Identification and covalent modification of a renal brush-border anion exchanger.
Brush-border membrane (BBM) proteins that bind the arginine-specific reagent phenylglyoxal (PG) and interact with stilbene disulfonic derivatives were identified in canine kidney cortex. Pretreatment of BBM vesicles with PG resulted in irreversible inhibition of anion exchange as assayed by 36Cl- influx mediated via Cl-/Cl- exchange. Cl-/Cl- exchange was reversibly inhibited by the disulfonic stilbene 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS). A stilbene-affinity matrix was prepared by immobilizing DNDS in polyacrylamide beads. Elution of the BBM proteins from a disulfonic stilbene (DNDS) affinity matrix revealed two proteins at 160 and 230 kDa that were significantly enriched compared to initial material. Radiolabeling of the eluted mixture with [14C]phenylglyoxal demonstrated covalent binding to several proteins, including the 160 kDa protein. Reconstitution of the proteins eluted from the affinity matrix into phosphatidylcholine demonstrated DIDS-sensitive 36Cl(-)-influx mediated via Cl-/Cl- exchange. Pretreatment of the BBM vesicles with PG selectively blocked binding of the 160 kDa protein to the DNDS affinity matrix. Radiolabelling of the PG-pretreated, affinity-purified membrane proteins showed selective prevention of [14C]phenylglyoxal binding to the 160 kDa protein. Reconstitution of the PG-pretreated proteins eluted from the affinity matrix demonstrated significant reduction in Cl-/Cl- exchange activity. These results suggest that a 160 kDa protein is a strong candidate for anion exchange transport in kidney proximal tubules.
Soleimani
M
M
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202-5116.
Hattabaugh
Y J
YJ
Bizal
G L
GL
D000818:Animals; D000838:Anions / Q000032:analysis; D002712:Chlorides / Q000032:analysis; D004285:Dogs; D005665:Furosemide; D007672:Kidney Cortex / Q000737:chemistry* / Q000187:drug effects; D008565:Membrane Proteins / Q000032:analysis*; D008871:Microvilli / Q000737:chemistry* / Q000187:drug effects; D010658:Phenylglyoxal / Q000494:pharmacology; D013267:Stilbenes / Q000494:pharmacology
D000838:Anions; D002712:Chlorides; D008565:Membrane Proteins; D013267:Stilbenes; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid; D005665:Furosemide; D010658:Phenylglyoxal