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.     

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.     

Specificity of hydrolysis of bovine kappa-casein by cell envelope-associated proteinases from Lactococcus lactis strains.

The cell envelope-associated proteinases from Lactococcus lactis subsp. cremoris H2 (a PI-type proteinase-producing strain) and SK11 (a PIII-type proteinase-producing strain) both actively hydrolyze the kappa-casein component of bovine milk but with significant differences in the specificity of peptide bond hydrolysis. The peptide bonds Ala-23-Lys-24, Leu-32-Ser-33, Ala-71-Gln-72, Leu-79-Ser-80, Met-95-Ala-96, and Met-106-Ala-107 were cleaved by both proteinase types, although the relative rates of hydrolysis at some of these sites were quite different for the two proteinases. Small histidine-rich peptides were formed as early products of the action of the cell envelope-associated proteinases on kappa-casein, implicating this casein as a possible significant source of histidine, which is essential for starter growth. The major difference between the two proteinase types in their action on kappa-casein was in their ability to cleave bonds near the C-terminal end of the molecule. The bond Asn-160-Thr-161 and, to a lesser extent, the bond Glu-151-Val-152 were very rapidly cleaved by the PIII-type proteinase, whereas hydrolysis of these bonds by the PI-type proteinase was barely detectable (even after 24 h of digestion). Differential hydrolysis of kappa-casein at these sites by the two different proteinase types resulted in the formation of distinctive, high-M(r) products detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the N-tosyl-L-phenylalanyl chloromethylketone modification site in Thermus thermophilus elongation factor Tu

EF-Tu from Thermus thermophilus was first labelled with N-[14C]tosyl-L-phenylalaninechloromethylketone and then cleaved by the combined action of CNBr and trypsin. The resulting peptides were separated by reversed-phase HPLC. Analysis of the isolated, labelled peptide led to the identification of a sequence which was identical to residues 76-88 in T. thermophilus EF-Tu. The TPCK reactive site is at Cys-82. Kinetic measurements of the incorporation of TPCK into native EF-Tu and EF-Tu nicked at position Arg-59 were performed. The results provide evidence that the cleavage of the peptide bond between Arg-59 and Gly-60 does not lead to a dramatic conformational change of EF-Tu at the aa-tRNA binding site.     

The structure of residues 7-16 of the A alpha-chain of human fibrinogen bound to bovine thrombin at 2.3-A resolution.

The tetradecapeptide Ac-D-F-L-A-E-G-G-G-V-R-G-P-R-V-OMe, which mimics residues 7f-20f of the A alpha-chain of human fibrinogen, has been co-crystallized with bovine thrombin from ammonium sulfate solutions in space group P2(1) with unit cell dimensions of a = 83.0 A, b = 89.4 A, c = 99.3 A, and beta = 106.6 degrees. Three crystallographically independent complexes were located in the asymmetric unit by molecular replacement using the native bovine thrombin structure as a model. The standard crystallographic R-factor is 0.167 at 2.3-A resolution. Excellent electron density could be traced for the decapeptide, beginning with Asp-7f and ending with Arg-16f in the active site of thrombin; the remaining 4 residues, which have been cleaved from the tetradecapeptide at the Arg-16f/Gly-17f bond, are not seen. Residues 7f-11f at the NH2 terminus of the peptide form a single turn of alpha-helix that is connected by Gly-12f, which has a positive phi angle, to an extended chain containing residues 13f-16f. The major specific interactions between the peptide and thrombin are 1) a hydrophobic cage formed by residues Tyr-60A, Trp-60D, Leu-99, Ile-174, Trp-215, Leu-9f, Gly-13f, and Val-15f that surrounds Phe-8f; 2) a hydrogen bond linking Phe-8f NH to Lys-97 O;3) a salt link between Glu-11f and Arg-173; 4) two antiparallel beta-sheet hydrogen bonds between Gly-14f and Gly-216; and 5) the insertion of Arg-16f into the specificity pocket. Binding of the peptide is accompanied by a considerable shift in two of the loops near the active site relative to human D-phenyl-L-prolyl-L-arginyl chloromethyl ketone (PPACK)-thrombin.     

Disulfide bond reduction in fibrinogen: calcium protection and effect on clottability

Fibrinogen contains 29 disulfide bonds. Limited reduction in buffers containing calcium led to cleavage of three of them: the two A alpha 442Cys-A alpha 472Cys intrapeptide disulfide bonds and the symmetrical A alpha 28Cys-A alpha 28Cys bond. The limited reduction did not affect clotting by thrombin. However, a prolongation of the thrombin clotting time occurred when the limited reduction took place in the absence of calcium. The bonds reduced under this condition included the three already mentioned and also the two gamma 326Cys-gamma 339Cys intrapeptide disulfide bonds located in the C-terminal ends of the gamma-chain. N-Terminal analysis of thrombin-treated samples showed that thrombin cleavage occurred at the normal A alpha 16-A alpha 17 site in fibrinogen that was partially reduced in the presence of calcium. By contrast, thrombin cleaved at the A alpha 19-A alpha 20 site in fibrinogen that was partially reduced in the absence of calcium, rendering the protein unclottable by removing the A alpha 17Gly-18Pro-19Arg peptide. The loss of thrombin clottability may have also come from gamma 326Cys-gamma 339Cys disulfide bond reduction since the structure supported by this bond may be important for the function of the C-terminal polymerization site. In samples of the partially reduced fibrinogen lacking the A alpha 17-19 residues, gel formation occurred through an oligomerization mechanism catalyzed by factor XIII.     

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.     

Hydrolysis of the C-terminal octapeptide of cholecystokinin by rat kidney membranes: characterization of the cleavage by solubilized endopeptidase-24.11

Rat kidney membranes were solubilized by Triton X-100 and the CCK-8 degrading peptidases were resolved by chromatography on DEAE-cellulose. Four proteases were detected: two phosphoramidon-sensitive endopeptidases (EC 3.4.24.11), a bestatin-sensitive aminopeptidase and an unidentified enzyme. The pattern of cleavage of CCK-8 and shorter C-terminal fragments by endopeptidase 24.11 was investigated and indicated that the Gly29-Trp30, Trp30-Met31 and Asp32-Phe33 were scissile bonds. However, the cleavage pattern differed markedly from one CCK peptide to another: in the penta- and hexapeptide of CCK the bonds hydrolyzed were either Asp-Phe and Trp-Met or, Asp-Phe and Gly-Trp, respectively. The presence of the sulfate group on the tyrosine residue of CCK-8 influence markedly the nature of the major cleavage fragments produced by the endopeptidase. The major bonds cleaved were Asp-Phe, Trp-Met and Gly-Trp for unsulfated CCK-8, whilst for the sulfated octapeptide, the Trp-Met bond became a minor cleavage site.     

Specificity of an extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein.

The specificity of the extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein was studied. Hydrolysis was monitored over time by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and urea-PAGE. The major pH 4.6-soluble peptides were isolated by high-performance liquid chromatography and identified by N-terminal amino acid sequencing and mass spectrometry. The time course of peptide formation indicated that His-8-Gln-9, Ser-161-Gly-162, and either Gln-172-Tyr-173 or Phe-23-Phe-24 were the first, second, and third bonds cleaved, respectively. Other cleavage sites included Asn-19-Leu-20, Phe-32-Gly-33, Tyr-104-Lys-105, Leu-142-Ala-143, Phe-150-Arg-151, Gln-152-Phe-153, Leu-169-Gly-170, and Thr-171-Gln-172. The proteinase had a broad specificity for the amino acid residues at the P1 and P'1 positions but showed a preference for hydrophobic residues at the P2, P3, P4, P'2, P'3, and P'4 positions.     

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).     

In vitro degradation of endothelin-1 by endopeptidase 24.11 (enkephalinase).

The breakdown of endothelin-1 by crude membrane preparations of human kidney and choroid plexus was investigated. 125I-labeled endothelin-1 was degraded by both tissues in a phosphoramidon-sensitive way, suggesting a role of endopeptidase 24.11 in the in vitro metabolism of this peptide. Identification of the cleavage sites of purified human renal endopeptidase 24.11 in the sequence of endothelin-1 revealed that bonds involving the amino side of the hydrophobic amino acids (Ser4, Leu6, Val12, Phe14, His16, Leu17, Ile19) were susceptible to cleavage. Endothelin-1 appears thus to be degraded at multiple sites by endopeptidase 24.11 in vitro, producing inactive fragments.     

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.     

Amino acid replacement studies of human cytochrome c by a complementation system using CYC1 deficient yeast

Various in vitro mutated human cytochrome c genes which encode displaced amino acid residues at the 14th, 17th, 28th, 37th, 38th, 56th, and/or 84th residues were constructed, and their degrees of complementation of yeast CYC1 deficiency were examined. Invariant Cys-17 and Arg-38 could not be replaced by alanine and tryptophan, respectively, without function impairment. Cytochrome c containing Ala-14 instead of conserved Cys-14, Gly-38 or Lys-38 instead of Arg-38, and Ser-84 instead of invariant Gly-84 were partly functional. These results indicate that these invariant or conserved residues are important. Cytochromes c containing Cys-56 instead of native Gly-56 was partly functional. Cytochrome c containing Arg-37 and Gly-38 instead of Gly-37 and Arg-38 was slightly functional. Replacement of variable Thr-28 and Gly-37 by Ile-28 and Arg-37, respectively, produced no effects. Our results are as a whole consistent with the view that conserved residues are important and variable residues are less important for cytochrome c to function.     

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.     

Determination of the cleavage site involved in C-terminal processing of penicillin-binding protein 3 of Escherichia coli.

Chromatographic peptide mapping of lysyl endopeptidase digests of penicillin-binding protein 3 (PBP 3) of Escherichia coli revealed peptides that differed in retention time between the precursor and mature forms. The peptides were purified from a processing-defective (prc) mutant and a wild-type (prc+) strain. These peptides were identified as the C-terminal region of the precursor form and mature PBP 3 by amino acid sequencing. Each of the C-terminal peptides was cleaved into two fragments by trypsin digestion. By sequencing the resultant carboxyl-side fragment derived from the mature form, it was concluded that the C-terminal residue of mature PBP 3 was Val-577, and thus the Val-577-Ile-578 bond is the cleavage site for processing. This conclusion was consistent with the amino acid compositions of the relevant peptides, which suggested that the peptide from the cleavage site to the end of the deduced sequence (Ile-578-Ser-588) was present in the precursor but absent in the mature form. One lysyl peptide bond resisted both lysyl endopeptidase and trypsin and remained uncleaved in the peptide analyzed above.     

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.     

Respective roles of kallikrein and endopeptidase 24.11 in the metabolic pathway of atrial natriuretic peptide in the rat.

The metabolism of atrial natriuretic peptide (ANP) and Cys-105-Phe-106-cleaved ANP (ANP) was studied during constant infusion of 125I-labelled peptides in rats. Analysis of circulating radioactivity indicated rapid clearance of ANP and ANP', with mean half-lives of 0.42 and 1.04 min respectively. H.p.l.c. fractionation of plasma taken during the infusion of labelled ANP revealed the presence of three radioactive fragments, the major one co-eluting with 125I-ANP'. These fragments correspond to cleavage products previously found to be generated in vitro by the action of endopeptidase 24.11 (E-24.11). On evaluating the effects of peptidase inhibitors, a significant increase in the half-life of ANP was observed with phosphoramidon (t1/2 7.8 min) and aprotinin (t1/2 5.4 min). A maximal inhibition of ANP degradation was obtained when both inhibitors were given simultaneously (t1/2 15 min). In blood samples taken during infusion of 125I-ANP and phosphoramidon, the intact peptide accounted for more than 90% of total circulating radioactivity, and no cleavage product was present in detectable amounts. Phosphoramidon had no effect on the metabolism of infused ANP'. In contrast, when 125I-ANP' was infused together with aprotinin, the rate of degradation of the infused peptide was reduced by more than 80%. It is proposed that two different peptidase activities, E-24.11 and a kallikrein-like proteinase, are responsible for the cleavage of ANP in the circulation. The Cys-Phe-cleaved ANP would in turn be degraded by kallikrein and not by E-24.11.     

Role of endopeptidase-24.11 in the inactivation of atrial natriuretic peptide

The circulating form of atrial natriuretic factor is a 28-residue peptide containing a 17-residue disulphide-linked ring. It has important actions on the kidney, largely on its haemodynamics, and at other sites including the adrenal cortex and CNS. It has a short half-life in vivo and is rapidly inactivated when incubated with kidney microvillar membranes. Of the battery of peptidases present in that membrane, only one, endopeptidase-24.11, is responsible for initiating the attack, and this commences with hydrolysis of the Cys7-Phe8 bond within the ring. Hydrolysis at this and other points has been shown to inactivate the peptide and this information has pointed the way to the synthesis of resistant analogues.     

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.