Characterization of an endopeptidase involved in pre-protein processing.
Proteolytic removal of the pre-segment from growing nascent chains of pre-human placental lactogen (hPL) occurred during in vitro translation of placental mRNA if crude membranes derived from ascites lysates, dog pancreas, or rat liver rough endoplasmic reticulum were added to the translation mixtures. The cotranslational proteolytic event was inhibited by the peptide protease inhibitor, chymostatin, but not by leupeptin, antipain, or elastatinal. The proteases involved in cleavage were solubilized with detergent and converted completed pre-hPL to hPL (post-translational processing). Direct assay of the solubilized membranes, with synthetic fluorogenic aminocoumarin peptide substrates, revealed no significant tryptic or elastase-like activity, but activity against a chymotrypsin substrate [(succinyl-Ala-Ala-Phe)-7-amino-4-methyl-coumarin] was found. This activity was dependent upon both an endopeptidase and an aminopeptidase. Although bestatin inhibited the aminopeptidase activity, it had no effect on the endopeptidase or on post-translational cleavage. Although this endopeptidase cleaved on the COOH side of an alanine residue, it was not inhibited by elastatinal. However, it was inhibited by high levels of chymostatin and by some serine protease inhibitors.
D000818:Animals; D002474:Cell-Free System; D004285:Dogs; D005260:Female; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism; D010928:Placental Lactogen / Q000378:metabolism*; D011247:Pregnancy; D011480:Protease Inhibitors / Q000494:pharmacology; D011498:Protein Precursors / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D013379:Substrate Specificity
D010446:Peptide Fragments; D011480:Protease Inhibitors; D011498:Protein Precursors; D012333:RNA, Messenger; D010928:Placental Lactogen; D010447:Peptide Hydrolases