Inhibition of macroautophagy and proteolysis in the isolated rat hepatocyte by a nontransportable derivative of the multiple antigen peptide Leu8-Lys4-Lys2-Lys-beta Ala.

Abstract

The multiple antigen peptide derivative, Leu8-Lys4-Lys2-Lys-beta Ala (Leu8-MAP), was synthesized by attaching the carboxyl of leucine 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 of the number of leucine substitutions), Leu8-MAP was as effective as leucine in suppressing macroautophagy and proteolysis; moreover, it exhibited the same apparent Km (about 0.1 mM). The effect was specific for leucine since Ile8-MAP was inactive. It is of interest, though, that Leu8-MAP did not elicit the multiphasic response typical of leucine but instead evoked the single site inhibition normally seen with leucine plus the co-regulator alanine. Some free leucine was produced from Leu8-MAP during hepatocyte incubations, but the amounts were insufficient to account for the inhibition. Although this degradation created species of Leu-MAP that had lost 1-3 residues of leucine, their inhibitory effectiveness was not diminished. Because the extracellular/intracellular distribution ratio of [3H]-Leu8-MAP was 100:1 or greater, the direct transport of Leu8-MAP across the plasma membrane into the cytosolic compartment can be excluded. Hence, cytosolic concentrations of Leu8-MAP will be at least 100-fold smaller than those of leucine under conditions of comparable proteolytic inhibition. For these and related reasons, effects attributable to the recognition of Leu8-MAP cannot be explained by signals generated within the cytosol. They could, however, be mediated from site(s) on the plasma membrane or within associated vesicles.