Inhibition of preprotein processing in ascites tumor lysates by incorporation of a leucine analog.
Leucine analogs were tested in the Krebs II ascites cell-free translation system for the ability to inhibit preprotein cleavage by replacing leucine in nascent chains of bovine preprolactin, rat preprolactin, human placental prelactogen (pre-hPL), and pre-alpha subunit of human chorionic gonadotropin (alpha-hCG). In the absence of analog, ascites microsomal membranes cleaved these preproteins to their mature forms and sequestered the processed products. Also, two asparagine residues in alpha-hCG were glycosylated. When 4 mM beta-DL-hydroxyleucine was added to the lysate instead of L-leucine, cotranslational processing and sequestration of both species of preprolactin and pre-hPL were inhibited. Sequential Edman degradation confirmed that pre-hPL was not cleaved. The inhibition of processing by beta-hydroxyleucine resulted from its incorporation into protein. This was shown by reversal of the effect by addition of leucine and by inhibition of [(3)H]leucine incorporation into protein. Of significance, the processing of pre-alpha-hCG was less sensitive to beta-hydroxyleucine because its prepeptide contains only four scattered leucine residues, whereas the presegments of hPL and the prolactins contain six to eight clustered leucine residues. These experiments demonstrate that translocation and processing of secretory proteins require structural features determined by the primary amino acid sequence.
D000818:Animals; D001692:Biological Transport; D002417:Cattle; D002474:Cell-Free System; D006063:Chorionic Gonadotropin / Q000378:metabolism*; D006801:Humans; D007930:Leucine / Q000031:analogs & derivatives*; D008970:Molecular Weight; D010928:Placental Lactogen / Q000378:metabolism*; D011388:Prolactin / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D051381:Rats; D013329:Structure-Activity Relationship
D006063:Chorionic Gonadotropin; D011498:Protein Precursors; C024584:beta-hydroxyleucine; D011388:Prolactin; D010928:Placental Lactogen; D007930:Leucine