The effect of protease inhibitors and decreased temperature on the degradation of different classes of proteins in cultured hepatocytes.
Leupeptin, chymostatin and antipain inhibited the degradation of long-lived proteins in cultured rat hepatocytes by 20-30%, probably by inhibiting lysosomal proteases: (1) Leupeptin and chymostatin decreased to a similar extent the degradation of an exogenous protein 125I-asialo fetuin, a process known to occur within lysosomes. (2) In extracts of cells treated with leupeptin, cathepsin B activity was inhibited by 35-50%. (3) Leupeptin, chymostatin and antipain inhibited proteolysis by homogenates of liver lysosomes but not by the supernatant fraction. These agents, however, do not appear to rapidly permeate the membrane of isolated lysosomes. Leupeptin, chymostatin and antipain did not inhibit the breakdown of short-lived normal cell proteins, and ones containing amino acid analogs. Even when the amount of abnormal proteins was increased, such that it comprised a large fraction of cell protein, the degradation of these polypeptides was still very rapid and not affected by these inhibitors. The pathway for the degradation of short-lived cell proteins thus appears distinct from that responsible for degradation of long-lived cell proteins. In accord with this conclusion, reduction of the temperature of cultures inhibited the breakdown of long-lived proteins to a much greater extent than it affected the breakdown of short-lived ones. Treatment of cultured hepatocytes with glucagon, or deprivation for serum or amino acids stimulated the degradation of the more stable cell proteins but did not affect the breakdown of 125I-asialo-fetuin. Under these conditions leupeptin and chymostatin inhibited the breakdown of long-lived cell proteins to the same extent as in control cultures. Thus, lysosomal enzymes seem to play an important role in protein breakdown both in fed hepatocytes and in cells where proteolysis is accelerated.
D000818:Animals; D000976:Antipain / Q000494:pharmacology; D002403:Cathepsins / Q000378:metabolism; D002478:Cells, Cultured; D007976:Leupeptins / Q000494:pharmacology; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D013696:Temperature
D007976:Leupeptins; D009842:Oligopeptides; D011480:Protease Inhibitors; D011506:Proteins; D000976:Antipain; C002101:chymostatin; D002403:Cathepsins