Production of angiotensin I-converting enzyme inhibitory peptides from defatted canola meal

To simplify the method of ACE-inhibitory peptide production, defatted canola meal was subjected to enzymatic proteolysis. Alcalase 2.4L and protease M “Amano” were found to be the most efficient enzymes in releasing ACE-inhibitory peptides from canola proteins among 13 tested enzymes. The IC₅₀ values of canola protein hydrolysates ranged from 18.1 to 82.5 μg protein/mL. Differences in ACE-inhibitory activities of various protein hydrolysates reflected varied enzyme specificities. A positive correlation was determined between ACE-inhibitory activity and the degree of hydrolysis (r = 0.5916, p < 0.001). Ion-exchange chromatography of canola protein hydrolysate increased the protein content greater than 95% without loss of ACE-inhibitory activity. This fraction was resistant to the degradation of gastrointestinal enzyme and ACE during in vitro incubation and may be a useful ingredient in the formulation of hypotensive functional food products.     

Production of angiotensin-I-converting enzyme inhibitory peptides from β-lactoglobulin- and casein-derived peptides: an integrative approach

Angiotensin I-converting enzyme (ACE) inhibition is one of the mechanisms by which reduction in blood pressure is exerted. Whey proteins are a rich source of ACE inhibitory peptides and have shown a blood pressure reduction effect i.e. antihypertensive activity. The aim of this work was to develop a simplified process using a combination of adsorption and microfiltration steps for the production of hydrolysates from whey with high ACE inhibitory activity and potency; the latter was measured as the IC50, which is the peptide concentration required to reduce ACE activity by half. This process integrates the selective separation of β-lactoglobulin- and casein-derived peptides (CDP) from rennet whey and their hydrolysis, which results in partially pure, less complex hydrolysates with high bioactive potency. Hydrolysis was carried out with protease N "Amano" in a thermostatically controlled membrane reactor operated in a batch mode. By applying the integrative approach it was possible to produce from the same feedstock two different hydrolysates that exhibited high ACE inhibition. One hydrolysate was mainly composed of casein-derived peptides with IC50=285 μg/mL. In this hydrolysate we identified the well-known potent ACE-inhibitor and antihypertensive tripeptide Ile-Pro-Pro (IPP) and another novel octapeptide Gln-Asp-Lys-Thr-Glu-Ile-Pro-Thr (QDKTEIPT). The second hydrolysate was mainly composed of β-lactoglobulin derived peptides with IC50=28 μg/mL. This hydrolysate contained a tetrapeptide (Ile-Ile-Ala-Glu) IIAE as one of the two major peptides. A further advantage to this process is that enzyme activity was substantially increased as enzyme product inhibition was reduced.     

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.     

Production of active angiotensin-I converting enzyme inhibitory peptides derived from bovine beta-casein by recombinant DNA technologies

A wide range of anti-hypertensive peptides potentially able to lower blood pressure through the inhibition of vasoactive enzymes such as angiotensin-I converting enzyme (ACE) are known. Currently, ACE-inhibitory peptides can be produced from precursor proteins via enzymatic hydrolysis by proteolytic enzymes, or food fermentation with proteolytic starter cultures. These approaches are neither selective nor easy. In this study a novel procedure has been developed, based on recombinant DNA technologies, for the production of highly purified fractions of three polypeptides derived from bovine beta-casein active as ACE inhibitors in vitro. The procedure includes peptide expression in Escherichia coli cells as recombinant fusion proteins, purification by affinity chromatography, cleavage by proteinase from a selected strain of Lactobacillus helveticus and isolation of bioactive peptides (BPs). The reported concentration of inhibitor needed to reduce at 50% ACE activity (IC(50)) values for single BP calculated in inhibiting the ACE enzyme gave results in agreement with the same parameters available in literature for other milk-derived BPs. This procedure could be used to obtain quantities of pure peptides to determine their interactions with ACE, with the aim of designing peptides that have stronger inhibitory properties and exhibit new pharmacological profiles. Moreover, its scale up would be of commercial interest for the production of functional foods, e.g., milk beverages with blood pressure-lowering effects.     

Purification and characterization of angiotensin I converting enzyme inhibitory peptides from the rotifer, Brachionus rotundiformis

Angiotensin I converting enzyme (ACE) inhibitory peptide was isolated from the marine rotifer, Brachionus rotundiformis. ACE inhibitory peptides were separated from rotifer hydrolysate prepared by Alcalase, α-chymotrypsin, Neutrase, papain, and trypsin. The Alcalase hydrolysate had the highest ACE inhibitory activity compared to the other hydrolysates. The IC₅₀ value of Alcalase hydrolysate for ACE inhibitory activity was 0.63 mg/ml. We attempted to isolate ACE inhibitory peptides from Alcalase prepared rotifer hydrolysate using gel filtration on a Sephadex G-25 column and high performance liquid chromatography on an ODS column. The IC₅₀ value of purified ACE inhibitory peptide was 9.64 μM, and Lineweaver–Burk plots suggest that the peptide purified from rotifer protein acts as a competitive inhibitor against ACE. Amino acid sequence of the peptide was identified as Asp-Asp-Thr-Gly-His-Asp-Phe-Glu-Asp-Thr-Gly-Glu-Ala-Met, with a molecular weight 1538 Da. The results of this study suggest that peptides derived from rotifers may be beneficial as anti-hypertension compounds in functional foods resource.     

Sugar Nucleotides from the Fungus Aureobasidium

Peptides showing inhibitory activity against the angiotensin I-converting enzyme (ACE) were investigated from the fibroin fraction of discarded silk fabric. Fibroin, which was hydrolyzed with alcalase after partial hydrolysis with hot aqueous 40% CaCl₂, released two major active peptides showing ACE-inhibitory activity. The two peptides were identified as glycyl-valyl-glycyl-tyrosine (GVGY) and glycyl-valyl-glycyl-alanyl-glycyl-tyrosine (GVGAGY) by analyses with a protein sequencer and LC/MS/MS. GVGY, whose ACE-inhibitory activity has not previously been reported, showed a blood pressure-depressing effect on spontaneously hypertensive rat (SHR).     

血管紧张素转换酶纯化与性质研究

为了深入了解猪肺血管紧张素转换酶 (angiotensin converting enzyme,ACE)的性质和功能 ,对猪肺 ACE的分离纯化以及部分酶学性质进行了研究 .猪肺组织匀浆经 1 .6～ 2 .6mol/L硫酸铵分级沉淀等步骤后 ,利用亲和胶进行亲和层析分离 .2 0 0 g猪肺组织中提纯出 0 .79mg ACE,比活力 38.8U/mg,SDS- PAGE电泳鉴定为一条带 ,分子量约 1 80 k D,等电点 (p I)为 p H4.5,糖含量约 2 3.6% ,氨基酸组成分析发现猪肺 ACE分子中含有 1 346个氨基酸 ,其中酸性氨基酸含量较高 ,碘乙酸的修饰结果表明猪肺 ACE中巯基基团未参与酶的催化反应 .酶反应动力学结果显示 ,ACE催化 Fa PGG底物反应时的最适 p H大约为 p H 7.6,反应活化能 Ea=4.37× 1 0 4 J/mol,酶活性部位附近的组氨酸和具有类似 α-氨基性质的氨基酸可能参与了 ACE催化反应 .有关猪肺 ACE的基本生化性质、氨基酸组成以及酶学性质的结果 ,为今后深入研究奠定了基础 .     

Ketomethylureas. A New Class of Angiotensin Converting Enzyme Inhibitors

Abstract

The design rationale for a new series of tripeptide derived angiotensin converting enzyme (ACE) inhibitors, which we term “ketomethylureas”, is described. Analogs of tripeptide substrates (i.e. N-benzoyl-Phe-Ala-Pro) in which the nitrogen atom of the scissile amide bond and the adjacent asymmetric carbon atom of the penultimate amino acid residue are formally transposed give rise to this novel class of inhibitors. The most potent ketomethylureas inhibit ACE wtih I₅₀ values in the nM range.     

Insight into the binding of ACE-inhibitory peptides to angiotensin-converting enzyme: a molecular simulation

Angiotensin-I-converting enzyme (EC 3.4.15.1; ACE) plays an important physiological role in the regulation of blood pressure by converting angiotensin I to angiotensin II, a potent vasoconstrictor. Therefore, ACE inhibition has become a major target control for hypertension. Pipefish, or hailong, is an essential traditional Chinese medicine that is widely used in anti-fatigue and anti-cancer. A recent study has found two ACE-inhibitory peptides (TFPHGP and HWTTQR) purified from the seaweed pipefish by Alcalase enzymatic hydrolysis. Two peptides exhibited different ACE-inhibitory activities; however, the molecular mechanism involved is poorly understood. Further investigations are necessary to elucidate the relationship between the inhibition mechanism and the peptides. The current study is focused on investigating the interactions between each ACE-inhibitory peptide and ACE by performing molecular docking and molecular dynamics (MD) simulations. ACE protein remained stable throughout the simulations. Furthermore, ACE-TFPHGP complex showed lower binding energy as compared to ACE-HWTTQR complex, which is in good agreement with the experimental results. Glu384 and Glu411 of ACE are key residues upon the ACE-inhibitory peptides binding. Molecular basis generated by this attempt could provide valuable information towards designing new medicine for ACE inhibitor.     

Parallel effects of genetic variation in ACE activity in baboons and humans

Like humans, savannah baboons (Papio sp.) show heritable interindividual variation in complex physiological phenotypes. One prominent example of such variation involves production of the homeostatic regulator protein angiotensin converting enzyme (ACE), which shows heritable variation in both baboons and humans. In humans, this phenotypic variation is associated with an Alu insertion-deletion polymorphism in the ACE gene, which explains approximately half of the variation in serum ACE activity. We identified a similar Alu insertion-deletion polymorphism in the baboon ACE homologue and measured its frequency in a wild population and a captive population of baboons. We also analyzed the contribution of ACE genotype at this indel to variation in serum ACE activity in the captive population. When conditioned on weight, a known factor affecting ACE activity in humans, age and ACE genotype both accounted for variance in ACE activity; in particular, we identified a significant nonadditive interaction between age and genotype. A model incorporating this interaction effect explained 21.6% of the variation in residual serum ACE activity. Individuals homozygous for the deletion mutation exhibited significantly higher levels of ACE activity than insertion-deletion heterozygotes at younger ages (10-14 years), but showed a trend towards lower levels of ACE activity compared with heterozygotes at older ages (> or =15 years). These results demonstrate an interesting parallel between the genetic architecture underlying ACE variation in humans and baboons, suggesting that further attention should be paid in humans to the relationship between ACE genetic variation and aging.     

Blood pressure-depressing activity of a peptide derived from silkworm fibroin in spontaneously hypertensive rats

Peptides showing inhibitory activity against the angiotensin I-converting enzyme (ACE) were investigated from the fibroin fraction of discarded silk fabric. Fibroin, which was hydrolyzed with alcalase after partial hydrolysis with hot aqueous 40% CaCl(2), released two major active peptides showing ACE-inhibitory activity. The two peptides were identified as glycyl-valyl-glycyl-tyrosine (GVGY) and glycyl-valyl-glycyl-alanyl-glycyl-tyrosine (GVGAGY) by analyses with a protein sequencer and LC/MS/MS. GVGY, whose ACE-inhibitory activity has not previously been reported, showed a blood pressure-depressing effect on spontaneously hypertensive rat (SHR).     

Met-enkephalin-Arg6-Phe7 metabolism: Conversion to Met-enkephalin by brain and kidney dipeptidyl carboxypeptidases

Enzymes degrading Met-enkephalin-Arg⁶-Phe⁷, an endogenous brain peptide with enhanced opiate activity in vivo, were isolated from membrane preparations of rabbit kidney and brain, and their specificity compared. A preparation from kidney or brain containing the angiotensin converting enzyme (EC 3.4.15.1) released with time Arg-Phe, Met-enkephalin, Phe-Met and Tyr-Gly-Gly. Kinetic analysis revealed a product precursor relationship with conversion of hepta- to pentapeptide (Met-enkephalin) followed by release of Tyr-Gly-Gly and Phe-Met indicating sequential cleavage at the Met⁵-Arg⁶ and Gly³-Phe⁴ bonds. A second preparation devoid of angiotensin converting enzyme activity released the same products and in addition a tetrapeptide Phe-Met-Arg-Phe. Release of products with time indicated cleavage at Gly³-Phe⁴ by an endopeptidase and at the Met⁵-Arg⁶ and Gly³-Phe⁴ bonds by a dipeptidyl carboxypeptidase. The dipeptidyl carboxypeptidases thus provide a mechanism for the formation of Met-enkephalin from a potential precursor.     

人源血管紧张素转化酶-C结构域在毕赤酵母中的表达

血管紧张素转化酶 (ACE,EC3.4.15.1) 在调节血压方面具有重要作用,研究证实,ACE-C结构域是体内使血管紧张素I (AngI) 分解的主要活性位点。PCR扩增ACE-C结构域基因片段,克隆至分泌表达载体pPIC9K,转化毕赤酵母GS115,阳性克隆再次电转,用G418筛选高拷贝的酵母菌落进行表达条件优化,获得0.5 g/L的蛋白表达量及7.178 U/mL的酶活力。经Ni+亲和层析纯化,获得纯度大于97％的目的蛋白。Captopril对酶的抑制试验证明ACE-C结构域可望成为新一代抗高血压药物ACE抑制剂筛选的理想酶靶。     

Purification of angiotensin I converting enzyme from pig lung using concanavalin-A sepharose chromatography

Angiotensin I converting enzyme (ACE) plays a major role in blood pressure regulation, catalyzing the conversion of angiotensin I to the vasoconstrictor angiotensin II. In this report we describe a two-step affinity chromatography method for preparative purification of ACE from pig lung using Concanavalin-A Sepharose 4B and affinity chromatography on Lisinopril Sepharose 6B. The same purification scheme was used to obtain Cobalt-ACE, where zinc ion located at the active site is replaced by cobalt. Cobalt-ACE visible spectrum shows a characteristic broad peak from 500 to 600 nm. The shape and maximum absorptivity of this peak changes in presence of ACE inhibitors that bind at the catalytic site.     

人粪便宏基因组α-L-鼠李糖苷酶的酶学性质及其对芦丁的生物转化

HFM-rha78是本实验室从健康人体粪便宏基因组DNA中获得的新型细菌源α-L-鼠李糖苷酶基因。该基因全长2 166 bp，编码蛋白质分子质量为83 kD，等电点5.58，其二级结构以α螺旋和无规卷曲为主，整体表现出亲水性，其氨基酸序列与GenBank收录的序列一致性仅为48%。系统进化树表明，该酶被分类至亚家族Ⅱ，其催化广义酸和广义碱均为谷氨酸，催化残基高度保守。为明确其酶学性质，以对硝基苯酚-α-L-吡喃鼠李糖苷（p-nitrophenyl-α-L-rhamnopyranoside，pNPR）为底物，通过紫外 可见分光光度法测定产物对硝基苯酚的生成量，得到HFM-Rha78的最适反应温度为50℃，最适pH为6.5，且在30～46℃下保温30 min，不影响活力。为初步研究其对芦丁的水解能力，首先对HFM-Rha78的有机溶剂耐受性进行了研究。结果表明，该酶能普遍耐受1%的有机溶剂，甚至1%～ 5%的异丙醇和1%的β 巯基乙醇，有提高其活性的能力。当DMSO浓度为10%时，仍能保持79%的酶活性。HFM Rha78对底物pNPR的Vmax为129.9 μmol min-1 mg-1，HFM-Rha78的KM较大，为26.0 mmol L-1。转换数kcat为185.1 s-1，催化效率kcat/KM为7.1×103 s-1 mol-1 L。采用高效液相色谱仪检测HFM-Rha78水解芦丁的能力。结果表明，37℃条件下，随着酶浓度和水解时间增加，芦丁转化率增大。反应10 h时，芦丁转化率达41.8%。本研究获得了HFM-Rha78的最适反应条件，并确定其可特异性生物转化芦丁为异槲皮素。     

Casein fermentate of Lactobacillus animalis DPC6134 contains a range of novel propeptide angiotensin-converting enzyme inhibitors

This work evaluated the angiotensin-converting-enzyme (ACE)-inhibitory activities of a bovine sodium caseinate fermentate generated using the proteolytic capabilities of the porcine small intestinal isolate Lactobacillus animalis DPC6134 (NCIMB deposit 41355). The crude 10-kDa L. animalis DPC6134 fermentate exhibited ACE-inhibitory activity of 85.51% (+/-15%) and had a 50% inhibitory concentration (IC50) of 0.8 mg protein/ml compared to captopril, which had an IC50 value of 0.005 mg/ml. Fractionation of the crude L. animalis DPC6134 fermentate by membrane filtration and reversed-phase high-performance liquid chromatography (HPLC) generated three bioactive fractions from a total of 72 fractions. Fractions 10, 19, and 43 displayed ACE-inhibitory activity percentages of 67.53 (+/-15), 83.71 (+/-19), and 42.36 (+/-11), respectively, where ACE inhibition was determined with 80 microl of the fractions with protein concentrations of 0.5 mg/ml. HPLC and mass spectrometry analysis identified 25 distinct peptide sequences derived from alpha-, beta-, and kappa-caseins. In silico predictions, based on the C-terminal tetrapeptide sequences, suggested that peptide NIPPLTQTPVVVPPFIQ, corresponding to beta-casein f(73-89); peptide IGSENSEKTTMP, corresponding to alpha(s1)-casein f(201212); peptide SQSKVLPVPQ, corresponding to beta-casein f(166-175); peptide MPFPKYPVEP, corresponding to beta-casein f(124133); and peptide EPVLGPVRGPFP, corresponding to beta-casein f(210-221), contained ACE-inhibitory activities. These peptides were chosen for chemical synthesis to confirm the ACE-inhibitory activity of the fractions. Chemically synthesized peptides displayed IC50 values in the range of 92 microM to 790 microM. Additionally, a simulated gastrointestinal digestion confirmed that the ACE-inhibitory 10-kDa L. animalis DPC6134 fermentation was resistant to a cocktail of digestive enzymes found in the gastrointestinal tract.     

Angiotensin I-converting-enzyme-inhibitory and antibacterial peptides from Lactobacillus helveticus PR4 proteinase-hydrolyzed caseins of milk from six species

Sodium caseinates prepared from bovine, sheep, goat, pig, buffalo or human milk were hydrolyzed by a partially purified proteinase of Lactobacillus helveticus PR4. Peptides in each hydrolysate were fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest angiotensin I-converting-enzyme (ACE)-inhibitory or antibacterial activity were sequenced by mass spectrum and Edman degradation analyses. Various ACE-inhibitory peptides were found in the hydrolysates: the bovine alpha(S1)-casein (alpha(S1)-CN) 24-47 fragment (f24-47), f169-193, and beta-CN f58-76; ovine alpha(S1)-CN f1-6 and alpha(S2)-CN f182-185 and f186-188; caprine beta-CN f58-65 and alpha(S2)-CN f182-187; buffalo beta-CN f58-66; and a mixture of three tripeptides originating from human beta-CN. A mixture of peptides with a C-terminal sequence, Pro-Gly-Pro, was found in the most active fraction of the pig sodium caseinate hydrolysate. The highest ACE-inhibitory activity of some peptides corresponded to the concentration of the ACE inhibitor (S)-N-(1-[ethoxycarbonyl]-3-phenylpropyl)-ala-pro maleate (enalapril) of 49.253 micro g/ml (100 micro mol/liter). Several of the above sequences had features in common with other ACE-inhibitory peptides reported in the literature. The 50% inhibitory concentration (IC(50)) of some of the crude peptide fractions was very low (16 to 100 micro g/ml). Some identified peptides were chemically synthesized, and the ACE-inhibitory activity and IC(50)s were confirmed. An antibacterial peptide corresponding to beta-CN f184-210 was identified in human sodium caseinate hydrolysate. It showed a very large spectrum of inhibition against gram-positive and -negative bacteria, including species of potential clinical interest, such as Enterococcus faecium, Bacillus megaterium, Escherichia coli, Listeria innocua, Salmonella spp., Yersinia enterocolitica, and Staphylococcus aureus. The MIC for E. coli F19 was ca. 50 micro g/ml. Once generated, the bioactive peptides were resistant to further degradation by proteinase of L. helveticus PR4 or by trypsin and chymotrypsin.     

Bio-functional pickles that reduce blood pressure of rats

Addition of γ-aminobutyric acid (GABA) and angiotensin converting enzyme (ACE)-inhibitory peptides to the pickles was studied in order to develop a new type of pickles that reduce blood pressure. Based on the outcome of these studies, a new type of fermentation bed composed of rice bran and white miso has been successfully developed. The advantage of such pickles is that they not only contain both GABA and ACE-inhibitory peptides, but also that their taste and flavor are excellent, with colors close to the original ones. The new type of pickles could temporarily reduce blood pressure in two types of rats, spontaneously hypertensive rats and NaCl-sensitive model rats. Thus, the newly developed pickles appear to be beneficial for pickle business.     

Characterization of Bovine Atrial Angiotensin-Converting Enzyme

Bovine atrial angiotensin-converting enzyme (ACE) was purified to electrophoretic homogeneity. The purification procedure included ion-exchange chromatography on DEAE-Toyopearl 650M, affinity chromatography on lisinopril-agarose and gel filtration on Sephadex G-100. The bovine atrial ACE exhibited similar sensitivities to inhibition by lisinopril and captopril as lung ACE (the K _i values for the atrial and lung enzymes differed insignificantly). However, the kinetic parameters of hydrolysis of some synthetic tripeptide substrates (FA-Phe-Gly-Gly, FA-Phe-Phe-Arg, Cbz-Phe-His-Leu, Hip-His-Leu) catalyzed by bovine atrial and lung ACE varied to a greater extent. The enzymes were also characterized by some differences in activation by chloride, nitrate, and sulfate anions. These data support the hypothesis of tissue specificity of ACEs.     

Antibacterial and proteolytic activity in venom from the endoparasitic wasp Pimpla hypochondriaca (Hymenoptera: Ichneumonidae)

Venom from the endoparasitic wasp, Pimpla hypochondriaca, is composed of a mixture of high and low molecular weight proteins, possesses phenoloxidase activity, has immunosuppressive properties, and induces paralysis in several insect species. In the present study we demonstrate that P. hypochondriaca venom also contains antibacterial and proteolytic activity. Antibacterial activity was detected against the Gram-negative bacteria Escherichia coli and Xanthamonas campestris but not against Pseudomonas syringae nor against two Gram-positive bacteria, Bacillus cereus and Bacillus subtilis. Endopeptidase and aminopeptidase activity in venom was detected using the synthetic fluorogenic substrates N-t-BOC-Phe-Ser-Arg-AMC, Arg-AMC and Leu-Arg. The aminopeptidase activity towards Arg-AMC was sensitive to amastatin (70% inhibition), an aminopeptidase inhibitor. Angiotensin-converting enzyme (ACE)-like enzyme activity was detected, by reverse-phase HPLC using the synthetic tripeptide Hip-His-Leu as a substrate. This activity was sensitive to captopril, an ACE inhibitor (IC(50) 3.8 x 10(-8) M). Using an antiserum raised against recombinant Drosophila melanogaster ACE-like enzyme, (rAnce), Western blot analysis revealed an immunoreactive protein, with a molecular weight estimate of 74 kDa, in P. hypochondriaca venom. The possibility that the endopeptidase, aminopeptidase and ACE are involved in the processing of peptide precursors in the venom sac is discussed.