EFFECT OF LEUPEPTIN ON THE LATERAL MOBILITY OF PROTEINS IN THE PLASMA-MEMBRANE OF HEPATOCYTES OF C57BL/6 MICE - FRAP STUDIES ON LIVER SMEARS

Four groups of male C57BL/6 mice (ages 2, 3.8, 11.8-12.5 and 24 months) were administered leupeptin at 5 mg/100 g body weight/day via intraperitoneally implanted osmotic minipumps. Both untreated and saline-treated mice served as controls. The time and dose-dependence of the effects of leupeptin on the lateral mobility of proteins in hepatocyte surface membranes were analyzed first in mice treated for 2, 5, 8, 11 and 14 days, respectively, by using the fluorescence recovery after photobleaching (FRAP) technique. The age dependence of the response to this treatment was also studied after 14 days of treatment in mice of various ages. The average lateral diffusion constant (D) and the fractional recovery (FR) were measured, and D x FR calculated. Leupeptin treatment at a daily dose of 5 mg/100 g for 2 weeks increased plasma glutamic-pyruvic transaminase levels 2-fold in all age groups. Leupeptin treatment caused a linear increase of D and a decrease of FR with respect to the duration of the treatment in adult mice. In all age groups about 30% of the membrane proteins became immobile after 14 days of treatment, whereas the still mobile fraction displayed a large increase of D. The values of D x FR in the leupeptin-treated groups were slightly higher than those in the untreated mice until the age of 15 months but decreased after this age. The results are interpreted in terms of the known inhibitory effect of leupeptin on cytoplasmic and lysosomal thiol proteases, causing a general slowing down of the protein turnover and, specifically, of proteins in the hepatocyte membrane.