Abstract
A novel 100-kDa ileal brush border membrane protein (I100) has been purified by anionic glycocholate affinity chromatography. Polyclonal antibodies raised against this protein were utilized to clone and characterize I100 in rats. A partial length human I100 cDNA was identified by hybridization screening. In the rat, the I100 protein is a 746-amino acid glycosylated (calculated core molecular mass of 80 kDa) type II integral membrane protein found on the apical surface of ileal villus enterocytes. Its 2.6-kilobase mRNA is expressed in distal small intestine in rats and in humans. The I100 cDNA is homologous to but distinct from human prostate-specific membrane antigen and rat brain N-acetylaspartylglutamate peptidase. It is expressed on both the basolateral and apical surfaces of stably transfected Madin Darby canine kidney cells. Analysis of these stably transfected Madin Darby canine kidney cells and I100 immunoprecipitates of rat ileal brush border membrane vesicles reveals that it has dipeptidyl peptidase IV activity. Future invesitgations will need to determine the exact substrate specificity of this novel peptidase.
Highlights
Intestinal reclamation of conjugated bile salts occurs primarily on the apical surface of ileal enterocytes by sodium-dependent carrier-mediated uptake [1, 2]
The I100 protein was immunoprecipitated from biotinylated rat ileal brush border membrane vesicles (BBMV) (Fig. 2B)
Various classical biochemical investigations demonstrated that the I100 protein is an integral membrane glycoprotein with an apparent molecular mass of approximately 90 kDa following deglycosylation, which localizes to the apical membrane of villus enterocytes
Summary
Animal Care—Sprague-Dawley rats (200 –250-g males) were obtained from Charles River (Raleigh, NC) and were exposed to 12-h day/night cycles. After several washes with TBSA, the bound antibody was released with 0.2 M glycine, pH 2.8, neutralized with 1 M Tris, pH 8.0, and concentrated using a Centricon100 device (Amicon, Beverly, MA) This purified antibody was used to immunoprecipitate the 100-kDa protein from 0.75 mg of ileal BBMV protein that had been solubilized in 1% Nonidet P-40, 0.8% bovine serum albumin, 150 mM NaCl, 10 mM Tris (pH 7.4). Analysis of the Stably Transfected MDCK Clones—The neomycinresistant colonies were lysed in 1.0% SDS and analyzed by SDS-PAGE and Western blotting to determine the presence and confirm the size of the expressed protein. Pilot studies were performed by labeling rat ileal BBMV with sulfo-NHS biotin (Pierce), and the 100-kDa protein was immunoprecipitated with the polyclonal antibody. Assays were performed using I100 that was immunoprecipitated from rat ileal BBMV or by analysis of stably transfected MDCK cells.
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