Identification of organic cation transporter in rat renal brush-border membrane by photoaffinity labeling.
As an approach to identification of the organic cation transport system in brush-border membranes, we designed a photoaffinity probe, 1-cyano-2-(4-azido[3,5-3H]benzoylethyl)-3-[2-[[(5-methyl-4-imidazo lyl ) methyl]thio]ethyl]-guanidine ([3H]AMC) based on the molecular structure of cimetidine, which is taken up by the organic cation transport system in brush-border membrane vesicles. The effect of nonradioactive 1-cyano-2-(4-azidobenzoylethyl)-3-[2-[[(5-methyl-4- imidazolyl)methyl]thio]ethyl]guanidine (AMC) on tetraethylammonium uptake was investigated in rat renal brush-border membrane vesicles. We examined the photolysis of AMC in which the azido group was converted to an active nitrene group using UV light at a wavelength of 254 nm and established a half-life of 7 s. This half-life duration did not significantly impair brush-border membrane vesicles during the exposure to light for photo-labeling. Photoaffinity labeling of brush-border membrane vesicles from the rat renal cortex with [3H]AMC resulted in the covalent incorporation of radioactivity into membrane polypeptides; an apparent 36 kDa polypeptide was predominantly labeled. Photolabeling specificity was shown by a reduction in the labeling of the 36 kDa polypeptide in the presence of organic cations, cimetidine, tetraethylammonium and N-methylnicotinamide whereas the organic anion, fur osemide, had no effect on labeling patterns. These data demonstrate that AMC, as well as organic cations, cimetidine, tetraethylammonium and N-methylnicotinamide, interact with a common 36 kDa membrane polypeptide, which may be the transport system or one of its brush-border membrane components.
Kimura
M
M
Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Japan.
Nabekura
T
T
Katsura
T
T
Takano
M
M
Hori
R
R
D000345:Affinity Labels / Q000138:chemical synthesis / Q000493:pharmacokinetics* / Q000494:pharmacology; D000818:Animals; D002412:Cations; D002851:Chromatography, High Pressure Liquid; D004347:Drug Interactions; D004591:Electrophoresis, Polyacrylamide Gel; D006146:Guanidines / Q000138:chemical synthesis / Q000493:pharmacokinetics* / Q000494:pharmacology; D017136:Ion Transport / Q000502:physiology; D007553:Isotope Labeling; D007668:Kidney / Q000378:metabolism* / Q000528:radiation effects / Q000648:ultrastructure*; D008297:Male; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism / Q000528:radiation effects; D010779:Photofluorography; D010782:Photolysis; D051381:Rats; D017208:Rats, Wistar; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000493:pharmacokinetics*; D014316:Tritium; D014466:Ultraviolet Rays
C095914:1-cyano-2-(4-azidobenzoylethyl)-3-(2-(((5-methyl-4-imidazolyl)methyl)thio)ethyl)guanidine; D000345:Affinity Labels; D002412:Cations; D006146:Guanidines; D008565:Membrane Proteins; D013757:Tetraethylammonium Compounds; D014316:Tritium; D019789:Tetraethylammonium