Characterization of Neuropeptidases Using Inhibitors

Abstract

It is now well established that the major mechanism for the termination of a neuropeptide signal is not internalization of the peptide, but rather its metabolism by one or more neuropeptidases (reviewed in refs. 1 and 2). By the very nature of their action in hydrolyzing peptides that have been released into the synaptic cleft, these neuropeptidases are ectoenzymes; that is, they are integral proteins of the plasma membrane, asymmetrically oriented with the bulk of the protein, including the active site, exposed at the extracytoplasmic surface (3). In addition to the termination of peptide signals, some neuropeptidases are involved in the activation or modulation of certain peptides, in which case the enzyme may be localized either extracellularly or intracellularly. Although 100 or more potential neuropeptides have now been discovered, there is a substantially smaller number of neuropeptidases (Table 1). The reason for this is that most of the neuropeptidases can act on more than one peptide substrate. For example, neprilysin (neutral endopeptidase-24.11; EC 3.4.24.11) can hydrolyze the enkephalins, tachykinins, cholecystokinin, natriuretic factors, brady-kinin, and so on, whereas peptidyl dipeptidase A (angiotensin converting enzyme; EC 3.4.15.1) can hydrolyze bradykinin, the enkephalins, substance P, and so on, in addition to angiotensin I (1). Whether a peptide is a substrate for a particular neuropeptidase in vivo will depend on whether the two are colocalized in the same neuronal pathway and whether the kinetics of hydrolysis are favorable. Thus, although neprilysin can hydrolyze lutemizing hormone-releasing hormone (LH-RH) in vitro, the kinetics for this reaction are so poor that even if the enzyme and peptide colocalized in the brain, it is unlikely that neprilysin would contribute in any appreciable way to the in vivo metabolism of LH-RH. As well as these broad-acting neuropeptidases, there are a few substrate-specific enzymes such as thyrotropin releasing hormone-degrading enzyme (4) and endothelin converting enzyme (5). This latter group of enzymes are potentially more difficult to identify because of the necessity to have the specific substrate available first.