THE RENAL CORTICAL NA+ HCO3- COTRANSPORTER .6. THE EFFECT OF CHEMICALMODIFICATION IN COTRANSPORTER ACTIVITY/

The Na+/HCO3- cotransporter is the main system that mediates bicarbona te removal out of the proximal tubule cell into the blood. We have pre viously partially purified this protein and showed that chemical modif ication of the alpha-amino groups by fluorescein isothiocyanate (FITC) inhibited the activity of the Na+/HCO3- cotransporter. The inhibition was prevented by the presence of Na and bicarbonate suggesting that t his compound binds at of near the substrate transport sites of the cot ransporter. We examined the effect of agents that modify the sulfhydry l group (dithiothreitol), carboxyl groups (n-n'dicyclohexyl carbodiimi de) and tyrosine residues (p-nitrobenzene sulfonyl fluoride, n-acetyl imidazole and tetranitromethane) on the activity of the cotransporter to gain insight into the chemical residues which may be important for transport function. The sulfhydryl residues modifier, carboxyl group m odifier, and tyrosine modifier significantly inhibited bicarbonate dep endent Na-22 uptake in basolateral membranes by 50-70% without alterin g the Na-22 uptake in the presence of gluconate indicating that these agents directly affected the cotransporter without affecting diffusive sodium uptake. The effect of the tyrosine modifier n-acetylimidazole was not prevented by the presence of Na and bicarbonate suggesting tha t the tyrosine residues are not at the substrate binding sites. To det ermine the presence and role of glycosylation on the Na+/HCO3- cotrans porter protein, we examined the effects of different glycosidases (end oglycosidase F and H, N-glycosidase F, O-glycanase) on the cotransport er activity. All glycosidases caused a significant 50-80% inhibition o f cotransporter activity. These data demonstrate that N-glycosylation as well as O-glycosylation are important for the function of the Na+/H CO3- cotransporter protein. Taken together, these results suggest that chemical modifiers of tyrosine, carboxyl and sulfhydryl groups as wel l as glycosylation are important for expression of full functional act ivity of the cotransporter.