Anion exchangers inhibited by disulfonic stilbenes have been described in brush border membrane vesicles isolated from mammalian renal cortex. While much is known about the kinetics of these transporters, there has been little progress to date toward isolation and biochemical characterization of the relevant transport proteins. For the past two years the applicant has been attempting to identify and purify stilbene-binding proteins from rabbit renal brush border membranes by use of affinity chromatography. A series of stilbene-binding proteins has been purified using an affinity matrix prepared by crosslinking the disulfonic stilbene SITS to aminoalkyl agarose. These highly purified stilbene-binding proteins were identified as integral membrane proteins and are excellent candidates to represent the structural components of anion transporters. The general goals over the next five years are to further characterize these putative anion transport proteins and to clone and sequence their structural genes. During Phase 1, monoclonal antibodies will be generated to stilbene-binding proteins and screened for their ability to inhibit anion exchange activity directly in intact membrane vesicles or by immunoprecipitation of transport activity from a reconstitution assay. Antibodies will also be screened for localization to the brush border membrane by immunohistochemical methods. Antibodies thereby identified will then be used to affinity-purify individual proteins. Partial amino acid sequences will be determined from proteolytic fragments of purified proteins. During Phase 2, monoclonal antibodies against specific transport proteins and/or oligonucleotide probes constructed on the basis of partial amino acid sequencing will be used to screen renal cortical cDNA libraries. The primary structure of anion transport proteins will be deduced from overlapping cDNAs. The function of cloned protein(s) will be confirmed by transient expression in Xenopus oocytes. Further studies will identify functionally important domains through construction of chimeric transporters and oligonucleotide- directed mutagenesis. Because anion exchangers are ubiquitous and involved in diverse aspects of cell physiology, the proposed studies will be of general biomedical significance. Most importantly, the project will provide training in a variety of techniques relevant to an investigative career in membrane physiology including subcellular fractionation,k protein isolation and biochemistry, generation of monoclonal antibodies an recombinant DNA technology.
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| McConnell, K R; Aronson, P S (1994) Effects of inhibitors on anion exchangers in rabbit renal brush border membrane vesicles. J Biol Chem 269:21489-94 |
