Identification of two unique polypeptides from dog kidney outer cortex and outer medulla that exhibit different Na +/ d-glucose cotransport functional properties

Abstract

The cloned Na +/ d-glucose cotransporter SGLT1 and an additional recently isolated human kidney cDNA Hu14/K15 belong to a family of similar cotransport proteins including the Na +-dependent nucleoside and Na +-dependent myo-inositol carrier SMIT1. For the present study we used two different polyclonal antibodies raised against the amino acid sequence 402–420 (Ab-E) and 565–574 (Ab-P) of SGLT1 to probe brush-border membrane fractions from different regions ( outer cortex a ̊ outer medulla ) of dog kidney. In Western blots both Ab-E and Ab-P react specifically (peptide blockable) with two distinct bands migrating on SDS-PAGE under reducing conditions at 75.5 kDa and 72.5 kDa. The higher molecular mass polypeptide is greatly enriched (13:1) in outer cortex and diminishes progressively towards outer medulla, whereas the lower molecular mass band is barely detectable in outer cortex but is enriched in outer medulla (4:1). Brush-border membrane vesicles (BBMV) prepared from the same outer cortical and outer medullary regions that were probed with Ab-E and Ab-P exhibit strikingly different Na +/ d-glucose functional transport behavior. The Na +/ d-glucose cotransport activity in outer cortical BBMV is a low-affinity system with K m  5.98 ± 1.01 mM, V max  13.05 ± 0.55 nmol/mg protein per min, and with 1:1 Na +: d-glucose stoichiometry. Outer medulla BBMV exhibit high-affinity K m  0.27 ± 0.03 mM V max  0.97 ± 0.04 nmol/mg protein per min and 2:1 Na +: d-glucose stoichiometry. Comparison of SGLT1, Hu14/K15, SNST1 and SMIT indicates that Ab-E could cross react with all four, but Ab-P would recognize SGLT1, Hu14/K15, SNST1 but not SMIT. Also SNST1 is not expressed in outer cortex. Based on currently available sequence data, and its marked enrichment in outer cortex, the 75.5 kDa band is a likely candidate protein responsible for low-affinity and 1:1 Na +: d-glucose stoichiometric Na +/ d-glucose cotransport activity (Hu14/K15) while the minor 72.5 kDa band in outer cortex is probably SGLT1. In outer medulla, the predominant band recognized by both Ab-E and Ab-P is the 72.5 kDa protein and this could be either SGLT1 or SNST1.