Role of thiols in degradation of proteins by cathepsins.
The effects of thiols on the breakdown of 125I-labelled insulin, albumin and formaldehyde-treated albumin by highly purified rat liver cathepsins B, D, H and L at pH 4.0 and 5.5 were studied. At both pH values degradation was strongly activated by the thiols cysteamine, cysteine, dithiothreitol, glutathione and 2-mercaptoethanol, and its rate increased with increasing thiol concentration. Preincubation of the protein substrates with 5 mM-glutathione did not affect concentration. Preincubation of the protein substrates with 5 mM-glutathione did not affect the rate of degradation by cathepsin D or L, and determination of free thiol groups after incubation of the proteins in the presence of glutathione but without cathepsin showed that their disulphide bonds were stable under the incubation conditions. Sephadex G-75 chromatography of the acid-soluble products of insulin digestion by cathepsin D or L suggested that thiols can reduce disulphide bonds in proteins after limited proteolysis. The resultant opening-up of the protein structure would lead to further proteolysis, so that the two processes (proteolysis and reduction) may act synergistically. By using the osmotic protection method it was shown that, at a physiological pH, cysteamine, and its oxidized form cystamine, can cross the lysosome membrane and thus may well be the physiological hydrogen donor for the reduction of disulphides in lysosomes. The results are discussed in relation to the lysosomal storage disease cystinosis.
D000118:Acetylglucosaminidase / Q000378:metabolism; D000818:Animals; D002403:Cathepsins / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D002850:Chromatography, Gel; D005978:Glutathione / Q000494:pharmacology; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism*; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology*; D011489:Protein Denaturation; D051381:Rats; D012711:Serum Albumin, Radio-Iodinated / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000494:pharmacology*; D014508:Urea / Q000494:pharmacology
D007328:Insulin; D012711:Serum Albumin, Radio-Iodinated; D013438:Sulfhydryl Compounds; D014508:Urea; D000118:Acetylglucosaminidase; D002403:Cathepsins; D005978:Glutathione