The hydrolysis of brain and atrial natriuretic peptides by porcine choroid plexus is attributable to endopeptidase-24.11.

The hydrolysis of the porcine 26-residue brain natriuretic peptide (BNP-26) and its counterpart human 28-residue atrial natriuretic peptide (alpha-hANP) by pig membrane preparations and purified membrane peptidases was studied. When the two peptides were incubated with choroid plexus membranes, the products being analysed by h.p.l.c., alpha-hANP was degraded twice as fast as BNP. The h.p.l.c. profiles of alpha-hANP hydrolysis, in short incubations with choroid plexus membranes, yielded alpha hANP' as the main product, this having been previously shown to be the result of hydrolysis at the Cys7-Phe8 bond. In short incubations this cleavage was inhibited 84% by 1 microM-phosphoramidon, a specific inhibitor of endopeptidase-24.11. BNP-26 was hydrolysed by choroid plexus membranes, kidney microvillar membranes and purified endopeptidase-24.11 in a manner that yielded identical h.p.l.c. profiles. In the presence of phosphoramidon, hydrolysis by the choroid plexus membranes was 94% inhibited. Captopril had no effect and, indeed, no hydrolysis of BNP-26 by peptidyl dipeptidase A (angiotensin-converting enzyme) was observed even after prolonged incubation with the purified enzyme. The stepwise hydrolysis of BNP-26 by endopeptidase-24.11 was investigated by sequencing the peptides produced during incubation. The initial product resulted from hydrolysis at Ser14-Leu15, thereby opening the ring. This product (BNP') was short-lived; further degradation involved hydrolysis at Ile12-Gly13, Arg8-Leu9, Gly17-Leu18, Val22-Leu23, Arg11-Ile12 and Cys4-Phe5. Thus endopeptidase-24.11 is the principal enzyme in renal microvillar and choroid plexus membranes hydrolysing BNP-26 and alpha-hANP.     

The metabolism of neuropeptides. Neurokinin A (substance K) is a substrate for endopeptidase-24.11 but not for peptidyl dipeptidase A (angiotensin-converting enzyme).

Both endopeptidase-24.11 and peptidyl dipeptidase A have previously been shown to hydrolyse the neuropeptide substance P. The structurally related peptide neurokinin A is also shown to be hydrolysed by pig kidney endopeptidase-24.11. The identified products indicated hydrolysis at two sites, Ser5-Phe6 and Gly8-Leu9, consistent with the known specificity of the enzyme. The pattern of hydrolysis of neurokinin A by synaptic membranes prepared from pig striatum was similar to that observed with purified endopeptidase-24.11, and hydrolysis was substantially abolished by the selective inhibitor phosphoramidon. Peptidyl dipeptidase A purified from pig kidney was shown to hydrolyse substance P but not neurokinin A. It is concluded that endopeptidase-24.11 has the general capacity to hydrolyse and inactivate the family of tachykinin peptides, including substance P and neurokinin A.     

Neurokinin B is hydrolysed by synaptic membranes and by endopeptidase-24.11 (enkephalinase) but not by angiotensin converting enzyme

The major site of hydrolysis was the Gly8-Leu9 bond. Angiotensin converting enzyme (peptidyl dipeptidase A, EC 3.4.15.1) from pig kidney hydrolysed substance P releasing the C-terminal tripeptide Gly-Leu-MetNH2 but failed to hydrolyse neurokinin B. Pig brain striatal synaptic membranes hydrolysed neurokinin B producing a similar pattern of products as did endopeptidase-24.11. Substantial inhibition of this activity was achieved with the selective inhibitor phosphoramidon. A combination of phosphoramidon and bestatin abolished the hydrolysis of neurokinin B by synaptic membranes. Thus, a bestatin-sensitive aminopeptidase may play a role in the synaptic metabolism of neurokinin B in addition to endopeptidase-24.11. This aminopeptidase appears to be distinct from aminopeptidase N (EC 3.4.11.2).     

The degradation of somatostatin by synaptic membrane of rat hippocampus is initiated by endopeptidase-24.11

Somatostatin was degraded by the synaptic membrane from rat hippocampus. Cleavage products were separated by reversed phase high performance liquid chromatography and identified by amino acid composition analyses and N-terminal amino acid and sequence determinations around the cleavage sites. Fragments produced from the cleavages at both or either sites between the Phe6-Phe7 and/or between the Thr10-Phe11, together with free phenylalanine and tryptophan, were major cleavage products, followed by that produced from the cleavage of the Asn5-Phe6 bond. The accumulation of the major cleavage products, as well as the initial cleavage of somatostatin, was strongly inhibited by metal chelators and also by specific inhibitors of endopeptidase-24.11 (EC 3.4.24.11), phosphoramidon and thiorphan. The inhibitor susceptibility of the synaptic membrane toward somatostatin was similar to that toward Leu-enkephalin, a natural substrate of endopeptidase-24.11. Furthermore, endopeptidase-24.11 purified from rat brain hydrolyzed somatostatin at the cleavage sites identical to those by the hippocampal synaptic membrane. Thus, it can be concluded that endopeptidase-24.11 plays a major role in the initial stage of somatostatin degradation in rat hippocampus.     

Metabolism of neuropeptides. Hydrolysis of the angiotensins, bradykinin, substance P and oxytocin by pig kidney microvillar membranes.

Microvillar membranes derived from the brush border of the renal proximal tubule are very rich in peptidases. Pig kidney microvilli contain endopeptidase-24.11 associated with a battery of exopeptidases. The manner by which some neuropeptides are degraded by the combined attack of the peptidases of this membrane has been investigated. The contribution of individual peptidases was assessed by including inhibitors (phosphoramidon, captopril, amastatin and di-isopropyl fluorophosphate) with the membrane fraction when incubated with the peptides. Substance P, bradykinin and angiotensins I, II and III and insulin B-chain were rapidly hydrolysed by kidney microvilli. Oxytocin was hydrolysed much more slowly, but no products were detected from [Arg8]vasopressin or insulin under the conditions used for other peptides. The peptide bonds hydrolysed were identified and the contributions of the different peptidases were quantified. For each of the susceptible peptides, the main contribution came from endopeptidase-24.11 (inhibited by phosphoramidon). Peptidyl dipeptidase A (angiotensin-I-converting enzyme) was of less importance, even in respect of angiotensin I and bradykinin. When [2,3-Pro3,4-3H]bradykinin was also investigated at a lower concentration (20 nM), the conclusions in regard to the contributions of the two peptidases were unchanged. The possibility that endopeptidase-24.11 might attack within the six-residue disulphide-bridged rings of oxytocin and vasopressin was examined by dansyl(5-dimethylaminonaphthalene-1-sulphonyl)ation and by reduction and carboxymethylation of the products after incubation. Additional peptides were only observed after prolonged incubation, consistent with hydrolysis at the Tyr2-Ile3 and Tyr2-Phe3 bonds respectively. These results show that a range of neuropeptides are efficiently degraded by microvillar membranes and that endopeptidase-24.11 plays a key role in this process.     

Hydrolysis of alpha-human atrial natriuretic peptide in vitro by human kidney membranes and purified endopeptidase-24.11. Evidence for a novel cleavage site.

alpha-Human atrial natriuretic peptide (hANP) is secreted by the heart and acts on the kidney to promote a strong diuresis and natriuresis. In vivo it has been shown to be catabolized partly by the kidney. Crude microvillar membranes of human kidney degrade 125I-ANP at several internal bonds generating metabolites among which the C-terminal fragments were identified. Formation of the C-terminal tripeptide was blocked by phosphoramidon, indicating the involvement of endopeptidase-24.11 in this cleavage. Subsequent cleavages by aminopeptidase(s) yielded the C-terminal dipeptide and free tyrosine. Using purified endopeptidase 24.11, we identified seven sites of hydrolysis in unlabelled alpha-hANP: the bonds Arg-4-Ser-5, Cys-7-Phe-8, Arg-11-Met-12, Arg-14-Ile-15, Gly-16-Ala-17, Gly-20-Leu-21 and Ser-25-Phe-26. However, the bonds Gly-16-Ala-17 and Arg-4-Ser-5 did not fulfil the known specificity requirements of the enzyme. Cleavage at the Gly-16-Ala-17 bond was previously observed by Stephenson & Kenny [(1987) Biochem. J. 243, 183-187], but this is the first report of an Arg-Ser bond cleavage by this enzyme. Initial attack of alpha-hANP by endopeptidase-24.11 took place at a bond within the disulphide-linked loop and produced a peptide having the same amino acid composition as intact ANP. The bond cleaved in this metabolite was determined as the Cys-7-Phe-8 bond. Determination of all the bonds cleaved in alpha-hANP by endopeptidase-24.11 should prove useful for the design of more stable analogues, which could have therapeutic uses in hypertension.     

Catabolism of substance P in the stomach wall of the rat

The aim of this study was to examine the catabolism of substance P (SP) in the stomach wall of the rat. Catabolism in vitro was investigated by incubation of unlabelled and tritiated SP (prolyl 2,4-3,4(n)-3H SP) with membrane bound-peptidases prepared from the rat gastric corpus. Catabolism was studied in vivo by use of a catheter chronically implanted in the stomach wall to deliver tritiated SP to the gastric tissues and implanted dialysis fibers to collect the catabolic products. The products from both experiments were separated by high pressure liquid chromatography and identified by their retention times or amino acid analysis. Membrane-bound peptidases in vitro hydrolyzed both unlabelled and tritiated SP and the products of hydrolysis were consistent with the cleavage of three bonds: Gln6-Phe7, Phe7-Phe8 and Gly9-Leu10. None of the peptide fragments would be expected to be biologically active. Only those fragments with tritiated Pro residues could be detected in vivo. The major identified products were SP(1-2) and SP(3-4), with smaller amounts of SP(1-4), SP(1-6), SP(1-7), SP(1-8) and SP(1-9). The enzymes that may be responsible for these cleavage patterns are discussed.     

Catabolism of substance P and neurotensin in the rat stomach wall is susceptible to inhibitors of angiotensin converting enzyme

The purpose of this investigation was to examine the pathway of substance P (SP) and neurotensin (NT) catabolism in the gastric wall of the rat and identify some of the enzymes involved. Under anaesthesia an infusion catheter and a bundle of dialysis fibres were implanted into the stomach wall of the rat. Experiments commenced on conscious rats 2 days after surgery. In control experiments [3H]-SP(Pro-2,4) or [3H]-NT(Tyr-3,11) were injected into gastric tissues through the catheter and catabolites were collected in the dialysis fibres and separated by high pressure liquid chromatography. In other studies captopril, MK422 (inhibitors of angiotensin converting enzyme) or phosphoramidon (an inhibitor of endopeptidase-24.11, 'enkephalinase') were injected into gastric tissues before the peptide label. SP1-11 was degraded to mainly SP1-2, SP3-4 with some SP1-6, SP1-7 and SP1-8. Catabolism was partially but significantly (5% level) inhibited by MK422 and captopril, but not by phosphoramidon. NT1-13 was degraded to NT1-8, NT9-13, NT1-11 and NT1-12. NT catabolism was partially but significantly (5% level) inhibited by MK422. It is concluded that an enzyme resembling angiotensin converting enzyme is involved in the initial stages of SP and NT catabolism in the rat stomach. The involvement of other peptidases cannot be excluded because inhibition of breakdown was not complete.     

Metabolic stability of the LHRH antagonist antide to cell-surface peptidases

The susceptibility to hydrolysis of LHRH and the decapeptide analogue Antide has been compared. The hydrolysis of LHRH by pig kidney brush border membranes is inhibited by phosphoramidon (I50 = 5.6 nM) implicating endopeptidase-24.11 in the initiation of hydrolysis. Under conditions in which LHRH is fully degraded by brush border membranes, Antide was completely resistant to hydrolysis. Similar results were obtained with purified preparations of both endopeptidase-24.11 and angiotensin converting enzyme. These data confirm that the remarkable duration of action of Antide is due principally to its stability to hydrolysis by cell-surface peptidases.     

The metabolism of neuropeptides. Both phosphoramidon-sensitive and captopril-sensitive metallopeptidases are present in the electric organ of Torpedo marmorata.

A membrane fraction from the electric organ of Torpedo marmorata hydrolyses the Gly3-Phe4 bond of [D-Ala2, Leu5]enkephalin as well as the Gly-His bond of benzoyl-Gly-His-Leu. The hydrolysis of benzoyl-Gly-His-Leu is completely inhibitable by Captopril (I50 = 19nM), consistent with peptidyl dipeptidase activity, but enkephalin hydrolysis is inhibited to a maximum of only 70%. The residual activity hydrolysing enkephalin is inhibited by phosphoramidon (I50 = 15nM) and therefore resembles endopeptidase-24.11, a mammalian plasma-membrane enzyme implicated in the metabolism of neuropeptides. Both enkephalin-hydrolysing activities in Torpedo electric organ are inhibited by 1,10-phenanthroline, like their mammalian counterparts. The peptidases may function in the hydrolysis of endogenous peptides or in neurotransmitter exocytosis in the electric organ.     

Endopeptidase-24.11 Is the Integral Membrane Peptidase Initiating Degradation of Somatostatin in the Hippocampus

Abstract: The membrane metalloenzyme endopeptidase-24.11 has been localized by immunocytochemistry in the porcine hippocampus in the stratum oriens and stratum radiatum. Endopeptidase-24.11 was found to be ∼10-fold more abundant in a striatal than a hippocampal membrane preparation. Both somatostatin-28 and somatostatin-14 were metabolized by endopeptidase-24.11, but the kinetics of hydrolysis markedly favoured the smaller form of the neuropeptide. After phase separation with Triton X-114 of striatal and hippocampal membrane preparations, and by using selective inhibitors, the major (>80%) somatostatin-metabolizing activity was found to partition into the detergent-rich phase and was attributable predominantly to endopeptidase-24.11. The residual activity observed in the presence of the selective endopeptidase-24.11 inhibitor phosphoramidon was blocked by Pro-Ile or N -[1-( RS )-carboxy-3-phenylpropyl]-Ala-Ala-Phe- p -aminobenzoate, inhibitors of endopeptidase-24.16 and endopeptidase-24.15, respectively. However, Pro-Ile, at comparable concentrations, was shown to inhibit endopeptidase-24.11, challenging the validity of its use as a selective inhibitor of endopeptidase-24.16. The immunocytochemical and Triton X-114 phase-separation data implicate endopeptidase-24.11, rather than endopeptidase-24.16 or endopeptidase-24.15, as the major physiological somatostatin-degrading neuropeptidase in the striatum and hippocampus.     

Proteolytic Inactivation of Substance P and Neurokinin A in the Longitudinal Muscle Layer of Guinea Pig Small Intestine

Membrane vesicles, showing a 21 +/- 2-fold enrichment in the activity of 5'-nucleotidase and a 11 +/- 4-fold enrichment in the activity of angiotensin-converting enzyme relative to homogenate, were prepared from the myenteric plexus-containing longitudinal muscle layer of guinea pig ileum. Incubation of the vesicles with substance P and neurokinin A led to degradation of the peptides, and metabolites were isolated by reverse-phase HPLC and identified by amino acid composition. Cleavages of substance P between Glu6-Phe7, Phe7-Phe8, and Gly9-Leu10 and of neurokinin A between Gly8-Leu9 were observed and could be inhibited in a dose-dependent manner by phosphoramidon, an inhibitor of neutral endopeptidase 24.11. Formation of these metabolites was not completely inhibited by this agent, indicating that a phosphoramidon-insensitive form of endopeptidase 24.11 was present in the gut. Substance P was resistant to degradation by aminopeptidases, but neurokinin A was a substrate for bestatin-sensitive aminopeptidase(s), so that the neurokinin A (3-10) fragment represented the predominant metabolite in the chromatograms. The rate of formation of all the metabolites was not inhibited by enalapril and not enhanced by an increased Cl- concentration, indicating that angiotensin-converting enzyme was unimportant in the degradation process. Degradation of neurokinin A by the vesicles (Km 30 microM; Vmax 7.2 +/- 0.8 nmol min-1 mg of protein-1) was more rapid than degradation of substance P (Km 25 microM; Vmax 4.4 +/- 0.4 nmol min-1 mg of protein-1).     

Substance P and neurokinin A metabolism by cultured human skeletal muscle myocytes and fibroblasts

A recent study determined that cultured human skeletal muscle adult myoblasts, myotubes, and fibroblasts degraded angiotensins and kinins via neutral endopeptidase-24.11 (NEP-24.11; EC 3.4.24.11) and aminopeptidase N (APN; EC 3.4.11.2). Due to the possible importance of other peptides to skeletal muscle blood flow and function, the present study looked specifically at the metabolism of the neurokinins substance P (SP) and neurokinin A (NKA) by skeletal muscle peptidases. The results show that SP is degraded not only by NEP-24.11, but also sequentially by dipeptidyl(amino)peptidase IV (DAP IV; EC 3.4.14.5)/APN. NKA is unaffected by DAP IV but is metabolized by NEP-24.11 and APN. NEP-24.11 was inhibited by phosphoramidon (IC₅₀ = 80 nM), thiorphan and ZINCOV, DAP IV by diprotin A (IC₅₀ = 8 μM), and APN by amastatin (IC₅₀ = 50 nM) and bestatin (IC₅₀ = 100 μM). Skeletal muscle myocyte and fibroblast metabolism of SP and NKA may regulate local skeletal muscle vascular and extravascular functions including SP- and NKA-mediated nerve-induced vasodilation. Inhibition of both NEP-24.11 and DAP IV/APN may increase skeletal muscle blood flow and decrease peripheral vascular resistance via potentiation of local neurokinin levels.     

Degradation and inactivation of rat atrial natriuretic peptide 1-28 by neutral endopeptidase-24.11 in rat pulmonary membranes.

Atrial natriuretic peptide (ANP), a 28-residue peptide with cardiovascular and renal effects, is rapidly cleared from the circulation. Beside renal clearance, an extra-renal metabolism by the enzyme neutral endopeptidase-24.11 (NEP-24.11) has been proposed, since specific NEP-24.11-inhibitors increase endogenous plasma-ANP. NEP-24.11 is present in rat lung but its significance for ANP hydrolysis within the lung is unclear. The aim of this study was to investigate a possible degradation of rat ANP in a membrane preparation from rat lung. Hydrolysis products of ANP were separated by HPLC and further characterized by a pulmonary artery bioassay, by radioimmunoassay with different antisera, by peptide sequencing and by masspectrometry. Rat pulmonary membranes degraded ANP to one main metabolite lacking biological activity and with poor cross-reactivity to an antiserum recognising the central ring-structure of the peptide. Formation of the hydrolysis product was prevented by the NEP-24.11-inhibitor phosphoramidon (1 microM). Peptide sequencing of the metabolite revealed a cleavage between Cys7 and Phe8, which was confirmed by mass-spectrometry. The metabolite had an HPLC elution time identical to that of the product formed by purified porcine NEP-24.11. These findings suggest that ANP is metabolized and inactivated by endopeptidase-24.11 in rat lungs, the first organ exposed to ANP released from the heart.     

Angiotensin converting enzyme (ACE) and neprilysin hydrolyze neuropeptides: a brief history, the beginning and follow-ups to early studies

Our investigations started when synthetic bradykinin became available and we could characterize two enzymes that cleaved it: kininase I or plasma carboxypeptidase N and kininase II, a peptidyl dipeptide hydrolase that we later found to be identical with the angiotensin I converting enzyme (ACE). When we noticed that ACE can cleave peptides without a free C-terminal carboxyl group (e.g., with a C-terminal nitrobenzylamine), we investigated inactivation of substance P, which has a C-terminal Met(11)-NH(2). The studies were extended to the hydrolysis of the neuropeptide, neurotensin and to compare hydrolysis of the same peptides by neprilysin (neutral endopeptidase 24.11, CD10, NEP). Our publication in 1984 dealt with ACE and NEP purified to homogeneity from human kidney. NEP cleaved substance P (SP) at Gln(6)-Phe(7), Phe(7)[see text]-Phe(8), and Gly(9)-Leu(10) and neurotensin (NT) at Pro(10)-Tyr(11) and Tyr(11)-Ile(12). Purified ACE also rapidly inactivated SP as measured in bioassay. HPLC analysis showed that ACE cleaved SP at Phe(8)-Gly(9) and Gly(9)-Leu(10) to release C-terminal tri- and dipeptide (ratio = 4:1). The hydrolysis was Cl(-) dependent and inhibited by captopril. ACE released only dipeptide from SP free acid. ACE hydrolyzed NT at Tyr(11)-Ile(12) to release Ile(12)-Leu(13). Then peptide substrates were used to inhibit ACE hydrolyzing Fa-Phe-Gly-Gly and NEP cleaving Leu(5)-enkephalin. The K(i) values in microM were as follows: for ACE, bradykinin = 0.4, angiotensin I = 4, SP = 25, SP free acid = 2, NT = 14, and Met(5)-enkephalin = 450, and for NEP, bradykinin = 162, angiotensin I = 36, SP = 190, NT = 39, Met(5)-enkephalin = 22. These studies showed that ACE and NEP, two enzymes widely distributed in the body, are involved in the metabolism of SP and NT. Below we briefly survey how NEP and ACE in two decades have gained the reputation as very important factors in health and disease. This is due to the discovery of more endogenous substrates of the enzymes and to the very broad and beneficial therapeutic applications of ACE inhibitors.     

The metabolism of neuropeptides. The hydrolysis of peptides, including enkephalins, tachykinins and their analogues, by endopeptidase-24.11.

Endopeptidase-24.11 (EC 3.4.24.11), purified to homogeneity from pig kidney, was shown to hydrolyse a wide range of neuropeptides, including enkephalins, tachykinins, bradykinin, neurotensin, luliberin and cholecystokinin. The sites of hydrolysis of peptides were identified, indicating that the primary specificity is consistent with hydrolysis occurring at bonds involving the amino group of hydrophobic amino acid residues. Of the substrates tested, the amidated peptide substance P is hydrolysed the most efficiently (Km = 31.9 microM; kcat. = 5062 min-1). A free alpha-carboxy group at the C-terminus of a peptide substrate is therefore not essential for efficient hydrolysis by the endopeptidase. A large variation in kcat./Km values was observed among the peptide substrates studied, a finding that reflects a significant influence of amino acid residues, remote from the scissile bond, on the efficiency of hydrolysis. These subsite interactions between peptide substrate and enzyme thus confer some degree of functional specificity on the endopeptidase. The inhibition of endopeptidase-24.11 by several compounds was compared with that of pig kidney peptidyldipeptidase A (EC 3.4.15.1). Of the inhibitors examined, only N-[1(R,S)-carboxy-2-phenylethyl]-Phe-p-aminobenzoate inhibited endopeptidase-24.11 but not peptidyldipeptidase. Captopril (D-3-mercapto-2-methylpropanoyl-L-proline), Teprotide (pGlu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro) and MK422 [N-[(S)-1-carboxy-3-phenylpropyl]-L-Ala-L-Pro] were highly selective as inhibitors of peptidyldipeptidase. Although not wholly specific, phosphoramidon was a more potent inhibitor of endopeptidase-24.11 than were any of the synthetic compounds tested.     

Metalloendopeptidase (EC 3.4.24.11) but not angiotensin converting enzyme is involved in the inactivation of substance P by synaptic membranes of the rat substantia nigra

Substance P is a neuropeptide released in vivo from the substantia nigra, the principal substance P nerve terminal region in the rat brain. Its inactivation was investigated in a purified nigral synaptic membrane preparation. The membrane-bound enzyme shares many features with the endopeptidase 24-11 (EC 3.4.24.11): 1) hydrolysis of peptide bonds Gln6-Phe7, Phe7-Phe8 and Gly9-Leu10, 2) sensitivity to the inhibition by phosphoramidon and 3) relative affinity for substance P. Bestatine and captopril inhibit only the hydrolysis of the metabolites. These results suggest that substance P is inactivated in substantia nigra by endopeptidase 24-11 and that a bestatin-sensitive aminopeptidase and angiotensin converting enzyme may play a role in subsequent degradation of the substance P metabolites.     

The metabolism of neuropeptides. Endopeptidase-24.11 in human synaptic membrane preparations hydrolyses substance P.

Synaptic membrane preparations from human striatum and human diencephalon were shown to contain a phosphoramidon-sensitive metalloendopeptidase that appeared identical with endopeptidase-24.11. The activity of endopeptidase-24.11 was determined with an enzymic assay employing [D-Ala2,Leu5]enkephalin as substrate, and its distribution in human brain was similar to that in pig brain, with the striatum containing the highest levels. The choroid plexus and pons also contained substantial activity. A good correlation (r = 0.97) was obtained for the distribution of the endopeptidase in pig brain and pituitary by the enzymic assay and by an immunoradiometric assay specific for pig endopeptidase-24.11. Synaptic membrane preparations from human striatum and diencephalon hydrolysed substance P at the same sites as did preparations of pig striatal synaptic membranes, and hydrolysis was substantially abolished by phosphoramidon. These results suggest that endopeptidase-24.11 is the principal enzyme hydrolysing substance P in human synaptic membrane preparations.     

Hydrolysis of substance p and neurotensin by converting enzyme and neutral endopeptidase

Angiotensin I converting enzyme (ACE) and neutral endopeptidase ("enkephalinase"; NEP), were purified to homogeneity from human kidney. NEP cleaved substance P (SP) at Gln6-Phe7,-Phe8, and Gly9-Leu10 and neurotensin (NT) at Pro10-Tyr11 and Tyr11-Ile12. NEP hydrolyzed 0.1 mM SP, NT and their C-terminal fragments at the following rates (mumol/min/mg): SP1-11 = 7.8, SP4-11 = 11.7, SP5-11 = 15.4, SP6-11 = 15.6, SP8-11 = 6.7, NT1-13 = 2.9, and NT8-13 = 4.0. Purified ACE rapidly inactivated SP as measured in bioassay. HPLC analysis showed that ACE cleaved SP at Phe8-Gly9 and Gly9-Leu10 to release C-terminal tri- and dipeptide (ratio = 4:1). The hydrolysis was Cl- dependent and inhibited by captopril. ACE released mainly C-terminal tripeptide from SP methyl ester, but only dipeptide from SP free acid. Modification of arginine residues in ACE with cyclohexanedione or butanedione similarly inhibited hydrolysis of SP, bradykinin and Bz-Gly-Phe-Arg (80-93%) indicating an active site arginine is required for hydrolysis of SP. ACE hydrolyzed NT at Tyr11-Ile12 to release Ile12-Leu13. SP, NT and their derivatives (0.1 mM) were cleaved by ACE at the following rates (mumol/min/mg): SP1-11 = 1.2, SP methyl ester = 0.7, SP free acid = 8.5, SP4-11 = 2.4, SP5-11 = 0.9, SP6-11 = 1.4, SP8-11 = 0, NT1-13 = 0.2, and NT8-13 = 1.3. Peptide substrates were used as inhibitors of ACE (substrate = FA-Phe-Gly-Gly) and NEP (substrate = Leu5-enkephalin).(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuropeptides and their peptidases: Functional considerations

The properties of the various brain membrane peptidases capable of hydrolysing released neuropeptides are reviewed, with particular emphasis on endopeptidase-24.11 and angiotensin converting enzyme. The substrate specificities of both enzymes are defined and their relative contribution to the degradation of tachykinins in vitro are considered. One approach to assessing the physiological roles of identified peptidases involves examining the protective effect of selective peptidase inhibitors on the degradation of peptides released from brain slices. This procedure has been applied to study the release of substance P-like immunoreactivity from slices of rat substantia nigra. Inhibition of endopeptidase-24.11, but not of angiotensin converting enzyme, produces a significant increase in recovery of substance P. The specificity and distribution of endopeptidase-24.11 would therefore not be inconsistent with a role in the physiological inactivation of tachykinins, as well as enkephalins. At peripheral sites, LHRH and atrial natriuretic peptide may be important substrates of the enzyme. The endogenous neuropeptide substrate(s) for striatal angiotensin converting enzyme remain unclear.