Purification and characterization of a rat liver cytosol neutral thiol peptidase that degrades glucagon, insulin, and isolated insulin A and B chains.
A thiol peptidase that catalyzes at near neutral pH the hydrolysis of insulin, the isolated A and B chains of insulin, and glucagon was purified from rat liver cytosol by fractionation on Sephadex G-200, Affi-Gel Blue, and Spherogel TSK-G 3000 SW. The purified enzyme showed a single component by chromatography on a Spherogel TSK column and by gel filtration on a Sephadex G-200 column. The native enzyme has a molecular weight of approximately 180,000 and consists of two subunits having pI's of 5.9 and 6.3. Studies on its substrate specificity showed that the purified enzyme degrades glucagon, insulin, insulin B chain, and insulin A chain, but it does not degrade proinsulin, ACTH, or denatured hemoglobin. Kinetic analyses were performed on three substrates. The Km values were: 34 nM for insulin, 276 nM for insulin B chain, and 3.5 microM for glucagon. The kcat and Vm/Km values were glucagon greater than B chain greater than insulin. Thus, the enzyme has the highest affinity/lowest efficiency for insulin, an intermediate affinity/intermediate efficiency for B chain of insulin and the lowest affinity/highest efficiency for glucagon. The effect of several potential activators and inhibitors on the enzyme's activity was investigated. The enzyme activity was markedly inhibited by N-ethylmaleimide, p-chloromercuribenzoic acid, iodoacetamide, and Np-tosyl-L-phenylalanine chloromethyl ketone (TPCK), and was partially inhibited by dithiothreitol, by the chelating agents EDTA and EGTA, and by phenylmethylsulfonyl fluoride (PMSF). Bacitracin inhibited the activity of the enzyme, but the protease inhibitors aprotinin, leupeptin, pepstatin, and phosphoramidon had little or no effect. Reduced glutathione, iodoacetate, and N alpha,p-tosyl-L-lysine chloromethyl ketone (TLCK) also had little or no effect on the enzyme activity.
D000818:Animals; D011232:Chemical Precipitation; D002845:Chromatography / Q000379:methods; D003546:Cysteine Endopeptidases; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D005934:Glucagon / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D010446:Peptide Fragments / Q000378:metabolism; D011480:Protease Inhibitors; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D007328:Insulin; D010446:Peptide Fragments; D011480:Protease Inhibitors; D005934:Glucagon; D010450:Endopeptidases; D003546:Cysteine Endopeptidases