Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1996-06-26
1999-02-09
Kemmerer, Elizabeth C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 691, 4353201, 435325, 4352523, 43525411, 536 235, 530350, G01N 3353, C12N 1512, C12N 510
Patent
active
058692657
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of sodium/bile acid cotransport systems in the ileum and kidney. Certain embodiments of the invention relate to the medically related fields of the control of blood cholesterol levels and treatments of diabetes, heart disease, liver disease and various digestive disorders. More particularly, the invention concerns the isolation and purification of bile acid cotransporter proteins and cDNA clones encoding the proteins and the use of these proteins and nucleic acids in therapeutic, preventative, genetic counseling and reagent screening applications.
2. Description of the Related Art
Bile acids are acidic sterols synthesized from cholesterol in the liver. Following synthesis, the bile acids are secreted into bile and enter the lumen of the small intestine, where they facilitate absorption of fat-soluble vitamins and cholesterol. Bile acids are then absorbed from the small intestine, returned to the liver via the portal circulation, and resecreted into bile. In the small intestine, bile acids are absorbed by both passive and active mechanisms (Dietschy, 1968). The active absorption of bile acids, first described by Lack and Weiner (1961), has been shown in man and experimental animals to be restricted to the ileum (Krag and Phillips, 1974; Schiff et al., 1972; Lack, 1979).
The first step in the active uptake of bile acids is mediated by a Na.sup.+ gradient-driven transporter located at the brush border (apical) membrane of the ileocyte (Wilson, 1981). Once inside the enterocyte, bile acids are transported across the cell to the basolateral membrane and secreted into the portal circulation via a Na.sup.+ -independent organic anion exchange system (Weinberg et al., 1986). The transport kinetics and specificity of this Na.sup.+ /bile acid cotransport system have been studied extensively using everted ileal gut sacs (Schiff et al., 1972; Lack, 1979), isolated ileocytes (Wilson et al., 1975; Schwenk et al., 1983), and ileal brush border membranes (Barnard and Ghishan, 1987; Kramer et al., 1992; Wilson and Treanor, 1979).
Although the mechanism of ileal bile acid transport has been characterized, the protein(s) responsible for this process have not been isolated and characterized. In an attempt to identify the proteins involved, photoaffinity studies have been performed using radiolabeled 7,7'-azo-derivatives of taurocholate with ileocytes and ileocyte membrane fractions (Kramer et. al., 1983; Lin et. al., 1990). These studies tentatively identified a brush border (apical) membrane protein of 99 kDa and basolateral membrane proteins of 54 and 59 kDa. More recently, lysylglycocholate-Sepharose affinity chromatography was used to isolate bile acid transporter-enriched ileal brush border membranes for polyclonal antibody production. In immunoblotting experiments, these antibodies detected a number of proteins including a 99 kDa protein in rat ileal brush border and kidney proximal tubule membranes. These antibodies also partially inhibited bile acid transport by isolated ileal brush border membranes (Gong et al., 1991). The tentative identification of a 90-99 kDa protein is also supported by chemical modification studies in rabbit ileum where agents that inhibited bile acid transport into ileal brush border membrane vesicles also blocked photoaffinity labeling of a 90 kDa protein (Kramer et al., 1992).
Notably lacking with the lysylglycocholate-Sepharose affinity chromatography and photoaffinity labeling studies was functional reconstitution of bile acid transport activity. Several candidate bile acid binding proteins previously identified by photoaffinity labeling have since been abandoned, illustrating the difficulties with this technique. For example, candidate 67 kDa and 43 kDa proteins were later shown to be albumin and actin, respectively (Fricker et al., 1982). Also, a candidate 54 kDa protein for the hepatic Na.sup.+ -independent multispecific anion transporter, has recently been shown to be Protein Disu
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Kaufman Claire M.
Kemmerer Elizabeth C.
Wake Forest University
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