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Characterization of Membrane-Bound Neuropeptide-Degrading Activities in Aplysia Californica

We are investigating the role of membrane-bound peptidases in the inactivation of neuropeptides in Aplysia. We found neuropeptide-degrading enzymes in all tested tissues of Aplysia. This activity was very sensitive to amastatin, suggesting that the major enzyme was an aminopeptidase. Accordingly, this enzyme hydrolysed [3H]leu-enkephalin at the tyr1-gly2 bond and FMRFamide at the Phc1-Met2 bond as determined by HPLC 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 mammalian aminopeptidase N. However, the enzyme from Aplysia was able to hydrolyse substrates known to be resistant to mammalian aminopeptidases due to the presence of a D-amino acid. Inhibition of this aminopeptidase with amastatin allowed us to characterize a second enkephalin-degrading enzyme in the kidney plasma membranes. This enzyme cleaved leu-enkephalin at the Gly3-Phe4 bond and its activity is sensitive to neutral endopeptidase inhibitors. After separation of kidney membrane proteins by SDS-PAGE, a specific radioiodinated inhibitor ([125I]RB104) which binds the Aplysia endopeptidase with high affinity recognized a protein band with an apparent molecular mass of 140 kDa. The labelling was abolished by specific NEP inhibitors.