Nucleoside transport in mammalian cell membranes

Abstract

Transport of the nucleoside analog cytosine-arabinoside (CAR) in transformed hamster cells in culture has been studied in conditions of minimal metabolic conversion. Uptake (zero-trans in) properties at 20 degrees C over a limited range of CAR concentrations were characterized by a Km of 350 micrometer and a maximal velocity (V) of 780 micrometer.min-1 (V/Km = 2.28 min-1). Equilibrium exhcange at 20 degrees C over a wider range of concentrations was best described by a saturable component with a Km of 500 micrometer and a v of 1230 micrometer.min-1 (V/Km = 2.26 min-1) and either a saturable component of high Km or a nonsaturable component of k = 0.3 min-1. For the saturable component, the v/Km values were similar in both procedures. CAR transport was inhibited by various metabolizable nucleosides. Uptake of some of these nucleosides was inhibited by CAR. CAR transport and uridine uptake were inhibited in a reversible but partially competitive fashion by high affinity probes like S-(p-nitrobenzyl-6-mercaptoinosine (NBMI) (Ki less than 0.5 nM) and in an irreversible fashion by SH reagents such as N-ethylmaleiimide (NEM). The organomercurial p-hydroxymercuribenzene sulfonate (pMBS) markedly stimulated transport of these nucleosides, but also markedly potentiated the inhibitory effects of either NBMI or NEM. The effects are interpreted either in terms of models which invoke allosteric properties or in terms of two transport systems which display distinct chemical susceptibilities to externally added probes.

Cytosine Arabinoside Influx and Nucleoside Transport Sites in Acute Leukemia

“…The growth fraction of leukemic cells is generally considered <1.0 and recent estimates based on nuclear DNA-polymerase activity suggest that less than half the leukemic blasts of marrow are in active proliferation (35,36). Moreover the growth fraction of blasts from peripheral blood may be still lower, since tritiated thymidine labeling indices are considerably less for circulating than for intramedullary blasts (37,38 It is established that araC is a substrate for the nucleoside-specific transport mechanism in the cell membrane (12)(13)(14)39) so that transport rate of araC should relate to the density of transport elements in the cell membrane. The validity of this concept is apparent in the correlation of Fig.…”

“…Movement of nu-cleoside and many of their analogs across the plasma membrane in many cell types is mediated by a nucleoside specific transport mechanism. Cytosine arabinoside is a substrate for this carrier system (11)(12)(13)(14)(15) that is reversible, nonconcentrative, and has a broad specificity for pyrimidine and purine nucleosides. However, the affinity of the nucleoside carrier in leukemic cells is less for araC than for its naturally occurring pyrimidine analog, deoxycytidine.…”

Binding of nitrobenzylthioinosine to high-affinity sites on the nucleoside-transport mechanism of HeLa cells

“…Concentration-effect curves for inhibition of uridine and adenosine transport are biphasic, raising the possibility of interaction of NBMPR with two or more classes of transport-inhibitory sites (Paterson, 1979;Paterson et al, 1980). Kinetic studies of NBMPR inhibition of uridine transport in hamster fibroblasts have indicated that interactions between NBMPR and nucleoside-transport mechanisms are also complex in this cell type (Eilam & Bibi, 1977;Eilam & Cabantchik, 1977;Heichal et al, 1978). Inhibition was partially competitive (K, = 3.7 nM), and a substantial fraction of transport activity (20-30%) was insensitive to increasing concentrations of inhibitor.…”

“…These data, together with results from studies of the effects of organomercurials on NBMPR inhibition of uridine transport, led Eilam & Cabantchik (1977) to propose that binding of NBMPR occurs at transporter sites different from substrate-binding sites. In a subsequent study (Heichal et al, 1978), four distinct classes of sites on the uridine carrier of hamster cells were postulated: a substrate-binding site, an NBMPR-binding site and two types of thiol-containing modifier sites (one stimulatory, the other inhibitory, after binding of organomercurials).…”

“…Also assessed in the present study was the ability of uridine and other nucleoside permeants, as well as that of nitrobenzylthiouridine {4-[(4-nitrobenzyl)thiol-1-fl-D-ribofuranosylpyrimidin-2-one I to compete with [G-3HINBMPR for binding to the transport-inhibitory sites. Because thiol groups are involved in nucleoside transport in hamster cells (Eilam & Bibi, 1977;Eilam & Cabantchik, 1977;Heichal et al, 1978), the effects of NEM and the organomercurial, p-CMBS, on uridine transport and NBMPR binding were examined to determine if thiol groups were also required for interaction of permeant or inhibitor with the nucleoside-transport mechanism of HeLa cells.…”