Inhibition of enkephalin-degrading enzymes from rat brain and of thermolysin by amino acid hydroxamates

Benzyloxycarbonyl derivatives (Z) of amino acid hydroxamates have been found to inhibit the bacterial metalloendopeptidase thermolysin and enkephalin-degrading enzymes from rat brain. The hydroxamate derivatives of glycine, leucine, phenylalanine and D-phenylalanine inhibit thermolysin with K_I values in the range of 3–23 μM. They also inhibit the enkephalin-degrading endopeptidase (enkephalinase) and aminopeptidase with different efficiencies, depending on the structure of the amino acid employed. Thus, Z-Gly-NHOH inhibits the enkephalinase and aminopeptidase with IC₅₀ values of 1 μM and 300 μM, respectively, whereas Z-D-Phe-NHOH inhibits the corresponding enzymes with IC₅₀ values of 0.2 μM and 1.5 μM.     

Enkephalin-degrading aminopeptidase in the longitudinal muscle layer of guinea pig small intestine: its properties and action on neuropeptides.

A membrane-bound enkephalin-degrading aminopeptidase was purified from the longitudinal muscle layer of the guinea pig small intestine by four steps of column chromatography using L-tyrosine beta-naphthylamide. The molecular weight of the enzyme was estimated to be 105,000 by gel filtration. The maximum activity was observed between pH 6.5 and 7.0. The Km value for leucine-enkephalin was 137 microM. The aminopeptidase activity toward aminoacyl beta-naphthylamide substrates was restricted to basic, neutral, and aromatic aminoacyl derivatives. No action was detected on acidic amino acid and proline derivatives. The enzyme was potently inhibited by the aminopeptidase inhibitors actinonin, amastatin, and bestatin, and bioactive peptides such as angiotensin III, substance P, and Met-Lys-bradykinin. The enzyme activity was also inhibited by the antibody against the purified serum enkephalin-degrading aminopeptidase of guinea pig at concentrations similar to those at which activity was observed toward serum enkephalin-degrading aminopeptidase and renal aminopeptidase M. The enzyme rapidly hydrolyzed Leu-enkephalin and Met-enkephalin with the sequential removal of the N-terminal amino acid residues. The enzyme also hydrolyzed two enkephalin derivatives, angiotensin III and neurokinin A. However, neurotensin, substance P, and bradykinin were not cleaved. These properties indicated that the membrane-bound enkephalin-degrading aminopeptidase in the longitudinal muscle layer of the small intestine is similar to the serum enkephalin-degrading aminopeptidase and resembles aminopeptidase M. It is therefore suggested to play an important role in the metabolism of some bioactive peptides including enkephalin in peripheral nervous systems in vivo.     

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.     

Characterization of Membrane-Bound Aminopeptidases from Rat Brain: Identification of the Enkephalin-Degrading Aminopeptidase

Rat brain aminopeptidase activity was solubilized from membranes by incubation with thiols. This novel procedure resulted in the release of the same two aminopeptidases (MI and MII) previously shown to be solubilized by the nonionic detergent Triton X-100. The solubilized aminopeptidases MI and MII were resolved by ion-exchange chromatography and further purified by hydroxylapatite chromatography. Aminopeptidase MI was shown to hydrolyze only the beta-naphthylamides of arginine and lysine whereas aminopeptidase MII exhibited a broad specificity with respect to amino acid beta-naphthylamides. Only aminopeptidase MII hydrolyzed Leu-enkephalin at a significant rate, indicating that this enzyme can account for the membrane-bound enkephalin aminopeptidase activity. The enkephalin-degrading aminopeptidase is potently inhibited by opioid (alpha-neo-endorphin and dynorphin) as well as nonopioid (substance P, somatostatin, and angiotensin I) peptides in the range of 0.2-2.0 microM. The regional distribution of aminopeptidases MI and MII in rat brain are rather different, with aminopeptidase MII distribution more closely paralleling the distribution of opiate receptors.     

Inhibition of a Membrane-Bound Enkephalin-Degrading Aminopeptidase by Bestatin Analogs

Abstract: A variety of bestatin analogs were examined as potent inhibitors of a membrane-bound enkephalin-degrading aminopeptidase that was purified from monkey brain. Bestatinyl amino acid derivatives showed strong inhibition of this enzyme. The most effective was bestatin- l -Arg AcOH, with a K _i value of 0.21 × 10⁻⁸ M with Leu-enkephalin as substrate. It exhibited competitive kinetics and was about 100-fold more potent than bestatin. This compound seems to be useful for pharmacological and other studies.     

Identification of dipeptidyl peptidase III in human neutrophils

We have found activity of dipeptidyl peptidase (DPP) III, one of the most important enkephalin-degrading enzymes in the central nervous system, in human neutrophils. HPLC analysis of the peptide fragments produced by treatment of leucine-enkephalin with isolated neutrophils in the presence of inhibitors of other enkephalin-degrading enzymes revealed that the enzyme in human neutrophils cleaved dipeptides from the NH(2) terminus of leucine-enkephalin, suggesting the presence of DPPIII activity in human neutrophils. Using a specific synthesized substrate and proteinase inhibitors, it was found that the neutrophils have 19.2 +/- 3.6 microM/h/5 x 10(6) cells of beta-naphthylamine for the enzyme. It was also confirmed that spinorphin and tynorphin, both reported to inhibit the activities of enkephalin-degrading enzymes, had potent inhibitory activities (IC(50): 4.0 and 0.029 microg/ml, respectively) against the enzyme. The presence of DPPIII activity in human neutrophils suggests that the biologically active peptides which are associated with enkephalin play a physiological role in regulating enkephalin or inflammatory mechanisms in peripheral tissues.     

Interaction mechanisms of imipramine and desipramine with enkephalin-degrading aminopeptidases in vitro

In the last few years, considerable evidence has appeared concerning the importance of the opioid systems in the action mechanism of some antidepressant drugs. This action mechanism could be mediated through the inhibition of the enzymes reponsible for enkephalin degradation. In this sense, imipramine treatment in vivo increases the enkephalin levels, and this effect is enhanced by inhibitors of enkephalin-degrading enzymes. The present work shows the effects in vitro of imipramine and its active metabolite desipramine on the activities of two membrane-bound enkephalin-degrading aminopeptidases present in rat brain. Imipramine and desipramine in vitro do not affect the aminopeptidase M activity, but they reversibly inhibits the aminoeptidase MII. The enzyme kinetic analysis shows that this enzyme molecule has two different binding sites for each drug, which exert a mixed type enzyme inhibition.     

Effect of Serine Hydroxamate on Phospholipid Synthesis in Escherichia coli

Serine hydroxamate, which inhibits the charging of seryl-transfer ribonucleic acid, reduced the synthesis of phospholipid and nucleic acids in Escherichia coli. This effect was analogous to depriving amino acid auxotrophs of their nutritional requirement and appears to be a manifestation of the stringent response shown by rel(+) strains of E. coli. Amino acid starvation (serine or methionine) alone or serine hydroxamate treatment alone results in 60 to 80% inhibition of lipid accumulation, 90% inhibition of ribonucleic acid accumulation, and an increase in guanosine tetraphosphate (ppGpp). These three effects were reversed by addition of chloramphenicol (CM). A combination of serine starvation and serine hydroxamate treatment resulted in inhibition of lipid and RNA accumulation as well as an increase in ppGpp, but the consequences of the double block were not reversed by CM. We conclude that a strong interrelationship exists among these processes and that CM acts to relax a stringent response by mechanisms other than interference with ppGpp formation. All species of phospholipid were affected by a stringent response evoked by amino acid starvation or addition of serine hydroxamate, but in all cases the synthesis of phosphatidylethanolamine was most severely inhibited. Serine hydroxamate was not incorporated into lipid but specifically caused phosphatidylserine accumulation. Serine starvation produced a dramatic alteration of the distribution of isotope incorporated into phospholipid, which resulted from the stringent response compounded with the limitation of a substrate for phosphatidylserine synthesis.     

Photolabile ligands for opiate receptors

The 2-nitro-4-azidophenyl(NAP)-D-Ala2-Leu5-Enkephalin derivatives: Try-D-Ala-Gly-Phe-Leu CONCH2CH2NH-NAP (E-NAP-EDA) and Try-D-Ala-Gly-Phe-Leu CONCH2CH2NH-COCH2CH2NHNAP(E-NAP- -Ala-EDA) were synthesized by conventional peptide methods. Their structure was determined by amino acid analysis, ultra violet, visible and infra red spectroscopy. Both peptides were shown a) to bind with high affinity to the opiate receptors of rat brain membranes and b) to inhibit strongly the contractions of electrically stimulated vas deferens and the adenyl cyclase of the NG 108-15 cell membranes. These effects were reversed by the antagonist naloxone. Photoloysis of the rat brain membranes-(E-NAP- -Ala-EDA) complex caused a 20-30% inactivation of the opiate receptors. Inactivation was prevented when the complex was irradiated in the presence of naloxone. The radio-labeled derivatives of these enkephalin analogs may prove useful photochemical labels of the opiate receptor.     

Effects of amino acid analogues on protein degradation in isolated rat hepatocytes

Analogues and derivatives of six of the amino acids which most effectively inhibit protein degradation in isolated rat hepatocytes (leucine, asparagine, glutamine, histidine, phenylalanine and tryptophan) were investigated to see if they could antagonize or mimic the effect of the parent compound. No antagonists were found. Amino alcohols and amino acid amides tended to inhibit protein degradation strongly, apparently by a direct lysosomotropic effect as indicated by their ability to cause lysosomal vacuolation. Amino acid alkyl esters and dipeptides inhibited degradation to approximately the same extent as did their parent amino acids, possibly by being converted to free amino acids intracellularly. Of several leucine analogues tested, four (L-norleucine, L-norvaline, D-norleucine and L-allo-isoleucine) were found to be as effective as leucine in inhibiting protein degradation. None of the analogues had any effect on protein synthesis. Since leucine appears to play a unique role as a regulator of bulk autophagy in hepatocytes, the availability of active leucine agonists may help tj elucidate the biochemical mechanism for control of this important process.     

Mass Fragmentographic Method for the Determination of Trace Amounts of Putative Amino Acid Neurotransmitters and Related Compounds from Brain Perfusates Collected In Vivo

Abstract: A mass fragmentographic method for the determination of trace amounts of amino acid neurotransmitter candidates from brain perfusates is described. The analytical procedure includes the measurements of glycine, β-alanine, γ-aminobutyric acid, proline, aspartic acid, and glutamic acid; αalanine, leucine, and sarcosine, undergoing gas chromatographic coelution, are detected simultaneously. Amino acids extracted from dried perfusate residues are converted to the corresponding N -pentafluoropropionyl hexafluoroisopropyl esters by a single-step procedure. Gas chromatographic separation of the amino acid derivatives is achieved on a packed glass column filled with trifluoropropylsilicone as stationary phase. The limit of detection for the different derivatives (signal-to-noise, 3:1) ranges from 50 femtomol to 1 picomol. Deuterium-labeled amino acid analogues are used as internal standards for quantitative measurements. The mass spectral characteristics of the derivatives are compared and discussed. The technique has been applied to the assay of amino acids released in vivo within the pigeon optic tectum, demonstrating the capabilities of the present analytical approach.     

Interaction of enkephalins and des-tyrosyl-enkephalins with synaptosomal plasma membrane vesicles: enkephalin binding and inhibition of proline transport

Leucine- and methionine-enkephalins inhibit the Na+-dependent transport of proline into plasma membrane vesicles derived from synaptosomes. Glycine transport is weakly inhibited by enkephalins whereas there is no inhibition of transport of glutamic acid, aspartic acid, or gamma-aminobutyric acid. The inhibition of proline uptake is observed with des-tyrosyl-enkephalins but not with morphine, dynorphin(1-13), or beta-endorphins. Furthermore, enkephalin-induced inhibition of proline transport is not antagonized by naloxone. [Leu]enkephalinamide and modified [Leu]enkephalins with greater selectivity for the delta-subclass of enkephalin binding sites are less effective than [Leu]enkephalin in the inhibition of proline transport. Specific binding of [3H]Leu-enkephalin to the plasma membrane vesicles is demonstrated, and des-Tyr-[Leu]enkephalin competes with Leu-enkephalin for [Leu]enkephalin binding sites. The similarity in the concentrations of des-Tyr-[Leu]enkephalin required to compete for specific [Leu]enkephalin binding and to inhibit proline transport suggests that a specific subclass of enkephalin binding sites, distinguished by their recognition of both the enkephalins and their des-tyrosyl derivatives, may be associated with the synaptic proline transport system.     

Enkephalin-degrading enzyme inhibitors. Crucial role of the C-terminal residue on the inhibitory potencies of retro-hydroxamate dipeptides

The retro-inversion of the amide bond in kelatorphan and analogs, the first series of complete inhibitors of enkephalin metabolism, led to compounds highly efficient only against the neutral endopeptidase 24-11 (NEP). In order to increase the recognition of the aminopeptidase N (APN) and dipeptidylaminopeptidase (DAP), without loss of affinity for NEP, the malonyl group of these retro-inhibitors was replaced by diversely substituted succinyl moieties. All the molecules synthesized are highly efficient NEP inhibitors with Ki's in the 0.2-1 nM range, indicating that NEP possesses a relatively large and not very selective S'2 subsite. In contrast, inhibition of DAP activity is crucially dependent on the size and the position of the substituent in the succinyl moiety. Inhibitory potencies in the nanomolar range are obtained with compounds containing a benzyl group in the alpha-position related to the retro amide bond. Finally, a relatively modest inhibition of APN was observed with Ki's in the 0.5-1 microM range for compounds with benzyl or cyclohexyl group in P'2 position. However, these data demonstrate that efficient and complete inhibition of enkephalin degradation can be obtained with hydroxamate dipeptides containing a retro amide bond. The analgesic potency of the most active inhibitors was measured using the hot plate test in mice. Significant antinociceptive responses were obtained but these effects were rather weaker than those expected from the in vitro inhibitory potencies of these compounds on the three enkephalin-degrading enzymes.     

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.     

Fluorogenic substrates for the enkephalin-degrading neutral endopeptidase (Enkephalinase)

Rat brain neutral endopeptidase ("Enkephalinase") was shown to hydrolyze a series of fluorogenic substrates of the general structure 2-aminobenzoyl-(amino acid)n- leucylalanylglycine -4- nitrobenzylamide . The hydrolysis of these substrates was competitively inhibited by Leu5-enkephalin, demonstrating that these are indeed substrates for the rat brain neutral endopeptidase. Cleavage of the fluorogenic substrates yielded leucylalanylglycine -4- nitrobenzylamide as a common product. In addition, a series of inhibitors previously shown to inhibit thermolysin-like enzymes inhibited the hydrolysis of both Leu5-enkephalin and the synthetic substrates. The results of this study (a) demonstrate that the enkephalin-degrading endopeptidase is similar in specificity to thermolysin, (b) provide a continuous sensitive assay system for the enzyme, and (c) point out the potential use of this substrate class for probing the specificity of the enzyme.     

Heterogeneous distribution of enkephalin-degrading peptidases between neuronal and glial cells

Cultured neurones, astroblasts and astrocytes from murine brain have been screened with specific tests for the presence of peptidases capable of degrading enkephalin. Bestatin-sensitive aminopeptidases represent the major enkephalin-degrading activity in all cases. The dipeptidylaminopeptidasic activity is much higher in the neuronal than the glial cultures, whereas the opposite is true for the metallopeptidase called "enkephalinase". Only trace amounts of the dipeptidylcarboxypeptidase "angiotensin-converting enzyme" have been found. We conclude that bestatin-sensitive aminopeptidases on nerve cells are probable candidates for enkephalin-inactivating enzymes, whereas the "enkephalinase" on glial cells more likely serves a scavenger function.     

Biochemical bases for the antimetabolite action of L-serine hydroxamate

The amino acid analogue l-serine hydroxamate, which is bacteriostatic for Escherichia coli, has been shown to inhibit protein synthesis. The antimetabolite is a competitive inhibitor of seryl-transfer ribonucleic acid (tRNA) synthetase with a K(i) value of 30 mum. Mutants resistant to l-serine hydroxamate have been selected, and three were shown to have seryl-tRNA synthetases with increased K(i) values. One mutant contains a 3-phosphoglycerate dehydrogenase which is insensitive to inhibition by l-serine.     

Isolation of human met-enkephalin and two groups of putative precursors (2K-pro-met-enkephalin) from an adrenal medullary tumour

Human met-enkephalin from an adrenal medullary tumour has been purified and characterised. Two groups of putative human met-enkephalin precursors (MW ～ 2000) were also isolated and were only opiate active after trypsinisation and yielded met-enkephalin on further treatment with carboxypeptidase B. Amino acid analyses of these peptides show they are not related to β-LPH nor any other reported putative enkephalin precursors. Their amino acid compositions suggest they are structurally closely related and may represent two groups of heterologous peptides.     

Purification and characterization of enkephalin convertase, an enkephalin-synthesizing carboxypeptidase

Enkephalin convertase, an enkephalin-synthesizing carboxypeptidase present in adrenal medulla chromaffin granules, has also been detected in brain and pituitary. To determine whether these three carboxypeptidase activities represent the same enzyme, we purified and characterized enkephalin convertase from adrenal medulla, whole brain, and whole pituitary. Enzyme from all three tissues co-purifies on DEAE-cellulose, gel filtration, concanavalin A, and L-arginine affinity columns, resulting in a 135,000-fold, 110,000-fold, and 2,800-fold purification for bovine adrenal medulla, brain, and pituitary, respectively. Purified enkephalin convertase appears homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showing a single band with an apparent molecular weight of 50,000 for enzyme isolated from all three tissues. Adrenal, brain, and pituitary enkephalin convertase are similarly inhibited by hexapeptide enkephalin precursors and active site-directed inhibitors. Both [Met]-and [Leu]enkephalin-Arg6 inhibit enkephalin convertase with Ki values between 50 and 80 microM, while [Met]-and [Leu]enkephalin-Lys6 are 3-fold less potent. Two active site-directed inhibitors, guanidinopropylsuccinic acid and guanidinoethylmercaptosuccinic acid, are potent inhibitors of all three enzymes with Ki values of 8-9 nM. A series of dansylated di-, tri-, and tetrapeptide substrates are hydrolyzed by enkephalin convertase with similar kinetic properties (Km, Vmax, and Kcat/Km) for the three enzymes. This evidence suggests that enkephalin convertase activity represents the same enzyme in adrenal medulla, brain, and pituitary. Enkephalin convertase may be involved in the production of other peptide neurotransmitters and hormones besides enkephalin.     

Inhibitors of the enkephalin degrading enzymes. Modulation of activity of hydroxamate containing compounds by modifications of the C-terminal residue

To further characterize the S'2 subsite of both the neutral endopeptidase (EC 3.4.24.11, NEP) and aminopeptidase N (EC 3.4.11.2, APN), two enzymes physiologically involved in enkephalin metabolism, a new series of hydroxamate inhibitors containing a cyclic amino acid as the P'2 component were synthesized. These amino acids differ by the size of the cycle, the relative position of the functional groups, and their absolute configuration. Highly efficient inhibitors of NEP were obtained whatever the modification on the P'2 component, while for APN inhibition, a cyclic beta-amino acid was preferred. The most active inhibitors contained a trans cyclopentyl beta-amino acid and a cis or a trans cyclohexyl beta-amino acid. When injected intracerebroventricularly in mice, these two latter compounds elicited potent antinociceptive responses on both the jump latency and the fore paw lick times.