Characterization of a distinct membrane bound dipeptidyl carboxypeptidase inactivating enkephalin in brain

A dipeptidyl carboxypeptidase distinct from the angiotensin converting enzyme (EC 3.4.15.1) was isolated from membrane preparations of rabbit brain. The enzyme cleaved enkephalin at the Gly-Phe bond, releasing either Phe-Leu from Leu-enkephalin or Phe-Met from Met-enkephalin, and also acted on bradykinin, releasing the terminal dipeptide Phe-Arg. In contrast to the converting enzyme, however, this dipeptidyl carboxypeptidase did not act on angiotensin-1, and it did not degrade hippuryl-His-Leu. Chloride ions did not affect its activity, but the enzyme was inhibited by metal chelating agents. The enzyme was not inhibited by captopril (SQ 14225) or by SQ 20881. Kinetic studies indicated a Km for this enzyme of 0.14 mM with Leu-enkephalin and 0.12 mM with bradykinin as substrates. Present data indicate that more than one enzyme is present in brain membrane fractions acting as dipeptidyl carboxypeptidases inactivating enkephalin; these data suggest multiple roles for such enzymes in the regulation of peptide metabolism.     

Liberation of enkephalins from enkephalin-containing peptides by brain endo-oligopeptidase A

Endo-oligopeptidase A, highly purified from the cytosol fraction of bovine brain by immunoaffinity chromatography, has been characterised as a thiol endopeptidase. This enzyme, known to hydrolyse the Phe5-Ser6 bond of bradykinin and the Arg8-Arg9 bond of neurotensin has been shown to produce, by a single cleavage, [Leu]enkephalin or [Met]enkephalin from small enkephalin-containing peptides. Enkephalin formation could be inhibited in a concentration dependent manner by the alternative substrate bradykinin. The optimal substrate size was found to be 8-13 amino acids, with enkephalin the only product released from precursors in which this sequence is immediately followed by a pair of basic residues. However, the specificity constants (kcat/Km) obtained for endo-oligopeptidase A hydrolysis of bradykinin, neurotensin and dynorphin B are of the same order. Taken together, these results indicate that the substrate amino acid sequence is not the only factor determining the cleavage site of this enzyme. Finally, endo-oligopeptidase A and metalloendopeptidase EC 3.4.24.15 are two different enzymes. The latter is not able to liberate enkephalins from metorphamide and dynorphin.     

Enkephalin degrading enzymes are present in the electric organ of Torpedo californica

Two proteolytic activities that degrade [Leu5]enkephalin were found in Torpedo californica electric organ. One is a soluble aminopeptidase that degrades enkephalin at the Tyr1-Gly2 peptide bond, and the second is an endopeptidase that degrades enkephalin at the Gly3-Phe4 peptide bond. The aminopeptidase is inhibited by low concentrations of puromycin and bestatin. More than 60% of the endopeptidase is associated with the particulate fraction and is almost completely inhibited by low concentrations of captopril (SQ 14225) or SQ 20881 (potent inhibitors of angiotensin converting enzyme). Thiorphan and phosphoramidon (potent enkephalinase inhibitors) are much less effective. The pattern of cleavage and inhibition of the particulate endopeptidase thus resembles that of angiotensin converting enzyme.     

Rat brain and kidney metalloendopeptidase: Enkephalin heptapeptide conversion to form a cardioactive neuropeptide,Phe-Met-Arg-Phe-Amide

Rat brain or kidney metalloendopeptidase purified from particulates cleaved Met-enkephalin-Arg⁶-Phe⁷ and its amide at the Gly³-Phe⁴ bond to release Phe-Met-Arg-Phe or the tetrapeptide amide. The latter, a neuropeptide with cardioactive properties, was relatively stable upon further incubation. The metallo-nature of the enzyme was established by inhibition with chelating agents (EDTA, o-phenanthroline) and its endopeptidase nature by cleavage at the Gly³-Phe⁴ bond of pentapeptide enkephalins or precursors such as the heptapeptide, or analogs bearing N- or C-terminal protective groups. Presence of C-terminal amides decreased the rate of hydrolysis. Thiorphan, (DL-3-mercapto-2-benzylpropanoyl)-glycine, competitively inhibited cleavage at the Gly³-Phe⁴ bond of enkephalin (Ki 10 nM). The thiorphan sensitive metalloendopeptidase provides a pathway for conversion of an enkephalin precursor to form a non-opioid peptide of biological interest.     

Inhibitors of a Rat Brain Enkephalin Aminopeptidase

Abstract: Eight protease inhibitors of microbiological origin were examined as potential inhibitors of a homogeneous rat brain enkephalin aminopeptidase. Bestatin [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]- l -leucine and analogs of bestatin having basic, acidic, and other neutral amino acids substituted for the Leu residue exhibited inhibition constants ranging from 3.3 ± 10⁻⁵ to 8.3 ± 10⁻⁸ m . The best inhibitor had a positively charged amino acid (Lys) substituted for Leu. A series of phenylalanyl dipeptides were examined as substrates with the aminopeptidase. The amino acid residue on the carboxyl side of the peptide bond undergoing cleavage was varied systematically in the dipeptides to include neutral, acidic, and basic residues. Again, a positively charged amino acid (Arg) adjacent to the bond undergoing scission was kinetically preferred. These results may be used to design highly specific inhibitors of the enkephalin aminopeptidase.     

Purification and characterization of enkephalinase B from rat brain membrane

Enkephalinase B from rat brain membrane which hydrolyzes enkephalin at the Gly-Gly bond was purified about 9400-fold to apparent electrophoretic homogeneity. The enzyme, which has a molecular weight of 82,000, consists of a single polypeptide chain. The enzyme has a pH optimum of 6.0-6.5 and is stable in the neutral pH region. The Km values of Met-enkephalin and Leu-enkephalin for this enzyme were 5.3 X 10(-5) M and 5.0 X 10(-5) M, respectively. The enzyme was inactivated by metal chelators, EDTA and o-phenanthroline and restored by the addition of divalent metal ions, Zn2+, Mn2+ or Fe2+, but was not inhibited by bestatin, amastatin, phosphoramidon or captopril. The enzyme hydrolyzed Met-enkephalin and Leu-enkephalin effectively. Although the enzyme belongs to the dipeptidyl aminopeptidase class, enkephalin-related peptides such as Leu-enkephalin-Arg, dynorphin (1-13) or alpha-endorphin and other biologically active peptides examined were hardly, or not at all, hydrolyzed. It was assumed that enkephalinase B functions mainly in enkephalin degradation in vivo.     

Antinociceptive effect of [Met5]enkephalin semicarbazide is not affected by dipeptidyl carboxypeptidase‐I

Dipeptidyl carboxypeptidase‐I is an enzyme involved in the biological degradation of enkephalins. It has been suggested that C‐terminal amidation of enkephalins enhances their resistance to dipeptidyl carboxypeptidase‐I‐mediated biodegradation. In this study, a novel [Met5]enkephalin amide (MEA) analogue [Met5]enkephalin (ME)‐semicarbazide synthesized by another laboratory in our group was assessed for its antinociceptive effects compared with ME‐ethylamide, MEA and ME, using tail flick test. To protect the administered drugs from biodegradation, rats were pretreated with peptidase inhibitors including amastatin, phosphoramidon and captopril. Then captopril (dipeptidyl carboxypeptidase‐I inhibitor) was deleted from the peptidase inhibitors' combination for evaluating in vivo resistance of the synthetic drugs to dipeptidyl carboxypeptidase‐I. According to the results, ME‐semicarbazide and MEA were resistant enough to dipeptidyl carboxypeptidase‐I to exert their strong antinociception following intrathecal administration even in the absence of captopril, whereas the antinociceptive effects produced by ME‐ethylamide (10 nmol) were abolished in rats not pretreated with captopril, indicating that significant amounts of the ME‐ethylamide were degraded by dipeptidyl carboxypeptidase‐I. Replacement of the amide moiety of MEA with semicarbazide provides a new ME derivative, with high analgesic effects as well as more resistance to dipeptidyl carboxypeptidase‐I‐mediated biodegradation. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Hydrolysis of substance P and its analogs by angiotensin-converting enzyme from rat lung. Characterization of endopeptidase activity of the enzyme

Hydrolysis of substance P and nine kinds of substance P analogs by angiotensin-converting enzyme highly purified from rat lung was examined by using amino-group fluorometry and high-performance liquid chromatography. The enzyme hydrolyzed substance P and several analogs, notwithstanding that they did not contain free C-terminal residues. The analyses of cleavage products separated by high-performance liquid chromatography indicated that the enzyme hydrolyzed substance P and its analogs mainly at the bond between Phe8-Gly9 and also at another bond, possibly between Gly9-Leu10, to a lesser extent by an endopeptidase action, followed by successive release of dipeptides by a dipeptidyl carboxypeptidase action. The analogs that had D-amino acid residues substituted at the presumed cleavage sites were scarcely hydrolyzed. It was further found that (Pyr6)-fragment (6-11) was hydrolyzed by the enzyme more efficiently than the other fragment-type analogs and was cleaved at a single bond by the endopeptidase activity of the enzyme. Therefore, this fragment was used as a substrate in order to characterized the endopeptidase activity of the enzyme by employing fluorometry. The activity was dependent on chloride ion, and was inhibited by captopril, MK-421, and EDTA. Thus, the endopeptidase activity of the enzyme showed properties similar to those of the dipeptidyl carboxypeptidase activity of the enzyme.     

Enkephalin degrading enzymes in cerebrospinal fluid

Enkephalins were rapidly degraded by specific enzyme systems in vivo. In cerebrospinal fluid (CSF), however, it has been undefined whether these enzyme systems existed. Our experiments showed enkephalins were hydrolyzed by the enzymatic activity in both CSF of human and monkey. The results by the thin layer chromatography and the high performance liquid chromatography revealed the reaction products of CSF and enkephalin were tyrosine, tyrosyl-glycine and tyrosyl-glycyl-glycine. Therefore, the enzymes in CSF were considered to be an aminopeptidase, a dipeptidyl aminopeptidase and a dipeptidyl carboxypeptidase. Our results suggest that in the assay of enkephalin in CSF, the effects of these enzymes should be considered.     

Hydrolysis of [Leu]enkephalin by chick brain in vitro.

Using high-performance liquid chromatography with electrochemical detection to measure substrate disappearance and metabolite accumulation following addition of [Leu]enkephalin to samples prepared from chick brain in vitro, the following were found: 1. [Leu]enkephalin hydrolysis by whole forebrain homogenates is almost solely attributable to aminopeptidase MII activity. 2. [Leu]enkephalin hydrolysis by whole forebrain P2 membrane fractions is attributable to both aminopeptidase MII and dipeptidyl carboxypeptidase activity. 3. Differences are apparent in both [Leu]enkephalin disappearance and Tyr-Gly-Gly accumulation in P2 membrane fractions, but not in homogenate fractions, prepared from several regions of the chick brain.     

Brain Endo-Oligopeptidase A, a Putative Enkephalin Converting Enzyme

Endo-oligopeptidase A, highly purified from the cytosol fraction of bovine brain by immunoaffinity chromatography, has been characterized as a thiol endopeptidase. This enzyme, known to hydrolyze the Phe5-Ser6 bond of bradykinin and the Arg8-Arg9 bond of neurotensin, has been shown to produce, by a single cleavage, Leu5-enkephalin or Met5-enkephalin from small enkephalin-containing peptides. Enkephalin formation could be inhibited in a concentration-dependent manner by the alternative substrate bradykinin. The optimal substrate size was found to be eight to 13 amino acids, with enkephalin the only product released from precursors in which this sequence is immediately followed by a pair of basic residues. However, the specificity constants (kcat/Km) obtained for endo-oligopeptidase A hydrolysis of bradykinin, neurotensin, and dynorphin B are of the same order, a result indicating that the substrate amino acid sequence is not the only factor determining the cleavage site of this enzyme.     

Degradation of Neurotensin by Rat Brain Synaptic Membranes: Involvement of a Thermolysin-Like Metalloendopeptidase (Enkephalinase), Angiotensin-Converting Enzyme, and Other Unidentified Peptidases

Neurotensin was inactivated by membrane-bound and soluble degrading activities present in purified preparations of rat brain synaptic membranes. Degradation products were identified by HPLC and amino acid analysis. The major points of cleavage of neurotensin were the Arg8-Arg9, Pro10-Tyr11, and Tyr11-Ile12 peptide bonds with the membrane-bound activity and the Arg8-Arg9 and Pro10-Tyr11 bonds with the soluble activity. Several lines of evidence indicated that the cleavage of the Arg8-Arg9 bond by the membrane-bound activity resulted mainly from the conversion of neurotensin1-10 to neurotensin1-8 by a dipeptidyl carboxypeptidase. In particular, captopril inhibited this cleavage with an IC50 (5.7 nM) close to its K1 (7 nM) for angiotensin-converting enzyme. Thiorphan inhibited the cleavage at the Tyr11-Ile12 bond by the membrane-bound activity with an IC50 (17 nM) similar to its K1 (4.7 nM) for enkephalinase. Both cleavages were inhibited by 1,10-phenanthroline. These and other data suggested that angiotensin-converting enzyme and a thermolysin-like metalloendopeptidase (enkephalinase) were the membrane-bound peptidases responsible for cleavages at the Arg8-Arg9 and Tyr11-Ile12 bonds, respectively. In contrast, captopril had no effect on the cleavage at the Arg8-Arg9 bond by the soluble activity, indicating that the enzyme responsible for this cleavage was different from angiotensin-converting enzyme. The cleavage at the Pro10-Tyr11 bond by both the membrane-bound and the soluble activities appeared to be catalyzed by an endopeptidase different from known brain proline endopeptidases. The possibility is discussed that the enzymes described here participate in physiological mechanisms of neurotensin inactivation at the synaptic level.     

Enkephalin hydrolysis in homogenates of various absorptive mucosae of the albino rabbit: similarities in rates and involvement of aminopeptidases

The systemic delivery of peptides and proteins from the nasal, rectal, vaginal, and buccal mucosae has been the subject of active investigation. The objective of this study was to determine the pathway and rate of hydrolysis of methionine enkephalin (TGGPM), leucine enkephalin (TGGPL), and [D-Ala2] met-enkephalinamide (TAGPM) in homogenates of these non-oral mucosae relative to the ileal mucosa. Aminopeptidases appeared to contribute over 85% to the hydrolysis of TGGPM and TGGPL, while dipeptidyl peptidase and dipeptidyl carboxypeptidase contributed much less. Overall, TGGPM was somewhat more susceptible to hydrolysis than TGGPL but was 10 times more so than TAGPM. These enkephalins were most rapidly hydrolyzed in the rectal and buccal homogenates, followed by the nasal and then the vaginal homogenates, but the differences in hydrolytic rates were small. Indeed, these rates did not differ substantially from the ileal mucosa, suggesting that the same enzymatic barrier to enkephalin absorption possibly exists in both the oral and the non-oral mucosae.     

Angiotensin III: A Potent Inhibitor of Enkephalin-Degrading Enzymes and an Analgesic Agent

Various angiotensins, bradykinins, and related peptides were examined for their inhibitory activity against several enkephalin-degrading enzymes, including an aminopeptidase and a dipeptidyl aminopeptidase, purified from a membrane-bound fraction of monkey brain, and an endopeptidase, purified from the rabbit kidney membrane fraction. Angiotensin derivatives having a basic or neutral amino acid at the N-terminus showed strong inhibition of the aminopeptidase. Dipeptidyl aminopeptidase was inhibited by angiotensins II and III and their derivatives, whereas the endopeptidase was inhibited by angiotensin I and its derivatives. The most potent inhibitor of aminopeptidase and dipeptidyl aminopeptidase was angiotensin III, which completely inhibited the degradation of enkephalin by enzymes in monkey brain or human CSF. The Ki values for angiotensin III against aminopeptidase, dipeptidyl aminopeptidase, endopeptidase, and angiotensin-converting enzyme, which degraded enkephalin, were 0.66 X 10(-6), 1.03 X 10(-6), 2.3 X 10(-4), and 1.65 X 10(-6) M, respectively. Angiotensin III potentiated the analgesic activity of Met-enkephalin after intracerebroventricular coadministration to mice in the hot plate test. Angiotensin III itself also displayed analgesic activity in that test. These actions were blocked by the specific opiate antagonist naloxone.     

Endooligopeptidase A activity in rabbit heart: Generation of enkephalin from enkephalin containing peptides

Two endopeptidases displaying similar specificities towards peptide hormone substrates but differing in molecular size have been identified in rabbit heart and isolated by a combination of ion-exchange chromatography, gel filtration and preparative gel electrophoresis. These two enzymes share several properties with the previously described rabbit brain endooligopeptidase A. They were shown to produce, by a single peptide bond cleavage, [Met⁵] enkephalin and [Leu⁵]enkephalin from small enkephalin containing peptides. They also hydrolyze the Phe⁵-Ser⁵ bond of bradykinin and the Arg⁸-Arg⁹ bond of neurotensin. Characteristically, the activity of both low and high Mr enzymes is restricted to oligopeptides. Both forms of heart endooligopeptidase A are inhibited by antibodies raised against the brain enzyme. When electrophoresed in SDS-polyacrylamide gel under denaturing conditions, the low Mr heart enzyme shows a major band of Mr=73,000, comparable in size to the brain enzyme. The SDS-PAGE of the high and low Mr enzymes analyzed by immunoblotting with an antibody raised against low Mr brain endooligopeptidase A, showed a major antigen band corresponding to Mr=72,000. In addition, immunoblotting has also demonstrated that a monoclonal antibody antitubulin reacts with a polypeptide corresponding to Mr=50,000 present in the purified high Mr endooligopeptidase A. Both enzymes are activated by dithiothreitol and inhibited by thiol reagents, but are not affected by leupeptin, DFP or EDTA, thus indicating that they should be classified as nonlysosomal cysteinyl-endooligopeptidase A.     

Peptidases that terminate the action of enkephalins. Consideration of physiological importance for amino-, carboxy-, endo-, and pseudoenkephalinase

The term "enkephalinase" has been frequently applied to enzyme activity in a variety of tissue preparations. In some cases there has been the implication that cleavage of a specific peptide bond in the enkephalin molecule results from the action of a single enzyme with the major responsibility of inactivating synaptic enkephalin. It is not known to what extent diverse enkephalin-degrading enzymes, with differing peptide bond specificities, may act in concert at any given synapse. There do exist, however, enzymes having known characteristic specificities with respect both to peptide substrates, including enkephalins, and to identifiable peptide bonds. Thus, at any given site of enkephalin release there probably resides a characteristic assembly of peptidases concerned with inactivation of this neuromediator. We propose that the term "enkephalinase" be used to encompass the entire family of enkephalin-degrading enzymes, and that "aminoenkephalinase", "carboxyenkephalinase", "endoenkephalinase" and "pseudoenkephalinase" should designate enzymes of known specificities with respect to both peptide substrates and particular peptide bonds.     

Proline-specific dipeptidyl peptidase from the blue blowfly Calliphora vicina hydrolyzes in vitro the ecdysiostatic peptide trypsin-modulating oostatic factor (Neb-TMOF)

To elucidate the mechanisms of inactivation of the ecdysiostatic peptide trypsin-modulating oostatic factor (Neb-TMOF) in the blue blowfly Calliphora vicina, we investigated its proteolytic degradation. In homogenates and membrane and soluble fractions, this hexapeptide (sequence: NPTNLH) was hydrolyzed into two fragments, NP and TNLH, suggesting the involvement of a proline-specific dipeptidyl peptidase. The dipeptidyl peptidase activity was highest in the late larval stage. It was purified 240-fold from soluble fractions of pupae of mixed age and classified on the basis of several catalytic properties as an invertebrate homologue of mammalian dipeptidyl peptidase IV (EC 3.4.14.5). Fly dipeptidyl peptidase IV has a molecular mass of 200 kDa, showed a pH optimum of 7.5–8.0 with the chromogenic substrate Gly-Pro-4-nitroanilide, and cleaved other chromogenic substrates with penultimate Pro or, with lower activity, Ala. It liberated Xaa-Pro dipeptides from the N-terminus of several bioactive peptides including substance P, neuropeptide Y, and peptide YY but not from bradykinin, indicating that the peptide bond between the two proline residues was resistant to cleavage. Fly dipeptidyl peptidase belongs to the serine class of proteases as the mammalian enzyme does; the fly enzyme, however, is not inhibited by several selective or nonselective inhibitors of its mammalian counterpart. It is suggested that dipeptidyl peptidases exert a regulatory role for the clearance not only of TMOF in flies but for other bioactive peptides in various invertebrates. Arch. Insect Biochem. Physiol. 37:146–157, 1998. © 1998 Wiley-Liss, Inc.     

Conformational selection and collagenolysis in Type III collagen

Matrix metalloproteases (MMPs) cleave native collagen at a single site despite the fact that collagen contains more than one scissile bond that can, in principle, be cleaved. For peptide bond hydrolysis to occur at one specific site, MMPs must (1) localize to a region near the unique scissile bond, (2) bind residues at the catalytic site that form the scissile bond, and (3) hydrolyze the corresponding peptide bond. Prior studies suggest that for some types of collagen, binding of noncatalytic MMP domains to amino acid sequences in the vicinity of the true cleavage site facilitates the localization of collagenases. In the present study, our goal was to determine whether binding to the catalytic site also plays a role in determining MMP specificity. To investigate this, we computed the conformational free energy landscape of Type III collagen at each potential cleavage site. The free energy profiles suggest that although all potential cleavage sites sample unfolded states at relatively low temperatures, the true cleavage site samples structures that are complementary to the catalytic site. By contrast, potential cleavage sites that are not cleaved sample states that are relatively incompatible with the MMP active site. Furthermore, our findings point to a specific role for arginine residues in modulating the structural stability of collagen near the collagenase cleavage site. These data imply that locally unfolded potential cleavage sites in Type III collagen sample distinct unfolded ensembles, and that the region about the true collagenase cleavage site samples states that are most complementary to the MMP active site. Proteins 2010. © 2009 Wiley‐Liss, Inc.