INHIBITION OF MACROAUTOPHAGY AND PROTEOLYSIS IN THE ISOLATED RAT HEPATOCYTE BY A NONTRANSPORTABLE DERIVATIVE OF THE MULTIPLE ANTIGEN PEPTIDE LEU(8)-LYS(4)-LYS(2)-LYS-BETA-ALA

The multiple antigen peptide derivative, Leu(8)-Lys(4)-Lys(2)-Lys-beta Ala (Leu(8)-MAP), was synthesized by attaching the carboxyl of leucin e to the NH2 termini of a branched lysine core, termed MAP, creating a molecule of about 1900 Da with 8 leucine residues. On a molar basis ( independent pendent of the number of leucine substitutions), Leu(8)-MA P was as effective as leucine in suppressing macroautophagy and proteo lysis; moreover, it exhibited the same apparent K-m (about 0.1 mM). Th e effect was specific for leucine since Ile(8)-MAP was inactive. It is of interest, though, that Leu(8)-MAP did not elicit the multiphasic r esponse typical of leucine but instead evoked the single site inhibiti on normally seen with leucine plus the coregulator alanine. Some free leucine was produced from Leu(8)-MAP during hepatocyte incubations, bu t the amounts were insufficient to account for the inhibition. Althoug h this degradation created species of Leu-MAP that had lost 1-3 residu es of leucine, their inhibitory effectiveness was not diminished. Beca use the extracellular/intracellular distribution ratio of [H-3]-Leu(8) -MAP was 100:1 or greater, the direct transport of Leu(8)-MAP across t he plasma membrane into the cytosolic compartment can be excluded. Hen ce, cytosolic concentrations of Leu(8)-MAP will be at least 100-fold s maller than those of leucine under conditions of comparable proteolyti c inhibition. For these and related reasons, effects attributable to t he recognition of Leu(8)-MAP cannot be explained by signals generated within the cytosol. They could, however, be mediated from site(s) on t he plasma membrane or within associated vesicles.