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Endoproteolytic activity in mammalian brain membranes cleaves 5-hydroxytryptamine-moduline into dipeptides
M Plantefol1, J C Rousselle, E Bernardi
1Unité de Pharmacologie, Neuro-Immuno-Endocrinienne, Institut Pasteur, Paris, France.
Abstract:
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.
Insights
Researchers identified specific enzymes in mammalian brain membranes that inactivate 5-hydroxytryptamine-moduline (5-HT-moduline). A metalloprotease and a bestatin-sensitive aminopeptidase were found to be responsible for its metabolism.
Area of Science:
- Neurochemistry
- Enzymology
- Molecular Biology
Background:
- 5-hydroxytryptamine-moduline (5-HT-moduline) is an endogenous peptide found in mammalian brain.
- Understanding the enzymes that metabolize 5-HT-moduline is crucial for elucidating its physiological role.
Purpose of the Study:
- To identify and characterize the enzymatic activities responsible for 5-HT-moduline inactivation in mammalian brain membranes.
- To determine the kinetic properties and specificity of the identified enzymes.
Main Methods:
- Development of an enzymatic assay using radiolabeled [3H]5-HT-moduline.
- High-performance liquid chromatography (HPLC) for metabolite identification and quantification.
- Enzyme inhibition studies using specific inhibitors like bestatin and metalloprotease inhibitors.
Main Results:
- 5-HT-moduline metabolism exhibited characteristics of metalloprotease activity, including sensitivity to chelators and Zn2+ reactivation.
- Two key enzymes were identified: a bestatin-sensitive aminopeptidase and a specific endoprotease.
- The endoprotease cleaved 5-HT-moduline into Leu-Ser and Ala-Leu dipeptides, with a Km of 37.1 μM and Vmax of 5.5 μmol/min/mg protein.
- This endoprotease showed specificity for tetrapeptides and was insensitive to inhibitors of ACE, ECE, and NEP.
Conclusions:
- A specific endoprotease is likely involved in the physiological metabolism of 5-HT-moduline in the brain.
- The identified enzymes contribute to the regulation of 5-HT-moduline levels and its interaction with serotonergic receptors.