Conversion of proinsulin to insulin: involvement of a 31,500 molecular weight thiol protease.
A lysed crude secretory granule fraction from rat islets of Langerhans was shown to process endogenous proinsulin to insulin with a pH optimum of 5.0--6.0. The converting activity in the lysed fraction was not inhibited by serine protease inhibitors (diisopropyl fluorophosphate, soybean trypsin inhibitor, and aprotinin) or metalloprotease inhibitors (EDTA and o-phenanthroline) but was inhibited by some thiol protease reagents (p-chloromercuribenzenesulfonic acid, antipain, and leupeptin) but not by others (N-ethylmaleimide and iodoacetamide). N alpha-p-Tosyl-L-lysyl chloromethyl ketone only mildly inhibited at higher concentrations, whereas L-alanyl-L-lysyl-L-arginyl chloromethyl ketone was a powerful inhibitor. L-Alanyl-L-lysyl-L-arginyl chloromethyl ketone was [125I]iodotyrosylated and used as an affinity labeling agent for the converting activity. Because the crude granule preparation contained contaminating lysosomes the affinity labeling of the granule preparation proteins was compared with that in liver lysosomes purified from rats injected with Triton WR1339. In the crude granule fraction the affinity label bound in a cysteine-enhanced manner to a single 31,500 molecular weight protein, but in purified liver lysosomes the major affinity-labeled protein had a molecular weight of 25,000 and minor 31,500 and 35,000 molecular weight proteins were also labeled. Evidence suggests that these proteins are thiol proteases and that in islets the 31,500 molecular weight thiol protease is involved in the conversion of proinsulin to insulin.
D000345:Affinity Labels; D000818:Animals; D003546:Cysteine Endopeptidases; D003594:Cytoplasmic Granules / Q000201:enzymology; D010450:Endopeptidases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000096:biosynthesis*; D007515:Islets of Langerhans / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology; D008297:Male; D008970:Molecular Weight; D011384:Proinsulin / Q000378:metabolism*; D011480:Protease Inhibitors; D051381:Rats
D000345:Affinity Labels; D007328:Insulin; D011480:Protease Inhibitors; D011384:Proinsulin; D010450:Endopeptidases; D003546:Cysteine Endopeptidases