Endoproteolytic activity in mammalian brain membranes cleaves 5-hydroxytryptamine-moduline into dipeptides.
This work was intended to determine which enzymatic activities from crude synaptosomal mammalian brain membranes could qualify for the status of 5-hydroxytryptamine-moduline (5-HT-moduline, LSAL, Leu-Ser-Ala-Leu) inactivating enzymes. An enzymatic assay for 5-HT-moduline metabolism was developed using [3H]5-HT-moduline measurement and high performance liquid chromatography (HPLC) technique to identify and quantify 5-HT-moduline metabolites. 5-HT-moduline metabolism displayed all characteristics of metalloprotease activity: sensitivity to divalent ion chelators, reactivation by Zn2+ ions and a pH optimum in the 7-8 range. Bestatin, an aminopeptidase inhibitor, allowed the identification of two enzymatic activities responsible for this metabolism: a bestatin-sensitive aminopeptidase and an endoprotease cleaving 5-HT-moduline into LS (Leu-Ser) and AL (Ala-Leu) dipeptides. This latter enzyme was shown to have a Km of 37.1 +/- 3.6 microM and a Vmax of 5.5 micromol min(-1) l(-1) per mg of protein. Moreover, this enzyme was insensitive to peptidyl dipeptidase A (angiotensin converting enzyme, EC 3.4.15.1), endothelin converting enzyme and neutral endopeptidase (neprylisin, EC 3.4.24.11) inhibitors and displayed some specificity among 5-HT-moduline-analogues and in particular recognized only tetrapeptides. These results, together with the isolation of the LS and AL metabolites [Rousselle, J.C., Massot, O., Delepierre, M., Zifa, E., Rousseau, B., Fillion, G., 1996. Isolation and characterization of an endogenous peptide from rat brain interacting specifically with the serotonergic 1B receptor subtypes. J. Biol. Chem. 271, 726-735] during the purification process of 5-HT-moduline are strong arguments for the physiological implication of this endoprotease in 5-HT-moduline metabolism.
Plantefol
M
M
Unité de Pharmacologie, Neuro-Immuno-Endocrinienne, Institut Pasteur, Paris, France.
Rousselle
J C
JC
Bernardi
E
E
Schoofs
A R
AR
Pourrias
B
B
Fillion
G
G
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D066298:In Vitro Techniques; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008024:Ligands; D008297:Male; D008566:Membranes; D009479:Neuropeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011480:Protease Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013329:Structure-Activity Relationship
C102005:5-HT-moduline; D004151:Dipeptides; D008024:Ligands; D009479:Neuropeptides; D009842:Oligopeptides; D011480:Protease Inhibitors; D010450:Endopeptidases; D007930:Leucine; C012211:ubenimex