Biosynthesis of intestinal microvillar proteins. Intracellular processing of lactase-phlorizin hydrolase.
The biosynthesis of pig small intestinal lactase-phlorizin hydrolase (EC 3.2.1.23-62) was studied by labelling of organ cultured mucosal explants with [35S]methionine. The earliest detactable form of the enzyme was an intracellular, membrane-bound polypeptide of Mr 225 000, sensitive to endo H as judged by its increased electrophoretic mobility (Mr 210 000 after treatment). The labelling of this form decreased during a chase of 120 min and instead two polypeptides of Mr 245 000 and 160 000 occurred, which both barely had their electrophoretic mobility changed by treatment with endo H. The Mr 160 000 polypeptide is of the same size as the mature lactase-phlorizin hydrolase and was the only form expressed in the microvillar membrane. Together, these data are indicative of an intracellular proteolytic cleavage during transport. The presence of leupeptin during labelling prevented the appearance of the Mr 160 000 form but not that of the Mr 245 000 polypeptide, suggesting that the proteolytic cleavage takes place after trimming and complex glycosylation. The proteolytic cleavage was not essential for the transport since the precursor was expressed in the microvillar membrane in the presence of leupeptin.
D000818:Animals; D001692:Biological Transport; D004792:Enzyme Precursors / Q000378:metabolism; D005696:Galactosidases / Q000096:biosynthesis*; D005959:Glucosidases / Q000096:biosynthesis*; D006596:Hexosaminidases / Q000378:metabolism; D007413:Intestinal Mucosa / Q000201:enzymology*; D007421:Intestine, Small / Q000201:enzymology; D010694:Lactase-Phlorizin Hydrolase / Q000096:biosynthesis*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008565:Membrane Proteins / Q000096:biosynthesis*; D008871:Microvilli / Q000201:enzymology; D008970:Molecular Weight; D009924:Organ Culture Techniques; D011499:Protein Processing, Post-Translational; D013552:Swine; D001616:beta-Galactosidase / Q000096:biosynthesis*
D004792:Enzyme Precursors; D008565:Membrane Proteins; D005696:Galactosidases; D005959:Glucosidases; D006596:Hexosaminidases; D001616:beta-Galactosidase; D010694:Lactase-Phlorizin Hydrolase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase