Co-and post-translational events in the biogenesis of pig small intestinal aminopeptidase N.
The biogenesis of pig small intestinal aminopeptidase N (EC 3. 4. 11. 2) was studied by cell-free translation of intestinal mRNA and by labelling of organ cultured intestinal explants. In cell-free translation, the primary mRNA translation product of aminopeptidase N was a polypeptide of Mr 115,000. When translation was performed in the presence of dog pancreatic microsomes, a Mr 140,000 polypeptide was also observed. A polypeptide of Mr 115,000 was seen for the enzyme, purified from tunicamycin exposed explants. This result suggests that aminopeptidase N is co-translationally inserted into the membrane without cleavage of the signal. Pulse-chase labelling of explants gave the following results: 1. Immediately after a 10 min pulse with [35S] methionine, aminopeptidase N was detected in the Ca2+-precipitated membrane fraction. 2. The earliest detectable form of the enzyme, a polypeptide of Mr 140,000, was "high mannose" glycosylated as judged by its sensitivity to endoglycosidase H. After 40 min of chase, a re-glycosylation, yielding the mature form of Mr 166,000, occurred. 3. Aminopeptidase N was expressed at the microvillar membrane after 60-90 min of chase. Monensin inhibited the conversion from high mannose to complex glycosylation and the appearance of the enzyme in the microvillar membrane, indicating a role of the Golgi complex in these processes. Colchicine prevented aminopeptidase N from reaching the microvillar membrane, suggesting the involvement of microtubules in the transport.
D000626:Aminopeptidases / Q000235:genetics* / Q000302:isolation & purification; D000818:Animals; D018826:CD13 Antigens; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D007421:Intestine, Small / Q000201:enzymology*; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics; D013552:Swine
D012333:RNA, Messenger; D000626:Aminopeptidases; D018826:CD13 Antigens