IDENTIFICATION AND CHARACTERIZATION OF A NEUTRAL ENDOPEPTIDASE ACTIVITY IN APLYSIA-CALIFORNICA

Kidney plasma membranes of Aplysia californica were shown to contain a n endopeptidase activity which cleaved [Leu]enkephalin (Tyr-Gly-Gly-Ph e-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 metabol ites. The optimal pH was shown to be 6.5. The bivalent cation chelatin g agent 1,10-phenanthroline protected [Leu]enkephalin from degradation , suggesting that this enzyme is a metallopeptidase. The degradation o f [Leu]enkephalin was also abolished by the neutral endopeptidase-24.1 1 inhibitors RB104 o-3-oxo-1-phenylmethyl)-propyl]amino-4-oxobutanoic acid), HABCO-Gly droxy-aminocarbonyl-2-benzyl-1-oxypropyl)glycine], ph osphoramidon and thiorphan, with IC50 values of 1 nM, 1 muM, 20 muM an d 30 muM respectively. By contrast, the angiotensin-converting enzyme inhibitor captopril and the serine proteinase inhibitor phenylmethanes ulphonyl fluoride were without effect. Phase separation experiments us ing Triton X-114 showed that about 64% of the neutral endopeptidase ac tivity in the Aplysia kidney membrane corresponds to an integral membr ane protein. A specific radioiodinated inhibitor ([I-125]RB 104) was s hown to bind the Aplysia endopeptidase with high affinity; the K(D) an d B(max) values were 21+/-5 pM and 20.3+/-5 fmol/mg of proteins respec tively. 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 wi th an apparent molecular mass of 140 kDa was observed. The labelling w as abolished by specific neutral endopeptidase inhibitors. This study provides the first biochemical characterization of an endopeptidase wi th catalytic properties similar to those of neutral endopeptidase-24.1 1 in the mollusc Aplysia californica.