Co-and Post-translational Events in the Biogenesis of Pig Small Intestinal Aminopeptidase N(MEMBRANE PROTEINS AND TRANSPORT-II)

  • DANIELSEN E. Michael
    Department of Biochemistry C, the Panum Institute, University of Copenhagen
  • NOREN Ove
    Department of Biochemistry C, the Panum Institute, University of Copenhagen
  • SJOSTROM Hans
    Department of Biochemistry C, the Panum Institute, University of Copenhagen

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説明

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 M_r 115,000. When translation was performed in the presence of dog pancreatic microsomes, a M_r 140,000 polypeptide was also observed. A polypeptide of M_r 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 [^<35>S] methionine, aminopeptidase N was detected in the Ca^<2+> -precipitated membrane fraction. 2. The earliest detectable form of the enzyme, a polypeptide of M_r 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 M_r 166,000, occured. 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 precesses. Colchicine prevented aminopeptidase N from reaching the microvillar membrane, suggesting the involvement of microtubules in the transport.

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