Enkephalinase enkephalins

Enkalon Enkastat Enkatherm Enkephalin analogues Enkephalinase Enkephalinase A Enkephalins... 

Neprilysin (enkephalinase, Endopeptidase-24.11, neutral endopeptidase, NEP, EC 3.4.24.11) bears considerable resemblance to other zinc-containing metallopeptidases it is an oligopeptidase that hydrolyzes enkephalins and a range of other active peptides. Enkephalins are endogenous ligands of opiate receptors, and the prolongation of their action via inhibition of enkephalinase... 

Bunnett, N.W., et al. 1988. Isolation of endopeptidase-24.11 (EC 3.4.24.11, enkephalinase ) from the pig stomach. Hydrolysis of substance P, gastrin-releasing peptide 10, [Leu5] enkephalin, and [Met5] enkephalin. Gastroenterology 95 952. 

The racemic inhibitor thiorphan 46) 25 inhibits enkephalinase and selectively supports the analgesic effect of enkephalins. 

Florentin, D., Sassi, A., and Roques, B.P. 1984 A highly sensitive fluorometric assay for enkephalinase, a neutral metalloendopeptidase that releases tryrosine-glycine-glycine from enkephalins. Anal. Biochem. 141, 62-69. 

Enkephalinase A (dipeptidyl carboxypeptidase) and enkephalinase B (dipep-tidyl aminopeptidase) are involved in the degradation of enkephalin, which has an opiate-like activity. Enkephalinase A cleaves methionine enkephalin (Tyr-Gly-Gly-Phe-Met) after the second glycine, whereas enkephalinase B cleaves after the first glycine. A postcolumn derivatization system is used to detect N-terminal tyrosine-containing peptides. 

The major intermediate of the Maillard reaction 38, having an ally lie system, seems to furnish a cytotoxic ester on metabolic sulfonation [298]. In contrast to the above findings, some Amadori products, such as pyrazines have antimutagenicity [299,300,301]. Enkastines, the Amadori products of 33 and dipeptides, beneficially prolong the action of enkephaline by inhibiting enkephalinase [302]. 

Enkephalinase is found in the brain as well as in other tissues and is localized in synaptic membranes enriched in opiate receptors (139). It is also a Zn(II) protease, with some sequence homology to carboxypep-tidase, and hydrolyzes the Gly -Phe bond of enkephalins (Tyr-Gly-Gly-Phe-Met and Tyr-Gly-Gly-Phe-Leu) into inactive fragments. Inhibitors are potentially useful as analgesic drugs. 

Enkephalins are pentapeptides that bind to opiate receptors. In the gut, enkephalins promote the absorption of sodium, chloride and water (Dobbins et al 1980). Racecadotril is an oral enkephalinase inhibitor used in France and the Philippines for the treatment of acute diarrhea. It prevents the degradation of endogenous opioids (enkephalins) and thus promotes absorption of water and electrolytes from the intestinal lumen (Matheson Noble 2000). Studies have demonstrated the efficacy of racecadotril in two models of hypersecretory diarrhea infusion of cholera toxin and castor oil induced diarrhea. Moreover, unlike loperamide, racecadotril did not prolong transit time in the small intestine or colon. Further experiments have shown that racecadotril does not promote bacterial overgrowth in the small intestine (Duval-Iflah et al 1999). There are no reports on the use of racecadotril in horses. 

The enkephalins, H—Tyr—Gly—Gly—Phe—X—OH (X=Leu, Met), or so-called opioid peptides because they mimic the action of the opiates, morphine and heroin, have a very short half life in the body because all four peptide bonds are prone to undergoing proteolysis. The Tyr—Gly bond can be hydrolysed by amino-peptidases, the Gly—Gly bond by dipeptidylaminopeptidases, the Gly—Phe bond by enkephalinase and the Phe—Met and Phe—Leu bonds by carboxypeptidases. An enormous number of analogues have been synthesised, especially with the object of producing compounds that exert potent analgaesic action but are free from side effects. Protection of the susceptible bonds by changing the amino-acid sequence is the obvious way to achieve this. The analogue H—Tyr—d—Met—Gly—Phe—... 

Enkephalins are endogenous opioids that are important enteric neurotransmitters. Enkephalins inhibit intestinal secretion without affecting motility. Racecadotril (acetor-phan), a dipeptide inhibitor of enkephalinase, reinforces the effects of endogenous enkephalins on the 5-opioid receptor to produce an antidiarrheal effect. 

Enkephalinase (EC 3.4.24.11). (membrane metalloendopeptidase, MEP neutral endopeptidase, NEP 24.11 enkephalinase A enkephalin-dipepti-dylcarboxypeptidase neutral proteinase from kidney brush border atrio-peptidase CDlO/CALLA/common acute lymphoblastic leukaemia antigen). This cleaves the enkephalin pentapeptides at the Gly -Phe bond. 

Figure 6.1. Diagramatic representation of the peptidases involved in the metabolism of the enkephalins. AP (aminopeptidases), DAP (dipeptidylaminopeptidase), ACE (angiotensin converting enzyme), ENK (enkephalinase) and CBXYP (non-specific carboxypeptidases). R represents methionine in f MetJ-enkephalin or leucine in [Leu]-enkephalin.

In washed brain slices, an in vitro model for the evaluation of effects of membrane-bound enzymes on enkephalin hydrolysis, the main hydrolysis products have been identified as Tyr and Tyr-Gly-Gly. The formation of Tyr-Gly-Gly is dependent on the action of enkephalinase and is reduced in the presence of thiorphan (an enkephalinase inhibitor). In the presence of bestatin (a general aminopeptidase inhibitor), the formation of Tyr is reduced Tyr is also reduced, to a lesser extent, in the presence of puromycin (an aminopeptidase Mil inhibitor). In the presence of thiorphan the formation of Tyr increases whereas in the presence of bestatin there is an increase in formation of Tyr-Gly-Gly. The level of Tyr-Gly in this model is low and is unaffected by either thiorphan or bestatin indicating that the action of DAP is unimportant. Recovery of endogenous enkephalins released by depolarisation of brain slices is enhanced in the presence of thiorphan or bestatin and is complete when both inhibitors are present. This does not occur in the case of puromycin or captopril (an ACE inhibitor) [31]. 

In vivo, both bestatin and thiorphan display antinociceptive properties in behavioural tests which are additive when the two inhibitors are coadministered [26,32]. Puromycin does not display similar activity. Taken together, these data strongly indicate that enkephalinase and puromycin insensitive aminopeptidase (aminopeptidase M) are involved in the metabolism of endogenous enkephalins. 

The biological activities of enkephalinase and aminopeptidase inhibitors have been found to be very weak when they are administered alone. However, enkephalinase inhibitors potentiate the effects of enkephalin analogues which are resistant to degradation by the aminopeptidases such... 

Both enkephalinase and membrane-bound aminopeptidases, particularly aminopeptidase M, are strong candidates as brain enkephalin-degrading neuropeptidases and fulfil the criteria laid down for this role [26]. The significance of aminopeptidase Mil has been insufficiently investigated for the enzyme to be disregarded as an enkephalin neuropeptidase. The role of DAP appears to be minor, at least in the CNS, although a peripheral role may be indicated [37], ACE is unlikely to be involved in deactivation of endogenously released enkephalin. 

Enkephalinase distribution is wide-spread in the brain and peripheral organs and soluble forms are present in human plasma and cerebrospinal fluid. In the central nervous system, its distribution largely parallels regional distribution of the enkephalins and opioid receptors (striatum > cortex > cerebellum) and it is localized in the particulate fraction of brain homogenates [26,50] in a non-xmiform fashion in mice, rats and humans. In the cortex or the striatum, opioid receptors and the enzyme display a parallel subcellular distribution pattern. Both enkephalinase and opioid receptors are present on the membranes from nerve-terminal regions, suggesting their involvement in synaptic transmission. 

Striatal levels of [Metj -enkephalin exhibit a 4-5-fold increase between birth and adulthood in rats [30]. The density of opiate receptors and striatal enkephalinase activity (measured by formation of Tyr-Gly-Gly) increase by about 2-fold between birth and adult-hood but the pattern of development appears to be different. The enkephalinase activity is significantly higher than in adults by days 15-21 of development whereas this is not the case for opiate receptor binding. The enkephalinase activity appears to reach adult level by day 15, possibly reflecting an early development of enzyme activity during brain maturation. The properties of the enzyme do not appear to change as its for [Leu] -enkephalin at 21 days is not significantly different from that obtained for the adult enzyme. 

Enkephalinase is not only involved in deactivation of the enkephalins but also in degradation of other peptides and hormones including Substance P, cholecystokinin, bradykinin, chemotactic peptide and atrial natriuretic factor (ANP) [67,69-72]. Peptides such as the dynorphins and )5-endorphin are not hydrolysed efficiently [73]. The specificity of the enzyme has been indirectly characterized by establishing the inhibitory potency of various peptides towards hydrolysis of H[Leu] -enkephalin [26]. 

C-Terminal carboxylic acid. The presence of a free terminal carboxylate in the P2 amino acid residue in a substrate increases the interaction with enkephalinase. Esterification or amidification of the C-terminal group of [Met] -enkephalin leads to a 6- and 30-fold decrease in binding [78], respectively. Replacement by a hydroxymethyl also leads to a drastic loss of activity [73,79]. This feature is also apparent in enkephalinase inhibitors [79,84] although the differences are smaller (Table 6.3). 

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