Inhibitory potency of various peptides on enkephalinase activity from mouse striatum

A variety of peptides chemically unrelated to enkephalins are relatively good inhibitors (IC₅₀ in the micromoLar range) of “enkephalinase” activity i.e. of the peptidase releasing Tyr-Gly-Gly from Leu-enkephalin. Its specificity has been also reinvestigated with a series of Met-enkephalin analogues. The poor recognition of several analogues by this inactivating enzyme might account for their enhanced biological activity.     

Differential recognition of "enkephalinase" and angiotensin-converting enzyme by new carboxyalkyl inhibitors

New carboxylalkyl compounds derived from Phe-Leu and corresponding to the general formula C6H5-CH2-CH(R)CO-L.Leu with R = -COOH, 3, R = -CH2-COOH, 4, R = -NH-CH2-COOH, 5, R = -NH-(CH2)2-COOH, 6, have been found to inhibit the breakdown of the Gly3-Phe4 bond of [3H] Leu-enkephalin or [3H]D.Ala2-Leu-enkephalin resulting from the action of the mouse striatal metallopeptidases: "enkephalinase" or angiotensin-converting enzyme (A.C.E.). The carboxyl coordinating ability of the Zn atom seems to be significantly higher in ACE than in "enkephalinase". Moreover, IC50 values against "enkephalinase" were found in the same range whatever the length of the chain bearing the carboxyl group whereas a well-defined position of this group with respect to the Zn atom is required for strong ACE inhibition. These features suggest a larger degree of freedom of the carboxyalkyl moieties within the active site of "enkephalinase". Therefore the differential recognition of active sites of both peptidases leads to: i) N-(carboxymethyl)-L-Phe-L-Leu, 5, a competitive inhibitor of "enkephalinase" (KI = 0.7 microM) and ACE (KI = 1.2 microM) which could be used as mixed inhibitor for both enzymes; ii) N-[(R,S)-2-carboxy, 3-benzylpropanoyl]-L-Leucine, 3, a full competitive inhibitor of "enkephalinase" (KI = 0.34 microM) which does not interact with ACE (IC50 greater than 10,000 microM). This compound can be considered as the first example of a new series of highly potent and specific "enkephalinase" inhibitors.     

New substrates for enkephalinase (neutral endopeptidase) based on fluorescence energy transfer

Novel fluorescent substrates for enkephalinase (neutral endopeptidase; EC 3.4.24.11) have been developed. These new assays are based on the disappearance of energy transfer between a tryptophan or a tyrosine residue and the 5-dimethylaminonaphthalene-1-sulfonyl group (dansyl) in the substrates dansyl-Gly-Trp-Gly or dansyl-Gly-Tyr-Gly upon hydrolysis of their Gly-Trp or Gly-Tyr amide bond by enkephalinase. No significant difference in Km or kcat values were found for dansyl-Gly-Trp-Gly and dansyl-Gly-Tyr-Gly, indicating that, in contrast to thermolysin, the active site of enkephalinase easily accommodates tryptophan residues. Both tryptophan and tyrosine-containing substrates can be used for continuous recording of enkephalinase activity and should prove useful for detailed study of the substrate specificity of this enzyme.     

Down-regulation and inactivation of neutral endopeptidase 24.11 (enkephalinase) in human neutrophils

Neutral endopeptidase (EC 3.4.24.11, NEP) is an integral membrane protein of human neutrophils. NEP is identical with the common acute lymphoblastic leukemia antigen (CALLA) of leukemic cells. The expression of NEP on the surface of neutrophils is down-regulated by endocytosis which can be induced by phorbol 12-myristate 13-acetate (PMA) at 37 degrees C. The activity of the enzyme on the surface of intact cells decreases by 76% within 5 min. The activity can be recovered, however, if the cells are lysed within 5 min of the endocytosis. After 30 min, only 32% of the NEP activity is present in the neutrophil lysates. The loss of activity is presumably due to proteolytic inactivation. Diacylglycerol and monoclonal antibody to CALLA/NEP also induce internalization of NEP. PMA induces endocytosis even at 4 degrees C, but NEP is not inactivated at that temperature. The disappearance of NEP activity after adding PMA was inhibited by various agents. Among the most active were the phospholipase inhibitor 4-bromophenacyl bromide and a combination of the serine protease and cathepsin inhibitors, diisopropylfluorophosphate and N-ethylmaleimide. The employment of fluorescent monoclonal antibody confirmed the down-regulation and internalization of NEP antigen on the neutrophils. Since NEP inactivates chemotactic peptides and thereby affects chemotaxis of neutrophils (Painter, R. G., Dukes, R., Sullivan, J., Carter, R., Erd?s, E. G., and Johnson, A. R. (1988) J. Biol. Chem. 263, 9456-9461), the down-regulation of NEP activity on the cell membrane may modulate the function of these cells in inflammation.     

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.     

Influenza infection causes airway hyperresponsiveness by decreasing enkephalinase

Ferret tracheal segments were infected with human influenza virus A/Taiwan/86 (H1N1) in vitro. After 4 days, the smooth muscle contractile responses to acetylcholine and to substance P were measured. The response to substance P was markedly accentuated, with a threefold increase in force of contraction at a substance P concentration of 10(-5) M, the highest concentration tested. In contrast, the response to acetylcholine was not affected by viral infection. Histological examination of tissues revealed extensive epithelial desquamation. Activity of enkephalinase (neutral metallo-endopeptidase, EC.3.4.24.11), an enzyme that degrades substance P, was decreased by 50% in infected tissues. Inhibiting enkephalinase activity by pretreating with thiorphan (10(-5) M) increased the response to substance P to the same final level in both infected and control tissues. Inhibiting other substance P-degrading enzymes including kininase II (angiotensin-converting enzyme), serine proteases, and aminopeptidases did not affect the response to substance P. Inhibiting cyclooxygenase and lipoxygenase activity using indomethacin and BW 755c did not affect hyperresponsiveness to substance P. Pretreating tissues with antagonists of alpha-adrenoceptors, beta-adrenoceptors, and H1 histamine receptors (phentolamine 10(-5) M, propranolol 5 X 10(-6) M, and pyrilamine 10(-5) M, respectively) had no effect on substance P-induced contraction. These results demonstrate that infection of ferret airway tissues with influenza virus increases the contractile response of airway smooth muscle to substance P. This effect is caused by decreased enkephalinase activity in infected tissues.     

Molecular cloning and amino acid sequence of rat enkephalinase

cDNA clones encoding rat enkephalinase (neutral endopeptidase, EC 3.4.24.11) have been isolated in lambda gt10 libraries from both brain and kidney mRNAs and the complete 742 amino acid sequence of rat enkephalinase is presented. The enzyme possesses a single transmembrane spanning domain near the N-terminal of the molecule but lacks a signal sequence. Because enkephalinase has it active site located extracellularly and is thus an ectopeptidase, we suggest that the N-terminal transmembrane region of the enzyme anchors the protein in membranes and that the majority of the protein, including the carboxy terminus, is extracellular. Enkephalinase, a zinc-containing metallo enzyme, displays homology with other zinc metallo enzymes such as carboxypeptidase A, B and E, suggesting enzymatic similarities in these enzymes.     

Effects of ES52, an enkephalinase inhibitor, on responses of ventrobasal thalamic neurons in rat

The effects of ES52, a highly potent derivative of Thiorphan, an inhibitor of enkephalinase, at doses of 5 and 10 mg/kg IV were studied on the responses to cutaneous stimuli of 18 “nociceptive” (N), 10 “convergent” (NNn) and 4 “non-nociceptive” (Nn) neurons recorded in the ventrobasal (VB) complex of the rat. The responses of neurons exclusively driven by noxious mechanical and thermal stimuli (N neurons) were depressed by 56% by ES52 15 min after the injection of 5 or 10 mg/kg IV. This depressive effect was reversed by naloxone for half the neurons. For the ten neurons driven by both noxious and non-noxious stimuli (convergent NNn neurons), the responses to noxious heat were decreased by 42% at 15 min. By contrast, there was a marked enlargement of their receptive fields to light tactile stimuli, which was not naloxone-reversible. The receptive fields of neurons exclusively driven by non-noxious stimuli (Nn neurons) were also greatly expanded by ES52. These results show that ES52 can depress the responses of VB thalamic neurons to noxious stimuli; the effects on receptive field size underlines the complexity of the endogenous opiate systems.     

The hydrolysis of endothelins by neutral endopeptidase 24.11 (enkephalinase)

Endothelins 1-3 are a family of 21-amino acid peptides whose structure consists of two rings formed by intra-chain disulfide bonds and a linear "COOH-terminal tail." These peptides were originally described on the basis of their potent vasoconstrictor activity. The hydrolytic inactivation of endothelin action has recently been implicated to be attributed, at least in part, to the enzyme neutral endopeptidase 24.11 (Scicli, A. G., Vijayaraghavan, J., Hersh, L., and Carretero, O. (1989) Hypertension 14, 353). The kinetic properties and mode of hydrolysis of the endothelins by this enzyme are reported in this study. The Km for endothelins 1 and 3 hydrolysis is approximately 2 microM while endothelin2 exhibits a 5-fold higher Km. Endothelins 1 and 2 exhibit similar Vmax values while endothelin3 is hydrolyzed considerably more slowly. The initial cleavage site in endothelin1 is at the Ser5-Leu6 bond located within one of the cyclic structures. Thermolysin, a bacterial neutral endopeptidase with a similar substrate specificity to neutral endopeptidase 24.11 initially cleaves endothelin1 between His16-Leu17 which lies within the COOH-terminal linear "tail" portion of the molecule. The cleavage of endothelins 2 and 3 by neutral endopeptidase 24.11 differs from that observed with endothelin1 in that cleavage of these endothelins occurs at Asp18-Ile19 within the linear COOH-terminal tail structure. These results demonstrate that the endothelins are good substrates for neutral endopeptidase 24.11 and suggest that their mode of cleavage is dependent upon both amino acid sequence as well as peptide conformation.     

Degradation of enkephalins: the search for an enkephalinase

Summary The opioid peptides methionine-enkephalin and leucine-enkephalin appear to exert their biological effects through a receptor mediated mechanism. There appears to be three potential mechanisms for enkephalin degradation which could serve to control enkephalin levels in the vicinity of enkephalin receptors. These are, 1) cleavage of the tyrosyl-glycine bond by aminopeptidases, 2) cleavage of the glycyl-glycine bond by a dipeptidyl aminopeptidase, and 3) cleavage of the glycyl-phenylalanine bond by a dipeptidyl carboxypeptidase. In this review the biochemical properties of these potential enkephalinases are described, and the evidence for each acting as an enkephalinase is reviewed.     

Mechanism of action of ethanol on enkephalinase A activity in the rat brain

The influence of ethanol, its metabolites and some opiates on enkephalinase A activity was studied in rat experiments in vitro after acute and chronic administration of ethanol. It was demonstrated that addition of ethanol to the reaction mixture activated enkephalinase A of the midbrain and hypothalamus of intact rats, the maximal effect being attained at an ethanol concentration of 10(-3) M. Multiple washings with buffer of the ethanol-preincubated membranous fraction of these brain structures in the control rats did not lead to a significant reduction in the activating effect of ethanol on enkephalinase A. No activation was recorded upon the use of an enzymatic preparation of the brain from chronically alcoholized animals. Morphine, naltrexon, beta-carbolines, salsolinol (10(-4) M) and acetaldehyde (10(-8)-10(-2) M) did not activate the enzyme. It is suggested that enkephalinase A activation in rats given ethanol is determined by a direct action of ethanol on the enzyme.     

Novel inhibitors of enkephalin-degrading enzymes. I: Inhibitors of enkephalinase by penicillins

Several penicillins have been found to have pro-antinociceptive properties and also to be enkephalinase (neutral endopeptidase-24.11) inhibitors, carfecillin being the most potent. Carfecillin i.c.v. (but not i.p.) had significant antinociceptive activity in the mouse tail immersion test and completely suppressed abdominal constrictions (acetic acid) in mice (IC50 = 23 micrograms/animal). In combination with (D-Ala2-D-leu5)-enkephalin (DADL) i.c.v. in the abdominal constriction test the complete protection observed was reversed by the opioid receptor antagonist naltrexone. Carfecillin was a competitive inhibitor of enkephalinase from mouse brain striata (IC50 = 207 + 57 nM, cf thiorphan 10.6 +/- 1.9 nM) but did not inhibit other known enkephalin- degrading enzymes. Carfecillin provides a new lead structure for the development of more potent enkephalinase inhibitors.     

Expression of enzymatically active enkephalinase (neutral endopeptidase) in mammalian cells

A cDNA encoding the rat enkephalinase protein (neutral endopeptidase; EC 3.4.24.11) has been constructed from overlapping lambda gt10 cDNA clones. This cDNA was inserted into an expression plasmid containing the cytomegalovirus enhancer and promoter. When transfected with this plasmid, Cos 7 cells transiently expressed the enkephalinase protein in a membrane-bound state. Recombinant enkephalinase recovered in solubilized extracts from transfected Cos 7 cells was enzymatically active and displayed properties similar to those of the native enzyme with respect to sensitivity to classical enkephalinase inhibitors.     

The use of a monoclonal antibody for the rapid purification of kidney neutral endopeptidase ("enkephalinase") solubilized in octyl glucoside

The neutral endopeptidase ("enkephalinase") of the rabbit brush border membrane has been purified to homogeneity by a rapid immunoaffinity method using a monoclonal antibody. In contrast with other methods used so far, a complete extraction of enkephalinase from the brush border membrane can be achieved with octyl glucoside, without loss of activity. The solubilized enzyme can be selectively separated from the other proteins in a single step using an immunoaffinity column consisting of the monoclonal antibody covalently linked to Sepharose CL-4B. It is demonstrated that enkephalinase can then be recovered in an active form by elution at low pH. The purified enzyme obtained by this method is completely inhibited by thiorphan and appears as a single 94,000 dalton protein after polyacrylamide gel electrophoresis under denaturing and reducing conditions.     

Molecular cloning and amino acid sequence of human enkephalinase (neutral endopeptidase)

We have isolated a cDNA clone encoding human enkephalinase (neutral endopeptidase, EC 3.4.24.11) in a lambda gt10 library from human placenta, and present the complete 742 amino acid sequence of human enkephalinase. The human enzyme displays a high homology with rat and rabbit enkephalinase. Like the rat and rabbit enzyme, human enkephalinase contains a single N-terminal transmembrane region and is likely to be inserted through cell membranes with the majority of protein, including its carboxy-terminus, located extracellularly.     

Influence of enkephalinase inhibitors on gastric emptying in mice depends on the nature of the meal

The effects of two enkephalinase inhibitors (thiorphan and acétorphan) and DALAMIDE on gastric emptying of fat or non-fat meals were evaluated in mice. When administered intraperitonally at low doses (0.1 and 0.2 mg/kg) 30 min prior to a fatty (milk) meal, both thiorphan and acetorphan increased significantly (P less than 0.01) gastric emptying; these effects were maximal for 0.2 and 0.1 mg/kg respectively and decreased progressively to be not significant for doses higher than 5 mg/kg for thiorphan and 0.5 mg/kg for acetorphan. Similarly DALAMIDE given IP increased significantly (P less than 0.05) gastric emptying at doses of 0.5 and 1 mg/kg while a slowing of gastric emptying was obtained for 10 times higher doses. The effects of thiorphan (0.2 mg/kg) and DALAMIDE (0.5 mg/kg) were blocked by previous administration of naloxone (0.3 mg/kg) and methyl-naloxone (0.5 mg/kg) while only naloxone (0.3 mg/kg) blocked the slowing effect of high dose of DALAMIDE. Administered prior to a non-fat meal, thiorphan (1 mg/kg) stimulated gastric emptying and inhibited it at higher dosage (10 mg/kg). Neither acetorphan nor DALAMIDE at similar dosages affected the gastric emptying of a non-fat meal and the effects of thiorphan (1 and 0.1 mg/kg) were not blocked by naloxone (0.3 mg/kg). It is concluded that enkephalinase inhibitors (thiorphan and acetorphan) administered systemically stimulate the gastric emptying of a fat meal by increasing enkephalin levels in peripheral tissues, while thiorphan exhibits non-opiate effects on gastric emptying of a non-fat meal.     

Effect of 5-Methylcytosine on the Structure and Stability of DNA. Formation of Triple-Stranded Concatenamers by Overlapping Oligonucleotides

Abstract

A triple helix can be formed upon binding of a pyrimidine oligonucleotide to the major groove of a homopurine-homopyrimidine (R·Y) double-stranded DNA target site. Here, we report that this reaction can be influenced by base methylation. The pyrimidine strand 5′- TmCTmCTmCTmCTTmCT (mY₁₂), whose cytosine residues are methylated at C5, does not bind the duplex 5′-AGAGAGAGAAGA·3′-TCTCTCTCTTCT (R₁₂·Y₁₂) to yield a 12-triad triplex, as would be expected from these DNA sequences. Rather, a complex of overlapping oligonucleotides, which we define concatenamer, is formed. The concatenamer is clearly evidenced by Polyacrylamide gel electrophoresis (PAGE) since it migrates with a smeared band of very low mobility. The stoichiometry of the concatenamer, determined by both UV mixing curves and electrophoresis, is surprisingly found to be (R₁₂· 2mY₁₂)_n, thus showing that the unmethylated Y₁₂ strand is excluded from the complex. Denaturation experiments performed by ultraviolet absorbance (UV) and differential scanning calorimetry (DSC) show that the concatenamers melt with a single and highly cooperative transition whose Tm strongly depends on pH. Overall, the data point to the conclusion that the concatenamers are in triple helix, where the methylated mY₁₂ strand is engaged in both Watson-Crick and Hoogsteen base pairings, thus displacing the Y₁₂ strand from the R₁₂·Y₁₂ duplex. A possible mechanism of concatenamer formation is proposed. The results presented in this paper show that 5-methylcytosine brings about a strong stabilizing effect on both double and triple DNA helices, and that pyrimidine oligonucleotides containing 5-methylcytosine can displace from R·Y duplexes the analogous non-methylated strand. The advantage of using methylated oligonucleotides in antisense technology is discussed.     

Biosynthesis of tetrahydrobiopterin: possible involvement of tetrahydropterin intermediates

The biosynthetic pathway of tetrahydrobiopterin (BH₄) from dihydroneopterin triphosphate (NH₂P₃) was studied in fresh as well as heat-treated human liver extracts. The question of NAD(P)H dependency for the formation of sepiapterin was examined. NH₂P₃ was converted by fresh extracts to sepiapterin in low quantities (2% conversion) in the absence of exogenously added NADPH as well as under conditions that ensured the destruction of endogenous, free NAD(P)H. The addition of NADPH to the fresh liver extracts stimulated the synthesis of BH₄ to a much higher yield (17% conversion), and the amount of sepiapterin formed was reduced to barely detectable levels. In contrast, the heat-treated extract (enzyme A2 fraction) formed sepiapterin (1.3% conversion) only in the presence and not in the absence of NADPH. These results indicate that sepiapterin may not be an intermediate on the pathway leading to BH₄ biosynthesis under normal $\text{in vivo}$ conditions. Rather, sepiapterin may result from the breakdown of an as yet unidentified intermediate that is actually on the pathway. It is speculated that NH₂P₃ may be converted to a diketo-tetrahydropterin intermediate (or an equivalent tautomeric structure) by a mechanism involving an intramolecular oxidoreduction reaction. A diketo-tetrahydropterin intermediate could be converted to 5,6-dihydrosepiapterin, which also has a tetrahydropterin ring system and can be converted directly to BH₄ by sepiapterin reductase. This proposed pathway can explain ho the tetrahydropterin ring system can be formed without sepiapterin, dihydrobiopterin, or dihydrofolate reductase being involved in BH₄ biosynthesis $\text{in vivo}$.     

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

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

Identification of a MHC class-II restricted epitope in carcinoembryonic antigen

Purpose The carcinoembryonic antigen (CEA) is extensively expressed on the vast majority of colorectal, gastric, and pancreatic carcinomas, and, therefore, is a good target for tumor immunotherapy. CD4+ T-helper (Th) cells play a critical role in initiation, regulation, and maintenance of immune responses. In this study, we sought to identify Th epitopes derived from CEA which can induce CEA-specific Th responses. The combined application with cytotoxic T lymphocyte (CTL) epitopes would be more potent than tumor vaccines that primarily activate CTL alone.Methods We utilized a combined approach of using a computer-based algorithm analysis TEPITOPE and in vitro biological analysis to identify Th epitopes in CEA.Results Initial screening of healthy donors showed that all five predicted peptides derived from CEA could induce peptide-specific T-cell proliferation in vitro. We characterized these CEA epitopes by establishing and analyzing peptide-specific T-cell clones. It was shown that CD4+ T-cells specific for the CEA₁₁₆ epitope can recognize and respond to naturally processed CEA protein and CEA₁₁₆ epitope can be promiscuously presented by commonly found major histocompatibility complex (MHC) alleles. Furthermore, it was demonstrated that immunization of human leukocyte antigen (HLA)-DR4 transgenic mice with CEA₁₁₆ peptide elicited antigen-specific Th responses which can recognize the antigenic peptides derived from CEA protein and CEA-positive tumors.Conclusion The MHC class II-restricted epitope CEA₁₁₆ could be used in the design of peptide-based tumor vaccine against several common cancers expressing CEA.