High affinity binding sites

FIGURE 4.18 Affinity of adenosine receptor agonists in whole cells (dark bars) and membranes (cross-hatched bars, high-affinity binding site). Data shown for (1) 2-phenylaminoadenosine, (2) 2-chloro adenosine, (3) 5 -N-ethylcarboxamidoadenosine, (4) N6-cyclohexyladenosine, (5) (-)-(R)-N6-phenylisopropyladenosine, and (6) N6-cyclopentyladenosine. Data redrawn from [15],... 

In cyclic nucleotide-regulated channels, this domain serves as a high-affinity binding site for 3-5 cyclic monophosphates. The CNBD of channels has a significant sequence similarity to the CNBD of most other classes of eukaryotic cyclic nucleotide receptors and to the CNBD of the prokaryotic catabolite activator protein (CAP). The primary sequence of CNBDs consists of approximately 120 amino acid residues forming three a-helices (oA-aC) and eight (3-strands ( 31- 38). 

Chemical modifications like alkylation with (A-ethylmaleimide (NEM) or oxidation with diamide that inhibit the phosphorylation activity of the enzyme did not seem to have any significant effect on the high affinity binding site when the enzyme was solubilized in the detergent decyl-PEG [69,41]. However, in the intact membrane these treatments reduced the affinity by a factor of 2-3. The reduction of the affinity was exclusively due to modification of the cysteine residue at position 384 in the B domain [69]. Apparently, the detergent effects the interaction between the B and C domains. 

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... 

Leukotriene B4 has been implicated in inflammatory processes and chemo-taxis. The G-protein-coupled receptor in human myeloid cells was postulated to possess two distinct binding sites. Photoaffinity studies using unmodified tritiated LTB4 as photoreactive species labeled two different proteins in the presence (53 kDa) and in the absence of a non-cleavable GTP analogue (56 kDa). This means that stabilization of the G-protein in the GTP-bound state resulted in an interconversion of the first high affinity binding site to an alternative low-affinity binding site [99]. 

L. De Petrocelis, G. Quagliarotti, L. Tomei, and G. Geraci, Structuring of HI histone. Evidence of high-affinity binding sites for phosphate ions, Eur. J. Biochem. 156, 143-148 (1986). 

Heltianu C, Simionescu M, Simionescu N. Histamine receptors of the micro vascular endothelium revealed in situ with a histamine-ferritin conjugate characteristic high-affinity binding sites in venules. J Cell Biol 1982 108 357-364. 

The agonist radioligands [ HJnicotine and [ HJepibatidine, which are most commonly used to monitor nAChR numbers, freely cross lipid bilayers. Therefore, they do not discriminate between intracellular and surface nAChRs and the majority of the increase in high-affinity binding sites appears to be intracellular (Whiteaker et al. 1998 Vallejo et al. 2005). It is now appreciated that nicotine can accumulate within cells and a new consensus is emerging that supports an intracellular action of nicotine that enhances assembly and/or maturation of nAChRs (Nashmi et al. 2003 Darsow et al. 2005 Kuryatov et al. 2005 Sallette et al. 2004, 2005 Nashmi and Lester 2007 Fig. 4). 

Geonzon, R., Exner, D.V., Woodman, R.C., Wang, L Feng, Z.P. and Duff, H.J. (1998) A high affinity binding site for [H-3]-dofetilide on human leukocytes. Journal of Molecular and Cellular Cardiology, 30, 1691-1701. 

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