Effects of denaturation and methylation on the degradation of proteins in cultured hepatoma cells and in reticulocyte cell-free systems.
Radioiodinated, native and denatured bovine serum albumin (albumin) beta-lactoglobulin and cytochrome c were introduced into hepatoma tissue culture cells by erythrocyte-ghost-mediated microinjection, and their rates of degradation were compared. Denatured albumin was degraded at 20% of the rate of undenatured albumin, denatured beta-lactoglobulin was degraded three times faster than undenatured beta-lactoglobulin, while denatured and undenatured cytochrome c were degraded at the same rate. Thus, denaturation does not affect the rates of intracellular breakdown of microinjected proteins in a simple predictable way. Exhaustive methylation did not inhibit the degradation of denatured beta-lactoglobulin or albumin, indicating that, like their undenatured counterparts, they are not degraded via the ubiquitin pathway. In reticulocyte lysates, in the presence of ATP, denatured albumin and beta-lactoglobulin were broken down at slightly slower rates than the parent proteins. Exhaustive methylation of both denatured and undenatured proteins completely abolished their ATP-dependent breakdown. This inhibition is consistent with the hypothesis that free -NH2 groups are required for the attachment of ubiquitin prior to degradation in this system. Removal of an ammonium sulfate fraction from reticulocyte lysates produces a proteolytic system markedly different from the whole lysate [Speiser, S. & Etlinger, J. D. (1983) Proc. Natl Acad. Sci. USA 80, 3577-3580]. In this system both denatured and undenatured albumin and beta-lactoglobulin were degraded essentially independently of ATP. Methylation only slightly decreased the breakdown of denatured proteins, suggesting that they are not degraded via the ubiquitin pathway. A possible explanation of these results is that removal of the ammonium sulfate fraction unmasks an ATP-independent proteolytic system unrelated to the ubiquitin pathway.
Katznelson
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Kulka
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D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002474:Cell-Free System; D002478:Cells, Cultured; D002918:Chymotrypsin; D003574:Cytochrome c Group / Q000378:metabolism*; D007782:Lactoglobulins / Q000378:metabolism*; D008113:Liver Neoplasms; D008745:Methylation; D011489:Protein Denaturation; D012156:Reticulocytes / Q000378:metabolism*; D012709:Serum Albumin / Q000378:metabolism*; D014357:Trypsin
D003574:Cytochrome c Group; D007782:Lactoglobulins; D012709:Serum Albumin; D002918:Chymotrypsin; D014357:Trypsin