Antimicrobial peptides with atypical structural features from the skin of the Japanese brown frog Rana japonica

Japonicin-1 (FFPIGVFCKIFKTC) and japonicin-2 (FGLPMLSILPKALCILLKRKC), two peptides with differential growth-inhibitory activity against the Gram-negative bacterium, Escherichia coli and the Gram-positive bacterium Staphylococcus aureus, were isolated from an extract of the skin of the Japanese brown frog Rana japonica. Both peptides show little amino acid sequence similarity to previously characterized antimicrobial peptides isolated from the skins of Ranid frogs. Circular dichroism studies, however, demonstrate that japonicin-2 adopts an alpha-helical conformation in 50% trifluoroethanol in common with many other cationic antimicrobial peptides synthesized in amphibian skin. Peptides belonging to the brevinin-1, brevinin-2, and tigerinin families, previously identified in the skins of Asian Ranid frogs, were not detected but a temporin-related peptide (ILPLVGNLLNDLL.NH(2); temporin-1Ja), that atypically bears no net positive charge, was isolated from the extract. The minimum inhibitory concentrations (MICs) of the peptides against E. coli were japonicin-1, 30 microM; japonicin-2, 12 microM; and temporin-1Ja > 100 microM. The MICs against S. aureus were japonicin-1, > 100 microM; japonicin-2, 20 microM; and temporin-1Ja, > 100 microM.     

Rubiscolin, a delta selective opioid peptide derived from plant Rubisco.

We found that the sequences YPLDL and YPLDLF in the large subunit of spinach D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) met the structure YP-aliphatic amino acid which might have opioid activity. We then synthesized these peptides to test their opioid activity. The IC(50) of these peptides in mouse vas deferens assay were 51.0 microM and 24.4 microM, respectively, and those in delta receptor binding assay using [(3)H]deltorphin II as radioligand were 2.09 microM and 0.93 microM, respectively. Both peptides were selective for delta receptor. We named them rubiscolin-5 and -6, respectively. Rubiscolin-5 and -6 have antinociceptive activity in mice after i.c.v. or oral administration. The enzymatic conditions to release rubiscolin were investigated using both spinach Rubisco and synthetic fragment peptides. This is the first example of bioactive peptides derived from plant Rubisco.     

Antimicrobial peptides from diverse families isolated from the skin of the Asian frog, Rana grahami

Seven peptides with antimicrobial activity were isolated in pure form from an extract of the skin of the Yunnanfu Kunming frog Rana grahami Boulenger, 1917. The peptides were identified as belonging to the nigrocin-2 (three peptides), brevinin-1 (one peptide), brevinin-2 (three peptides), and esculentin-1 (one peptide) families. Nigrocin-2GRb (GLFGKILGVGKKVLCGLSGMC) containing three lysine residues, represented the peptide with highest potency against microorganisms (MIC = 3 microM against Escherichia coli, 12.5 microM against Staphylococcus aureus and 50 microM against Candida albicans) and the greatest hemolytic activity against human erythrocytes (LD50 = 40 microM). In contrast, nigrocin-2GRa (GLLSGILGAGKHIVCGLSGLC) and nigrocin-2GRc (GLLSGILGAGKNIVCGLSGLC), with only a single lysine residue, showed weak antimicrobial and hemolytic activity. Phylogenetic relationships among Eurasian ranid frogs are less well understood than those of North American ranids but the primary structures of the R. grahami antimicrobial peptides suggest a close relationship of this species with the Japanese pond frogs R. nigromaculata and R. porosa brevipoda.     

Peptides with antimicrobial activity of the brevinin-1 family isolated from skin secretions of the southern leopard frog, Rana sphenocephala.

Three peptides with growth-inhibitory activity towards the gram-negative bacterium Eschericia coli were isolated from electrically stimulated secretions from the skin of the southern leopard frog, Rana sphenocephala. Structural characterization demonstrated that the peptides [brevinin-1Sa, minimum inhibitory concentration (MIC) = 55 microM; brevinin-1Sb, MIC = 17 microM; brevinin-1Sc, MIC = 14 microM] represent new members of the brevinin-1 family of antimicrobial peptides, previously isolated from several other species of frogs of the genus Rana. Their high concentration in skin secretions and extreme variability in amino acid sequence suggest that the brevinin family of peptides may be of value as molecular markers for the identification and taxonomic classification of Ranid frogs.     

Activity of cecropin P1 and FA-LL-37 against urogenital microflora

Two mammalian antimicrobial peptides, FA-LL-37 and cecropin P1, were tested for activity against six uropathogens and five Lactobacillus strains by broth microdilution assay. Both peptides inhibited Escherichia coli at 25 microM (FA-LL-39), and 1.56 microM (cecropin P1), Pseudomonas aeruginosa (12.5 microM, and 25 microM), and Klebsiella pneumoniae, (50 microM, and 1.56 microM), but not Enterococcus faecalis and Staphylococcus epidermidis. FA-LL-37 acted bacteriocidally against E. coli and bacteriostatically against the other two Gram-negative organisms. Cecropin P1 was bacteriocidal to all susceptible bacteria. Lactobacilli were resistant to both peptides, with the exception of poultry isolate Lactobacillus fermentum B-54, which was susceptible to FA-LL-37 at 100 microM. The differential activities of these peptides toward Gram-negative uropathogens versus urogenital lactobacilli demonstrate their potential as a topical treatment for urinary tract infections. In addition, production of such peptides in vivo could be a natural mechanism to aid in the maintenance of the lactobacilli-dominated urogenital flora at the expense of pathogens.     

Analysis of novel angiotensin-I-converting enzyme inhibitory peptides from protease-hydrolyzed marine shrimp Acetes chinensis.

Acetes chinensis is an underutilized shrimp species thriving in the Bo Hai Gulf of China. In a previous study, we had used the protease from Bacillus sp. SM98011 to digest this kind of shrimp and found that the oligopeptide-enriched hydrolysate possessed antioxidant activity and high angiotensin I-converting enzyme (ACE) inhibitory activity with an IC50 value of 0.97 mg/ml. In this paper, by ultrafiltration, gel permeation chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC), five peptides with high ACE inhibitory activity were purified from the shrimp hydrolysates and their sequences were identified by amino acid composition analysis and molecular weight (MW) analysis. Three of them, FCVLRP (a), IFVPAF (f) and KPPETV (j), were novel ACE inhibitory peptides. Their IC50 values were 12.3 microM, 3.4 microM and 24.1 microM, respectively, and their recoveries were 30 mg/100 g (solid basis of shrimp), 19 mg/100 g and 33 mg/100 g, respectively. Lineweaver-Burk plots for the three novel peptides showed that they are all competitive inhibitors. To test the ACE inhibitory activity of peptide a, f, j after they were digested by digestive enzymes in vivo, 12 derived peptides from FCVLRP and IFVPAF were synthesized based on their amino acid sequences and the cleavage sites of digestive enzymes. No digestive enzyme cleavage site was found in KPPETV. The IC50 values of the derived peptides were determined and the result showed that except for VPAF, FC and FCVL, the ACE inhibitory activity of the other nine derived peptides did not significantly change when compared with their original peptides. Surprisingly, five peptides had lower IC50 values than their original peptides, particularly for RP (IC50 value = 0.39 microM), which is about 30 times lower than its original peptide and almost the lowest IC50 value for ACE inhibitory peptides reported. Therefore, the novel peptides identified from A. chinensis hydrolysates probably still maintain a high ACE inhibitory activity even if they are digested in vivo. This is the first report about novel ACE inhibitory peptides from hydrolysates of marine shrimp A. chinensis. The novel peptides from hydrolysate of A. chinensis and some of their derived peptides with high ACE inhibitory activity probably have potential in the treatment of hypertension or in clinical nutrition.     

Development of an at-line method for the identification of angiotensin-I inhibiting peptides in protein hydrolysates

A fast at-line method was developed for the identification of ACE inhibiting (ACEI) peptides in protein hydrolysates. The method consists of activity measurements of fractions collected from a two-dimensional HPLC fractionation of the peptide mixture followed by MS identification of the peptides in the inhibiting fractions. The inhibition assay is based on the inhibiting effect of ACEI peptides on the hydrolytic scission of the substrate Hippuric acid-His-Leu (HHL) during the ACE-catalysed hydrolysis reaction. A fast LC method was developed for the quantification of Hippuric acid (H) and Hippuric acid-Histidine-Leucine (HHL), allowing a large number of fractions to be analysed within a reasonable time period. The method is sensitive and uses only standard laboratory equipment. The limit of detection is 0.34 microM for the known ACEI peptide IPP. This is sufficiently sensitive for the identification of only moderately active peptides and/or ACEI peptides present at low concentrations. The relative standard deviation of the inhibition assay was 12% measured over a time period of 2 months. The IC50 value of IPP measured with the assay was 5.6 microM, which is comparable to the values of 5 microM and 5.15 microM reported in literature for the standard Matsui method. The assay was successfully applied in the identification of ACEI peptides in enzymatically hydrolysed caseinate samples. Two new, not earlier published ACEI peptides were identified; MAP (beta-casein f102-104) and ITP (alpha-s2-casein f119-121) with IC50 values of 3.8 microM and 50 microM, respectively.     

Novel angiotensin I-converting enzyme inhibitory peptides isolated from Alcalase hydrolysate of mung bean protein.

Mung bean protein isolates were hydrolyzed for 2 h by Alcalase. The generated hydrolysate showed angiotensin I-converting enzyme (ACE) inhibitory activity with the IC(50) value of 0.64 mg protein/ml. Three kinds of novel ACE inhibitory peptides were isolated from the hydrolysate by Sephadex G-15 and reverse-phase high performance liquid chromatography (RP-HPLC). These peptides were identified by amino acid composition analysis and matrix assisted-laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS), as Lys-Asp-Tyr-Arg-Leu, Val-Thr-Pro-Ala-Leu-Arg and Lys-Leu-Pro-Ala-Gly-Thr-Leu-Phe with the IC(50) values of 26.5 microM, 82.4 microM and 13.4 microM, respectively.     

Specificities of autoinhibitory domain peptides for four protein kinases. Implications for intact cell studies of protein kinase function

Synthetic peptides corresponding to the autoinhibitory domains of calcium/calmodulin-dependent protein kinase II (CaMK-(281-309)), smooth muscle myosin light chain kinase (MLCK-(480-501)), and protein kinase C (PKC-(19-36)) as well as a peptide derived from the heat-stable inhibitor of cAMP-dependent protein kinase (PKI-tide) were tested for their inhibitory specificities. The inhibitory potencies of the four peptides were determined for each of the four protein kinases using both peptide substrates (at approximate Km concentrations) and protein substrates (at concentrations less than Km). In agreement with previous studies PKI-tide was a specific and potent inhibitor of only cAMP kinase, and none of the other inhibitory peptides gave significant inhibition of cAMP kinase at concentrations of less than 100 microM. With synthetic peptide substrates, PKC-(19-36) strongly inhibited native PKC (IC50 less than 1 microM) but also significantly inhibited autophosphorylated CaMK-II (IC50 = 30 microM) and proteolytically activated MLCK (IC50 = 35 microM). MLCK-(480-501) potently inhibited MLCK (IC50 = 0.25 microM) and also strongly inhibited both PKC and CaMK-II (IC50 = 1.4 and 1.7 microM, respectively). CaMK-(281-309) inhibited autophosphorylated CaMK-II, PKC, and proteolyzed MLCK almost equally (IC50 = 10, 38, and 48 microM, respectively). Qualitatively similar results were obtained with protein substrates. These studies validate the use of PKI-tide as a specific inhibitor of cAMP kinase in intact cell studies and suggest that PKC-(19-36) can also be used but only within a narrow concentration range. However, the autoinhibitory domain peptides from MLCK and CaMK-II are not sufficiently specific to be used in similar investigations.     

Structural determinants of alpha-bungarotoxin binding to the sequence segment 181-200 of the muscle nicotinic acetylcholine receptor alpha subunit: effects of cysteine/cystine modification and species-specific amino acid substitutions

The sequence segment 181-200 of the Torpedo nicotinic acetylcholine receptor (nAChR) alpha subunit forms a binding site for alpha-bungarotoxin (alpha-BTX) [e.g., see Conti-Tronconi, B. M., Tang, F., Diethelm, B. M., Spencer, S. R., Reinhardt-Maelicke, S., & Maelicke, A. (1990) Biochemistry 29, 6221-6230]. Synthetic peptides corresponding to the homologous sequences of human, calf, mouse, chicken, frog, and cobra muscle nAChR alpha 1 subunits were tested for their ability to bind 125I-alpha-BTX, and differences in alpha-BTX affinity were determined by using solution (IC50S) and solid-phase (KdS) assays. Panels of overlapping peptides corresponding to the complete alpha 1 subunit of mouse and human were also tested for alpha-BTX binding, but other sequence segments forming the alpha-BTX site were not consistently detectable. The Torpedo alpha 1(181-200) and the homologous frog and chicken peptides bound alpha-BTX with higher affinity (KdS approximately 1-2 microM, IC50s approximately 1-2 microM) than the human and calf peptides (Kds approximately 3-5 microM, IC50s approximately 15 microM). The mouse peptide bound alpha-BTX weakly when attached to a solid support (Kd approximately 8 microM) but was effective in competing for 125I-alpha-BTX in solution (IC50 approximately 1 microM). The cobra nAChR alpha 1-subunit peptide did not detectably bind alpha-BTX in either assay. Amino acid substitutions were correlated with alpha-BTX binding activity peptides from different species. The role of a putative vicinal disulfide bound between Cys-192 and -193, relative to the Torpedo sequence, was determined by modifying the peptides with sulfhydryl reagents. Reduction and alkylation of the peptides decreased alpha-BTX binding, whereas oxidation of the peptides had little effect. Modifications of the cysteine/cystine residues of the cobra peptide failed to induce alpha-BTX binding activity. These results indicate that while the adjacent cysteines are likely to be involved in forming the toxin/alpha 1-subunit interface a vicinal disulfide bound was not required for alpha-BTX binding.     

Species-specific inhibition of fertilization by a peptide derived from the sperm protein bindin.

The sperm protein bindin is responsible for the species-specific adhesion of the sperm to the egg. The regions of the bindin molecule responsible for forming the contact between the sperm and the egg were investigated by measuring the ability of peptides representing various regions of the bindin sequence to inhibit fertilization. Twenty-four peptides were studied: 7 based on the Strongylocentrotus purpuratus bindin sequence, 11 based on the S. franciscanus bindin sequence, and 6 control peptides. Values for the concentration of peptide required to inhibit 50% of the productive sperm contacts (IC50) were extracted from experimental measurements of the extent of fertilization in the presence of various concentrations. of these peptides. The IC50 value averaged 220 microM for the control peptides. Active peptides representing certain specific subregions of the bindin sequence displayed IC50 values < 10% of the average value for control peptides, and the IC50 for the most potent of the peptides tested was only approximately 1% of the control peptide value (IC50 = 2.2 microM). Furthermore, we found that a peptide representing a particular region of the S. franciscanus bindin sequence that differs from the S. purpuratus bindin sequence inhibits fertilization species specifically. For the reaction of S. purpuratus sperm and eggs, the IC50 of this peptide was approximately 120 microM, whereas for the reaction of S. franciscanus sperm and eggs it was only 8.6 microM. These results demonstrate that a few specific regions of the bindin molecule are involved in the sperm-egg contact and that certain of these regions mediate the species specificity of the interaction in a sequence-specific manner.     

Immunoreceptor transmembrane peptides and their effect on natural killer (NK) cell cytotoxicity

Short peptides derived from the transmembrane sequence of NK activating receptors and associated molecules were tested in vitro for inhibition of NK cell cytotoxicity using a standard (51)Cr release assay in the absence or presence of peptides. NKL23 cell line was used as the NK effector and the target was the NKL23 sensitive 721.221 cell line. NKp46, NKp30, NKG2D and CD3-zeta peptides inhibited NK activity at higher concentration (100 microM) compared to controls by 6-13% (p<0.05). Modification of one non-effective peptide (NKP44) significantly enhanced inhibition by 30%, 17% and 11% at 100 microM, 50 microM and 10 microM respectively compared to controls. A T-cell antigen receptor-alpha chain transmembrane sequence derived peptide (CP) significantly inhibited NKL cell activation by 20-30% (p<0.05) at 50 microM and 100 microM concentrations compared to the control. The structural similarities between these immuno-receptors, and in particular the need for transmembrane electrostatic interactions for receptor function, provides the basis for current and future targeted therapeutic strategies.     

Purification and in vitro antioxidative effects of giant squid muscle peptides on free radical-mediated oxidative systems

Low molecular weight peptides obtained from ultrafiltration (UF) of giant squid (Dosidicus gigas) muscle protein were studied for their antioxidative effects in different in vitro oxidative systems. The most potent two peptides, Asn-Ala-Asp-Phe-Gly-Leu-Asn-Gly-Leu-Glu-Gly-Leu-Ala (1307 Da) and Asn-Gly-Leu-Glu-Gly-Leu-Lys (747 Da), exhibited their antioxidant potential to act as chain-breaking antioxidants by inhibiting radical-mediated peroxidation of linoleic acid, and their activities were closer to highly active synthetic antioxidant, butylated hydroxytoluene. Addition of these peptides could enhance the viability of cytotoxic embryonic lung fibroblasts significantly (P<.05) at a low concentration of 50 microg/ml, and it was presumed due to the suppression of radical-induced oxidation of membrane lipids. Electron spin trapping studies revealed that the peptides were potent scavengers of free radicals in the order of carbon-centered (IC(50) 396.04 and 304.67 microM), hydroxyl (IC(50) 497.32 and 428.54 microM) and superoxide radicals (IC(50) 669.34 and 573.83 microM). Even though the exact molecular mechanism for scavenging of free radicals was unclear, unusually high hydrophobic amino acid composition (more than 75%) of giant squid muscle peptides was presumed to be involved in the observed activities.     

Inhibition of Calmodulin-Stimulated Phosphodiesterase Activity by Vasoactive Intestinal Peptide

The effects of certain peptides of the glucagon family on calmodulin activity were determined from their capacity to inhibit a calmodulin-dependent form of phosphodiesterase. Vasoactive intestinal peptide and secretin were potent inhibitors of calmodulin activity, having IC50 values of 0.5 microM and 2 microM, respectively. By contrast, glucagon failed to inhibit calmodulin activity even at concentrations of 100 microM. None of these compounds significantly inhibited the basal activity of phosphodiesterase at concentrations up to 100 microM. These findings support the suggestion that important structural features of peptides for anticalmodulin activity include a net positive charge and a hydrophobic surface.     

A potent, non-toxic insulin-releasing peptide isolated from an extract of the skin of the Asian frog, Hylarana guntheri (Anura:Ranidae)

Peptides in extract of the skin of the Asian frog Hylarana guntheri Boulenger,1882 were purified by reversed-phase HPLC and individual components analysed for their ability to release insulin from the rat BRIN-BD11 clonal beta cell line. The most potent peptide identified in the extract belonged to the brevinin-2 family (brevinin-2GUb; GVIIDTLKGAAKTVAAELLRKAHCKLTNSC). Other peptides with weaker insulin-releasing activity belonged to the brevinin-1 (2 peptides), brevinin-2 (2 peptides) and temporin (3 peptides) families. Only the brevinin-1 peptides showed cytolytic activity against the BRIN-BD11 cells, as demonstrated by an increased rate of release of the cytosolic enzyme, lactate dehydrogenase. A synthetic replicate of brevinin-2GUb produced a significant stimulation of insulin release (139% of basal rate; P<0.05) at a concentration of 100 nM with a maximum response of 373% of basal rate at a concentration of 3 microM) by a mechanism that did not involve mobilization of intracellular calcium. Brevinin-2GUb also inhibited the growth of microorganisms (MIC against Escherichia coli=32 microM, Staphylococcus aureus=64 microM, and Candida albicans=64 microM) but had only weak hemolytic activity against human erythrocytes (LC(50)=700 microM). Administration of brevinin-2GUb (75 nmol/kg body weight) into mice significantly (P<0.05) improved glucose tolerance following a intraperitoneal injection of glucose, thereby demonstrating that the peptide shows potential for development into a therapeutically valuable agent for the treatment of Type 2 diabetes.     

Design, synthesis and properties of novel powerful antioxidants, glutathione analogues

Glutathione (GSH) is the major low-molecular weight antioxidant in mammalian cells. Thus, its analogues carrying similar and/or additional positive properties might have clinical perspectives. Here, we report the design and synthesis of a library of tetrapeptidic GSH analogues called UPF peptides. Compared to cellular GSH our designed peptidic analogues showed remarkably higher hydroxyl radical scavenging ability (EC(50) of GSH: 1,231.0 +/- 311.8 microM; EC(50) of UPF peptides: from 0.03 to 35 microM) and improved antiradical efficiency towards a stable alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) radical. The best of UPF peptides was 370-fold effective hydroxyl radical scavengers than melatonin (EC(50): 11.4 +/- 1.0 microM). We also found that UPF peptides do not influence the viability and membrane integrity of K562 human erythroleukemia cells even at 200 microM concentration. Dimerization of GSH and UPF peptides was compared in water and in 0.9% saline solutions. The results, together with an earlier finding that UPF1 showed protective effects in global cerebral ischemia model in rats, suggest that UPF peptides might serve both as potent antioxidants as well as leads for design of powerful non-peptidic antioxidants that correct oxidative stress-driven events.     

Cyclic and linear peptides derived from alpha-amylase inhibitory protein tendamistat

Tendamistat is a strong inhibitory protein of porcine pancreatic alpha-amylase (PPA) with a K(i) value of 0.2 nM. To develop potent alpha-amylase inhibitors, we synthesized six odd-length cyclic peptides (5-15 residues) and four even-length cyclic peptides (10 and 12 residues) having the inhibitory sequence of tendamistat. Their PPA inhibitory activities were evaluated, and, among them, the 11-residue cyclic peptide Ten(15-23) (K(i) = 0.27 microM) exhibited the strongest inhibitory activity (K(i) = 0.27-1.41 microM). To examine the effect of cyclic structure on PPA inhibition, ten linear peptides corresponding to the cyclic peptides were also synthesized, and their PPA inhibitory activities were evaluated (K(i) = 0.28-1.00 microM). Interestingly, the 11-residue linear peptide Ten(15-23) exhibited almost the same inhibitory activity (K(i) = 0.28 microM) as that of cyclic Ten(15-23). The results of a circular dichroism study indicated that stabilization of the beta-hairpin structure occurred only for cyclic Ten(15-23). Also, the results of proteolytic digestion experiments of the cyclic and linear Ten(15-23) peptides by trypsin and chymotrypsin suggested no differences in protease resistance between the cyclic and linear structures. Therefore, we demonstrated that both cyclic and linear peptides containing the inhibitory sequence of tendamistat exhibit potent PPA inhibitory activity.     

Isolation and characterization of Psalmopeotoxin I and II: two novel antimalarial peptides from the venom of the tarantula Psalmopoeus cambridgei

Two novel peptides that inhibit the intra-erythrocyte stage of Plasmodium falciparum in vitro were identified in the venom of the Trinidad chevron tarantula, Psalmopoeus cambridgei. Psalmopeotoxin I (PcFK1) is a 33-residue peptide and Psalmopeotoxin II (PcFK2) has 28-amino acid residues; both have three disulfide bridges and belong to the Inhibitor Cystine Knot superfamily. The cDNAs encoding both peptides were cloned, and nucleotide sequence analysis showed that the peptides are synthesized with typical signal peptides and pro-sequences that are cleaved at a basic doublet before secretion of the mature peptides. The IC(5O) of PcFK1 for inhibiting P. falciparum growth was 1.59+/-1.15 microM and that of PcFK2 was 1.15+/-0.95 microM. PcFK1 was adsorbed strongly to uninfected erythrocytes, but PcFK2 was not. Neither peptide has significant hemolytic activity at 10 microM. Electrophysiological recordings in isolated frog and mouse neuromuscular preparations revealed that the peptides (at up to 9.3 microM) do not affect neuromuscular transmission or quantal transmitter release. PcFK1 and PcFK2 do not affect the growth or viability of human epithelial cells, nor do they have any antifungal or antibacterial activity at 20 microM. Thus, PcFK1 and PcFK2 seem to interact specifically with infected erythrocytes. They could therefore be promising tools for antimalaria research and be the basis for the rational development of antimalarial drugs.     

Metabolism of opioid peptides by cerebral microvascular aminopeptidase M

Aminopeptidase M (EC 3.4.11.2), which can degrade low molecular weight opioid peptides, has been reported in both peripheral vasculature and in the CNS. Thus, we have studied the metabolism of opioid peptides by membrane-bound aminopeptidase M derived from cerebral microvessels of hog and rabbit. Both hog and rabbit microvessels were found to contain membrane-bound aminopeptidase M. At neutral pH, microvessels preferentially degraded low molecular weight opioid peptides by hydrolysis of the N-terminal Tyr1-Gly2 bond. Degradation was inhibited by amastatin (I50 = 0.2 microM) and bestatin (10 microM), but not by a number of other peptidase inhibitors including captopril and phosphoramidon. Rates of degradation were highest for the shorter peptides (Met5- and Leu5-enkephalin) whereas beta-endorphin was nearly completely resistant to N-terminal hydrolysis. Km values for the microvascular aminopeptidase also decreased significantly with increasing peptide length (Km = 91.3 +/- 4.9 and 28.9 +/- 3.5 microM for Met5-enkephalin and Met5-enkephalin-Arg6-Phe7, respectively). Peptides known to be present within or in close proximity to cerebral vessels (e.g., neurotensin and substance P) competitively inhibited enkephalin degradation (Ki = 20.4 +/- 2.5 and 7.9 +/- 1.6 microM, respectively). These data suggest that cerebral microvascular aminopeptidase M may play a role in vivo in modulating peptide-mediated local cerebral blood flow, and in preventing circulating enkephalins from crossing the blood-brain barrier.