Peripheral inactivation of neurotensin. Isolation and characterization of a metallopeptidase from rat ileum

A peptidase that inactivated neurotensin by cleaving the peptide at the Pro10-Tyr11 bond, generating the biologically inactive fragments neurotensin(1-10) and neurotensin(11-13) was purified from whole rat ileum homogenate. The purified enzyme behaved as a 70-75-kDa monomer as determined by SDS-PAGE analysis in reducing or non-reducing conditions and gel permeation on Ultrogel AcA34. The peptidase was insensitive to thiol-blocking agents and acidic and serine protease inhibitors but could be strongly inhibited by 1,10-phenanthroline, EDTA, dithiothreitol and heavy metal ions such as zinc, copper and cobalt. Zinc was the only divalent cation able potently to reactivate the apoenzyme. This enzyme could be distinguished from endopeptidases EC 3.4.24.15 and EC 3.4.24.11, angiotensin-converting enzyme, proline endopeptidase, aminopeptidase and pyroglutamyl-peptide hydrolase since it was not affected by micromolar concentrations of their specific inhibitors. The peptidase displayed a high affinity for neurotensin (1.6 microM). Studies concerning the specificity of the enzyme towards the sequence of neurotensin established the following. (a) Neurotensin(9-13) was the shortest partial sequence that fully inhibited tritiated neurotensin degradation; shortening the C-terminal part of the neurotensin molecule led to inactive fragments. (b) Amidation of the C-terminal end of the peptide did not prevent the recognition by the peptidase. (c) There existed a strong stereospecificity of the peptidase for the residues in positions 8, 9 and 11 of the neurotensin molecule. (d) Pro-Xaa dipeptides (where Xaa represented aromatic or hydrophobic residues) were the most potent inhibitors of tritiated neurotensin degradation while all the Xaa-Pro dipeptides tested were totally ineffective. (e) The neurotensin-related peptides: neuromedin N, xenopsin and [Lys8-Asn9]neurotensin(8-13), as well as angiotensins I and II and dynorphins(1-8) and (1-13) were as potent as neurotensin in inhibiting [3H]neurotensin hydrolysis.     

Inactivation of Neurotensin by Rat Brain Synaptic Membranes. Cleavage at the Pro10-Tyr11 Bond by Endopeptidase 24.11 (Enkephalinase) and a Peptidase Different from Proline-Endopeptidase

It was shown previously that the tridecapeptide neurotensin is inactivated by rat brain synaptic membranes and that one of the primary inactivating cleavages occurs at the Pro10-Try11 peptide bond, leading to the formation of NT1-10 and NT11-13. The present study was designed to investigate the possibility that this cleavage was catalyzed by proline endopeptidase and/or endopeptidase 24.11 (enkephalinase). Purified rat brain synaptic membranes were found to contain a N-benzyloxycarbonyl-Gly-Pro-4-methyl-coumarinyl-7-amide-hydrolyzin g activity that was markedly inhibited (93%) by the proline endopeptidase inhibitor N-benzyloxycarbonyl-Pro-Prolinal and partially blocked (25%) by an antiproline endopeptidase antiserum. In contrast, the cleavage of neurotensin at the Pro10-Tyr11 bond by synaptic membranes was not affected by N-benzyloxycarbonyl-Pro-Prolinal and the antiserum. When the conversion of NT1-10 to NT1-8 by angiotensin converting enzyme was blocked by captopril and when the processing of NT11-13 by aminopeptidase(s) was inhibited by bestatin, it was found that thiorphan, a potent endopeptidase 24.11 inhibitor, partially decreased the formation of NT1-10 and NT11-13 by synaptic membranes. In conclusion: (1) proline endopeptidase, although it is present in synaptic membranes, is not involved in the cleavage of neurotensin at the Pro10-Tyr11 bond; (2) endopeptidase 24.11 only partially contributes to this cleavage; (3) there exists in rat brain synaptic membranes a peptidase different from proline endopeptidase and endopeptidase 24.11 that is mainly responsible for inactivating neurotensin by cleaving at the Pro10-Tyr11 bond.     

Peptidases in dog-ileum circular and longitudinal smooth-muscle plasma membranes. Their relative contribution to the metabolism of neurotensin

We established the content in neuropeptide-metabolizing peptidases present in highly purified plasma membranes prepared from the circular and longitudinal muscles of dog ileum. Activities were measured by the use of fluorigenic substrates and the identities of enzymes were confirmed by the use of specific peptidase inhibitors. Endopeptidase 24.11, angiotensin-converting enzyme, post-proline dipeptidyl aminopeptidase and aminopeptidases were found in both membrane preparations. Proline endopeptidase was only detected in circular smooth muscle plasma membranes while pyroglutamyl-peptide hydrolase was not observed in either tissue. The relative contribution of these peptidases to the inactivation of neurotensin was assessed. The enzymes involved in the primary inactivating cleavages occurring on the neurotensin molecule were as follows. In both membrane preparations, endopeptidase 24.11 was responsible for the formation of neurotensin-(1-11) and contributed to the formation of neurotensin-(1-10); a recently purified neurotensin-degrading neutral metallopeptidase was also involved in the formation of neurotensin-(1-10). A carboxypeptidase-like activity hydrolysed neurotensin at the Ile12-Leu13 peptide bond, leading to the formation of neurotensin-(1-12). Proline endopeptidase and endopeptidase 24.15 only occurred in circular muscle plasma membranes, yielding neurotensin-(1-7) and neurotensin-(1-8), respectively. In addition, the secondary processing of neurotensin degradation products was catalyzed by the following peptidases. In circular and longitudinal muscle membranes, angiotensin-converting enzyme converted neurotensin-(1-10) into neurotensin-(1-8) and tyrosine resulted from the rapid hydrolysis of neurotensin-(11-13) by bestatin-sensitive aminopeptidases. A post-proline dipeptidyl aminopeptidase activity converted neurotensin-(9-13) into neurotensin-(11-13) in circular muscle plasma membranes. The mechanism of neurotensin inactivation occurring in these membranes will be compared to that previously established for membranes from central origin.     

Neurotensin-Metabolizing Peptidases in Rat Fundus Plasma Membranes

The mechanisms by which neurotensin (NT) was inactivated by rat fundus plasma membranes were characterized. Primary inactivating cleavages occurred at the Arg8-Arg9, Pro10-Tyr11, and Ile12-Leu13 peptidyl bonds. Hydrolysis at the Arg8-Arg9 bond was fully abolished by the use of N-[1(R,S)-carboxy-2-phenylethyl]-alanyl-alanyl-phenylalanine-p- aminobenzoate, a result indicating the involvement at this site of a recently purified soluble metallopeptidase. Hydrolysis of the Pro10-Tyr11 bond was totally resistant to N-benzyloxycarbonyl-prolyl-prolinal and thiorphan, an observation suggesting that the peptidase responsible for this cleavage was different from proline endopeptidase and endopeptidase 24.11 and might correspond to a NT-degrading neutral metallopeptidase recently isolated from rat brain synaptic membranes. The enzyme acting at the Ile12-Leu13 bond has not yet been identified. Secondary cleavages occurring on NT degradation products were mainly generated by bestatin-sensitive aminopeptidases and post-proline dipeptidyl aminopeptidase. The content in NT-metabolizing peptidases present in rat fundus plasma membranes is compared with that previously established for purified rat brain synaptic membranes.     

High-Affinity Receptor Sites and Rapid Proteolytic Inactivation of Neurotensin in Primary Cultured Neurons

The present article describes the interaction of neurotensin with specific receptors in pure primary cultured neurons and the mechanisms by which this peptide is inactivated by these cells. Neurotensin binding sites are not detectable in nondifferentiated neurons and appear during maturation. The binding at 37 degrees C of [monoiodo-Tyr3]neurotensin to monolayers of neurons 96 h after plating is saturable and characterized by a dissociation constant of 300 pM and a maximal binding capacity of 178 fmol/mg of protein. The binding parameters as well as the specificity of these receptors toward neurotensin analogues reveal close similarities between the binding sites present in primary cultured neurons and those described in other membrane preparations or cells. Neurotensin is rapidly degraded by primary cultured neurons. The sites of primary inactivating cleavages are the Pro7-Arg8, Arg8-Arg9, and Pro10-Tyr11 bonds. Proline endopeptidase is totally responsible for the cleavage at the Pro7-Arg8 bond and contributes to the hydrolysis mainly at the Pro10-Tyr11 site. However, the latter breakdown is also generated by a neurotensin-degrading neutral metallopeptidase. The cleavage at the Arg8-Arg9 bond is due to a peptidase that can be specifically inhibited by N-[1(R,S)-carboxy-2-phenylethyl]-alanyl-alanyl-phenylalanyl-p- aminobenzoate. The secondary processing occurring on neurotensin degradation products are: a bestatin-sensitive aminopeptidasic conversion of neurotensin11-13 to free Tyr11, and a rapid cleavage of neurotensin8-13 by proline endopeptidase. A model for the inactivation of neurotensin in primary cultured neurons is proposed and compared to that previously described for purified rat brain synaptic membranes.     

Neurotensin metabolism in various tissues of central and peripheral origins: ubiquitous involvement of a novel neurotensin degrading metalloendopeptidase

The metabolism of neurotensin in vitro, in various membrane preparations and cell lines of central and peripheral origins was studied. Neurotensin degradation products were separated by HPLC and identified by either amino acid analysis or by their retention times. Peptidases responsible for the cleavages were identified by means of specific fluorigenic substrates or inhibitors. Although the patterns of neurotensin inactivation varied according to the tissue source in all cases, a major primary cleavage occurred at the Pro10-Tyr11 bond, leading to the biologically inactive fragments NT1-10 and NT11-13. A novel neurotensin-degrading metallopeptidase was responsible for this cleavage. Interestingly, it was the only peptidase that was ubiquitously detected. In addition, endopeptidase 24.11 (EC 3.4.24.11) contributed to this cleavage in rat brain synaptic membranes as well as in circular and longitudinal smooth muscle plasma membranes from dog ileum.     

Catabolism of neurotensin in the epithelial layer of porcine small intestine

The mammalian small intestine is both a source and a site of degradation of neurotensin. Metabolites produced by incubation of the peptide with dispersed enterocytes from porcine small intestine were isolated by high-performance liquid chromatography and identified by amino-acid analysis. The principal sites of cleavage were at the Tyr-11-Ile-12 bond, generating neurotensin-(1-11), and at the Pro-10-Tyr-11 bond, generating neurotensin-(1-10). The corresponding COOH-terminal fragments, neurotensin-(11-13) and -(12-13) were metabolized further. Formation of neurotensin-(1-11) and -(1-10) was completely inhibited by phosphoramidon (Ki = 6 nM), an inhibitor of endopeptidase 24.11, but not by captopril, an inhibitor of peptidyl dipeptidase A. Incubation of neurotensin with purified endopeptidase 24.11 from pig stomach also resulted in cleavage of the Tyr-11-Ile-12 and Pro-10-Tyr-11 bonds. A minor pathway of cell-surface-mediated degradation was the phosphoramidon-insensitive cleavage of the Tyr-3-Glu-4 bond, generating neurotensin-(1-3) and neurotensin-(4-13). No evidence for specific binding sites (putative receptors) for neurotensin was found either on the intact enterocyte or on vesicles prepared from the basolateral membranes of the cells. Neurotensin-(1-8), the major circulating metabolite, was not formed when neurotensin(1-13) was incubated with cells, but represented a major metabolite (together with neurotensin-(1-10] when neurotensin-(1-11) was used as substrate. The study has shown that degradation of neurotensin in the epithelial layer of the small intestine is mediated principally through the action of endopeptidase 24.11, but this enzyme is probably not responsible for the production of the neurotensin fragments detected in the circulation.     

Degradation of Neurotensin by Rat Brain Synaptic Membranes: Involvement of a Thermolysin-Like Metalloendopeptidase (Enkephalinase), Angiotensin-Converting Enzyme, and Other Unidentified Peptidases

Neurotensin was inactivated by membrane-bound and soluble degrading activities present in purified preparations of rat brain synaptic membranes. Degradation products were identified by HPLC and amino acid analysis. The major points of cleavage of neurotensin were the Arg8-Arg9, Pro10-Tyr11, and Tyr11-Ile12 peptide bonds with the membrane-bound activity and the Arg8-Arg9 and Pro10-Tyr11 bonds with the soluble activity. Several lines of evidence indicated that the cleavage of the Arg8-Arg9 bond by the membrane-bound activity resulted mainly from the conversion of neurotensin1-10 to neurotensin1-8 by a dipeptidyl carboxypeptidase. In particular, captopril inhibited this cleavage with an IC50 (5.7 nM) close to its K1 (7 nM) for angiotensin-converting enzyme. Thiorphan inhibited the cleavage at the Tyr11-Ile12 bond by the membrane-bound activity with an IC50 (17 nM) similar to its K1 (4.7 nM) for enkephalinase. Both cleavages were inhibited by 1,10-phenanthroline. These and other data suggested that angiotensin-converting enzyme and a thermolysin-like metalloendopeptidase (enkephalinase) were the membrane-bound peptidases responsible for cleavages at the Arg8-Arg9 and Tyr11-Ile12 bonds, respectively. In contrast, captopril had no effect on the cleavage at the Arg8-Arg9 bond by the soluble activity, indicating that the enzyme responsible for this cleavage was different from angiotensin-converting enzyme. The cleavage at the Pro10-Tyr11 bond by both the membrane-bound and the soluble activities appeared to be catalyzed by an endopeptidase different from known brain proline endopeptidases. The possibility is discussed that the enzymes described here participate in physiological mechanisms of neurotensin inactivation at the synaptic level.     

Specific inhibition of endopeptidase 24.16 by dipeptides.

The inhibitory effect of various dipeptides on the neurotensin-degrading metallopeptidase, endopeptidase 24.16, was examined. These dipeptides mimick the Pro10-Tyr11 bond of neurotensin that is hydrolyzed by endopeptidase 24.16. Among a series of Pro-Xaa dipeptides, the most potent inhibitory effect was elicited by Pro-Ile (Ki approximately 90 microM) with Pro-Ile greater than Pro-Met greater than Pro-Phe. All the Xaa-Tyr dipeptides were unable to inhibit endopeptidase 24.16. The effect of Pro-Ile on several purified peptidases was assessed by means of fluorigenic assays and HPLC analysis. A 5 mM concentration of Pro-Ile does not inhibit endopeptidase 24.11, endopeptidase 24.15, angiotensin-converting enzyme, proline endopeptidase, trypsin, leucine aminopeptidase, pyroglutamyl aminopeptidase I and carboxypeptidase B. The only enzyme that was affected by Pro-Ile was carboxypeptidase A, although it was with a 50-fold lower potency (Ki approximately 5 mM) than for endopeptidase 24.16. By means of fluorimetric substrates with a series of hydrolysing activities, we demonstrate that Pro-Ile can be used as a specific inhibitor of endopeptidase 24.16, even in a complex mixture of peptidase activities such as found in whole rat brain homogenate.     

Specificity of action on neuropeptides of an endopeptidase from the synaptosomal membranes of guinea pig brain

An endopeptidase was solubilized and highly purified from the synaptosomal membrane fraction of guinea pig brain, and its specificity of action on various neuropeptides was investigated. It hydrolyzed specifically the Pro10-Tyr11 bond of neurotensin and showed a marked specificity toward Pro-X bonds present in the interior parts of various neuropeptides and related peptides. No cleavage, however, was observed at the first and second peptide bonds from the NH2-termini or from the COOH-termini of the peptides examined, suggesting that the enzyme requires both NH2- and COOH-terminal extentions of at least 3 residues from the scissile bond for its action. In addition, a limited number of other peptide bonds were cleaved, indicating that the enzyme is not strictly specific to Pro-X bonds. These results suggest the possible implication of this enzyme in the specific degradation of neurotensin and other peptide neurotransmitters in the synaptic cleft.     

Mechanism of degradation of LH-RH and neurotensin by synaptosomal peptidases

The products of degradation of LH-RH and neurotensin by synaptosomes isolated from rat hypothalamus and cortex have been identified. LH-RH is cleaved at Tyr5-Gly6 and Pro9-Gly10 giving rise to LH-RH (1-5), LH-RH (6-10) and LH-RH (1-9). Neurotensin is cleaved at Arg8-Arg9, Pro10-Tyr11 and Ile12-Leu13, giving neurotensin (1-8), neurotensin (1-10), neurotensin (1-12) and neurotensin (9-13) as major products. While most of the peptidase activity is localized in the cytoplasmic fraction, a small but significant proportion is membrane bound. For LH-RH, the specificity of the membrane-bound activity is similar to that in the cytosol fraction; for neurotensin, the membrane fraction preferentially gives rise to the (1-10) and (1-11) peptides. The most potent inhibitors of the LH-RH and neurotensin degrading enzymes in synaptosomes are heavy metal ions (mercury and copper), p-chloromercuribenzoate and 1,10 phenanthroline.     

Further characterization of a neurotensin-degrading neutral metalloendopeptidase from rat brain

A peptidase inactivating neurotensin at the Pro¹⁰-Tyr¹¹ peptidyl bond, leading to the biologically inactive fragments neurotensin_1–10 and neurotensin_11–13 was purified from rat brain homogenate. The peptidase was characterized as a 70 kDa monomer and could be classified as a metaliopeptidase with respect to its sensitivity to o-phenanthroline, EDTA and divalent cations. The enzyme was also strongly inhibited by dithiothreitol but appeared totally insensitive to thiol-blocking agents, acidic and serine protease inhibitors. Experiments performed with a series of highly specific peptidase inhibitors clearly indicated that the peptidase was a novel enzyme distinct from previously purified cerebral peptidases. The enzyme displayed a rather high affinity for neurotensin (Km = 2.3 itM). Studies on its specificity indicated that: (i) neurotensin_9–13 was the shortest neurotensin fragment with full inhibitory potency of [³H]neurotensin degradation. Shortening the C-terminal end of the neurotensin molecule progressively led to inactive analogs; (ii) the peptidase exhibited a strong stereospecificity towards the residues in positions 8, 9 and 11. By contrast, neither introduction of a steric hindrance in position 11 nor amidation of the C-terminal end of the neurotensin molecule affected the ability of the corresponding analog to inhibit [³H]neurotensin degradation; (iii) Pro-Phe was the most potent dipeptide to compete for [³H]neurotensin degradation; (iv) the peptidase could not be described as an exclusive “neurotensinase” activity since, in addition to the neurotensin natural analogs (neuromedin N and xenopsin), non related natural peptides such as angiotensins I and II, dynorphins 1–8 and 1–13, atriopeptin III and bradykinin potently inhibited [³H]neurotensin degradation. Most of these peptides behaved as substrates for the enzyme.     

Purification of the main somatostatin-degrading proteases from rat and pig brains, their action on other neuropeptides, and their identification as endopeptidases 24.15 and 24.16.

The main somatostatin-degrading proteases were purified from rat and pig brain homogenates and characterized as thiol- and metal-dependent endoproteases. Two types of proteases with apparent native and subunit molecular masses of 70 kDa and 68 kDa could be differentiated in both species. Beside somatostatin, both hydrolyzed several other neuropeptides with chain lengths between 8 and 30 amino acid residues. Cleavage sites were generally similar or identical, but some clear exceptions were observed for enzymes from both species which could be used to differentiate between the two proteases. The 68-kDa protease cleaved somatostatin at three bonds (Asn5-Phe6, Phe6-Phe7 and Thr10-Phe11) and neurotensin only at the Arg8-Arg9 bond, whereas the 70-kDa protease digested somatostatin at only two bonds (Phe6-Phe7 and Thr10-Phe11) and neurotensin as well as acetylneurotensin-(8-13) additionally (pig protease) or almost exclusively (rat protease) at the Pro10-Tyr11 bond. Relative rates for the digestions of various peptides were, however, more dependent on the species than on the type of protease. Cleavage sites for angiotensin II, bradykinin, dynorphin, gonadoliberin and substance P were, apart from different rates, identical for both proteases. In both species the 68-kDa protease was found to be mainly, but not exclusively, soluble and not membrane-associated, whereas the inverse was detected for the 70-kDa protease. Based on distinct molecular and catalytic properties, the 68-kDa protease is supposed to be congruent with the endopeptidase 24.15 (EC 3.4.24.15), the 70-kDa protease with endopeptidase 24.16 (EC 3.4.24.16, neurotensin-degrading endopeptidase). This investigation demonstrates that both proteases hydrolyze various neuropeptides with similar cleavage sites, but with species-dependent activity. Species-independent distinctions are the exclusive action of endopeptidase 24.16 on acetylneurotensin-(8-13) and liberation of free Phe from somatostatin only by endopeptidase 24.15.     

Peptidases involved in the catabolism of neurotensin: inhibitor studies using superfused rat hypothalamic slices

In order to identify which peptidases are involved in the catabolism of neurotensin in the CNS, [3H-Tyr3,11]-neurotensin was superfused over rat hypothalamic slices in the presence and absence of peptidase inhibitors. The degree of degradation of the peptide was determined by reverse phase HPLC separation of 3H-labelled neurotensin from 3H-labelled products. Very little degrading activity was released from the slice into the medium during the superfusion. In the absence of inhibitors, 20 to 50% of 3H-neurotensin was degraded giving mainly 3H-Tyr along with other unidentified 3H-labelled products. Inhibitors of endopeptidase 24.11 (phosphoramidon) and proline endopeptidase (antibody) had no effect on the degradation. Captopril, an inhibitor of angiotensin converting enzyme, had a small inhibitory effect. In contrast, dynorphin(1-13), an inhibitor of a soluble, thiol dependent metallopeptidase which hydrolyses neurotensin at Arg8-Arg9, gave greater than 80% inhibition of 3H-neurotensin degradation in the slice preparation. 1,10-Phenanthroline, an inhibitor of metallopeptidases, was also an effective inhibitor. The dynorphin sequence responsible for the inhibition contains the Arg6-Arg7 bond. Other peptides (bradykinin and angiotensin) which are substrates of the soluble metallopeptidase also inhibited neurotensin breakdown by the slice. This evidence suggests that this thiol dependent metalloendopeptidase is the major neurotensin catabolizing enzyme in hypothalamic slices.     

Neurotensin and Neuromedin N Are Differently Metabolized in Ventral Tegmental Area and Nucleus Accumbens

Whole homogenates and membrane-bound and cytosoluble fractions prepared from rat ventral tegmental area (VTA) and nucleus accumbens were examined for their content of peptidasic activities and for their ability to metabolize neurotensin and its natural related hexapeptide neuromedin N. No qualitative differences were observed between these two brain regions concerning the presence and the subcellular distribution of a series of activities able to hydrolyze various specific fluorimetric enzymatic substrates. However, aminopeptidase B, endopeptidase 24-15, and endopeptidase 24-11 were significantly lower in the VTA than in the nucleus accumbens membrane preparations, while proline endopeptidase was detected in significantly higher amount only in the cytosolic fraction prepared from nucleus accumbens. Both neurotensin and neuromedin N were metabolized more rapidly in the nucleus accumbens than in the VTA. Furthermore, the degradation rate of neuromedin N was considerably faster than that of neurotensin whatever the cerebral area examined. Studies carried out with highly specific peptidase inhibitors revealed that endopeptidase 24-15 mainly contributed to the catabolism of neurotensin in homogenates and membrane-bound preparations of nucleus accumbens and VTA, while aminopeptidase B appeared predominantly responsible for the rapid disappearance of neuromedin N in both cerebral tissues. The possibility that the different metabolic processes of the two peptide congeners could explain their distinct pharmacological profiles observed after their microinjection in the nucleus accumbens and in the VTA is discussed.     

A membrane-bound metallo-endopeptidase from rat kidney. Characteristics of its hydrolysis of peptide hormones and neuropeptides.

A membrane-bound metallo-endopeptidase that hydrolyzes human parathyroid hormone (1-84) and reduced hen egg lysozyme between hydrophilic amino acid residues was isolated from rat kidney [Yamaguchi et al. (1991) Eur. J. Biochem. 200, 563-571]. In this study, the hydrolyses of various peptide hormones and neuropeptides by the metallo-endopeptidase were examined using an automated gas-phase protein sequencer. The purified enzyme hydrolyzed the oxidized insulin B chain and substance P most rapidly, followed by big endothelin 1, neurotensin, angiotensin 1, endothelin 1, rat alpha-atrial natriuretic peptide and bradykinin, in this order. The enzyme mainly cleaved these peptides at bonds involving a hydrophilic amino acid residue. However, it cleaved bonds between less hydrophilic amino acid pairs in several short peptides, e.g. at the His5-Leu6 bond in oxidized insulin B chain, the Ile28-Val29 bond in big endothelin-1 and the Ile5-His6 and Phe8-His9 bonds in angiotensin 1. The enzyme cleavage sites of oxidized insulin B chain and angiotensin 1 were different from the reported sites cleaved by meprin and by endopeptidase 2, respectively. Kinetic determination of bradykinin hydrolysis by the purified enzyme yielded values of Km = 18.1 microM and kcat = 0.473 s-1, giving a ratio of kcat/Km = 2.62 x 10(4) s-1.M-1. The Km value was about 20-fold lower than that reported for meprin and endopeptidase 2. These results indicate that the membrane-bound metallo-endopeptidase from rat kidney is distinguished from meprin and endopeptidase 2 in its substrate specificity and is not parathyroid hormone specific, but has potential capacities to inactivate various biologically active peptide hormones and neuropeptides in vivo.     

Enkephalinase from rat kidney. Purification, characterization, and study of substrate specificity

"Enkephalinase," a membrane-bound peptidase hydrolyzing the Gly3-Phe4 amide bond of enkephalins, initially characterized in brain, was purified from a rat kidney microsomal fraction. After differential solubilization with Triton X-100, the use of DEAE-Sephadex, concanavalin A, and hydroxylapatite chromatography led to a 2000-fold purification, close to homogeneity. Renal enkephalinase appears to be a glycoprotein Mr = 92,000-95,000 with catalytic properties and sensitivity to chelating agents and inhibitors (Thiorphan, phosphoramidon) very similar to those of the cerebral enzyme. The enzyme co-purified until the final step with "renal brush-border neutral proteinase" (EC 3.4.24.11) activity assayed with 125I-insulin B chain as substrate and displaying similar sensitivity to inhibitors. The specificity of the purified enkephalinase has been studied using either peptides derived from the enkephalins or model peptides of general formula (Ala)m-Tyr-(Ala)n as substrates. In all cases the bond cleaved was that involving the amino group of an aromatic residue, specificity being also defined by the nature of the neighboring residue on the COOH-terminal side. A free carboxyl in the latter residue was essential in the two series of substrates, indicating that enkephalinase more efficiently functions as a dipeptidyl carboxypeptidase than as an endopeptidase.     

Neutral endopeptidase modulates neurotensin-induced airway contraction

To determine the role of endogenous neutral endopeptidase (NEP) (also called enkephalinase, EC 3.4.24.11) in regulating neurotensin-induced airway contraction, we used phosphoramidon, a specific NEP inhibitor, in the guinea pig. In studies in vitro, neurotensin and the COOH-terminal fragment neurotensin-(8-13) contracted strips of bronchial smooth muscle in a concentration-dependent fashion (P less than 0.001). In contrast, the NH2-terminal fragment neurotensin-(1-11) and the COOH-terminal fragment neurotensin-(12-13), the main fragments of neurotensin hydrolysis by NEP, had no effect. Phosphoramidon (10(-5) M) did not change resting tension but shifted the concentration-response curves to neurotensin to lower concentrations (P less than 0.001), whereas inhibitors of kininase II, aminopeptidases, serine proteases, and carboxypeptidase N were without effect. Removing the epithelium increased the contractile response to neurotensin (P less than 0.001), and phosphoramidon further increased the response to neurotensin in these tissues (P less than 0.001). Similar results were obtained in studies in vivo using aerosolized neurotensin and phosphoramidon. These results suggest that endogenous NEP in the airways modulates the effects of neurotensin on airway smooth muscle contraction by inactivating the peptide.     

Specificity of Neurotensin Metabolism by Regional Rat Brain Slices

Regional differences in neurotensin metabolism and the peptidases involved were studied using intact, viable rat brain microslices and specific peptidase inhibitors. Regional brain slices (2 mm x 230 microns) prepared from nucleus accumbens, caudate-putamen, and hippocampus were incubated for 2 h in the absence and presence of phosphoramidon, captopril, N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate, and o-Phenanthroline, which are inhibitors of neutral endopeptidase 24.11, angiotensin-converting enzyme, metalloendopeptidase 24.15, and nonspecific metallopeptidases, respectively. Neurotensin-degrading proteolytic activity varied by brain region. Significantly less (35.0 +/- 1.6%) neurotensin was lost from hippocampus than from caudate-putamen (45.4 +/- 1.0%) or nucleus accumbens (47.8 +/- 1.1%) in the absence of inhibitors. Peptidases responsible for neurotensin metabolism on brain slices were found to be predominantly metallopeptidases. Metalloendopeptidase 24.15 is of major importance in neurotensin metabolism in each brain region studied. The relative contribution of specific peptidases to neurotensin metabolism also varied by brain region; angiotensin-converting enzyme and neutral endopeptidase 24.11 activities were markedly elevated in the caudate-putamen as compared with the nucleus accumbens or hippocampus. Interregional variation in the activity of specific peptidases leads to altered neurotensin fragment formation. The brain microslice technique makes feasible regional peptide metabolism studies in the CNS, which are impractical with synaptosomes, and provides evidence for regional specificity of neurotensin degradation.     

Endopeptidases 24.16 and 24.15 Are Responsible for the Degradation of Somatostatin, Neurotensin, and Other Neuropeptides by Cultivated Rat Cortical Astrocytes

Abstract: Several neuropeptides, including neurotensin, somatostatin, bradykinin, angiotensin II, substance P, and luteinizing hormone-releasing hormone but not vasopressin and oxytocin, were actively metabolized through proteolytic degradation by cultivated astrocytes obtained from rat cerebral cortex. Because phenanthroline was an effective degradation inhibitor, metalloproteases were responsible for neuropeptide fragmentation. Neurotensin was cleaved by astrocytes at the Pro¹⁰-Tyr¹¹ and Arg⁸- Arg⁹ bonds, whereas somatostatin was cleaved at the Phe⁶-Phe⁷ and Thr¹⁰-Phe¹¹ bonds. These cleavage sites have been found previously with endopeptidases 24.16 and 24.15 purified from rat brain. Addition of specific inhibitors of these proteases, the dipeptide Pro-He and N -[1-( RS )-carboxy-3-phenylpropyl]-Ala-Ala-Phe-4-aminobenzoate, significantly reduced the generation of the above neuropeptide fragments by astrocytes. The presence of endopeptidases 24.16 and 24.15 in homogenates of astrocytes could also be demonstrated by chromatographic separations of supernatant solubilized cell preparations. Proteolytic activity for neurotensin eluted after both gel and hydroxyapatite chromatography at the same positions as found for purified endopeptidase 24.16 or 24.15. In incubation experiments or in chromatographic separations no phosphoramidon-sensitive endopeptidase 24.11 (enkephalinase) or captopril-sensitive peptidyl dipeptidase A (angiotensin-converting enzyme) could be detected in cultivated astrocytes. Because astrocytes embrace the neuronal synapses where neuropeptides are released, we presume that the endopeptidases 24.16 and 24.15 on astrocytes are strategically located to contribute significantly to the inactivation of neurotensin, somatostatin, and other neuropeptides in the brain.