title: Sensitivity to inhibition by N-ethylmaleimide: a property of nitrobenzylthioinosine-sensitive equilibrative nucleoside transporter of murine myeloma cells.
abstract: Murine myeloma SP2/0-Ag14 cells possess both nitrobenzylthioinosine (NBMPR)-sensitive and NBMPR-insensitive equilibrative uridine transport systems. No Na(+)-dependent uridine transport system was detected. The NBMPR-insensitive transport system is similarly insensitive to inhibition by dilazep and dipyridamole. Dose-response curve for the inhibition of equilibrative uridine transport by N-ethylmaleimide (NEM), a sulfhydryl reagent, in these cells was biphasic. About 30-40% of the uridine transport was inhibited by NEM at IC50 value of 0.15 mM. The other 60-70% of the transport activity remained insensitive to NEM at concentration as high as 3 mM. The decrease in NBMPR-sensitive uridine transport in the presence of 0.3 mM NEM was due to a 3-fold decrease in transport affinity. Apparent Km values of 500 and 1600 microM and Vmax values of 13 and 12 microM/s were obtained for untreated and NEM-treated cells, respectively. NEM (0.3 mM) has little effect on the Km of NBMPR-insensitive transporter, with apparent Km values of 100 and 110 microM and Vmax values of 3.0 and 2.5 microM/s for untreated and NEM-treated cells, respectively. High sensitivity of NBMPR-sensitive transporter to NEM inhibition was also observed in HL-60 and MCF-7 cells. Decrease in specific 3H-NBMPR equilibrium binding affinity in myeloma cells was observed after treatment with 0.3 mM NEM. Apparent Kd values of 0.32 and 2.3 nM with Bmax values of 48,000 and 44,000 sites/cell were obtained for untreated and NEM-treated cells, respectively. NBMPR, dilazep and dipyridamole at 30 microM, and uridine at 10 mM failed to protect the NBMPR-sensitive transporter against NEM inhibition. It is possible that a critical sulfhydryl residue is closed to substrate binding/transporting site of the NBMPR-sensitive transporter. NEM, a sulfhydryl reagent containing an activated double bond, hinders the affinity of this transporter by forming a stable thiol ether bond with the reactive residue.
authors.affiliation: Department of Physiology, National University of Singapore.
authors.forename: C W
authors.initials: CW
authors.lastname: Lee
authors.forename: L B
authors.initials: LB
authors.lastname: Goh
authors.forename: Y
authors.initials: Y
authors.lastname: Tu
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D005033:Ethylmaleimide / Q000494:pharmacology*; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D033703:Nucleoside Transport Proteins; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors; D014407:Tumor Cells, Cultured; D014529:Uridine / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine; D005033:Ethylmaleimide; D014529:Uridine