An evaluation of the role of a pyroglutamyl peptidase, a post-proline cleaving enzyme and a post-proline dipeptidyl amino peptidase, each purified from the soluble fraction of guinea-pig brain, in the degradation of thyroliberin in vitro

The degradation of thyroliberin (less than Glu-His-Pro-NH2) to its component amino acids by the soluble fraction of guinea pig brain is catalysed by four enzymes namely a pyroglutamate aminopeptidase, a post-proline cleaving enzyme, a post-proline dipeptidyl aminopeptidase and a proline dipeptidase. 1. The pyroglutamate aminopeptidase was purified to over 90% homogeneity with a purification factor of 2868-fold and a yield of 5.7%. In addition to catalysing the hydrolysis of thyroliberin, acid thyroliberin and pyroglutamate-7-amido-4-methylcoumarin the pyroglutamate aminopeptidase catalysed the hydrolysis of the peptide bond adjacent to the pyroglutamic acid residue in luliberin, neurotensin bombesin, bradykinin-potentiating peptide B, the anorexogenic peptide and the dipeptides pyroglutamyl alanine and pyroglutamyl valine. Pyroglutamyl proline and eledoisin were not hydrolysed. 2. The post-proline cleaving enzyme was purified to apparent electrophoretic homogeneity with a purification factor of 2298-fold and a yield of 10.6%. The post-proline cleaving enzyme catalysed the hydrolysis of thyroliberin and N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. It did not catalyse the hydrolysis of glycylproline-7-amido-4-methylcoumarin or His-Pro-NH2. 3. The post-proline dipeptidyl aminopeptidase was partially purified with a purification factor of 301-fold and a yield of 8.9%. The post-proline dipeptidyl aminopeptidase catalysed the hydrolysis of His-Pro-NH2 and glycylproline-7-amido-4-methylcoumarin but did not exhibit any post-proline cleaving endopeptidase activity against thyroliberin or N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. 4. Studies with various functional reagents indicated that the pyroglutamate aminopeptidase could be specifically inhibited by 2-iodoacetamide (100% inhibition at an inhibitor concentration of 5 microM), the post-proline cleaving enzyme by bacitracin (IC50 = 42 microM) and the post-proline dipeptidyl aminopeptidase by puromycin (IC50 = 46 microM). Because of their specific inhibitory effects these three reagents were key elements in the elucidation of the overall pathway for the metabolism of thyroliberin by guinea pig brain tissue enzymes.     

Purification of and kinetic studies on a narrow specificity synaptosomal membrane pyroglutamate aminopeptidase from guinea-pig brain

A pyroglutamate aminopeptidase activity, distinct from that of cytoplasm, was released from a synaptosomal membrane preparation of guinea-pig brain by papain treatment. This activity was further purified 3560-fold relative to the homogenate with a yield of 17% by a procedure involving gel filtration chromatography, calcium phosphate cellulose chromatography and hydrophobic interaction chromatography on phenyl-Sepharose CL-4B. The purified synaptosomal pyroglutamate aminopeptidase hydrolysed only thyroliberin, acid-thyroliberin, the luliberin N-terminal tripeptide (Glp-His-Trp) and, only slightly, Glp-His-Gly. No hydrolysis was observed with dipeptides containing N-terminal pyroglutamic acid (Glp) or with pyroglutamyl peptides containing more than three amino acids. A Km value of 40 microM was recorded when thyroliberin was used as substrate; however, luliberin was found to inhibit the hydrolysis of thyroliberin competitively with a Ki value of 20 microM.     

Active Site Studies on a Narrow-Specificity Thyroliberin-Hydrolysing Pyroglutamate Aminopeptidase Purified from Synaptosomal Membrane of Guinea-Pig Brain

The effect of protein-modifying reagents on the activity of a purified preparation of a thyroliberin-hydrolysing pyroglutamate aminopeptidase, solubilised from synaptosomal membranes of guinea-pig brain by treatment with papain, was investigated. The results indicated that tyrosine, histidine, arginine, and possibly lysine residues were necessary for expression of catalytic activity and that these tyrosine, histidine, and arginine residues were probably located at the active site of the enzyme. Cysteine, serine, glutamate, and aspartate residues were not involved in the expression of catalytic activity.     

Catabolism of thyroliberin by rat adenohypophyseal tissue extract

Rapid fragmentation of thyroliberin (less than Glu-His-Pro-NH2) by rat adenohypophyseal tissue enzymes could be demonstrated. Based on the identification of the metabolic products and by the demonstration that the individual enzymatic reactions can be preferentially blocked by enzyme inhibitors, specific and sensitive biochemical tests could be developed in order to monitor the enzymatic activities after gel chromatographic fractionation of the tissue extracts. These findings are in agreement with the interpretation that the observed degradation of thyroliberin by hypophyseal tissue extracts may follow the proposed pathways. The primary enzymatic cleavage of thyroliberin is either initiated by the action of a 'thyroliberin-deamidating enzyme' (thyroliberin leads to less than Glu-His-Pro-OH + NH3), or by the action of a pyroglutamate aminopeptidase (thyroliberin leads to less than Glu + His-Pro-NH2). While the pyroglutamate aminopeptidase also catalyzes the subsequent degradation of deamidated thyroliberin (less than Glu-His-Pro-OH leads to less than Glu + His-Pro-OH), the enzymatic deamidation of His-Pro-NH2 is not catalyzed by the 'thyroliberin-deamidating enzyme; but by a post-proline dipeptidyl aminopeptidase. Hydrolysis of the common intermediary metabolite His-Pro-OH to the free amino acids is apparently catalyzed by a proline dipeptidase. In addition to these enzymatic events rapid cyclization of His-Pro-NH2 to histidyl-proline-diketopiperazine His-Pro could be observed. This reaction however is mainly due to the non-enzymatic intramolecular condensation reaction which is characteristic for proline-containing dipeptide derivatives. An enzymatic activity which catalyzes this reaction could not be observed when the enzyme fractions were tested. Enzymatic degradation of His-Pro by hypophyseal tissue extracts could also not be observed.     

Developmental changes in the distribution of rat brain pyroglutamate aminopeptidase,a possible determinant of endogenous cyclo(His-Pro) concentrations

The distribution of cyclo(His-Pro), thyrotropin-releasing hormone (TRH) and Pyroglutamate aminopeptidase activity in adult and developing rat brains were studied. A comparison of the subcellular distribution of Pyroglutamate aminopeptidase activity in hypothalamic and cerebral cortical extracts from adult rats exhibited remarkable differences. In hypothalamus, the enzyme activity was mainly associated with the soluble fraction whereas in cortex it was predominantly associated with the particulate fractions. During postnatal development, the brain concentrations of cyclo(His-Pro) and Pyroglutamate aminopeptidase activities declined with age. These data suggest that Pyroglutamate aminopeptidase activity, but not TRH, plays an active role in determining the levels of endogenous cyclo(His-Pro) concentrations in brain.     

The purification and characterization of a proline dipeptidase from guinea pig brain

A proline dipeptidase (EC 3.4.13.9) from guinea pig brain was purified to over 90% homogeneity by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, calcium phosphate-cellulose chromatography, chromatofocusing, and gel filtration on Sephadex G-200. A purification factor of 2718-fold was obtained with a yield of 7%. The purified enzyme was found to have an apparent molecular weight of 132,000 and to consist of two dissimilar subunits of molecular weights 64,000 and 68,000. The substrate specificity of the enzyme is not that of a strict proline dipeptidase. Although it preferentially hydrolyzes proline dipeptides (Leu-Pro) it also hydrolyzes prolyl dipeptides (Pro-Leu) and dipeptides not containing proline (Leu-Leu). The purified enzyme preparation exhibited weak aminoacylproline aminopeptidase activity against Arg-Pro-Pro but it did not exhibit any post-proline dipeptidyl aminopeptidase, post-proline cleaving endopeptidase, proline iminopeptidase, prolyl carboxypeptidase or carboxypeptidase P activities when tested with a large variety of peptides and arylamides. With all of the proline and prolyl dipeptides examined the enzyme exhibited biphasic kinetics (two distinct slopes on Lineweaver-Burk plots). However, with Leu-Leu as substrate normal Michaelis-Menten kinetics were obeyed.     

Distribution and characterization of cyclo(His-Pro)-like immunoreactivity in human cerebrospinal fluid

The distribution of cyclo(His-Pro), thyrotropin-releasing hormone and pyroglutamate aminopeptidase activity was examined in the CSF of human and a number of other mammalian species. Cyclo(His-Pro)-like immunoreactivity was present in the CSF of all species examined, and was immunologically and chromatographically identical with the authentic cyclo(His-Pro). Cyclo(His-Pro) concentration in CSF had no significant correlation with CSF TRH or pyroglutamate aminopeptidase.     

The narrow specificity pyroglutamate amino peptidase degrading TRH in rat brain is an ectoenzyme

In order to determine the pathway of extracellular metabolism of the thyrotropin releasing hormone (pyroglu-his-proNH₂) in brain, the topographical organization of pyroglutamate aminopeptidase II on the plasma membrane was investigated. Its activity was only slightly increased when intact brain synaptosomes were lysed by osmotic shock or detergent treatment. Trypsin treatment of intact synaptosomes destroyed 70–80% of enzyme activity without affecting lactate dehydrogenase. Pyroglutamate aminopeptidase II activity was present in primary cultures of foetal mice cortical cells. It was detected in intact cells, was not released by the cells and its activity was not increased by saponin pretreatment. Trypsin treatment of the cells reduced pyroglutamate aminopeptidase II by 70% but did not affect pyroglutamate aminopeptidase I and lactate dehydrogenase. These data support that brain pyroglutamate aminopeptidase II is an ectoenzyme. They suggest that this enzyme could be responsible for thyrotropin releasing hormone extracellular catabolism in brain.     

Purification and properties of guanylate cyclase from the synaptosomal soluble fraction of rat brain.

Guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) was purified 2250-fold from the synaptosomal soluble fraction of rat brain. The specific activity of the purified enzyme reached 41 nmol cyclic GMP formed per min per mg protein at 37 degrees C. In the purified preparation, GTPase activity was not detected and cyclic GMP phosphodiesterase activity was less than 4% of guanylate cyclase activity. The molecular weight was approx. 480 000. Lubrol PX, hydroxylamine, or NaN3 activated the guanylate cyclase in crude preparations, but had no effect on the purified enzyme. In contrast, NaN3 plus catalase, N-methyl-N'-nitro-N-nitrosoguanidine or sodium nitroprusside activated the purified enzyme. The purified enzyme required Mn2+ for its activity; the maximum activity was observed at 3-5 mM. Cyclic GMP activated guanylate cyclase activity 1.4-fold at 2 mM, whereas inorganic pyrophosphate inhibited it by about 50% at 0.2 mM. Guanylyl-(beta,gamma-methylene)-diphosphonate and guanylyl-imidodiphosphate, analogues of GTP, served as substrates of guanylate cyclase in the purified enzyme preparation. NaN3 plus catalase or N-methyl-N'-nitro-N-nitrosoguanidine also remarkably activated guanylate cyclase activity when the analogues of GTP were used as substrates.     

Subcellular distribution and activation by non-ionic detergents of guanylate cyclase in cerebral cortex of rat

Non-ionic detergents stimulated particulate guanylate cyclase activity in cerebral cortex of rat 8- to 12-fold while stimulation of soluble enzyme was 1.3- to 2.5-fold. Among various detergents, Lubrol PX was the most effective one. The subcellular distribution of guanylate cyclase activity was examined with or without 0.5% Lubrol PX. Without Lubrol PX two-thirds of the enzyme activity was detected in the soluble fraction. In the presence of Lubrol PX, however, two-thirds of guanylate cyclase activity was recovered in the crude mitochondrial fraction. Further fractionation revealed that most of the particulate guanylate cyclase activity was associated with synaptosomes. The sedimentation characteristic of the particulate guanylate cyclase activity was very close to those of choline acetyltransferase and acetylcholine esterase activities, two synaptosomal enzymes. When the crude mitochondrial fraction was subfractionated after osmotic shock, most of guanylate cyclase activity as assayed in the absence of Lubrol PX was released into the soluble fraction while the rest of the enzyme activity was tightly bound to synaptic membrane fractions. The total guanylate cyclase activity recovered in the synaptosomal soluble fraction was 6 to 7 times higher than that of the starting material. The specific enzyme activity reached more than 1000 pmol per min per mg protein, which was 35-fold higher than that of the starting material. The membrane bound guanylate cyclase activity was markedly stimulated by Lubrol PX. Guanylate cyclase activity in the synaptosomal soluble fraction, in contrast, was suppressed by the addition of Lubrol PX. The observation that most of guanylate cyclase activity was detected in synaptosomes, some of which was tightly bound to the synaptic membrane fraction upon hypoosmotic treatment, is consistent with the concept that cyclic GMP is involved in neural transmission.     

Thyrotropin-releasing hormone-degrading enzyme in human serum is classified as type II of pyroglutamyl aminopeptidase: influence of thyroid status

The characteristics of thyrotropin-releasing hormone (TRH)-degrading enzyme in human serum were studied. Serum was incubated in 0.1 M phosphate buffer containing [proline-3H]TRH at 37 degrees C. A thin layer chromatography analysis of TRH degradation did not show any radioactive peak located in an acid TRH position, but apparent radioactive peaks corresponding to His-Pro and His-ProNH2 occurred in the presence of p-hydroxymercuriphenyl sulfonic acid, an inhibitor of proline dipeptidase. With ion exchange paper chromatography, the formation of 3H-labeled His-Pro and His-ProNH2 was estimated as an end point in the measurement of pyroglutamyl aminopeptidase (pGlu-peptidase) activity. An assay using p-hydroxymercuriphenyl sulfonic acid was developed to sensitively quantitate the pGlu-peptidase. Neither bacitracin nor p-chloromercuribenzoic acid increased the activity of pGlu-peptidase. The addition of EDTA, dithiothreitol, and o-phenanthroline significantly inhibited pGlu-peptidase activity, but neither iodoacetamide nor ethylmaleimide altered its activity. The pGlu-peptidase had a stereotypic specificity for the tripeptide, pGlu-His-ProNH2 of TRH, and its Km was 44.9 microM. The pGlu-peptidase activity was not changed by either hyper- or hypothyroidism. The present data indicate that a TRH-degrading enzyme in human serum possesses a nature identical to type II of pGlu-peptidase which is not altered by thyroid status.     

Degradation of Melanotropin Inhibiting Factor by Brain

Degradation of melanotropin inhibiting factor (MIF) was measured by fluorometry, using pareptide as an internal standard, following the separation of the dansyl derivatives of MIF and its metabolites by HPLc. MIF was not split by carboxypeptidases A and B, prolidase, or pyroglutamate aminopeptidase. It was hydrolyzed by leucine aminopeptidase, aminopeptidase M, and carboxypeptidase Y. Rat brain hydrolyzed 159 nmol of MIF per mg of protein per h; the activity was linear with enzyme concentration. Hydrolysis start from the N-terminal end, as shown by the appearance of proline as the first metabolite of the MIF degradation, followed by leucine, glycinamide, leucylglycine, and glycine. Activity in the rat brain regions was in the order striatum, medulla oblongata > cortex, hippocampus, midbrain > hypothalamus, cerebellum, and pituitary. The enzyme was mostly in the supernatant, with significant amounts in the myelin and synaptosomal fractions. MIF aminopeptidase could be separated from carboxypeptidase by centrifugation at 30,000 x g for 20 min and precipitation with 45--75% (NH4)2SO4. It showed pH optima in the alkaline range (8.25 and 8.75) and was inhibited by EDTA, EGTA, SQ 14,225, puromycin, bacitracin, and bestatin.     

Neuronal Localization of Pyroglutamate Aminopeptidase II in Primary Cultures of Fetal Mouse Brain

Pyroglutamate aminopeptidase II is a highly specific membrane-bound ectopeptidase proposed to inactivate thyrotropin releasing hormone (TRH) in brain extracellular space. Its activity was measured in primary cell cultures of fetal brain in an attempt to define its cellular localization. Enzyme activity was detected in hypothalamic or cortical cell membrane fractions from 4- to 12-day-old cultures. When proliferation of nonneuronal cells was abolished by cytosine arabinoside treatment, pyroglutamate aminopeptidase II specific activity was increased as compared to untreated cultures, the opposite was observed for pyroglutamate amino-peptidase I activity. Treatment of cortical cells with the neurotoxic agent glutamate reduced simultaneously pyroglutamate aminopeptidase II and glutamate decarboxylase activities. Glial cell cultures expressed pyroglutamate aminopeptidase I or glutamate synthase activities but not pyroglutamate aminopeptidase II. The data suggest that pyroglutamate aminopeptidase II is predominantly localized in neuronal cells. This is consistent with a role for pyroglutamate aminopeptidase II in TRH-ergic synaptic transmission.     

Cyclic nucleotide phosphodiesterase from a particulate fraction of rat brain. Evidence for an activator deficient form.

A cyclic nucleotide phosphodiesterase from a particulate fraction of rat brain was partially purified after solubilization with a non-ionic detergent. Influence of divalent ions, of phosphodiesterase inhibitors and of the activator protein from different sources were tested. Determination of K_m-values shows two enzymes with different values, one at 7.3 μM and the other at 15 mM. Two distinct activity peaks were determined after resolution by isoelectric focusing. It was concluded that this particulate enzyme is regulated in a way opposite to that of the soluble enzyme and is independent from calcium and the activator protein.     

In vitro inhibition of stable 1,3-beta-D-glucan synthase activity from Neurospora crassa.

Glucan synthase activity of Neurospora crassa was isolated by treatment of protoplast lysates with 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4, containing 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 200 mM inorganic phosphate, 10 microM GTP, 1 mM DTT, 10 mM sodium fluoride, and 600 mM glycerol. Resulting activity was partially purified by sucrose gradient density sedimentation. Approximately 70% of enzyme activity in the sucrose gradient peak fraction was soluble and enzyme activity was purified 7.3-fold. Partially purified enzyme activity had a half-life of several weeks at 4 degrees C, and a Km(app) of 1.66 +/- 0.28 mM. Inhibitors (Cilofungin, papulacandin B, aculeacin A, echinocandin B, sorbose and UDP) of 1,3-beta-D-glucan synthase activity were tested against crude particulate and detergent treated enzyme fractions and the Ki(app) of each inhibitor determined. It seems likely that this stable preparation of glucan synthase activity may be useful for in vitro enzyme screens for new glucan synthase inhibitors.     

Neuropeptide-hydrolysing activities in synaptosomal fractions from dog ileum myenteric, deep muscular and submucous plexi. Their participation in neurotensin inactivation

The mapping of neuropeptidases in synaptosomal fractions prepared from dog ileum myenteric, deep muscular and submucous plexus was established by means of fluorigenic substrates and specific inhibitors. Endopeptidase 24.11, angiotensin-converting enzyme and aminopeptidases were found in all tissues, the highest amounts being recovered in the submucous preparation. Post-proline dipeptidyl aminopeptidase was obtained in high quantities whatever the tissue source while proline endopeptidase was detected in low amounts and pyroglutamyl-peptide hydrolase was never detectable. The above peptidases were examined for their putative participation in the inactivation of neurotensin by monitoring the effect of specific inhibitors on the formation of the metabolites of labeled neurotensin separated by HPLC. Endopeptidases 24.11, 24.15 and 24.16 were respectively responsible for the formation of neurotensin(1-11), neurotensin(1-8) and neurotensin(1-10) that are devoid of biological activity. The secondary attacks occurring on neurotensin degradation products were the following: cleavage of neurotensin(1-10) into neurotensin(1-8) by angiotensin-converting enzyme; conversion of neurotensin(9-13) into neurotensin(11-13) by post-proline dipeptidyl aminopeptidase; hydrolysis of neurotensin(11-13) into free tyrosine by aminopeptidase(s).     

Solubilization and characterization of the partially purified penicillin sensitive D-alanine carboxypeptidase of Neisseria gonorrhoeae.

Treatment of crude gonococcal cell envelopes with a solution of 2 M KCl + 1% Brij 36T resulted in the solubilization of a portion of the D-alanine carboxypeptidase activity of Neisseria gonorrhoeae envelopes. This soluble enzyme preparation was partially resolved by chromatography on a column of DEAE-cellulose. The partially purified enzyme eluted from the column with a gradient of NaCl (0-1 M), catalysed the release of D-alanine from a radioactively labelled UDP-N-acetylmuramyl-pentapeptide with a pH optimum of 8.6. The Km for the soluble enzyme acting on this substrate was 0.18 mM. The enzyme activity was sensitive to inhibition by low concentrations of the beta-lactam antibiotics, penicillin G, ampicillin, oxacillin and mecillinam.     

Pyroglutamate aminopeptidase in rat submaxillary gland.

A significant amount of pyroglutamate aminopeptidase (PGAP) activity was found to be present in 27,000 x g supernatant of rat submaxillary gland, maximum activity being at pH 6.5. EDTA stimulated the enzyme activity by 95% at pH 8.0 while at pH 6.5 it did not have any significant effect. On comparison of its properties submaxillary PGAP appears to be different from brain, pituitary and other reported PGAPs. Submaxillary PGAP could also catalyze efficiently the formation of cyclo (His-Pro) from TRH. Cyclo (His-Pro) formation by submaxillary enzyme was more pronounced than that by liver PGAP.     

Purification and characterization of a protein-phosphotyrosine phosphatase from rat spleen which dephosphorylates and inactivates a tyrosine-specific protein kinase

A particulate form of protein-phosphotyrosine phosphatase was solubilized and purified over 2,000-fold from the particulate fraction of rat spleen. Phosphorylated poly(Glu, Tyr), a random copolymer of glutamic acid and tyrosine, was used as substrate for measuring protein-phosphotyrosine phosphatase activity. Nonionic detergents like Triton X-100 increased the protein-phosphotyrosine phosphatase activity of the particulate fraction (but not of the soluble fraction) by 4-8-fold. Chromatography of the Triton extract of the particulate fraction on DEAE-Sephacel gave three peaks of protein-phosphotyrosine phosphatase activity. The major peak of activity was further purified on Bio-Gel HTP, Sephadex G-75, and phosphocellulose columns. On polyacrylamide gel electrophoresis in the presence of Na-dodecyl-SO4 the purified enzyme showed a major protein band of Mr 36,000 which comigrated with enzyme activity on the phosphocellulose column. The apparent Vmax and Km for phosphorylated poly(Glu,Tyr) were 6,150 nmol min-1 mg-1 and 1.6 microM, respectively. This enzyme was strongly inhibited by microM concentrations of orthovanadate and zinc acetate. Fluoride (50 mM) inhibited this enzyme only by 30-40%. Divalent metal ions Ca2+, Mg2+, and Mn2+ were inhibitory at 1-10 mM concentration. EDTA had no effect on the activity of the purified enzyme. This phosphatase could dephosphorylate and inactivate the phosphorylated form of a tyrosine-specific protein kinase (TK-I) previously purified from rat spleen. Dephosphorylation and inactivation of TK-I by purified phosphatase were inhibited by orthovanadate. After dephosphorylation and inactivation by phosphatase, TK-I could be rephosphorylated and reactivated on incubation with ATP. These results suggest that this protein-phosphotyrosine phosphatase may be involved in the regulation of the kinase activity of TK-I.     

Purification and characterization of an enkephalin-degrading dipeptidyl-aminopeptidase from porcine brain

A porcine brain dipeptidyl-aminopeptidase (DAP) has been purified more than 2400-fold from a crude mitochondrial fraction containing synaptosomes. This enzyme catalyzes the release of free Tyr-Gly from Leu-enkephalin (Km = 2.5 microM) with an optimal activity between pH 6.0 and pH 8.0. The enzyme appears homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis devoid of detectable contaminating aminopeptidase activities. The native enzyme is a monomeric protein with a molecular weight of 51,000 +/- 1,000 and an isoelectric point of 4.6 +/- 0.1. This enzyme cosediments with synaptosomes on a Ficoll-sucrose gradient and is partially associated with synaptic plasma membranes. Its activity is inhibited by the metal-chelating agents ethylenediaminetetraacetate and o-phenanthroline. It is not inhibited by the OH-reactive agent phenylmethanesulfonyl fluoride and SH-reactive agents such as p-(chloromercuri)benzoate and N-ethylmaleimide. Among the various biologically active peptides tested, the purified enzyme releases efficiently the N-terminal dipeptide moiety from enkephalins, Trp-Met-Asp-Phe-NH2 (CCK4), and Gly-Trp-Met-Asp-Phe-NH2 (CCK5). At variance, the native peptides CCK8, substance P, neurotensin, and angiotensin II are not cleaved by the DAP. This enzyme is different from other unspecific DAPs, as well as from enkephalin-degrading DAPs previously reported, by its molecular weight and substrate specificity.