Characterization of mammalian equilibrative nucleoside transporters (ENTs) by mass spectrometry.
Equilibrative nucleoside transporters (ENTs) are integral membrane proteins that facilitate the movement of nucleosides and hydrophilic nucleoside analog (NA) drugs across cell membranes. ENTs are also targets for cardioprotectant drugs, which block re-uptake of the purine nucleoside adenosine, thereby enhancing purinergic receptor signaling pathways. ENTs are therefore important contributors to drug bioavailability and efficacy. Despite this important clinical role, very little is known about the structure and regulation of ENTs. Biochemical and structural studies on ENT proteins have been limited by their low endogenous expression levels, hydrophobicity and labile nature. To address these issues, we developed an approach whereby tagged mammalian ENT1 protein was over-expressed in mammalian cell lines, confirmed to be functional and isolated by affinity purification to sufficient levels to be analyzed using MALDI-TOF and tandem MS mass spectrometry. This proteomic approach will allow for a more detailed analysis of the structure, function and regulation of ENTs in the future.
Reyes
German
G
Department of Biology, York University, 4700 Keele St., Toronto, Ontario, Canada M3J 1P3.
Naydenova
Zlatina
Z
Abdulla
Parween
P
Chalsev
Maria
M
Villani
Anita
A
Rose
Jennifer B
JB
Chaudary
Naz
N
DeSouza
Leroi
L
Siu
K W Michael
KW
Coe
Imogen R
IR
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D033705:Equilibrative Nucleoside Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009842:Oligopeptides; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism; D014357:Trypsin
D033705:Equilibrative Nucleoside Transport Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; D013868:Thioinosine; C085706:FLAG peptide; D014357:Trypsin; C001789:4-nitrobenzylthioinosine