Cell-free synthesis of fish preproinsulin, and processing by heterologous mammalian microsomal membranes.
Poly(A)-containing mRNA isolated from the islets of Langerhans obtained from two species of fish, angler fish (Lophius americanus) and sea raven (Hemitripterus americanus), stimulated protein synthesis 16-fold in a wheat germ cell-free system. Characterization of the translation products by polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed a major polypeptide weighing 11,500 daltons that was specifically precipitated by an antibody against angler fish insulin. Partial sequence analysis of the amino terminal revealed that this polypeptide is preproinsulin, in which the amino terminus of proinsulin is preceded by either 23 (angler fish) or 25 (sea raven) amino acid residues. Translation of fish islet mRNA in a wheat germ cell-free system in the presence of dog pancreas microsomal membranes led to the correct cleavage of the nascent preproinsulin, resulting in the synthesis of authentic fish proinsulin, as verified by partial sequence analysis. Moreover, the synthesized fish proinsulin was segregated, presumably into the luminal space of the dog pancreas microsomal vesicles, because it was found to be resistant to proteolysis by added trypsin and chymotrypsin. Our data thus suggest that the mechanisms and information for the transfer of secretory proteins across the microsomal membrane are highly conserved during evolution.
D000818:Animals; D002474:Cell-Free System; D004285:Dogs; D005399:Fishes / Q000378:metabolism*; D007515:Islets of Langerhans / Q000032:analysis; D008566:Membranes / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism / Q000648:ultrastructure; D008970:Molecular Weight; D010179:Pancreas / Q000378:metabolism*; D011061:Poly A / Q000302:isolation & purification; D011384:Proinsulin / Q000096:biosynthesis / Q000378:metabolism*; D011498:Protein Precursors / Q000096:biosynthesis / Q000378:metabolism*; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism; D013045:Species Specificity; D014908:Triticum
D011498:Protein Precursors; D012333:RNA, Messenger; D011061:Poly A; D011384:Proinsulin