Various lysosomotropic amines have two parallel effects in human lymphocytes: they inhibit t h e degradation of cellular proteins and increase the migration of nonhistone proteins (NHP) from the cytoplasm to t h e nucleus. The increased nuclear level of N H P is associated with increased cellular binding of [3H] actinomycin D, indicating an altered structure of chromatin. The agents inhibit t h e degradation of short-and long-lived proteins equally. Fractionation of the [3H] N H P of the nucleus according to pH 2.5-6.5 shows that [3H] N H P with a high rate of degradation in untreated cells correspond to [3H] N H P with a high rate of migration in cells treated with the agents. Eserine, amantadine, nicotine, atropine, benzylamine, and propranolol inhibit cathepsin D in concentrations causing proteolytic inhibition in cell cultures or in concentrations believed to be attained in lysosomes. The agents strongly inhibit t h e cellular accumulation of [3H] chloroquine. The data support the proposal that the migration of N H P from the cytoplasm to t h e nucleus is the direct consequence of inhibited degradation of these proteins in lysosomes by the amines. 0 1985 ALAN R. LISS, INC. hydrochloride, eserine sulfate, nicotine, methylamine hydrochloride, morpholine, propranolol hydrochloride, and trimethylamine hydrochloride were from Sigma Chemical Co., St. Louis, MO. Human hemoglobin was from Calbiochem Behring, San Diego, CA; [3H] Leucine (55 Cilmmole) and r3H] uridine (55 Ci/mmole) were from ICN, Irvine, CA: [3H] Actinomycin D (['HI AD) (5.1 Ci/ mmole) was from Schwartz-Mann, Cambridge, MA; [3H]chloroquine Q3H] CQ) (130 mCi/mmole) and [3H] acetic anhydride (50 mCi/mmole) were from New England Nuclear Corporation, Boston, MA: L3H] propranolol (26 Cilmmole) was from Amersham, Arlington Heights,
IL.
Labeling of lymphocytes with [3Hl leucine
Labeling of lymphocytes with [3H] leucine has been described in detail previously (Polet, 1983). Briefly, to label the long-lived proteins, the cells were incubated in medium containing 0.1 mM leucine, 5 pCi/ml [3H] leucine, and 5 x lo6 celldm1 for 30 h, followed by incubation in isotope-free medium containing 2 mM leucine for 5 h. To label the short-lived proteins, the cells, after incubation overnight in unlabeled medium, were incubated in medium without plasma containing 0.01 mM leucine, 10 pWml [3H] leucine, 2 mg/ml BSA, and 10 x lo6 celldml for 1 h at 37ยฐC. Other methods of labeling are described in the text. At the end of each labeling