Substrate-transporter affinity

Relationship between Substrate-Transporter Affinity and Rate ofTransport... 

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... 

In summary, ATP hydrolysis by P-gp correlates well with the intrinsic rate of substrate transport. A complete characterization of the interaction of a compound with P-gp is obtained by measuring the ATPase activity as a function of concentration. The rate of intrinsic substrate transport first increases with increasing concentration, reaches a maximum, and decreases again at high concentrations. The rate of intrinsic transport by P-gp depends not only on the substrate concentration but also on its affinity to the transporter substrates with high affinities for P-gp are transported more slowly than those with low affinities. 

Transporters have binding sites for their substrate. Transport processes, just like the kinetic quantities of transport speed and exchange rate, are characterized by the affinity of the substrate to the transporter protein and the number of its binding sites. The transport speed (e.g., in rM substrate/min) reaches a maximum value with increasing substrate concentration, the maximum transport speed (Vmax). The exchange rate (in sec ) measures the number of substrate molecules transported by a transporter molecule per second. 

Substrate specificity is determined by high affinity for the cognate neurotransmitter substrate. However, low affinity uptake does also have a part in the clearance of transmitters from the interstitial space (e.g., in uptake mediated by the extraneuronal monoamine transporter, EMT) and in the intestinal absoiption of glycine and glutamate. It is obvious that there is an evolutionary relation of neurotransmitter transporters and amino acid and cation transporters in epithelia. 

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. 

Binding to transport proteins may be of particular interest, since binding not only assays the affinities of the binding site on the transporter protein but also the translocation equilibria [67], In terms of enzyme catalysis, a transport protein transforms a substrate, a molecule located at one side of the membrane, into a product, the same molecule at the other side of the membrane, without chemical modification. Substrate must bind to a particular conformation of the enzyme with the binding sites accessible only from, for example, the outside. Similarly, the release of the product has to occur from a conformation which opens the binding site to the inside only this implies at least one transition step between the two types of conformations (see Fig. 

In Saccharomyces cerevisiae, as in most eukaryotic cells, the plasma membrane is not freely permeable to nitrogenous compounds such as amino acids. Therefore, the first step in their utilization is their catalyzed transport across the plasma membrane. Most of the transported amino acids are accumulated inside the yeast cells against a concentration gradient. When amino acids are to be used as a general source of nitrogen, this concentration is crucial because most enzymes which catalyze the first step of catabolic pathways have a low affinity for their substrates. 

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