Characterization of aminopeptidases responsible for inactivating endogenous (Met5)enkephalin in brain slices using peptidase inhibitors and anti-aminopeptidase M antibodies.
In addition to "enkephalinase" (EC 3.4.24.11), two enkephalin-hydrolyzing aminopeptidases recently identified in cerebral membranes--aminopeptidase M (EC 3.4.11.2) and a "puromycin-sensitive" aminopeptidase (also designated "MII" or "aminoenkephalinase")--are potentially involved in endogenous enkephalin inactivation. Their participation in the hydrolysis of the endogenous (Met5)enkephalin released by depolarization of slices from rat globus pallidus was assessed, using three inhibitory agents: bestatin, puromycin, and anti-aminopeptidase M antibodies. The selectivity and potency of these agents were first determined by evaluating their IC50 values for inhibition of [3H](Met5)enkephalin hydrolysis by increasingly complex preparations comprising semipurified aminopeptidases, pallidal membranes, and pallidal slices. Bestatin was a fairly potent inhibitor but lacked selectivity, as there was only a 3-fold difference between its IC50 values for the two aminopeptidases, and it displayed restricted diffusion and degradation in the slice preparation. Puromycin discriminated well between the two aminopeptidases (30-fold difference in IC50 values) and did not show any apparent restricted diffusion in the slice preparation. Antiaminopeptidase M antibodies were highly discriminant (greater than 300-fold difference in IC50 values for the two aminopeptidases) but displayed restricted diffusion. Analysis of the concentration-protection curves of the three agents for recovery of the (Met5)enkephalin released from pallidal slices in the presence of the "enkephalinase" inhibitor, thiorphan, indicated that both aminopeptidases participated in enkephalin degradation but that the role of aminopeptidase M was largely predominant, in contrast with its low relative activity in the preparation.
Giros
B
B
Gros
C
C
Solhonne
B
B
Schwartz
J C
JC
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000502:physiology*; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001921:Brain / Q000201:enzymology*; D018826:CD13 Antigens; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004744:Enkephalin, Methionine / Q000378:metabolism*; D005917:Globus Pallidus / Q000201:enzymology; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008297:Male; D011691:Puromycin / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains
D000906:Antibodies; D011691:Puromycin; D004744:Enkephalin, Methionine; D000626:Aminopeptidases; D018826:CD13 Antigens; D007930:Leucine; C012211:ubenimex