Oxidation of sulfhydryl groups of ribonuclease inhibitor in epithelial cells is sufficient for its intracellular degradation.
Ribonuclease inhibitor (RI) is a cytoplasmic protein (50 kDa) that inhibits a variety of pancreatic type RNases. The porcine inhibitor contains 30 cysteine residues, all of which occur in the reduced state. It is well known that in vitro modification of the thiol groups inactivates the protein and greatly increases its susceptibility to proteolysis. Here we show that oxidation of thiol groups in RI can also occur within the cell. Induction of an oxidative insult in cultured LLC-PK1 cells, either with a general oxidant, H2O2, or with a thiol-specific oxidant, diamide, led to the loss of RI activity. By using specific antibodies it was demonstrated that the decrease correlated with a decline in the amount of RI protein in the cells. Furthermore, analysis of RI mRNA levels and half-life of the protein excluded inhibition of the synthesis of RI as the cause of its depletion. The results indicate that oxidation of thiol groups in RI is sufficient to cause its rapid inactivation and disappearance from the cell. Most likely this results from intracellular degradation of the protein.
Blázquez
M
M
Friedrich Miescher-Institut, CH-4002 Basel, Switzerland.
Fominaya
J M
JM
Hofsteenge
J
J
D000818:Animals; D002417:Cattle; D003958:Diamide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D018374:LLC-PK1 Cells; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D011506:Proteins / Q000378:metabolism; D012260:Ribonucleases / Q000037:antagonists & inhibitors*; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013552:Swine
D004791:Enzyme Inhibitors; D016877:Oxidants; D011506:Proteins; D013438:Sulfhydryl Compounds; D003958:Diamide; D006861:Hydrogen Peroxide; D007770:L-Lactate Dehydrogenase; D012260:Ribonucleases