The characterization of endosomal insulin degradation intermediates and their sequence of production.
Insulin degradation within isolated rat liver endosomes was studied in vitro with the aid of three 125I-insulin isomers specifically labelled at tyrosine (A14, B16 and B26). Chloroquine and 1,10-phenanthroline were used to minimize insulin proteolysis during endosome preparation, whereas the manipulation of endosomal processing of insulin in vitro by Co2+ ions (to activate) and 1,10-phenanthroline (to inhibit) permitted the study of degradation intermediates and their time-dependent production. Structural and kinetic analysis of intermediates isolated from both intra- and extra-endosomal compartments allowed the determination of major cleavage sites and the probable sequence of proteolytic events. It was found that 125I-tyrosine is the ultimate labelled degradation product of all iodo-insulin isomers, suggesting that endosomal proteases are able to degrade insulin to the level of its constituent amino acids. 125I-tyrosine was also the only radiolabelled product able to cross the endosomal membrane. Intra-endosomal insulin degradation proceeds via two inter-related cleavage routes after metalloendoprotease cleavage of the B-chain. One pathway results from an initial cleavage in the centre region of the B-chain (B7-19), probably at B14-15, whereas the major route results from a cleavage at B24-25. B24-25 cleavage removes the B-chain C-terminal hexapeptide (B25-30), which is subsequently cleaved by an aminopeptidase activity to produce first the pentapeptide B26-30 and then 125I-tyrosine. The isolation of intact radiolabelled A-chain from the degradation of 125I-[A14]-insulin suggests that further degradation of proteolytic intermediates containing cleaved B-chain proceeds via interchain disulphide reduction. The A-chain is then processed by several cleavages, one of which occurs at A13-14.
Seabright
P J
PJ
University of Cambridge, Department of Clinical Biochemistry, Addenbrooke's Hospital, U.K.
Smith
G D
GD
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D002738:Chloroquine / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003035:Cobalt / Q000494:pharmacology; D003198:Computer Simulation; D010450:Endopeptidases / Q000378:metabolism; D011992:Endosomes / Q000201:enzymology / Q000378:metabolism*; D000939:Epitopes / Q000737:chemistry; D007328:Insulin / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D014443:Tyrosine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007328:Insulin; D010618:Phenanthrolines; D011480:Protease Inhibitors; D003035:Cobalt; D014443:Tyrosine; D002738:Chloroquine; D010450:Endopeptidases; C025205:1,10-phenanthroline