Isolation, chemical characterization, and immunohistochemical localization of a protein from the basolateral plasma membrane of the rat intestinal absorptive cell.
The protein pattern of the basolateral membrane (BLM) of the rat small intestinal absorptive cell shows about 20 major and a multitude of minor bands. A simple and efficient method is described for isolation and purification of a major protein in the 17 kDa molecular weight (MW)-range called Prot 17. The isolated BLM of intestinal epithelial cells was dissolved in buffer 1 (Tris/HCl, 2% SDS, 10% glycerol, 5% beta-mercaptoethanol, pH 6.8) and subsequently dialyzed for 4 h against buffer 2 (Tris/glycine, pH 8.3) and then for 12 h against buffer 2 containing 25% methanol. The resulting precipitate contained Prot 17 and phospholipids in the form of liposomes. All other BLM proteins remained dissolved in the supernatant. Chemical characterization of Prot 17 suggested that it is an integral membrane protein amounting to about 5% of the total BLM protein. Amino acid analysis revealed a MW of 17.6 kDa. The Prot 17 molecule did not contain any PAS-positive carbohydrates. In its isolated form, and apparently also in the BLM, Prot 17 occurred as a polymerized structure with a MW of about 90 kDa. By dissolution in buffer 1 and heating to 100 degrees C for 1 min the complex was split into its 17 kDa subunits. By oxidation with performic acid it was also broken down into its subunits. A specific antiserum against Prot 17 was obtained from immunized Balb/c mice. Immunofluorescence labelling of rat small intestinal sections with this serum showed that Prot 17 was not a BLM-specific protein. It occurred in both plasma membrane domains of the intestinal absorptive cell.
Schiechl
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Institute of Histology and Embryology, University of Graz, School of Medicine, Austria.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D007106:Immune Sera; D007408:Intestinal Absorption; D007421:Intestine, Small / Q000378:metabolism*; D008051:Lipid Bilayers; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains
D007106:Immune Sera; D008051:Lipid Bilayers; D008565:Membrane Proteins