Biosynthesis, glycosylation, and intracellular transport of intestinal lactase-phlorizin hydrolase in rat.

摘要: The biosynthesis of rat intestinal lactase-phlorizin hydrolase was studied by pulse-labeling jejunal explants from 5-day-old suckling rats in organ culture. Explants were either continuously labeled with [35S] methionine for 15, 30, and 60 min or pulse-labeled 30 chased various periods time up to 6 h the presence absence protease inhibitors (PI), leupeptin, phenylmethylsulfonyl fluoride, soybean trypsin inhibitor. Lactase-phlorizin immunoprecipitated microvillus membrane (MVM) ER-Golgi fractions monoclonal antibodies. After pulse-labeling, fraction appeared on SDS-PAGE as one band approximately 220 kDa, regardless PI culture media. 220-kDa protein could also be after incubation [2-3H]mannose. In PI, MVM chase, simultaneously a 180-kDa band, chase an additional 130 kDa seen. With increasing relative intensity 130-kDa increased, whereas that decreased, suggesting precursor-product relationship. When added medium, formation not affected, but conversion virtually blocked. These findings suggest is initially synthesized glycosylated precursor which transported MVM. There it undergoes following two cleavages: first, form, prevented used these experiments, second, form inhibited PI.