TY - JOUR
T1 - Nucleoside transport in mammalian cell membranes
T2 - A specific inhibitory mechanism of high affinity probes
AU - Eilam, Yael
AU - Cabantchik, Z. Ioav
PY - 1977/8
Y1 - 1977/8
N2 - The nucleoside transport of systems of hamster cells are susceptible to inhibition by S‐6‐substituted derivatives of mercaptonucleosides. The mechanism of interaction between the most potent inhibitor of the class, 6‐nitrobenzyl mercaptoinosine (NBMI) and the uridine transport system of hamster fibroblasts is studied in the present work. A kinetic description of the interaction is presented. Uridine transport is inhibited in a partially competitive fashion, leaving a substantial free fraction of the transport (20–30%) virtually insensitive to increasing concentrations of inhibitor. One interpretation compatible with the kinetic and chemical properties of the system assumes that binding of the inhibitor to carriers occurs at sites different from the substrate binding sites (allosteric binding). Such a binding induces a conformational change in the carrier as manifested in a reduced affinity to the substrate and a susceptibility to inhibition by organomercurials. The alternative interpretation postulates two parallel transport systems which display distinctly different susceptibilities to NBMI and to organomercurials. Experiments performed with non‐penetrating organomercurials show that the sulfhydryl groups related either to the alleged allosteric components or to additional carriers, are located superficially on the membrane. The binding of NBMI is reversible, the affinity is extremely high (Ki = 0.15 nMolar) and the rate of reaction could probably be diffusionally limited at low concentrations of reagent (activation energy 250 cal/mole, rate k = 1.3.108 min−1. Molar−1). The high affinity properties of the probes are used to determine the number of NBMI binding sites. A value of 47,000 and 77,000 sites/cell was obtained by two separate methods. The possibility that allosteric properties are present in carrier systems are discussed in terms of current concepts of modulation of transport functions in biological membranes.
AB - The nucleoside transport of systems of hamster cells are susceptible to inhibition by S‐6‐substituted derivatives of mercaptonucleosides. The mechanism of interaction between the most potent inhibitor of the class, 6‐nitrobenzyl mercaptoinosine (NBMI) and the uridine transport system of hamster fibroblasts is studied in the present work. A kinetic description of the interaction is presented. Uridine transport is inhibited in a partially competitive fashion, leaving a substantial free fraction of the transport (20–30%) virtually insensitive to increasing concentrations of inhibitor. One interpretation compatible with the kinetic and chemical properties of the system assumes that binding of the inhibitor to carriers occurs at sites different from the substrate binding sites (allosteric binding). Such a binding induces a conformational change in the carrier as manifested in a reduced affinity to the substrate and a susceptibility to inhibition by organomercurials. The alternative interpretation postulates two parallel transport systems which display distinctly different susceptibilities to NBMI and to organomercurials. Experiments performed with non‐penetrating organomercurials show that the sulfhydryl groups related either to the alleged allosteric components or to additional carriers, are located superficially on the membrane. The binding of NBMI is reversible, the affinity is extremely high (Ki = 0.15 nMolar) and the rate of reaction could probably be diffusionally limited at low concentrations of reagent (activation energy 250 cal/mole, rate k = 1.3.108 min−1. Molar−1). The high affinity properties of the probes are used to determine the number of NBMI binding sites. A value of 47,000 and 77,000 sites/cell was obtained by two separate methods. The possibility that allosteric properties are present in carrier systems are discussed in terms of current concepts of modulation of transport functions in biological membranes.
UR - http://www.scopus.com/inward/record.url?scp=0017754613&partnerID=8YFLogxK
U2 - 10.1002/jcp.1040920207
DO - 10.1002/jcp.1040920207
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C2 - 881432
AN - SCOPUS:0017754613
SN - 0021-9541
VL - 92
SP - 185
EP - 201
JO - Journal of Cellular Physiology
JF - Journal of Cellular Physiology
IS - 2
ER -