Kidney plasma membranes of Aplysia californica were shown to contain an endopeptidase activity which cleaved [Leu]enkephalin (Tyr-Gly-Gly-Phe-Leu) and [Leu]enkephalinamide (Tyr-Gly-Gly-Phe-Leu-NH2) at the Gly3-Phe4 bond, as determined by reverse-phase h.p.l.c. analysis of metabolites. The optimal pH was shown to be 6.5. The bivalent cation chelating agent, 1,10-phenanthroline protected [Leu]enkephalin from degradation, suggesting that this enzyme is a metallopeptidase. The degradation of [Leu]enkephalin was also abolished by the neutral endopeptidase-24.11 inhibitors RB104 (2-[(3-iodo-4-hydroxyl)-phenylmethyl]-4-N-[3-(hydroxyamino-3-oxo-1- phenylmethyl)-propyl]amino-4-oxobutanoic acid), HABCO-Gly [(3-hydroxy-aminocarbonyl-2-benzyl-1-oxypropyl)glycine], phosphoramidon and thiorphan, with IC50 values of 1 nM, 1 microM, 20 microM and 30 microM respectively. By contrast, the angiotensin-converting enzyme inhibitor captopril and the serine proteinase inhibitor phenylmethanesulphonyl fluoride were without effect. Phase separation experiments using Triton X-114 showed that about 64% of the neutral endopeptidase activity in the Aplysia kidney membrane corresponds to an integral membrane protein. A specific radioiodinated inhibitor ([125I]RB104) was shown to bind the Aplysia endopeptidase with high affinity; the KD and Bmax. values were 21 +/- 5 pM and 20.3 +/- 5 fmol/mg of proteins respectively. This inhibitor was used to determine the molecular form of the enzyme, after separation of solubilized membrane proteins on SDS/PAGE and transfer on to nitrocellulose membranes. A single protein band with an apparent molecular mass of 140 kDa was observed. The labelling was abolished by specific neutral endopeptidase inhibitors. This study provides the first biochemical characterization of an endopeptidase with catalytic properties similar to those of neutral endopeptidase-24.11 in the mollusc Aplysia californica.
Identification and characterization of a neutral endopeptidase activity in Aplysia californica
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W Bawab, R S Aloyz, P Crine, B P Roques, L DesGroseillers; Identification and characterization of a neutral endopeptidase activity in Aplysia californica. Biochem J 1 December 1993; 296 (2): 459–465. doi: https://doi.org/10.1042/bj2960459
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