Apparent affinity

Weiss, J. M., Morgan, P. H., Lutz, M. W., and Kenakin, T. P. (1996b). The cubic ternary complex receptor-occupancy model. II. Understanding apparent affinity. J. Theroet. Biol. 178 169-182. 

VMAT1 is expressed in the adrenal medulla, by small intensely fluorescent cells in sympathetic ganglia, and by other nonneural cells that release monoamines. In contrast, VMAT2 is expressed by neuronal populations in the nervous system. The substrate specificity for the two isoforms is similar, but VMAT2 has a somewhat higher apparent affinity for all monoamines than VMAT1. In addition, only VMAT2 appears able to transport histamine, consistent with its expression by mast cells. 

For simple noncompetitive inhibition, E and EI possess identical affinity for substrate, and the EIS complex generates product at a negligible rate (Figure 8-10). More complex noncompetitive inhibition occurs when binding of the inhibitor does affect the apparent affinity of the enzyme for substrate, causing the tines to intercept in either the third or fourth quadrants of a double reciprocal plot (not shown). 

Kulanthaivel et al. [28] found that the apical Na /H exchanger in human placenta (sensitive-type) was sensitive to phenylarsine oxide, a reagent specific for dithiols that are situated in close proximity (vicinal dithiols). Moreover, the effect of phenylarsine oxide was to decrease without affecting apparent affinity for... 

Wilson S, Wilkinson G, Milligan G. The CXCR1 and CXCR2 receptors form constitutive homo- and heterodimers selectively and with equal apparent affinities. J Biol Chem 2005 280(31) 28663-28674. 

In Chapter 3 we saw that inhibitors of different modalities respond differently to the concentration of substrate used in an enzymatic reaction. Recall that the apparent affinity of the free enzyme for substrate was diminished in the presence of a competitive inhibitor, and vice versa, the apparent affinity of a competitive inhibitor could be abrogated at high substrate concentrations. On the other hand, the appar-... 

Enalaprilat and SQ27,519 are angiotensin-converting enzyme (ACE) inhibitors with poor oral absorption. Enalapril and fosinopril are dipeptide and amino acid derivatives of enalaprilat and SQ27,519, respectively [51] (Fig. 10). Both prodrugs are converted via deesterification to the active drug by hepatic biotransformation. In situ rat perfusion of enalapril indicated a nonpassive absorption mechanism via the small peptide carrier-mediated transport system. In contrast to the active parent, enalapril renders enalaprilat more peptide-like, with higher apparent affinity for the peptide carrier. The absorption of fosinopril was predominantly passive. Carrier-mediated transport was not demonstrated, but neither was its existence ruled out. 

A very simple technique is to use a radiolabeled ligand (usually a well-known substrate) of the specific transporter of interest. A recent suggestion for functional quantitation of the apparent affinities (fQ values) to P-gp using Caco-2 cells and the substrate taxol has been published [143], The method can be described simply as (1) determination of b —> a transport of3 H -taxol in normal, untreated Caco-2 cells and (2) determination of b —> a transport of 3H-taxol in the presence of verapamil (0.2 mM). The difference between these two components represents the active transport via P-gp. The two concentrations of the test compound are chosen as approximately 0.25 x K, and 4.0 x K, and for the inhibition of taxol transport, and in the study of Gao et al. [143], 16 pM and 250 pM of the test compound were used... 

The cellular and subcellular distributions of a-subunit isoforms provide clues to their different physiological functions. The four isoforms exhibit about 85% sequence identity. The most substantial differences occur in their N-terminal regions and in an 11-residue sequence of the large cytoplasmic loop. When measured in cell cultures, the isoforms differ in their apparent affinities for intracellular Na+ (al < a2 < a3) [21 ] and extracellular K+ (a3 < a2 = al) [22], In adult tissues, al is the major iso form in... 

Interestingly, we have recently identified a mutation of a tyrosine in the third intracellular loop of the hDAT that causes a major alteration in the conformational equilibrium of the transport cycle, and thus as such is comparable to mutants on G protein-coupled receptors causing constitutive isomerization of the receptor to the active state (66). Most importantly, this conclusion is based on the observation that mutation of the tyrosine completely reverts the effect of Zn2+ at the endogenous Zn2+ binding site in the hDAT (50,51) from potent inhibition of transport to potent stimulation of transport (Fig. 6). In the absence of Zn2+, transport capacity is reduced to less than 1% of that observed for the wild-type, however, the presence of Zn2+ in only micromolar concentrations causes a close to 30-fold increase in uptake (66). Moreover, it is found that the apparent affinities for cocaine and several other inhibitors are substantially decreased, whereas the apparent affinities for substrates are markedly increased (66). Notably, the decrease in apparent cocaine affinity was around 150-fold and thus to date the most dramatic alteration in cocaine affinity reported upon mutation of a single residue in the monoamine transporters (66). 

The criteria we used for inclusion of a mutation in this section are (1) that binding or uptake was detected or the presence of transporter in the membrane was confirmed by immunoblotting or immunos-taining, and (2) mutation must have produced a decrease in apparent affinity for substrates or affinity for... 

Finally, loop regions have also been implicated in ligand interactions or their modulation. Chimeras generated between NET and DAT were assayed for ethanol sensitivity and revealed that Glyl302.68 and Ilel373.3i in IL1 are important for ethanol modulation of DAT activity (78). In addition, mutation to alanine of Prol363 30 in IL1, as well as Pro553i2.43 in EL6, decreased the apparent affinity for dopamine uptake (59). 

G Op-coupling lowers the apparent affinity of PLC for Ca2+ to about 0.1 - that is, the level found in the cytosol of resting cells. 

Chang Y, Covey DF, Weiss DS. 2000. Correlation of the apparent affinities and efficacies of Y-aminobutyric acidc receptor agonists. Mol Pharmacol 58 (6) 1375. 

---