Bestatin-sensitive Aminopeptidase Research Articles

Electroosmotic sampling. Application to determination of ectopeptidase activity in organotypic hippocampal slice cultures.

We hypothesize that peptide-containing solutions pulled through tissue should reveal the presence and activity of peptidases in the tissue. Using the natural zeta-potential in the organotypic hippocampal slice culture (OHSC), physiological fluids can be pulled through the tissue with an electric field. The hydrolysis of the peptides present in the fluid drawn through the tissue can be determined using capillary HPLC with electrochemical detection of the biuret complexes of the peptides following a postcolumn reaction. We have characterized this new sampling method by measuring the flow rate, examining the use of internal standards, and examining cell death caused by sampling. The sampling flow rate ranges from 60 to 150 nL/min with a 150 microm (ID) sampling capillary with an electric field (at the tip of the capillary) from 30 to 60 V/cm. Cell death can be negligible with controlled sampling conditions. Using this sampling approach, we have electroosmotically pulled Leu-enkephalin through OHSCs to identify ectopeptidase activity in the CA3 region. These studies show that a bestatin-sensitive aminopeptidase may be critical for the hydrolysis of exogenous Leu-enkephalin, a neuropeptide present in the CA3 region of OHSCs.

Metabolism of dynorphins by peptidases of pulmonary artery endothelial cells.

Degradation of several dynorphins by peptidases expressed in cultured porcine pulmonary artery endothelial cells was studied by incubation of the peptide in cell suspensions followed by electrospray ionization and tandem mass spectrometric analyses. Under the in vitro conditions applied, only the metabolism of dynorphin A1-8 occurred in a significant extent. Studies involving specific peptidase inhibitors indicated that mainly bestatin-sensitive aminopeptidases, thiorphan-sensitive endopeptidases, and cFPAAF-pAB-sensitive endopeptidases expressed by the endothelial cells were involved in the process that converted dynorphin A1-8 to dynorphin A2-8, dynorphin A1-6, and leucine enkephalin (dynorphin A1-5), respectively. These peptidases may form a metabolic barrier for the cellular penetration of intact dynorphin A1-8 and/or control effects of the circulating peptide on endothelial opioid receptors of the cells.

Endoproteolytic activity in mammalian brain membranes cleaves 5-hydroxytryptamine-moduline into dipeptides

This work was intended to determine which enzymatic activities from crude synaptosomal mammalian brain membranes could qualify for the status of 5-hydroxytryptamine-moduline (5-HT-moduline, LSAL, Leu-Ser-Ala-Leu) inactivating enzymes. An enzymatic assay for 5-HT-moduline metabolism was developed using [ 3 H ]5-HT-moduline measurement and high performance liquid chromatography (HPLC) technique to identify and quantify 5-HT-moduline metabolites. 5-HT-moduline metabolism displayed all characteristics of metalloprotease activity: sensitivity to divalent ion chelators, reactivation by Zn 2+ ions and a pH optimum in the 7–8 range. Bestatin, an aminopeptidase inhibitor, allowed the identification of two enzymatic activities responsible for this metabolism: a bestatin-sensitive aminopeptidase and an endoprotease cleaving 5-HT-moduline into LS (Leu-Ser) and AL (Ala-Leu) dipeptides. This latter enzyme was shown to have a K m of 37.1±3.6 μM and a V max of 5.5 μmol min −1 l −1 per mg of protein. Moreover, this enzyme was insensitive to peptidyl dipeptidase A (angiotensin converting enzyme, EC 3.4.15.1), endothelin converting enzyme and neutral endopeptidase (neprylisin, EC 3.4.24.11) inhibitors and displayed some specificity among 5-HT-moduline-analogues and in particular recognized only tetrapeptides. These results, together with the isolation of the LS and AL metabolites [Rousselle, J.C., Massot, O., Delepierre, M., Zifa, E., Rousseau, B., Fillion, G., 1996. Isolation and characterization of an endogenous peptide from rat brain interacting specifically with the serotonergic 1B receptor subtypes. J. Biol. Chem. 271, 726–735] during the purification process of 5-HT-moduline are strong arguments for the physiological implication of this endoprotease in 5-HT-moduline metabolism.

Interferon-γ Can Stimulate Post-proteasomal Trimming of the N Terminus of an Antigenic Peptide by Inducing Leucine Aminopeptidase

Most antigenic peptides presented on major histocompatibility complex class I molecules are generated during protein breakdown by proteasomes, whose specificity is altered by interferon-gamma (IFN-gamma). When extended versions of the ovalbumin-derived epitope SIINFEKL are expressed in vivo, the correct C terminus is generated by proteasomal cleavage, but distinct cytosolic protease(s) generate its N terminus. To identify the other protease(s) involved in antigen processing, we incubated soluble extracts of HeLa cells with the 11-mer QLESIINFEKL, which in vivo is processed to the antigenic 8-mer (SIINFEKL) by a proteasome-independent pathway. This 11-mer was converted to the 9-mer by sequential removal of the N-terminal residues, but surprisingly the extract showed little or no endopeptidase or carboxypeptidase activity against this precursor. After treatment of cells with IFN-gamma, this N-terminal trimming was severalfold faster and proceeded to the antigenic 8-mer. The IFN-treated cells also showed greater aminopeptidase activity against many model fluorogenic substrates. Upon extract fractionation, three bestatin-sensitive aminopeptidase peaks were detected. One was induced by IFN-gamma and was identified immunologically as leucine aminopeptidase (LAP). Purified LAP, like the extracts of IFN-gamma-treated cells, processed the 11-mer peptide to SIINFEKL. Thus, IFN-gamma not only promotes proteasomal cleavages that determine the C termini of antigenic peptides, but also can stimulate formation of their N termini by inducing LAP. This enzyme appears to catalyze the trimming of the N terminus of this and presumably other proteasome-derived precursors. Thus, susceptibility to LAP may be an important influence on the generation on immunodominant epitopes.

Production and characterization of an antiserum against the 5-HT 1B receptor for immunocytochemical localization

This work was intended to determine which enzymatic activities from crude synaptosomal mammalian brain membranes could qualify for the status of 5-hydroxytryptamine-moduline (5-HT-moduline, LSAL, Leu-Ser-Ala-Leu) inactivating enzymes. An enzymatic assay for 5-HT-moduline metabolism was developed using [3H]5-HT-moduline measurement and high performance liquid chromatography (HPLC) technique to identify and quantify 5-HT-moduline metabolites. 5-HT-moduline metabolism displayed all characteristics of metalloprotease activity: sensitivity to divalent ion chelators, reactivation by Zn2+ ions and a pH optimum in the 7–8 range. Bestatin, an aminopeptidase inhibitor, allowed the identification of two enzymatic activities responsible for this metabolism: a bestatin-sensitive aminopeptidase and an endoprotease cleaving 5-HT-moduline into LS (Leu-Ser) and AL (Ala-Leu) dipeptides. This latter enzyme was shown to have a Km of 37.1±3.6 μM and a Vmax of 5.5 μmol min−1 l−1 per mg of protein. Moreover, this enzyme was insensitive to peptidyl dipeptidase A (angiotensin converting enzyme, EC 3.4.15.1), endothelin converting enzyme and neutral endopeptidase (neprylisin, EC 3.4.24.11) inhibitors and displayed some specificity among 5-HT-moduline-analogues and in particular recognized only tetrapeptides. These results, together with the isolation of the LS and AL metabolites [Rousselle, J.C., Massot, O., Delepierre, M., Zifa, E., Rousseau, B., Fillion, G., 1996. Isolation and characterization of an endogenous peptide from rat brain interacting specifically with the serotonergic 1B receptor subtypes. J. Biol. Chem. 271, 726–735] during the purification process of 5-HT-moduline are strong arguments for the physiological implication of this endoprotease in 5-HT-moduline metabolism.

Effect of some peptidase inhibitors on exogenous and endogenous opioid actions in guinea-pig ileum.

Met-enkephalin concentration-dependently and transiently inhibited the ileal twitch contraction and this inhibition gradually recovered with time. Bacitracin, phosphoramidon, thiorphan and captopril did not influence the twitch inhibition of met-enkephalin, but bestatin increased the twitch inhibitory potency of met-enkephalin and terminated it in a manner which almost paralleled that of untreated tissue. Transient inhibition of twitch contraction after tetanic stimulation (post-tetanic twitch inhibition) was obtained. Bestatin increased the potency of met-enkephalin and this was terminated within 2 min. Phosphoramidon tended to increase the potency and delayed the termination of post-tetanic twitch inhibition. Bacitracin, thiorphan and captopril did not influence either the potency or the termination of post-tetanic twitch inhibition. Morphine-induced twitch inhibition was not influenced by bacitracin, bestatin or phosphoramidon. These results suggest that bestatin-sensitive aminopeptidase and phosphoramidon-sensitive enkephalinase take part in post-tetanic twitch inhibition, acting in a different mode of action, and have an important role in the termination of the pharmacological action of endogenous opioids (post-tetanic twitch inhibition) in MPLM. This different mode of response of bestatin and phosphoramidon upon post-tetanic twitch inhibition may underlie that aminopeptidase is a more soluble enzyme and enkephalinase is membrane-bound in myenteric plexus-longitudinal muscle (MPLM).

Characterization of an aminopeptidase in cerebrospinal fluid: Structure elucidation of enzyme hydrolysis products of synthetic methionine-enkephalin by reversed-phase high-performance liquid chromatography and mass spectrometry

An aminopeptidase was found in canine cerebrospinal fluid via the presence of two products: Y, which has an [M + H] + ion at m/z 182; and GGFM, which has an [M + H] + ion at m/z 411. The linked scan at a constant ratio of the magnetic field to the electric field of the GGFM [M + H] + ion at m/z 411 generates product ions at m/z 120, 150, 266, 297, 354, 357, and 411. That aminopeptidase was bestatin-sensitive (BSAP = bestatin-sensitive aminopeptidase), and had a half-time for disappearance of 60 min, maximum velocity of 1.08 nmol ml −1 min −1, and Michaelis constant of 0.26 n M.

Enkephalin hydrolysis by mouse plasma [formula omitted

Hydrolysis of [Leu]- and [Met] enkephalin was determined in samples of pooled whole mouse plasma in vitro by using HPLC-ECD to measure accumulation of Tyr-containing metabolites. More Tyr-Gly-Gly accumulated from [Met] enkephalin than from [Leu] enkephalin hydrolysis, and [Met] enkephalin's half-life in mouse plasma was approximately half that of [Leu] enkephalin. Comparisons of metabolite formation in the presence versus the absence of inhibitors with high selectivity for various peptidases demonstrated that a bestatin-sensitive aminopeptidase, presumably aminopeptidase M, as well as enkephalinase and angiotensin converting enzyme, participate in the hydrolysis of enkephalin in mouse plasma.

N-terminal degradation of ACTH(4–10) and its synthetic analog semax by the rat blood enzymes

Degradation of a regulatory peptide ACTH(4-10) and its synthetic analog semax in rat blood and serum was studied using high-performance liquid chromatography. About one third to one half of the serum degrading activity could be ascribed to bestatin-sensitive aminopeptidase which cleaved first and second N-terminal residues Met and Glu producing relatively stable intermediates. Comparable areas under the degradation/accumulation curves for intact peptides and intermediates implied that the latter can contribute to effects of intact peptides. Semax turned out to be more stable than ACTH(4-10) against the action of other enzymes that took part in degradation.

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 Gln 6-Phe 7, Phe 7-Phe 8 and Gly 9-Leu 10, 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.

Novel Inhibitors of Enkephalin-Degrading Enzymes II: N5-Substituted-4-Thioxohydantoic Acids as Aminopeptidase Inhibitors

Some 2-substituted-(2'-aminophenyl)-4-thioxohydantoic acids (o-amino PTC-amino acids) have antinociceptive activity when administered (icv) alone (IC50 = 0.04-0.87 microM/animal) and show a striking prolongation of the antinociceptive action of (D-Ala-2 D-Leu5)-enkephalin (DADL) in combination. The effects are thought to be mediated via opioid receptors since they are naloxone-reversible. Although inhibitors of the enkephalin degrading puromycin-insensitive, bestatin-sensitive aminopeptidase (possibly aminopeptidase M) their action is weak (IC50 = 32 microM leucine, 536 microM, glycine) and they might be considered to have a direct antinociceptive effect on opioid receptors. The titled compounds constitute novel 'lead' compounds for the development of potent aminopeptidase M inhibitors.

Degradation of endogenous proteins and internalized asialofetuin in primary cultured hepatocytes of rats

1. 1. The effects of various metabolic and enzyme inhibitors on the degradation of endogenous proteins and internalized asialofetuin were examined in the primary cultured hepatocytes of rats. 2. 2. The results showed the important physiological role of bestatin-sensitive aminopeptidases in the degradation of endogenous and internalized proteins as well as in the degradation of both long- and short-lived proteins.

Potentiation by thiorphan and bestatin of the naloxone-insensitive analgesic effects of neurotensin and neuromedin n

Neurotensin induced significant antinociceptive activity as measured in a variety of nociceptive tests 10 and 30 min following intracerebroventricular (i.c.v.) injection in mice. The lowest effective peptide doses were 25 ng in the writhing test, 25–50 ng in the tail-flick test, 50–100 ng in the hot-plate test and 2000 ng in the tail electrical stimulation test. The neurotensin related hexapeptide neuromedin N also displayed antinociceptive properties but only in the writhing and tail-flick tests. Furthermore, as compared to neurotensin, the neuromedin effects required higher doses. ED 50's for neurotensin and neuromedin in the writhing test were 70 ng and 1070 ng, respectively. Separate or combined injections of the endopeptidase 24.11 (enkephalinase) inhibitor thiorphan (l0μg) and the aminopeptidase inhibitor bestatin (50μg) did not affect tail-flick latencies. In contrast, i.c.v. injection of thiorphan together with an ineffective dose of neurotensin (25 ng) resulted in a significant antinociceptive effect. Bestatin did not modify tail-flick latencies in neurotensin-treated mice whether in the absence or presence of thiorphan. On the contrary, each of these peptidase inhibitors promoted antinociceptive effects of subthreshold doses of neuromedin (lμg) in the tail-flick test. Maximal antinociception was obtained by combining both inhibitors, thus conferring antinociceptive effects to neuromedin doses that were as low as 10 ng. Naloxone (0.5–2 mg/kg, s.c.) did not significantly reduced the antinociceptive effects of combinations of neurotensin and thiorphan and of neuromedin, thiorphan and bestatin. The data show that both neurotensin and neuromedin elicit analgesia in mice through an opiate independent mechanism. Furthermore, like enkephalin, neuromedin is readily degraded by brain endopeptidase 24.11 and bestatin sensitive aminopeptidase(s), whereas the resistance of neurotensin to aminopeptidase attack confers to this peptide a broader spectrum and longer duration of action than its congener neuromedin.

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.

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.

Binding of vasoactive intestinal polypeptide to dispersed enterocytes results in rapid removal of the NH 2-terminal histidyl residue

Specific binding sites for 125I-labelled vasoactive intestinal polypeptide (VIP) (half-maximal inhibition at 1.5 ± 0.2 nM VIP ) were identified on dispersed porcine enterocytes. Radioactivity bound to the cell surface was internalized. At 37 ° C, a steady state was achieved after 45 min with a ratio of internalized to cell surface-bound radioactivity of approximately 1:2 but at 10 ° C, no radioactivity appeared intracellularly. Incubation of VIP with cells in the absence of inhibitors of proteolysis for as short a time as 30 s at 37 °C led to the formation of [des-His 1]VIP by the action of amastatin- and bestatin-sensitive aminopeptidase(s). This metabolite was formed in the presence of sodium azide and when incubations were performed at 10°C suggesting that internalization was not a prerequisite for degradation. As [des-His 1]-VIP has only 1% of the bioactivity of VIP, formation of this metabolite will effectivily terminate the action of VIP in the epithelial layer of the intestine.

Enkephalin-degrading enzymes and angiotensin-converting enzyme in human and rat meninges

The neutral endopeptidase NEP 24.11 (enkephalinase) has been visualized in human spinal cord by in vitro autoradiography using [ 3H]HACBO-Gly as a radiolabelled probe. The specific binding was present in the substantia gelatinosa and particularly dense in meninges surrounding the spinal cord. Enzymatic studies using [ 3H][D-Ala 2, Leu]enkephalin as substrate confirmed the presence of NEP in dura and pia mater of human tissue. In addition, the human meninges were shown to contain high concentrations of angiotensin-converting enzyme (ACE) and aminopeptidases. The three enzymes have also been detected in rat tissues but their distribution pattern differs from that of human tissue. In dura mater, 45% of the [Leu]enkephalin hydrolysis was due to enkephalinase and 38% to bestatin-sensitive aminopeptidases. In contrast in pia mater aminopeptidases were more efficient in hydrolyzing enkephalin. The possible role of these enzymes in the meninges could be to maintain the homeostatic concentration of neuropeptides in the central nervous system.

Catabolism of neurotensin by neural (neuroblastoma clone N1E115) and extraneural (HT29) cell lines

The mechanisms by which neurotensin (NT) was inactivated by differentiated neuroblastoma and HT29 cells were characterized. In both cell lines, the sites of primary cleavages of NT were Pro 7-Arg 8, Arg 8-Arg 9 and Pro 10-Tyr 11 bonds. The cleavage at the Pro 7-Arg 8 bond was totally inhibited by N-benzyloxycarbonyl-Prolyl-Prolinal and therefore resulted from the action of proline endopeptidase. This peptidase also contributed in a major way to the cleavage at the Pro 10-Tyr 11 bond. However the latter breakdown was partly due to an NT-degrading neutral metallopeptidase. Finally, we demonstrated the involvement of a recently purified rat brain soluble metalloendopeptidase at the Arg 8-Arg 9 site by the use of its specific inhibitor N-[1(R,S)-carboxy-2-Phenylethyl]-alanylalanylphenylalanine-p-aminobenzoate. The secondary processing of NT degradation products revealed differences between HT29 and N1E115 cells. Angiotensin converting enzyme was shown to degrade NT 1–10 and NT 1–7 in N1E115 cells but was not detected in HT29 cells. A post-proline dipeptidyl aminopeptidase activity converted NT 9–13 into NT 11–13 in HT29 cells but not in N1E115 cells. Finally bestatin-sensitive aminopeptidases rapidly broke down NT 11–13 to Tyr in both cell lines. Models for the inactivation of NT in HT29 and N1E115 cells are proposed and compared to that previously described for purified rat brain synaptic membranes.

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).

The Inactivation of [Met5]-Enkephalin by Bestatin-Sensitive Aminopeptidase, Captopril-Sensitive Peptidyl Dipeptidase A and Thiorphan-Sensitive Endopeptidase-24.11 in Mouse Vas Deferens

The enkephalin-hydrolyzing peptidases in mouse vas deferens were studied and compared with those in guinea pig ileum which had been characterized in the previous study. The present results showed that three distinct peptidases, bestatin-sensitive aminopeptidase, captopril-sensitive peptidyl dipeptidase A, and thiorphan-sensitive endopeptidase-24.11, played a critical role in the inactivation of enkephalin in mouse vas deferens, being consistent with the previous results obtained with guinea pig ileum. However, the data in both previous and present studies showed that the activity of the bestatin-sensitive aminopeptidase relative to that of either the captopril-sensitive peptidyl dipeptidase A or the thiorphan-sensitive endopeptidase-24.11 in guinea pig ileum was higher than that in mouse vas deferens, while the activity of either peptidyl dipeptidase A or endopeptidase-24.11 relative to that of aminopeptidase in mouse vas deferens was higher than that in guinea-pig ileum. In contrast to these three enzymes, both L-tyrosyl-L-tyrosine-sensitive dipeptidyl aminopeptidase and D-phenylalanine-sensitive carboxypeptidase were suggested not to be involved significantly in the inactivation of enkephalin in mouse vas deferens as well as guinea pig ileum.