Binding stoichiometry

MacrocycHc receptors 31a and 31b were found to bind both dihydrogen phosphate and chloride in exclusively 1 1 host/guest stoichiometries. Binding constants were calculated for 31a and 31b with dihydrogen phosphate and chloride and revealed that dihydrogenphosphate was bound more strongly (2.5 X 10 M for 31a and 4.0 x 10 M for 31b) than chloride (500 for 31a and <50 M for 31b). 

Thus, NMR spectroscopy has the potential to provide almost complete information on ligand-CD interactions (stoichiometry, binding constants, free energy, enthalpy and entropy of complex formation, dynamics and structure of the complexes) in a solution. The kinetics of complex formation is usually too fast on the NMR time scale and therefore difficult to follow using this technique. However, an information on the kinetics of complex formation may be also obtained from NMR data. 

Organic host Spectroscopy Stoichiometry Binding constants Years References... 

Our work exploited the reported polarity-sensitive fluorescence of curcumin to allow for accurate determinations of host-guest stoichiometries, binding constants, and solubility enhancements for its inclusion into the three parent CD as well as their HP-modified derivatives [94]. We were able to demonstrate... 

Fig. 1. The GP Ib-IX-V complex. The complex consists of seven transmembrane polypeptides denoted GP Iba (mol wt 145,000), GP IbP (mol wt 24,000), GPIX (mol wt 17,000) and GP V (mol wt 82,000), in a stoichiometry of 2 2 2 1. The hatched region represents the plasma membrane. The area above the hatched region represents the extracellular space that below represents the cytoplasm. The complex is a major attachment site between the plasma membrane and the cytoskeleton. Two molecules associated with the cytoplasmic domain are depicted a 14-3-3 dimer, which may mediate intracellular signaling, and actin-binding protein, which connects the complex to the cortical cytoskeleton and fixes its position and influences its function.

The often fast binding step of the inhibitor I to the enzyme E, forming the enzyme inhibitor complex E-I, is followed by a rate-determining inactivation step to form a covalent bond. The evaluation of affinity labels is based on the fulfillment of the following criteria (/) irreversible, active site-directed inactivation of the enzyme upon the formation of a stable covalent linkage with the activated form of the inhibitor, (2) time- and concentration-dependent inactivation showing saturation kinetics, and (3) a binding stoichiometry of 1 1 of inhibitor to the enzyme s active site (34). 

FIGURE 10.8 A schematic diagram of the Na, K -ATPase in mammalian plasma membrane. ATP hydrolysis occurs on the cytoplasmic side of the membrane, Na ions are transported out of the cell, and ions are transported in. The transport stoichiometry is 3 Na out and 2 in per ATP hydrolyzed. The specific inhibitor ouabain (Figure 7.12) and other cardiac glycosides inhibit Na, K -ATPase by binding on the extracellular surface of the pump protein. 

Ryanodine is a neutral plant alkaloid from Ryania speciosa and was used as an insecticide. It also has been well known by the characteristic action on mammalian skeletal muscle of slowly developing, and intensive and irreversible contracture. Ryanodine binds specifically to the open RyR channel at the stoichiometry of 1 mol/mol homotetramer with a high affinity (ATD nM) and leads the channel to ryanodine modified state characteristic of long-lasting subconductance ( 50% of normal) opening. At higher concentration, it blocks the channel. 

Griffith M.C., Risen L.M., Greig M.J., Lesnik E.A., Sprankle K.G., Griffey R.H., Kiely J.S., Eeier S.M. Single and bis peptide nucleic acids as triplex-ing agents binding and stoichiometry. 

These CD studies confirmed the binding stoichiometries of our aPNA-DNA complexes and provided further support for our binding model. Comparisons between the CD spectra of the individual components and the aPNA DNA complex suggest a template effect (not unhke that observed with certain DNA-binding proteins) where the components induce mutual conformational changes upon their interaction with each other. 

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". ... 

It has been established by substitution of for Mg that, prior to phosphorylation, the divalent cation binds at a cytosolic site with a stoichiometry of about 1 mol per phosphorylation site [124,125]. These experiments also demonstrated that the phosphorylation rate is sensitive to the nature of the divalent cation bound. With Mg bound, the phosphorylation rate is about 20 times faster than with Ca bound. The divalent cation dissociates after dephosphorylation, suggesting that it is tightly bound to the phosphoenzyme during the reaction cycle. It was also demonstrated that the type of divalent cation that occupies the divalent cation site required for phosphorylation is important for the step 2K E2-P to 2K E2 P to 2K E2 [124,125]. With Mg bound, the 2K E2-P conformer is -sensitive, whereas with Ca bound, the intermediate is -insensitive. 

NCD-4 is a nonfluorescent carbodiimide derivative that forms a fluorescent adduct with the Ca -ATPase, accompanied by inhibition of ATPase activity and phos-phoenzyme formation [376-378]. Ca protected the enzyme against the inhibition by NCD-4 and reduced the extent of labeling, suggesting that the reaction may involve the Ca " " binding site. The stoichiometry of the Ca -protected labeling was i 2mole/mol ATPase. The fluorescence emission of the modified Ca -ATPase is consistent with the formation of a protein bound A-acylurea adduct in a relatively hydrophobic environment. After tryptic proteolysis of the NCD-4 labeled ATPase the fluorescence was associated with the A2 band of 24 kDa [376,379]. 

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