Purification and reconstitution of a 75-kilodalton protein identified as a component of the renal Na+/glucose symporter

A 75-kilodalton (kDa) protein was purified from solubilized renal brush border membranes by using high-pressure liquid chromatography (HPLC) and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Functional and immunological properties identified the 75-kDa protein as a component of the Na+/glucose symport system. The purified protein was specifically recognized by a monoclonal antibody that functionally interacts with the Na+/glucose symporter. Na+-dependent phlorizin binding activity was associated with fractions containing the 75-kDa protein during HPLC fractionation on the anion exchanger Mono-Q and was greatly increased after reconstitution into egg yolk phosphatidylcholine vesicles. The final purified preparation contained glucosamine and a blocked N-terminus.