title: Hydrophobic labeling, isolation, and partial characterization of the NH2-terminal membranous segment of sucrase-isomaltase complex.
abstract: A photogenerated carbene, 3-trifluoromethyl-3-(m-[125I]iodophenyl)carbene (Brunner, J., and Semenza G. (1981) Biochemistry, 20, 7174-7182), was used to label the hydrophobic core of small intestinal brush-order membrane vesicles. Reaction of the carbene with sucrase-isomaltase complex was restricted to a polypeptide segment which is essential for binding the enzyme complex to the native membrane or to liposomes. The same labeling selectivity was obtained when purified sucrase-isomaltase complex was labeled either in Triton X-100 solution or when it was incorporated in egg-lecithin liposomes. During cleavage of sucrase-isomaltase with papain, the radiolabel remained covalently associated with the anchor peptide. It was thus possible to detect easily the polypeptide in the course of subsequent separation and purification operations. The molecular weight of the peptide was determined by gel filtration on Sephadex LH-60 in ethanol-formic acid (Takagaki, Y., Gerber, G. E., Nihei, K., and Khorana, H. G. (1980) J. Biol. Chem. 255, 1536-1541). The figure thereby obtained, 6500, is somewhat lower than that obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (approximately 8000). Circular dichroism of the peptide indicates a secondary structure of high alpha-helical content. A possible structure of the membranous segment is discussed.
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D002942:Circular Dichroism; D007421:Intestine, Small / Q000201:enzymology*; D008081:Liposomes; D008871:Microvilli / Q000201:enzymology*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism*; D010743:Phospholipids / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D013394:Sucrase-Isomaltase Complex / Q000378:metabolism*
chemical_list: D008081:Liposomes; D009097:Multienzyme Complexes; D010743:Phospholipids; D013394:Sucrase-Isomaltase Complex