Hydrolysis and association of leucine enkephalin to lymphomic and erythroleukaemic cell lines--II.
The relationship between the hydrolysis of labelled leucine enkephalin and the association of its radioactive label to the cells of lymphomic and erythroleukaemic cell lines have been studied using intact cells and resealed membranes obtained from these cells as models. Hydrolysis by cell enzymes and its effect on association have been analysed using protease inhibitors and non-hydrolysable enkephalin analogues. Results obtained confirm that hydrolysis of the pentapeptide is a prerequisite for association of the radiolabel to cells. The same results provide evidence of marked differences between enkephalin hydrolysis by whole cells and hydrolysis measured in the presence of resealed membranes, suggesting the existence in intact cells of proteolytic enzymes other than those bound to the membranes. The lack of reversibility of association and the intracellular localization of the radioactive label suggest that the association measured is prevailingly caused by internalization of a hydrolysis fragment, and not by binding to receptors. In order to determine the nature of the active fragment, association was measured in the presence of all four labelled N-terminal hydrolysis fragments of leu-enkephalin under conditions of nearly-total inhibition of proteolytic enzymes. Under these conditions, the label carried by Tyr, but not that carried by the other N-terminal fragments, was associated with cells. Free Tyr, furthermore, inhibits the association to cells of both labelled Tyr and leu-enkephalin. Data summarized above are consistent with the hypothesis that the radioactive label is taken up by the cells as Tyr, freed from the parent peptide by cell-related enzymes. The same data tend to exclude that a relevant fraction of the intact pentapeptide is bound to membrane receptors or that the radioactive label is carried into the cell by a N-terminal fragment other than Tyr.
bongiorno
L
L
Istituto per lo Studio delle Malattie Ereditarie e Carenziali, Cosenza, Italy.
Urbani
A
A
Marini
M
M
Gilardi
E
E
Roda
L G
LG
D002855:Chromatography, Thin Layer; D004743:Enkephalin, Leucine / Q000378:metabolism*; D004745:Enkephalins / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism*; D008223:Lymphoma / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D014316:Tritium; D014407:Tumor Cells, Cultured
D004745:Enkephalins; D011480:Protease Inhibitors; D014316:Tritium; D004743:Enkephalin, Leucine