Effects of leupeptin and pepstatin on protein turnover in adult rat hepatocytes in primary culture

Addition of pepstatin, an inhibitor of acid protease, to 2-day cultures of rat hepatocytes rapidly inhibited the activity to hydrolyze hemoglobin (Hb), but did not affect the activity to hydrolyze α-N-benzoyl-dl-arginine-β-naphthylamide (BANA). On the other hand, addition of leupeptin, an inhibitor of thiol protease, inhibited the activity of BANA hydrolase and caused a sixfold increase in the activity of Hb hydrolase within 1 day. Neither protease inhibitor affected the rate of protein synthesis. Release of amino acids from hepatocytes into Hanks' salt solution was measured by the ninhydrin method. Pepstatin inhibited the release only 15% within 2 days, but leupeptin inhibited it 65% within 10 h. These two inhibitors had additive inhibitory effects on the release, suggesting that they inhibit the degradations of different groups of proteins. The inhibitory effect of leupeptin gradually decreased after 10 h, which is consistent with the observed induction of a protease activity mentioned above. A preferential involvement of leupeptin-sensitive protease in the degradation of proteins with longer half-lives was suggested from studies on ¹⁴C]leucine release from hepatocytes prelabeled for 30 h. On the other hand, the two inhibitors had similar effects on the release of ¹⁴C]leucine from hepatocytes labeled for only 1 h. Their inhibitory effects were again additive, but there was no reduction in the inhibition by leupeptin on prolonged incubation, suggesting that proteins with short half-lives were not substrates for the induced protease. These results suggest that in hepatocytes, proteins with longer half-lives are degraded more by cathepsin B than by cathepsin D, while those with short half-lives are degraded equally by these two proteases.