CHARACTERIZATION OF MEMBRANE-BOUND NEUROPEPTIDE-DEGRADING ACTIVITIES IN APLYSIA-CALIFORNICA

We are investigating the role of membrane-bound peptidases in the inac tivation of neuropeptides in Aplysia. We found neuropeptide-degrading enzymes in all tested tissues of Aplysia. This activity was very sensi tive to amastatin, suggesting that the major enzyme was an aminopeptid ase. Accordingly, this enzyme hydrolysed [H-3]leunkephalin al the tyr- gly(2) bond and FMRFamide at the Phe(1)-Met(2) bond as determined by H PLC analysis of cleavage products. Enzymatic studies in the absence or presence of aminopeptidase inhibitors revealed that this enzyme is a metallopeptidase with the same enzymatic characteristics as the mammal ian aminopeptidase N. However, the enzyme from Aplysia was able to hyd rolyse substrates known to be resistant to mammalian aminopeptidases d ue to the presence of a D-amino acid. Inhibition of this aminopeptidas e with amastatin allowed us to characterize a second enkephalin-degrad ing enzyme in the kidney plasma membranes. This enzyme cleaved leu-enk ephalin al the Gly(3)-Phe(4) bond and its activity is sensitive to neu tral endopeptidase inhibitors. After separation of kidney membrane pro teins by SDS-PAGE, a specific radioiodinated inhibitor ([I-125]RB104,) which binds the Aplysia endopeptidase with high affinity recognized a protein band with an apparent molecular mass of 140 kDa. The labellin g was abolished by specific NEP inhibitors.