Examination of the molecular mechanism of SH reagent-induced inhibition of the intestinal brush-border membrane Na+/phosphate cotransporter.

SH residues on the rabbit intestinal brush-border membrane Na+/phosphate cotransporter were examined using a variety of SH specific reagents, proteolytic digestion and HPLC separation of SH-labeled cotransporter, and partial reaction assays. Of the seven SH-containing peptide fragments on the non-denatured non-reduced cotransporter six peptides were labeled: five SH-containing peptides were labeled with acrylodan or IAF (iodoacetamidofluorescein) and three peptides were labeled with IAEDANS. One SH-containing peptide was labeled with IAEDANS or fluorescein maleimide only. Selective SH labeling conditions employing acrylodan and IAEDANS were used to identify the environments of these SH-containing peptides in the native cotransporter. The nature of SH reagent-induced inhibition of Na(+)-dependent phosphate uptake was examined using substrate-induced conformational changes, and substrate-induced changes in IAEDANS and acrylodan fluorescence of the SH-labeled Na+/phosphate cotransporter. The results indicate that five of the SH-labeled peptides sense the Na(+)-induced conformational change, three peptides sense the Na++ difluorophosphate-induced conformational change, and one peptide senses only the Na++ monofluorophosphate-induced conformational change. Five of the SH-labeled peptides are passive participants in the substrate-induced conformational changes with only SH 51 involved in cotransporter function. Alkylation of SH 51 resulted in a cotransporter conformation which differed from the substrate-mediated conformations and was characterized by increased monofluorophosphate sensitivity.

Reconstitution of intestinal Na(+)-phosphate cotransporter.

The rabbit intestinal brush-border membrane Na(+)-phosphate cotransporter was purified from sodium dodecyl sulfate (SDS)-brush-border membrane vesicles (BBMV) protein (SDS-treated Ca(2+)-precipitated BBMV) by a three-column chromatography protocol. The purification included a preparative scale chromatofocusing chromatography column over the pH range from 7.4 to 4 after solubilization in 3-[(3-cholamidopropyl)-diamethylammonia]-1-propanesulfonate (CHAPS), a chromatofocusing column over the pH range from 5.6 to 4 after solubilization in n-octyl glucoside, and gel filtration chromatography on a Sephacryl S-200 column. Verification of Na(+)-phosphate cotransporter purification involved substrate affinities, substrate stoichiometry, and inhibitor sensitivity after proteoliposome reconstitution and SDS-polyacrylamide gel electrophoresis (PAGE). After gel filtration Na(+)-dependent phosphate uptake was 3,300-fold enriched compared with the cell homogenate. A single 130-kDa polypeptide was visualized by SDS-PAGE under reducing conditions using silver stain. The coenrichment of this 130-kDa polypeptide and proteoliposome reconstituted Na(+)-dependent phosphate uptake suggest that the intestinal brush-border membrane Na(+)-phosphate cotransporter has been purified and proteoliposome reconstituted.

Role of carboxyl and sulfhydryl residues on rabbit small intestinal brush-border membrane Na(+)-glucose cotransporter.

The role of sulfhydryl (SH) and carboxylic acid residues in Na(+)-dependent glucose uptake, Na(+)-dependent phlorizin binding, and substrate exchange by the rabbit small intestinal brush-border membrane (BBM) Na(+)-glucose cotransporter was examined in sodium dodecyl sulfate-BBM vesicles. The sulfhydryl reagent p-chloromercuribenzoate (PCMB) inhibited all three measures of cotransporter function in a dithiothreitol-sensitive manner with similar K0.5 values (concn of PCMB resulting in 50% inhibition). PCMB sulfonate had no effect on Na(+)-glucose cotransporter function < 250 microM. The carboxylic acid reagent 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide no effect on Na(+)-glucose cotransporter function. N,N'-dicyclohexylcarbodiimide (DCCD) inhibited all three measures of cotransporter function with similar K0.5 values for inhibition. Inhibition by DCCD did not require addition of a nucleophile. In contrast, PCMB-pretreated cotransporter was insensitive to DCCD in the absence of added nucleophile with respect to substrate transport (Na(+)-dependent glucose uptake) but not Na(+)-dependent phlorizin binding. These results indicate an intravesicular or lipophilic environment for both the PCMB-reactive SH residue and the DCCD-reactive carboxylic acid residues, suggesting that a SH residue may act as an endogenous nucleophile for interaction of DCCD with the Na(+)-glucose cotransporter and suggesting that different carboxylic acid residues may be involved in Na(+)-dependent glucose uptake and Na(+)-dependent phlorizin binding.