EFFECT OF CROSS-LINKERS ON THE STRUCTURE AND FUNCTION OF PIG-RENAL SODIUM-GLUCOSE COTRANSPORTERS AFTER PAPAIN TREATMENT

Kidney brush-border membranes contain two sodium-dependent glucose tra nsporters, one with low and one with high affinity for phlorizin, the specific inhibitor of these transporters. Using Scatchard analysis of phlorizin binding and Western blotting with specific antibodies agains t these transporters, we demonstrate in this study that although both transporters were proteolysed by papain treatment, only the high-affin ity phlorizin-binding sites were decreased. Papain treatment followed by cross-linking with homobifunctional disuccinimidyl tartarate restor ed only the structure of the low-affinity phlorizin-binding protein (a pprox. molecular mass 70 kDa) without modifying the phlorizin-binding sites. When disuccinimidyl tartarate was replaced with dithiobis(succi nimidyl acetate), another homobifunctional cross-linker with a higher spacer arm, the low-and high-affinity sites were both restored, with r eappearance of two phlorizin-binding proteins with approx. molecular m asses of 70 and 120 kDa. We conclude that high-affinity phlorizin-bind ing sites depend on the presence of the heterodimeric 120 kDa protein.