| pubmed-article:2478543 | pubmed:abstractText | Two endopeptidases are involved in the conversion of proinsulin; a type I activity directed at the B chain, Arg31,Arg32, C-peptide junction, and type II which cleaves the C-peptide, Lys64,Arg65, A chain junction. To define further the substrate specificities of these enzymes, a series of mutant preproinsulin cDNAs were generated by site-directed and deletion mutagenesis. These were inserted into pT7 plasmids and capped cRNA transcripts synthesized, that were then microinjected into Xenopus oocytes. Oocytes were biosynthetically radiolabeled with [3H]leucine and the secreted peptides (greater than 95% present as unprocessed proinsulins) then incubated with types I and II endopeptidase activities prepared from isolated insulinoma secretory granules. The reaction products were analyzed by high performance liquid chromatography. Des-38-62-proinsulin, in which all but six amino acids of C-peptide were deleted was not processed by either enzyme. The mutant Lys64,Arg65 to Thr64,Arg65 was not cleaved by the type II enzyme but was still a substrate for the type I enzyme. The mutant Arg31,Arg32 to Arg31,Gly32 correspondingly was not cleaved by the type I enzyme; however, in this case it was not attacked by the type II enzyme. These results indicate that not only is the presence of a dibasic sequence essential, but also that the secondary structure of the protein is important in determining whether the prohormone is susceptible to proteolytic processing. | lld:pubmed |
