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. 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.     

The hydrolysis of alpha-human atrial natriuretic peptide by pig kidney microvillar membranes is initiated by endopeptidase-24.11.

alpha-Human atrial natriuretic peptide, a 28-amino-acid-residue peptide, was rapidly hydrolysed by pig kidney microvillar membranes in vitro, with a t1/2 of 8 min, comparable with the rate observed with angiotensins II and III. The products of hydrolysis were analysed by h.p.l.c., the pattern obtained with membranes being similar to that with purified endopeptidase-24.11 (EC 3.4.24.11). No hydrolysis by peptidyl dipeptidase A (angiotensin I converting enzyme, EC 3.4.15.1) was observed. The contribution of the various microvillar membrane peptidases was assessed by including specific inhibitors. Phosphoramidon, an inhibitor of endopeptidase-24.11, caused 80-100% suppression of the products. Captopril and amastatin (inhibitors of peptidyl dipeptidase A and aminopeptidases respectively) had no significant effect. Hydrolysis at an undefined site within the disulphide-linked ring occurred rapidly, followed by hydrolysis at other sites, including the Ser25--Phe26 bond.     

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.     

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 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.     

Catabolism of gastrin releasing peptide and substance P by gastric membrane-bound peptidases

The catabolism of two gastric neuropeptides, the C-terminal decapeptide of gastrin releasing peptide-27 (GRP10) and substance P (SP), by membrane-bound peptidases of the porcine gastric corpus and by porcine endopeptidase-24.11 ("enkephalinase") has been investigated. GRP10 was catabolized by gastric muscle peptidases (specific activity 1.8 nmol min-1 mg-1 protein) by hydrolysis of the His8-Leu9 bond and catabolism was inhibited by phosphoramidon (I50 approx. 10(-8) M), a specific inhibitor of endopeptidase-24.11. The same bond in GRP10 was cleaved by purified endopeptidase-24.11, and hydrolysis was equally sensitive to inhibition by phosphoramidon. SP was catabolized by gastric muscle peptidases (specific activity 1.7 nmol min-1 mg-1 protein) by hydrolysis of the Gln6-Phe7, Phe7-Phe8 and Gly9-Leu10 bonds, which is identical to the cleavage of SP by purified endopeptidase-24.11. The C-terminal cleavage of GRP10 and SP would inactivate the peptides. It is concluded that a membrane-bound peptidase in the stomach wall catabolizes and inactivates GRP10 and SP and that, in its specificity and sensitivity to phosphoramidon, this peptidase resembles endopeptidase-24.11.     

Pyridinedicarboxylates, the first mechanism-derived inhibitors for prolyl 4-hydroxylase, selectively suppress cellular hydroxyprolyl biosynthesis. Decrease in interstitial collagen and Clq secretion in cell culture.

Peptidyl-dipeptidase A (angiotensin converting enzyme; ACE, EC 3.4.15.1), has been purified from pig kidney and striatum by affinity chromatography employing the selective inhibitor lisinopril as ligand. The inclusion of a 2.8 nm spacer arm improved the yield of the enzyme compared with the 1.4 nm spacer arm described in previous work. Two forms of striatal ACE (Mr 180,000 and 170,000), but only a single form of kidney ACE (Mr 180,000), were isolated by this procedure. Both forms of striatal ACE were recognized by a polyclonal antibody to kidney ACE. No significant differences in substrate specificity or inhibitor sensitivity between kidney and striatal ACE could be detected. In particular, the amidated neuropeptide, substance P, was hydrolysed identically by both preparations and no significant hydrolysis of the related tachykinin peptides neurokinin A and neurokinin B could be detected. After chemical or enzymic deglycosylation, kidney and both forms of striatal ACE migrated identically on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent Mr of 150,000. We suggest that the two detectable forms of ACE in pig brain are not isoenzymes but are the result of differential glycosylation in different cell types in the brain. It appears that ACE, unlike endopeptidase-24.11, does not have the general capacity to hydrolyse and inactivate the tachykinin peptides at a significant rate in brain.     

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.     

Regional Metabolism of Met-Enkephalin and Cholecystokinin on Intact Rat Brain Slices: Characterization of Specific Peptidases

Abstract: The metabolism of Met-enkephalin and cholecystokinin (CCK) 8-(sulfated) by intact microslices was studied in rat brain regions. Incubation of brain slices with Met-enkephalin (400 µ M ) resulted in a linear rate of disappearance of parent peptide and appearance of metabolic fragments whose rate of accumulation was specific to brain region. The degradative rate (pmol/min/mg of protein) of Met-enkephalin was high in caudate-putamen (5,160 ± 120) and lower in nucleus accumbens (3,630 ± 110) and frontal cortex (3,180 ± 120). Inhibition of aminopeptidases decreased Met-enkephalin degradation (50–97% vs. control) in frontal cortex but was less effective in caudate-putamen (20–34%). Tyr-Gly-Gly and Phe-Met were recovered in caudate-putamen and nucleus accumbens, whereas negligible quantities of these fragments were recovered from frontal cortex. Phosphoramidon, an inhibitor of neutral endopeptidase 24.11, decreased Met-enkephalin degradation in caudate-putamen (14%) but had no effect on that in frontal cortex. A cocktail of bestatin or leuhistin (inhibitors of aminopeptidases), phosphoramidon, and captopril (an inhibitor of angiotensin converting enzyme) protected Met-enkephalin from degradation (recovery >95%) in caudate-putamen. CCK 8-(sulfated) degradation on slices from caudate-putamen, nucleus accumbens, and frontal cortex was not altered by inhibitors of neutral endopeptidase 24.11, metalloendopeptidase 24.15, angiotensin converting enzyme, or thiol proteases. Inhibitors of either aminopeptidases or serine proteases produced small reductions (13–30%) in CCK degradation in each region. These data provide evidence for regional and structural specificity in terminating the actions of neuropeptides.     

Synaptosomal Membrane-Bound Form of Endopeptidase-24.15 Generates Leu-Enkephalin from Dynorphin 1-8, α- and β-Neoendorphin, and Met-Enkephalin from Met-Enkephalin-Arg6-Gly7-Leu

Brain contains a membrane-bound form of endopeptidase-24.15, a metalloendopeptidase predominantly associated with the soluble protein fraction of brain homogenates. Subcellular fractionation of the enzyme in rat brain showed that 20-25% of the total activity is associated with membrane fractions including synaptosomes. Solubilization of the enzyme from synaptosomal membranes required the use of detergents or treatment with trypsin. The specific activity of the enzyme in synaptosomal membranes measured with tertiary-butoxycarbonyl-Phe-Ala-Ala-Phe-p-aminobenzoate as substrate was higher than that of endopeptidase-24.11 ("enkephalinase"), a membrane-bound zinc-metalloendopeptidase believed to function in brain neuropeptide metabolism. Purified synaptosomal membranes converted efficiently dynorphin1-8, alpha- and beta-neoendorphin into leucine enkephalin and methionine-enkephalin-Arg6-Gly7-Leu8 into methionine enkephalin in the presence of captopril, bestatin, and N-[1-(R,S)-carboxy-2-phenylethyl]-Phe-p-aminobenzoate, inhibitors of angiotensin converting enzyme (EC 3.4.15.1), aminopeptidase (EC 3.4.11.2), and membrane-bound metalloendopeptidase (EC 3.4.24.11), respectively. The conversion of enkephalin-containing peptides into enkephalins was virtually completely inhibited by N-[1-(R,S)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate, a specific active-site-directed inhibitor of endopeptidase-24.15, indicating that this enzyme was responsible for the observed interconversions. The data indicate that synaptosomal membranes contain enzymes that can potentially generate and degrade both leucine- and methionine-enkephalin.     

Endopeptidase-24.15 Is the Primary Enzyme that Degrades Luteinizing Hormone Releasing Hormone Both In Vitro and In Vivo

The concentration of luteinizing hormone releasing hormone (LHRH) (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), which reaches the anterior pituitary via the hypothalamo-hypophyseal portal system, appears to be controlled in part by the rate of LHRH degradation within the hypothalamus and/or pituitary. Specific, active site-directed endopeptidase inhibitors synthesized in our laboratory were used to identify the enzyme(s) involved in LHRH degradation by hypothalamic and pituitary membrane preparations, and by an intact anterior pituitary tumor cell line (AtT20). Incubation of LHRH with pituitary and hypothalamic membrane preparations led to the formation of pGlu-His-Trp (LHRH1-3) as the main reaction product. Under the same conditions, addition to the incubation mixtures of captopril, an inhibitor of the angiotensin converting enzyme, led to accumulation of pGlu-His-Trp-Ser-Tyr (LHRH1-5) and, to a lesser extent, pGlu-His-Trp-Ser-Tyr (LHRH1-6). The degradation of LHRH and the formation of the N-terminal tri- and pentapeptides was blocked by N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFP-AAF-pAB), a specific, active site directed inhibitor of endopeptidase-24.15. Some inhibition of LHRH degradation and formation of the N-terminal hexapeptide was also obtained in the presence of N-[1-carboxy-2-phenylethyl]-Phe-p-aminobenzoate (cFE-F-pAB), an inhibitor of endopeptidase-24.11. Similar results were obtained with AtT20 cell membranes and with intact AtT20 cells in monolayer culture. Following cleavage by endopeptidases the C-terminal part of LHRH was rapidly degraded by aminopeptidases. Superactive analogs of LHRH in which Gly6 was replaced by a D-amino acid are resistant to degradation by both endopeptidase-24.11 and -24.15. In vivo, when LHRH was injected directly into the third ventricle of rats, the presence of cFP-AAF-pAB inhibited LHRH degradation. It is concluded that LHRH degradation is primarily initiated by the membrane-bound form of endopeptidase-24.15 to yield pGlu-His-Trp-Ser-Tyr and to a lesser extent by endopeptidase-24.11 to yield pGlu-His-Trp-Ser-Tyr-Gly.     

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.     

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.     

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.     

Peptidyl dipeptidases (Ance and Acer) of Drosophila melanogaster: major differences in the substrate specificity of two homologs of human angiotensin I-converting enzyme

Drosophila melanogaster angiotensin converting enzyme (Ance) and angiotensin converting enzyme related (Acer) are single domain homologs of mammalian peptidyl dipeptidase A (angiotensin I-converting enzyme) whose physiological substrates have not as yet been identified. We have investigated the in vitro substrate specificities of the two peptidases towards a variety of insect and mammalian peptides. Ance was generally much better than Acer at hydrolyzing peptides of 5-13 amino acids in length. Only two of the peptides, [Leu(5)]enkephalinamide and leucokinin-I were cleaved faster by Acer. Increasing NaCl concentration had opposite affects on the cleavage of [Leu(5)]enkephalin and [Leu(5)]enkephalinamide by Acer, decreasing the activity towards [Leu(5)]enkephalin but increasing the activity towards [Leu(5)]enkephalinamide. Of the insect peptides tested, the tachykinin-related peptide, Lom TK-1, proved to be the best substrate for Ance with a k(cat)/K(m) ratio of 0.122s(-1) microM(-1). However, in comparison, the D. melanogaster tachykinins, DTK-1, DTK-2, DTK-3 and DTK-4 were poor Ance substrates. DTK-5 was the best substrate of this family, but the apparent high K(m) for hydrolysis by Ance suggested that this peptide would not be a natural Ance substrate. This low affinity for DTK-5 is the likely reason why the peptide was not rapidly degraded in D. melanogaster hemolymph, where Ance was shown to be a major peptide-degrading activity.     

Enkephalinases

Enkephalins can be degraded by a variety of peptidases. We have characterized several membrane-associated brain peptidases in an effort to determine which if any are concerned with the physiological inactivation of synaptically released enkephalin. We have distinguished two carboxyl-directed dipeptidylpeptidases, designated enkephalinase A1 and A2, that give rise to the Tyr-Gly-Gly fragment. Both enzymes are physically separable from angiotensin converting enzyme. Regional variations in enkephalinase A1 activity and opiate receptors are similar. A novel amino-terminal-directed dipeptidylpeptidase, enkephalinase B, which generates Tyr-Gly, has been identified. All of these enzymes as well as aminopeptidase have been solubilized from brain membranes by detergent treatment and have been mutually resolved by DEAE column chromatography. Enkephalinase A1 has been purified 1500-fold, to apparent homogeneity.     

Membrane Localization of Endopeptidase-24.11 and Peptidyl Dipeptidase A (Angiotensin Converting Enzyme) in the Pig Brain: A Study Using Subcellular Fractionation and Electron Microscopic Immunocytochemistry

Brains from piglets were dissected and a block of tissue including the substantia nigra, globus pallidus, and entopeduncular nucleus was homogenized and then fractionated on discontinuous Percoll gradients. Ligand-binding assays using (-)-[3H]nicotine and [3H]quinuclidinyl benzilate served to delineate fractions containing nicotinic and muscarinic acetylcholine receptors. In this system endopeptidase-24.11 exhibited a biphasic distribution, consistent with its presence on both pre- and postsynaptic membranes. Peptidyl dipeptidase A (angiotensin converting enzyme; ACE) was associated with membrane fractions containing muscarinic receptors. An immunoblot of these fractions with an affinity-purified polyclonal antibody to ACE revealed only the neuronal form of ACE (Mr 170,000), the endothelial form (Mr 180,000) being undetectable. Electron microscopic immunoperoxidase staining of the substantia nigra, with an affinity-purified antibody to endopeptidase-24.11 at the preembedding stage, showed this antigen to be confined to the plasma membranes of boutons, axons, and some dendrites. Both pre- and postsynaptic membranes were stained, and occasionally other regions of the dendritic membrane were positive. No staining of synaptic vesicles within the boutons was observed. Thus, two independent approaches indicate that endopeptidase-24.11 is present on both pre- and postsynaptic membranes in the pig substantia nigra. The subcellular fractionation suggests that neuronal ACE is confined to dendritic membranes.     

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.