Essential disulfide and sulfhydryl groups for organic cation transport in renal brush-border membranes.
The disulfide reducing agent, dithiothreitol (DTT) and the sulfhydryl-modifying reagents p-chloromercuribenzenesulfonic acid and N-ethylmaleimide (NEM) were employed to assess the role of disulfide and sulfhydryl groups in organic cation transport. The transport of N1-[3H]methylnicotinamide (NMN), a prototypic organic cation, was examined employing brush-border membrane vesicles isolated from the outer cortex of canine kidneys. DTT inhibited NMN transport reversibly with an IC50 of 250 microM/mg of protein. 5 mM NMN protected against DTT inactivation. The specificity of substrate protection was demonstrated by showing that D-glucose had no effect on the DTT inactivation of NMN transport and conversely that NMN had no effect on the DTT inactivation of D-glucose transport. Disulfide bonds reduced by DTT could be reoxidized by washing with excess buffer or by addition of 0.02% H2O2 thereby restoring NMN transport. p-Chloromercuribenzenesulfonic acid reversibly inactivated NMN transport with an IC50 of 25 microM/mg of protein. 5mM NMN protected against inactivation. NEM irreversibly inactivated transport with an IC50 of 250 microM/mg of protein. The rate of NMN inactivation by NEM followed pseudo-first order reaction kinetics. A replot of the data gave a linear relationship between the apparent rate constants and the NEM concentration with a slope of 1.3. The data are consistent with a simple bimolecular reaction mechanism and imply that one molecule of NEM inactivates 1 sulfhydryl group/active transport unit. The presence of 5 mM NMN affected the rate of NEM (2.5 mM) inactivation: the t1/2 values for inactivation in the presence and absence of substrate were 7.3 and 2.0 min, respectively. The results demonstrate an essential requirement for disulfide and sulfhydryl groups.
D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002412:Cations / Q000378:metabolism*; D004220:Disulfides / Q000032:analysis*; D004229:Dithiothreitol / Q000494:pharmacology; D004285:Dogs; D005033:Ethylmaleimide / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D007672:Kidney Cortex / Q000378:metabolism / Q000648:ultrastructure*; D008871:Microvilli / Q000032:analysis / Q000378:metabolism; D009536:Niacinamide / Q000031:analogs & derivatives / Q000378:metabolism; D009994:Osmolar Concentration; D010622:Phencyclidine / Q000031:analogs & derivatives / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000032:analysis*; D013997:Time Factors
D002412:Cations; D004220:Disulfides; D013438:Sulfhydryl Compounds; C036590:1-(1-phenylcyclohexyl)-3-methylpiperidine; D009536:Niacinamide; D005947:Glucose; D010622:Phencyclidine; D005033:Ethylmaleimide; D004229:Dithiothreitol; C008472:N-methylnicotinamide