Biosynthesis, glycosylation, and intracellular transport of intestinal lactase-phlorizin hydrolase in rat.
The biosynthesis of rat intestinal lactase-phlorizin hydrolase was studied by pulse-labeling of 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 for 30 min and chased for various periods of time up to 6 h in the presence or absence of protease inhibitors (PI), leupeptin, phenylmethylsulfonyl fluoride, and soybean trypsin inhibitor. Lactase-phlorizin hydrolase was immunoprecipitated from microvillus membrane (MVM) and ER-Golgi fractions with monoclonal antibodies. After pulse-labeling, lactase-phlorizin hydrolase from the ER-Golgi fraction appeared on SDS-PAGE as one band of approximately 220 kDa, regardless of the presence or absence of PI in the culture media. The 220-kDa protein band could also be labeled after incubation with [2-3H]mannose. In the absence of PI, the 220-kDa band appeared in the MVM by 30 min chase, simultaneously with a 180-kDa band, and by 60 min of chase an additional band of 130 kDa was seen. With increasing time of chase, the relative intensity of the 130-kDa band increased, whereas that of the 220-kDa band decreased, suggesting a precursor-product relationship. When PI were added to the medium, the formation of the 180-kDa band was not affected, but the conversion of the 180-kDa protein to the 130-kDa protein was virtually blocked. These findings suggest that lactase-phlorizin hydrolase is initially synthesized as a glycosylated precursor of 220 kDa, which is transported to the MVM. There it undergoes the following two cleavages: first, to the 180-kDa form, which is not prevented by PI used in these experiments, and second, to the 130-kDa form inhibited by PI.
D000818:Animals; D000833:Animals, Suckling; D000911:Antibodies, Monoclonal; D004591:Electrophoresis, Polyacrylamide Gel; D005470:Fluorometry; D005696:Galactosidases / Q000096:biosynthesis*; D005959:Glucosidases / Q000096:biosynthesis*; D007422:Intestines / Q000201:enzymology*; D010694:Lactase-Phlorizin Hydrolase / Q000096:biosynthesis* / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000378:metabolism; D009924:Organ Culture Techniques; D051381:Rats; D011919:Rats, Inbred Strains; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D009097:Multienzyme Complexes; D008715:Methionine; D005696:Galactosidases; D005959:Glucosidases; D001616:beta-Galactosidase; D010694:Lactase-Phlorizin Hydrolase