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.
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.
Miotto
G
G
Dipartimento di Chimica Biologica, Università Degli Studi Padova, Italy.
Venerando
R
R
Marin
O
O
Siliprandi
N
N
Mortimore
G E
GE
D000818:Animals; D001343:Autophagy / Q000187:drug effects*; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D004355:Drug Stability; D006868:Hydrolysis / Q000187:drug effects; D007930:Leucine / Q000378:metabolism*; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D008297:Male; D010455:Peptides / Q000494:pharmacology*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D014617:Vacuoles / Q000378:metabolism
D010455:Peptides; D011506:Proteins; C089941:multiple antigen peptide, leucyl(8)-lysyl(4)-lysyl(2)-lysyl-beta-alanine; D007930:Leucine