydrogen between the two sites induced by such factors as a change in temperature, application of an electric field, and irradiation with light both tautomers may be present at symmetry-independent sites in the crystal different tautomers may be present in different crystal modifications and the presence of molecular substituents that do not directly affect the properties of the hydrogen bond may influence the tautomerism via the crystal structure. 

Complete clustering, or segregation, of tautomers may occur during crystal growth. This can lead, for example, to crystals that consist of two tautomers in equal proportions and located at crystallographically independent sites. This is the case for isocytosine, 47 (104). Similarly, in crystalline anthranilic acid the neutral molecule and the zwitterion coexist (10S). Another example, 39, has been mentioned already (82). 

We shall present here an intuitively plausible argument on how to construct the probabilities of the various events of our new system, having m independent sites, opened with respect to the ligand. Each site, say j, can be in one of two states empty, with probability Pj 0), and occupied, with probability P,(l). Since the system is at equilibrium with the ligand at some fixed chemical potential, it is reasonable to assume that the probability ratio Pj(l)/Pj(0) is proportional to two factors one that measures the affinity of the site to the ligand, which we denote by q., and the second that depends on the concentration C of the ligand in the reservoir, i.e.. 

In Eq. (1.1.17) we derived the GPF of a system having m independent sites. Statistical mechanics provide the recipe for constructing the GPF for more general systems. This is discussed in Section 1.4. Here, we present the general form of the GPF of a single adsorbent molecule with m (identical or different) binding sites, namely,... 

In Section 3.5 we discussed the phenomenon of spurious cooperativity in single-site systems. Since cooperativity, as defined in this book, is undefinable for single-site systems, any apparent cooperative behavior must be due to the presence of different and independent sites. In Section 4.4 we encounter the same phenomenon in two-site systems with different sites. This was shown to be equivalent to the system in Section 3.5. 

The first, 7(0, can be determined from the experimental data for any finite value of C. The second, 7,<0, may be interpreted as the same integral 7(0, but computed for a hypothetical system of independent sites. As we shall see below, this interpretation is somewhat risky and should be avoided. The function 7,.(Q is better viewed as defined in Eq. (5.8.10), with the binding constants determined in Eq. (5.8.8). 

For all the above reasons we have defined g(C) without reference to any hypothetical, independent-site system. One simply extracts both 1(C) and all from the experimental data, and then constructs the quantity g(C). When the sites are identical in a weak sense, i.e., all k = k, some of the correlations for a given / might differ. For example, four identical subunits arranged in a square will have only one intrinsic binding constant k, but two different pair correlation functions. For this particular example we have four nearest-neighbor pair correlations g (2), and two second-nearest-neighbor pair correlations gJJ)- The average correlation for this case is... 

The unit cell contains 12 molecules in three crystallographically independent sites. The space group is P2i/c. The bond characteristics are... 

A ligand binding plot (1/n versus l/[Aj) that permits one to determine the number of ligand binding sites and the dissociation constant for the protein-ligand complex. The typical binding equation for -independent sites is... 

In either case, polyuridylic acid or polythymidylic acid, the rate of dimer formation will not be that calculated for an assemblage of independent sites, as in a solution of mononucleotide or dinucleotide, given by eq. (11). The sites are not independent but are in a connected sequence along a one-dimensional chain, and the rate of dimer formation will begin to deviate from the rate law of eq. (11) as soon as the location of unreacted sites becomes an important factor, certainly when... 

The influence of the nonuniform character of the interior of zeolites on the photophysics of adsorbed guest molecules has been observed. Pyrene molecules included in zeolite faujasites show both monomer and excimer emission [232,233]. As in the case of silica surfaces, the excitation spectra of the emission corresponding to the monomer and the excimer differ (Figure 36), suggesting that there are at least two independent sites, each responsible for monomer emission and excimer emission. Time-resolved emission studies of pyrene included in Na + -X and Na + -Y (<0.1 molecule per cage) indicate... 

The quantitative formulation of the site sorption mode, on the other hand, has the virtue of simplicity, but is undoubtedly rather highly idealised. Ideally, Eq. (9) refers to a collection of distinct, permanent and independent sites each accomodating one penetrant molecule Sq measures the concentration of these sites in the membrane and K2 their affinity for the penetrant assuming them to be isoenergetic 35,36). On this basis, the temperature dependence of K2 should yield a constant enthalpy for this sorption mode AH2 35,36). Consistency with the physical picture presented above requires moreover that AH2 be more exothermic than AH, U). 

The technique is temperature programmed desorption and reaction, or more generally, thermal desorption and reaction. The principle of the technique is simple. A reactant molecule is adsorbed at low temperatures on an active site. The temperature is then raised in a controlled manner and the evolution of the reaction products is monitored. If the various products are evolved independent of each other, they must be formed on independent sites. 

A) or adding H2PO4" into host 4 solution at 0.69 mM (panel B). The lines represent the best fit to a two-independent-site-model. 

Parker and Dalgleish (1977a,b) considered aSi-casein to have / equally reactive and independent sites (functionalities) per monomer, enabling the casein to aggregate and form a coagulum of potentially infinite molecular weight, according to the condition... 

The Independent Site Alive and Well. CenterWatch. Vol. 3, Issue 4. August 1, 1996. 

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