A survey of peptidase activity in rumen bacteria.

Twenty-nine strains of 14 species of rumen bacteria were screened for their ability to hydrolyse Ala2, Ala5, GlyArg-4-methoxy-2-naphthylamide (GlyArg-MNA) and Leu-MNA. Several species, notably Megasphaera elsdenii, were active against Ala2, and a smaller number, including Bacteroides ruminicola, Butyrivibrio fibrisolvens, Ruminococcus flavefaciens, Lachnospira multipara and Ruminobacter amylophilus, broke down Ala5. Streptococcus bovis had an exceptionally high leucine arylamidase activity. However, only Ba. ruminicola hydrolysed GlyArg-MNA. Further investigation revealed that only Ba. ruminicola and Bu. fibrisolvens hydrolysed Ala5 to Ala3 and Ala2, with little ALa4 being produced, in a manner similar to rumen fluid. The activity of Ba. ruminicola against synthetic peptidase substrates, including GlyArg-MNA, LysAla-MNA, ArgArg-MNA, GlyPro-MNA, LeuVal-MNA, and Ala3-p-nitroanilide, was similar to that of rumen fluid, whereas the activity of Bu. fibrisolvens was quite different. Since the main mechanism by which peptides are broken down in the rumen is similar to dipeptidyl aminopeptidase type I, for which GlyArg-MNA is a diagnostic substrate, it was concluded that Ba. ruminicola was the most important single species in peptide breakdown in the rumen.     

Purification and Properties of a Type II-like Dipeptidyl Peptidase from the Ruminal Peptidolytic Bacterium,Prevotella albensis M384

A dipeptidyl peptidase which hydrolyses the synthetic dipeptidyl peptidase (DPP) substrate, Ala₂- p -nitroanilide, was purified 193-fold from the ruminal peptidolytic bacterium, Prevotella albensis M384. The enzyme was a homodimer of molecular mass 91 kDa. Its activity against Ala₂- p -nitroanilide had optimal pH and temperature of 7.2 and 40°C respectively. Enzyme activity was inhibited by the serine protease inhibitors, PMSF and dichloroisocoumarin, but not by inhibitors of other categories of proteases. Synthetic substrates for DPP-1 (GlyArg- p -nitroanilide, GlyArg-4-methoxy-naphthylamide), DPP-3 (ArgArg-4-methoxynaphthylamide) and DPP-4 (GlyPro-4-methoxynaphthylamide) or for leucine or alanine aminopeptidase were not hydrolysed, nor were di- or tripeptides. N-Acetyl-Ala₂- p -nitroanilide was not hydrolysed. Oligopeptides with Ala, Ile, Ser or Val adjacent to the N-terminal amino acid were all hydrolysed, while peptides with basic or acidic residues in the same position were not. The purified DPP from P. albensis is therefore most similar in its catalytic properties to mammalian DPP-2.     

Analysis of peptide metabolism by ruminal microorganisms

Methods were developed for the determination of oligoalanine and other short-chain peptides and peptide analogs in ruminal fluid by using reverse-phase high-pressure liquid chromatography. Chromatographic analysis of the breakdown of (Ala)3 and (Ala)4 in ruminal fluid in vitro revealed that the predominant mechanism of hydrolysis was a dipeptidyl peptidase-like activity. Hydrolysis proceeded from the N terminal of the peptide chain; N-acetyl-(Ala)3 was broken down at 11% of the rate of breakdown of (Ala)3 or (Ala)3-p-nitroanilide. (Ala)2-p-nitroanilide was hydrolyzed most rapidly of the arylamide substrates tested, but fluorogenic 4-methoxy-2-naphthylamide (MNA) compounds were more convenient and potentially more versatile substrates than p-nitroanilides. Gly-Arg-MNA was the most rapidly hydrolyzed dipeptidyl peptidase substrate, suggesting that ruminal peptidase activity was predominantly of a type I specificity.     

Analysis of peptide metabolism by ruminal microorganisms.

Methods were developed for the determination of oligoalanine and other short-chain peptides and peptide analogs in ruminal fluid by using reverse-phase high-pressure liquid chromatography. Chromatographic analysis of the breakdown of (Ala)3 and (Ala)4 in ruminal fluid in vitro revealed that the predominant mechanism of hydrolysis was a dipeptidyl peptidase-like activity. Hydrolysis proceeded from the N terminal of the peptide chain; N-acetyl-(Ala)3 was broken down at 11% of the rate of breakdown of (Ala)3 or (Ala)3-p-nitroanilide. (Ala)2-p-nitroanilide was hydrolyzed most rapidly of the arylamide substrates tested, but fluorogenic 4-methoxy-2-naphthylamide (MNA) compounds were more convenient and potentially more versatile substrates than p-nitroanilides. Gly-Arg-MNA was the most rapidly hydrolyzed dipeptidyl peptidase substrate, suggesting that ruminal peptidase activity was predominantly of a type I specificity.     

Peptidase activity of human colonic bacteria

Aminopeptidase activities of mixed faecal suspensions from four human donors and 12 of the most numerous species of human colonic bacteria were measured using alanine oligopeptides and various dipeptidyl- and amino acyl-arylamidase substrates. The pattern of hydrolysis of Ala(4)and Ala(5)in faecal suspensions, whereby Ala(2)was the first breakdown product, suggested that the main mechanism of peptide hydrolysis was dipeptidyl peptidase. Dipeptidyl p-nitroanilides and 4-methoxynaphthylamides were broken down more rapidly than amino acyl derivatives in three out of four individuals tested, consistent with this conclusion. The predominant Bacteroides spp. of the intestine also had greater dipeptidyl peptidase activity than amino acyl aminopeptidase activity, while Bifidobacterium, Clostridium, Enterococcus and Propionibacterium spp. had a more variable pattern of peptidase activities. Thus, peptide hydrolysis in the human intestine, as in the rumen, appears to be mainly a two-stage process which is initiated by dipeptidyl peptidases present in the most numerous Bacteriodes spp.     

Selective isolation of bacteria with dipeptidyl aminopeptidase type I activity from the sheep rumen

Five-hundred-and-six fresh isolates of rumen bacteria were tested for their ability to hydrolyse the synthetic substrate for dipeptidyl aminopeptidase type I, GlyArg-4-methoxy-2-naphthylamide (GlyArg-MNA), using a gel overlay technique. Twelve positive isolates were small Gram-negative rods which resembled Bacteroides ruminicola in their biochemical and morphological properties. SDS-PAGE of whole cell extracts indicated that two were similar to B. ruminicola strain B14, six resembled B. ruminicola strain M384, and four were similar to B. ruminicola GA33. All hydrolysed GlyArg-MNA, Ala2 and Ala5, and showed no activity against Leu-MNA. Ala3 and Ala2, but no Ala4, was produced from Ala5. The different groups had different, distinctive activity profiles. The two remaining positive isolates were Lactobacillus spp. with an exceptionally high Leu-MNA activity. It was concluded that, although different strains may only be distantly related, B. ruminicola forms the most important group of bacteria in the rumen to possess a dipeptidyl aminopeptidase type I activity.     

Separation of rat muscle aminopeptidases.

By means of chromatography on DEAE-Sephadex, two arylamidases (hydrolysing L-arginine 2-naphthylamide) and three dipeptidyl peptidases (hydrolysing dipeptide 2-naphthylamides) were distinguished in extracts of rat muscle. However, the arylamidase from the larger peak also hydrolysed the dipeptide 2-naphthylamides. Glycyl-L-arginine amide, an alternative substrate for dipeptidyl peptidase I, was not hydrolysed by arylamidase. L-Leucine amide was hydrolysed by an enzyme, presumed to be leucine aminopeptidase, from a separate peak, but was also hydrolysed by arylamidase. Arylamidase, dipeptidyl peptidase III and most of the leucine aminopeptidase could be extracted from the muscle with a neutral salt solution, but dipeptidyl peptidase I was extracted only in the presence of Triton X-100; dipeptidyl peptidase II showed an intermediate extraction behaviour.     

Ala657 and conserved active site residues promote fibroblast activation protein endopeptidase activity via distinct mechanisms of transition state stabilization

Fibroblast activation protein (FAP) and dipeptidyl peptidase-4 (DPP-4) are highly homologous serine proteases of the prolyl peptidase family and therapeutic targets for cancer and diabetes, respectively. Both proteases display dipeptidyl peptidase activity, but FAP alone has endopeptidase activity. FAP Ala657, which corresponds to DPP-4 Asp663, is important for endopeptidase activity; however, its specific role remains unclear, and it is unknown whether conserved DPP-4 substrate binding residues support FAP endopeptidase activity. Using site-directed mutagenesis and kinetic analyses, we show here that Ala657 and five conserved active site residues (Arg123, Glu203, Glu204, Tyr656, and Asn704) promote FAP endopeptidase activity via distinct mechanisms of transition state stabilization (TSS). The conserved residues provide marked TSS energy for both endopeptidase and dipeptidyl peptidase substrates, and structural modeling supports their function in binding both substrates. Ala657 also stabilizes endopeptidase substrate binding and additionally dictates FAP reactivity with transition state inhibitors, allowing tight interaction with tetrahedral intermediate analogues but not acyl-enzyme analogues. Conversely, DPP-4 Asp663 stabilizes dipeptidyl peptidase substrate binding and permits tight interaction with both transition state analogues. Structural modeling suggests that FAP Ala657 and DPP-4 Asp663 confer their contrasting effects on TSS by modulating the conformation of conserved residues FAP Glu204 and DPP-4 Glu206. FAP therefore requires the combined function of Ala657 and the conserved residues for endopeptidase activity.     

Cleavage of di- and tripeptides by Prevotella ruminicola

The final step in the conversion of protein to amino acids by the common Gram-negative rumen bacterium, Prevotella (formerly Bacteroides) ruminicola , is the cleavage of di- and tripeptides. Dipeptidase and tripeptidase activities were predominantly cytoplasmic, and toluene treatment increased the rate of Ala2 and Ala3 hydrolysis by whole cells, suggesting that transport limited the rate of hydrolysis of extracellular di- and tripeptides. The hydrolysis of Ala2 and Ala3 by whole cells was not affected by protonophores, ionophores or dicyclohexylcarbodiimide, but Ala2 hydrolysis by EDTA-treated cells was inhibited by the Ca2+/H+ ionophore, tetronasin. Ala3 hydrolysis was not affected by protonophores or ionophores in EDTA-treated cells. The dipeptidase of strain M384 was inhibited > 99% by 1,10-phenanthroline and 39% by EDTA but not other protease inhibitors, consistent with the enzyme being a metalloprotease. Tripeptidase was insensitive to protease inhibitors, except for a 33% inhibition by EDTA. Cleavage of tripeptides occurred at the bond adjacent to the N-terminal amino acid. Distinct di-, tri- and oligopeptidase peaks were obtained by anion-exchange liquid chromatography of disrupted cells. Banding patterns on native PAGE using activity staining also indicated that P. ruminicola M384 had separate single dipeptidase and tripeptidase enzymes which hydrolysed a range of peptides. The dipeptidase of strain M384 was different from other strains of P. ruminicola: strains GA33 and B(1)4 had activities which ran at the same R(f); strain GA33 had another band of lower activity; strain 23 had two bands different from those of the other strains. The tripeptidases ran at the same R(f) for the different strains. Dipeptidase activity of all strains was inhibited by 1,10-phenanthroline on gels. Gel permeation chromatography indicated that the M(r) of the dipeptidases from strains M384 and B(1)4 were 115,000 and 114,500 respectively, and 112,500 and 121,500 for the corresponding tripeptidases. Thus the metabolism of small peptides by P. ruminicola involves separate permeases and intracellular peptidases for di- and tripeptides.     

Purification of Two Dipeptidyl Aminopeptidases II from Rat Brain and Their Action on Proline-Containing Neuropeptides

From the soluble and membrane fractions of rat brain homogenate, two enzymes that liberate dipeptides of the type Xaa-Pro from chromogenic substrates were purified to homogeneity. The two isolated dipeptidyl peptidases had similar molecular and catalytic properties: For the native proteins, molecular weights of 110,000 were estimated; for the denatured proteins, the estimate was 52,500. Whereas the soluble peptidase yielded one band of pI 4.2 after analytical isoelectric focusing, two additional enzymatic active bands were detected between pI 4.2 and 4.3 for the membrane-associated form. As judged from identical patterns after neuraminidase treatment, both peptidases contained no sialic acid. A pH optimum of 5.5 was estimated for the hydrolysis of Gly-Pro- and Arg-Pro-nitroanilide. Substrates with alanine instead of proline in the penultimate position were hydrolyzed at comparable rates. Acidic amino acids in the ultimate N-terminal position of the substrates reduced the activities of the peptidases 100-fold as compared with corresponding substrates with unblocked neutral or, especially, basic termini. The action of the dipeptidyl peptidase on several peptides with N-terminal Xaa-Pro sequences was investigated. Tripeptides were rapidly hydrolyzed, but the activities considerably decreased with increasing chain length of the peptides. Although the tetrapeptide substance P 1-4 was still a good substrate, the activities detected for the sequential liberation of Xaa-Pro dipeptides from substance P itself or casomorphin were considerably lower. Longer peptides were not cleaved. The peptidases hydrolyzed Pro-Pro bonds, e.g., in bradykinin 1-3 or 1-5 fragments, but bradykinin itself was resistant. The enzymes were inhibited by serine protease inhibitors, like diisopropyl fluorophosphate or phenylmethylsulfonyl fluoride, and by high salt concentrations but not by the aminopeptidase inhibitors bacitracin and bestatin. Based on the molecular and catalytic properties, both enzymes can be classified as species of dipeptidyl peptidase II (EC 3.4.14.2) rather than IV (EC 3.4.14.5). However, some catalytic properties differentiate the brain enzyme from forms of dipeptidyl peptidase II of other sources.     

Selective inhibition of fibroblast activation protein protease based on dipeptide substrate specificity

Fibroblast activation protein (FAP) is a transmembrane serine peptidase that belongs to the prolyl peptidase family. FAP has been implicated in cancer; however, its specific role remains elusive because inhibitors that distinguish FAP from other prolyl peptidases like dipeptidyl peptidase-4 (DPP-4) have not been developed. To identify peptide motifs for FAP-selective inhibitor design, we used P(2)-Pro(1) and acetyl (Ac)-P(2)-Pro(1) dipeptide substrate libraries, where P(2) was varied and substrate hydrolysis occurs between Pro(1) and a fluorescent leaving group. With the P(2)-Pro(1) library, FAP preferred Ile, Pro, or Arg at the P(2) residue; however, DPP-4 showed broad reactivity against this library, precluding selectivity. By contrast, with the Ac-P(2)-Pro(1) library, FAP cleaved only Ac-Gly-Pro, whereas DPP-4 showed little reactivity with all substrates. FAP also cleaved formyl-, benzyloxycarbonyl-, biotinyl-, and peptidyl-Gly-Pro substrates, which DPP-4 cleaved poorly, suggesting an N-acyl-Gly-Pro motif for inhibitor design. Therefore, we synthesized and tested the compound Ac-Gly-prolineboronic acid, which inhibited FAP with a K(i) of 23 +/- 3 nm. This was approximately 9- to approximately 5400-fold lower than the K(i) values for other prolyl peptidases, including DPP-4, DPP-7, DPP-8, DPP-9, prolyl oligopeptidase, and acylpeptide hydrolase. These results identify Ac-Gly-BoroPro as a FAP-selective inhibitor and suggest that N-acyl-Gly-Pro-based inhibitors will allow testing of FAP as a therapeutic target.     

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

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

Novel tetrahydropyran analogs as dipeptidyl peptidase IV inhibitors: Profile of clinical candidate (2R,3S,5R)-2-(2,5-difluorophenyl)-5-[2-(methylsulfonyl)-2,6-dihydropyrrolo[3,4-c]pyrazol-5(4H)-yl]tetrahydro-2H-pyran-3-amine (23)

A series of novel tri-2,3,5-substituted tetrahydropyran analogs were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-4) for the treatment of type 2 diabetes. Optimization of the series provided inhibitors with good DPP-4 potency and selectivity over other peptidases (QPP, DPP8, and FAP). Compound 23, which is very potent, selective, efficacious in the diabetes PD model, and has an excellent pharmacokinetic profile, is selected as a clinical candidate.     

Mechanism of proline-specific proteinases: (I) Substrate specificity of dipeptidyl peptidase IV from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum

The substrate specificity of dipeptidyl peptidase IV (dipeptidyl peptide hydrolase, EC 3.4.14.5) from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum, was investigated with a series of N-terminal unprotected (dipeptidyl peptidases IV) and succinylated dipeptidyl-p-nitroanilides (proline-specific endopeptidase). Both enzymes are specific for the S configuration of the amino-acid residue in P1 and P2 position if the penultimate residue is proline. In the case of alanine substrates (Ala in P1, dipeptidyl peptidase IV hydrolyzes such compounds where the configuration of the P2 residue is R. The penultimate residue with dipeptidyl peptidase IV can be, beside proline and alanine, dehydroproline, hydroxyproline and pipecolic acid. Proline substrates (Pro in P1) with an R configuration in P2 are inhibitors of the hydrolysis of proline substrates with an S,S configuration in an uncompetitive (dipeptidyl peptide IV) or mixed inhibition type (proline-specific endopeptidase). Derivatives of Gly-Pro-pNA where the N-terminal amino group is methylated are hydrolyzed by dipeptidyl peptidase IV.     

Extended investigation of the substrate specificity of dipeptidyl peptidase IV from pig kidney.

The substrate specificity of dipeptidyl peptidase IV (dipeptidyl peptide hydrolase, EC 3.4.14.5) from pig kidney was investigated, using a series of substrates, in which the amino-acid residue in position P1, a structural derivative of proline, was altered with respect to ring size and substituents. It was demonstrated that dipeptidyl peptidase IV hydrolyses substrates of the type Ala-X-pNA, where X is proline (Pro), (R)-thiazolidine-4-carboxylic acid (Thz), (S)-pipecolic acid (Pip), (S)-oxazolidine-4-carboxylic acid (Oxa), or (S)-azetidine-2-carboxylic acid (Aze). The ring size and ring structure of the residue in the P1 position influence the rate of enzyme-catalysed hydrolysis of the substrate. The highest kcat value (814 s-1) was found for Ala-Aze-pNA. In contrast, the kcat value for Ala-Pro-pNA is nearly 55 s-1. With all substrates of this series, the rate-limiting step of the hydrolysis by dipeptidyl peptidase IV is the deacylation reaction. Compounds of substrate-like structure, in which the P2 residue has an R-configuration, are not hydrolysed by dipeptidyl peptidase IV.     

Multiple intracellular peptidases in Neurospora crassa.

Neurospora crassa possesses multiple intracellular peptidases which display overlapping substrate specificities. They were readily detected by an in situ staining procedure for peptidases separated in polyacrylamide gels, within which the auxilliary enzyme, l-amino acid oxidase, was immobilized. Eleven different intracellular peptidases were identified by electrophoretic separation and verified by their individual patterns of substrate specificities. Most peptide substrates tested were hydrolyzed by several different peptidases. The multiple intracellular peptidases may play overlapping roles in several basic cell processes which involve peptidase activity. The amount of peptidase activity for leucylglycine present in crude extracts of cells grown under widely different conditions was relatively constant, suggesting that this enzyme may be constitutive, although alterations in the amounts of individual peptidase isozymes may occur. A single enzyme, designated peptidase II, was partially purified and obtained free from the other peptidase species. Peptidase II was found to be an aminopeptidase with activity toward many peptides of varied composition and size. It was more active with tripeptides than homologous dipeptides and showed strong activity toward methionine-containing peptides. This enzyme, with a molecular weight of about 37,000, was thermolabile at 65 degrees C and was strongly inhibited by p-hydroxymercuribenzoate, Zn(2+), Co(2+), and Mn(2+), but was insensitive to the serine protease inhibitor phenylmethylsulfonyl fluoride. Peptidase II apparently possesses an essential sulfhydryl group and may be a metalloenzyme.     

Comparative peptide specificity of cell wall, membrane and intracellular peptidases of group N streptococci

Solubilized cell walls of group N streptococci contain two electrophoretically distinct peptidases, one of which hydrolysed trileucine only, while the second hydrolysed a wide range of di- and tripeptides. Neither enzyme possessed leucine aminopeptidase or endopeptidase activity. Four and three peptidases, respectively, were separated in intracellular extracts of Streptococcus lactis subsp. lactis and Strep, lactis subsp. cremoris produced by osmotic lysis of spheroplasts. In contrast with the cell-wall extracts, two of the peptidases had broad specificites, though only one of these hydrolysed trileucine. Purified membranes of Strep. lactis subsp. lactis contained only one electrophoretically distinct peptidase of very narrow specificity. There were small differences between the numbers of peptides hydrolysed by cell wall preparations from milk-grown or broth-grown cells.     

Comparative peptide specificity of cell wall, membrane and intracellular peptidases of group N streptococci

Solubilized cell walls of group N streptococci contain two electrophoretically distinct peptidases, one of which hydrolysed trileucine only, while the second hydrolysed a wide range of di- and tripeptides. Neither enzyme possessed leucine aminopeptidase or endopeptidase activity. Four and three peptidases, respectively, were separated in intracellular extracts of Streptococcus lactis subsp. lactis and Strep. lactis subsp. cremoris produced by osmotic lysis of spheroplasts. In contrast with the cell-wall extracts, two of the peptidases had broad specificites, though only one of these hydrolysed trileucine. Purified membranes of Strep. lactis subsp. lactis contained only one electrophoretically distinct peptidase of very narrow specificity. There were small differences between the numbers of peptides hydrolysed by cell wall preparations from milk-grown or broth-grown cells.     

A dipeptidyl carboxypeptidase in brain synaptic membranes active in the metabolism of enkephalin containing peptides

A dipeptidyl carboxypeptidase activity has been localized in synaptic plasma membranes which have been prepared from isolated rat brain cortical synaptosomes. The specificity of this proteolytic activity towards various synthetic and biological active peptides is compared to the peptidase activities of intact synaptosomes. In contrast to the synaptosomal peptidases which are capable of cleaving all peptide bonds of Met-enkephalin-Arg6-Phe7 the peptidase activity associated with the synaptic plasma membrane exclusively hydrolyses a dipeptide from the carboxyl terminus of all hepta- and hexapeptides tested. The fact that this dipeptidyl carboxypeptidase does not cleave the Gly3-Phe4 peptide bond of Met-enkephalin suggests that this enzyme is different from "enkephalinase". The synaptic membrane dipeptidyl carboxypeptidase is inhibited by metal chelating agents and thiols but is not affected by compounds known to inhibit serine proteases, thermolysin and "enkephalinase".     

Aminopeptidase P from rat brain. Purification and action on bioactive peptides.

Aminopeptidase P (EC 3.4.11.9) was purified from rat brain cytosol. A subunit Mr of 71,000 was determined for the reduced, denaturated protein whereas an Mr of 143,000 was determined for the native enzyme. The purified aminopeptidase P selectively liberated all unblocked, preferentially basic or hydrophobic ultimate amino acids from di-, tri- and oligopeptides with N-terminal Xaa-Pro- sequences. Corresponding peptides with penultimate Ala instead of Pro were cleaved with much lower rates; oligopeptides with residues other than Pro or Ala in the penultimate position appeared not to be substrates for the enzyme. Several bioactive peptides with Xaa-Pro sequences, especially bradykinin, substance P, corticortropin-like intermediate lobe peptide, casomorphin and [Tyr]melanostatin were shortened by the N-terminal amino acid by aminopeptidase P action. Rat brain aminopeptidase P was optimally active at pH 7.6-8.0 in the presence of Mn2+. Chelating agents and SH-reacting reagents inhibited the enzyme, but common inhibitors of aminopeptidases, like amastatin or bestatin, of prolidase or of dipeptidyl peptidases II and IV, like N-benzoyloxycarbonyl-proline or epsilon-benzyl-oxycarbonyl-lysyl-proline, as well as antibiotics like beta-lactam ones, bacitracin or puromycin, had little or no effect.