A bioluminescent probe for in vivo imaging of pyroglutamate aminopeptidase in a mouse model of inflammation.

Pyroglutamate aminopeptidase (PGP) specifically cleaves the peptide bond of pyroglutamic acid linked to the N-terminal end of a polypeptide or protein. Previous studies showed that PGP was associated with several physiological processes and diseases especially those involving inflammation. Utilizing a 'caging' strategy, we designed and synthesized a bioluminescence probe (PBL) with a limit-of-detection of 3.7 * 10-4 mU/mL. In vivo imaging in a mouse model of inflammatory liver disease revealed that the probe has excellent sensitivity and selectivity and provides a powerful tool for studying the physiological and pathological processes involving PGP.

Crystal structure analysis of pyrrolidone carboxyl peptidase from Thermus thermophilus.

Pyrrolidone carboxyl peptidase (PCP) hydrolytically removes the L-pyroglutamic acid from the amino terminal region of pyroglutamyl proteins or peptides. So far, only a limited number of structures of PCP have been solved. Here we report the crystal structure of pyrrolidone carboxyl peptidase from Thermus thermophilus (TtPCP) which has been solved using the molecular replacement method and refined at 1.9 Å resolution. TtPCP follows the α/ß/α architecture in which the central ß-sheets are surrounded by α-helices on both sides. The inter subunit contact between two monomers consists of two short antiparallel ß-strands and part of a long protrusion loop. By comparing the TtPCP with its structural homologs, we identified the putative catalytic triad residues as Glu76, Cys139 and His160. A unique disulfide link found in some homologs of TtPCP, formed between two monomers that provide thermal stability to the protein, is not observed in TtPCP. Hence, being a thermophilic protein, the putative thermal stability of TtPCP could be due to more intra and inter-molecular hydrogen bonds, hydrophobic and ion pair interactions when compared with its mesophilic counterpart. The structural details of TtPCP will be helpful to understand the basis of the intrinsic stability of thermophilic proteins. Also, it could be useful for protein engineering.

Circ-PGPEP1 augments renal cell carcinoma proliferation, Warburg effect, and distant metastasis.

Circular RNAs (circRNAs) contribute to the malignant phenotype and progression of several types of human cancers, including renal cell carcinoma (RCC). This study probed the molecular mechanism of circPGPEP1 regulating RCC proliferation, Warburg effect, and distant metastasis by targeting the miR-378a-3p/JPT1 axis. Here identified higher circPGPEP1 expression in RCC tissues and cells by RT-qPCR, and high levels of circPGPEP1 were positively correlated with high histological grade and distant metastasis in RCC patients. Furthermore, patients with high levels of circPGPEP1 had a worse survival prognosis. Functional assays presented that knockdown of circPGPEP1 inhibited RCC proliferation, invasion, migration, EMT, and Warburg effect. Dual-luciferase reporter assay, RNA immunoprecipitation, nucleoplasmic RNA isolation, and functional rescue experiments confirmed that circPGPEP1 induced JPT1 expression by sponging miR-378a-3p, thereby promoting RCC malignant phenotype. Xenograft assays and metastasis models further demonstrated that down-regulation of circPGPEP1 effectively inhibited tumor growth and distant metastasis of RCC. Taken together, circPGPEP1, a prognostic circRNA in RCC, acts through the miR-378a-3p/JPT1 axis to regulate RCC progression.

A ratiometric fluorescent sensor for rapid detection of the pyroglutamate aminopeptidase-1 in mouse tumors.

Pyroglutamate aminopeptidase-1 (PGP-1) is an important enzyme that plays an indispensable role in the process of inflammation. Up to now, few reports have been reported on the detection of PGP-1 activity in vivo and in vitro, and there are no reports on ratiometric detection. Here, the first red-emitting ratiometric fluorescent sensor (DP-1) for the specific detection of PGP-1 both in vivo and in vitro was designed and synthesized by using DCD-NH2 as the luminescent parent and pyroglutamate as a recognition group. After interacting with PGP-1, the amide bond is hydrolyzed by the enzyme and the color of the solution changes from yellow (λabs = 420 nm) to red (λabs = 520 nm), accompanied by obvious fluorescence emission wavelength change (from ∼564 nm to ∼616 nm). The probe has high specificity and sensitivity towards PGP-1 in about 10 min, and the DL is as low as 0.25 ng mL-1. Interestingly, under the stimulation of Freund's incomplete adjuvant and lipopolysaccharide, the imaging of DP-1 in HepG2 and RAW264 cells shows that the expression of PGP-1 is associated with inflammation. What's more, for the first, the imaging of a mouse tumor model confirms that the enzyme is closely related to the occurrence of some inflammation and tumor diseases. These results indicate that DP-1 can be used as an effective tool for real-time monitoring of PGP-1 levels both in vivo and in vitro and the study of inflammatory tumor pathology.

Two-photon fluorogenic probe for visualizing PGP-1 activity in inflammatory tissues and serum from patients.

A PGP-1-specific one/two-photon fluorogenic probe (BH1), capable of high sensitivity, super selectivity, and visual imaging of endogenous PGP-1 activity from live mammalian cells and serum/skin tissues from patients by using one/two-photon fluorescence microscopy (O/TPFM).

The Role of High Fat Diets and Liver Peptidase Activity in the Development of Obesity and Insulin Resistance in Wistar Rats.

High-fat diets (HFD) have been widely associated with an increased risk of metabolic disorders and overweight. However, a high intake of sources that are rich in monounsaturated fatty acids has been suggested as a dietary agent that is able to positively influence energy metabolism and vascular function. The main objective of this study was to analyze the role of dietary fats on hepatic peptidases activities and metabolic disorders. Three diets: standard (S), HFD supplemented with virgin olive oil (VOO), and HFD supplemented with butter plus cholesterol (Bch), were administered over six months to male Wistar rats. Plasma and liver samples were collected for clinical biochemistry and aminopeptidase activities (AP) analysis. The expression of inducible nitric oxide synthase (iNOS) was also determined by Western blot in liver samples. The diet supplement with VOO did not induce obesity, in contrast to the Bch group. Though the VOO diet increased the time that was needed to return to the basal levels of plasma glucose, the fasting insulin/glucose ratio and HOMA2-%B index (a homeostasis model index of insulin secretion and valuation of ß-cell usefulness (% ß-cell secretion)) were improved. An increase of hepatic membrane-bound dipeptidyl-peptidase 4 (DPP4) activity was found only in VOO rats, even if no differences in fasting plasma glucagon-like peptide 1 (GLP-1) were obtained. Both HFDs induced changes in hepatic pyroglutamyl-AP in the soluble fraction, but only the Bch diet increased the soluble tyrosyl-AP. Angiotensinase activities that are implicated in the metabolism of angiotensin II (AngII) to AngIV increased in the VOO diet, which was in agreement with the higher activity of insulin-regulated-AP (IRAP) in this group. Otherwise, the diet that was enriched with butter increased soluble gamma-glutamyl transferase (GGT) and Leucyl-AP, iNOS expression in the liver, and plasma NO. In summary, VOO increased the hepatic activity of AP that were related to glucose metabolism (DPP4, angiotensinases, and IRAP). However, the Bch diet increased activities that are implicated in the control of food intake (Tyrosine-AP), the index of hepatic damage (Leucine-AP and GGT), and the expression of hepatic iNOS and plasma NO. Taken together, these results support that the source of fat in the diet affects several peptidases activities in the liver, which could be related to alterations in feeding behavior and glucose metabolism.

Improved recovery of proteome-informative, protein N-terminal peptides by combined fractional diagonal chromatography (COFRADIC).

We previously described a proteome-wide, peptide-centric procedure for sorting protein N-terminal peptides and used these peptides as readouts for protease degradome and xenoproteome studies. This procedure is part of a repertoire of gel-free techniques known as COmbined FRActional DIagonal Chromatography (COFRADIC) and highly enriches for alpha-amino-blocked peptides, including alpha-amino-acetylated protein N-terminal peptides. Here, we introduce two additional steps that significantly increase the fraction of such proteome-informative, N-terminal peptides: strong cation exchange (SCX) segregation of alpha-amino-blocked and alpha-amino-free peptides and an enzymatic step liberating pyroglutamyl peptides for 2,4,6-trinitrobenzenesulphonic acid (TNBS) modification and thus COFRADIC sorting. The SCX step reduces the complexity of the analyte mixture by enriching N-terminal peptides and depleting alpha-amino-free internal peptides as well as proline-starting peptides prior to COFRADIC. The action of pyroglutamyl aminopeptidases prior to the first COFRADIC peptide separation results in greatly diminishing numbers of contaminating pyroglutamyl peptides in peptide maps. We further show that now close to 95% of all COFRADIC-sorted peptides are alpha-amino-acetylated and, using the same amount of starting material, our novel procedure leads to an increased number of protein identifications.

Microbiological characteristics, presumptive identification, and antibiotic susceptibilities of Staphylococcus lugdunensis.

This study validated abbreviated methods for the presumptive identification of Staphylococcus lugdunensis and studied the antibiotic susceptibilities of 106 isolates. The combination of positive responses to ornithine and pyrrolidonyl arylamidase identified all S. lugdunensis isolates. Resistance to penicillin and methicillin was detected in 27 and 5% of isolates, respectively.

Chronic ethanol intake modifies pyrrolidon carboxypeptidase activity in mouse frontal cortex synaptosomes under resting and K+ -stimulated conditions: role of calcium.

Pyrrolidon carboxypeptidase (Pcp) is an omega peptidase that removes pyroglutamyl N-terminal residues of peptides such as thyrotrophin-releasing hormone (TRH), which is one of the neuropeptides that has been localized into many areas of the brain and acts as an endogenous neuromodulator of several parameters related to ethanol (EtOH) consumption. In this study, we analysed the effects of chronic EtOH intake on Pcp activity on mouse frontal cortex synaptosomes and their corresponding supernatant under basal and K+ -stimulated conditions, in presence and absence of calcium (Ca2+) to know the regulation of Pcp on TRH. In basal conditions, chronic EtOH intake significantly decreased synaptosomes Pcp activity but only in absence of Ca2+. However, supernatant Pcp activity is also decreased in presence and absence of calcium. Under K+-stimulated conditions, chronic EtOH intake decreased synaptosomes Pcp activity but only in absence of Ca2+, whereas supernatant Pcp activity was significantly decreased only in presence of Ca2+. The general inhibitory effect of chronic EtOH intake on Pcp activity suggests an inhibition of TRH metabolism and an enhancement of TRH neurotransmitter/neuromodulator functions, which could be related to putative processes of tolerance to EtOH in which TRH has been involved. Our data may also indicate that active peptides and their degrading peptidases are released together to the synaptic cleft to regulate the neurotransmitter/neuromodulator functions of these peptides, through a Ca2+ -dependent mechanism.

Unfolding of the immunoglobulin light and heavy chains is required for the enzymatic removal of N-terminal pyroglutamyl residues.

To enable Edman sequencing of pyroglutamylated immunoglobulins, enzymatic deblocking by pyroglutamate aminopeptidase is performed, often with variable yield and compromised solubility. Recently, enzymatic deblocking of immunoglobulins without denaturation was described. Although the conditions ensured efficient removal of pyroglutamyl residues, we conclude that deblocking is preceded by denaturation, which results in aggregation of the immunoglobulins. To study the effect of folding status on deblocking we developed a methanol based deblocking solution, which preserved the enzymatic activity of pyroglutamate aminopeptidase, provided conditions compatible with sequencing and enhanced deblocking of electroblotted samples, as well. At 50 degrees C and 35% (v/v) methanol the immunoglobulin chains were completely aggregated, but the degree of deblocking was comparable to that obtained with the previously described method. At 37 degrees C, the immunoglobulins were partly aggregated, but the deblocked chains were completely in the insoluble fractions, whereas the soluble fractions had retained pyroglutamylation in both chains, suggesting that unfolding of the immunoglobulins is required for the excision of the pyroglutamates. Inspection of the structures of pyroglutamylated immunoglobulin and pyroglutamate aminopeptidase P. furiosus indicates that the enzyme requires the substrate in an extended conformation, a criterium, which we conclude not to be fulfilled in the native form of immunoglobulins. Unfolding of the N-terminus would disrupt the immunoglobulin fold by breaking interactions between secondary structure elements and expose surfaces prone to aggregation.

The expression of TRH, its receptors and degrading enzyme is differentially modulated in the rat limbic system during training in the Morris water maze.

TRH administration induces arousal, improves cognition, and modulates glutamatergic and cholinergic transmission in hippocampal neurons. To study the possible involvement of TRH neurons in learning and memory processes, gene expression of TRH, its receptors, and pyroglutamyl peptidase (PPII), were measured in limbic regions of water-maze trained rats. Hypothalamus and amygdala showed changes related to the task but not specific to spatial learning while in hippocampus, pro-TRH and TRH-R1 mRNA levels were specifically increased in those animals trained to find a hidden platform. Variation of TRH content and mRNA levels of pro-TRH, TRH-R1, TRH-R2 and PPII are observed in conditions known to activate TRH hypophysiotropic neurons. Changes in some of these parameters could indicate the activation of TRHergic neurons and their possible involvement in some memory related process. Male Wistar rats were immersed (10 times) for 1, 3 or 5 days in a Morris water-maze containing, or not (yoked control) a platform and sacrificed 5, 30 and 60 min after last trial. TRH content and TSH serum levels were determined by radioimmunoassay; mRNA levels of pro-TRH, TRH-R1, TRH-R2, and PPII, by RT-PCR. Exclusive changes due to spatial training were observed in posterior hippocampus of rats trained for 5 days sacrificed after 60min: decreased TRH content and increased mRNA levels of pro-TRH and TRH-R1, particularly in CA3 region (measured by in situ hybridization). The hypothalamus-pituitary axis responded in both yoked and trained animals (increasing serum TSH levels and pro-TRH expression, due to swim-stress); in the amygdala of both groups, pro-TRH expression increased while diminished that of both receptors and PPII. Differential expression of these parameters suggests involvement of TRH hippocampal neurons in memory formation processes while changes in amygdala could relate to TRH anxiolytic role. The differential modulation in anterior and posterior portions of the hippocampus is discussed.

Applications of microwave-assisted proteomics in biotechnology.

Biotechnology has recently celebrated 30 years both as a science and as a multi-billion dollar industry. One application of biotechnology is to use human genetic information to discover, develop, manufacture, and commercialize biotherapeutics. Recombinant proteins can be produced in large quantities at high purity. High-throughput proteomic analysis is at the heart of the biotechnology research and development process, and the industry is constantly striving to streamline and automate the analytical processes involved. Microwave-assisted proteomics has recently emerged as a tool for increasing the bio-catalysis of several processes including tryptic digestions lipase selectivities, identification of metal-catalyzed oxidation sites on proteins, identification of protein N- and C-termini and enzyme catalyzed N-linked deglycosylation. Here, we explore the above mentioned methods, and describe our experiences evaluating microwave-technology for other common proteomic protocols including: removal of N-terminal pyroglutamyl for antibody characterization, beta elimination and Michael addition for identification of phosphorylation sites on recombinant proteins and enzyme mediated O-linked deglycosylation.

Ontogeny of prolyl endopeptidase and pyroglutamyl peptidase I in rat tissues.

Prolyl endopeptidase and pyroglutamyl peptidase I are enzymes which participate in the degradation of thyrotropin-releasing hormone (TRH), a hormone which is thought to play an important role in the development of organs and tissues. Here, we have characterized the ontogeny of TRH degrading enzyme activity in the brain cortex, lung, heart, kidney and liver. Overall, prolyl endopeptidase activity was found to be 2 to 5 fold higher in newborn vs. adult rat tissues, with the exception of the soluble form in the liver and the particulate form in the lung. In contrast, the developmental profile of pyroglutamyl peptidase I activity was found to be more variable and tissue dependent. These results corroborate the idea that both enzymes play important, tissue-specific roles during the development and maturation of rat organs.

Pyroglutamyl peptidase type I from Trypanosoma brucei: a new virulence factor from African trypanosomes that de-blocks regulatory peptides in the plasma of infected hosts.

Peptidases of parasitic protozoans are emerging as novel virulence factors and therapeutic targets in parasitic infections. A trypanosome-derived aminopeptidase that exclusively hydrolysed substrates with Glp (pyroglutamic acid) in P1 was purified 9248-fold from the plasma of rats infected with Trypanosoma brucei brucei. The enzyme responsible was cloned from a T. brucei brucei genomic DNA library and identified as type I PGP (pyroglutamyl peptidase), belonging to the C15 family of cysteine peptidases. We showed that PGP is expressed in all life cycle stages of T. brucei brucei and is expressed in four other blood-stream-form African trypanosomes. Trypanosome PGP was optimally active and stable at bloodstream pH, and was insensitive to host plasma cysteine peptidase inhibitors. Native purified and recombinant hyper-expressed trypanosome PGP removed the N-terminal Glp blocking groups from TRH (thyrotrophin-releasing hormone) and GnRH (gonadotropin-releasing hormone) with a k(cat)/K(m) value of 0.5 and 0.1 s(-1) x microM(-1) respectively. The half-life of TRH and GnRH was dramatically reduced in the plasma of trypanosome-infected rats, both in vitro and in vivo. Employing an activity-neutralizing anti-trypanosome PGP antibody, and pyroglutamyl diazomethyl ketone, a specific inhibitor of type I PGP, we demonstrated that trypanosome PGP is entirely responsible for the reduced plasma half-life of TRH, and partially responsible for the reduced plasma half-life of GnRH in a rodent model of African trypanosomiasis. The abnormal degradation of TRH and GnRH, and perhaps other neuropeptides N-terminally blocked with a pyroglutamyl moiety, by trypanosome PGP, may contribute to some of the endocrine lesions observed in African trypanosomiasis.

Differentiation of Leishmania major is impaired by over-expression of pyroglutamyl peptidase I.

Pyroglutamyl peptidases I (PPI) are cysteine peptidases of the clan CF, family C15, which hydrolyse N-terminal l-pyroglutamyl residues (l-pGlu). The l-pGlu modification is a post-transcriptional modification that confers relative aminopeptidase resistance and, in some cases, is essential to the modified peptides' biological activity. PPIs have been identified in a variety of organisms, although definitive biological functions have yet to be attributed to them. The L. major PPI was expressed in Escherichia coli as active recombinant enzyme, and shown to have biochemical properties more similar to mammalian than bacterial PPIs. The LmPPI active site catalytic triad of E101, C210, and H234 was confirmed by mutagenesis. PPI activity was detected in L. major promastigotes, and the enzyme localised to the parasite cytosol. No detectable phenotype could be observed for L. major PPI-deficient mutants, which retained infectivity to macrophages in vitro and mice. However, over-expression of the active PPI, but not inactive PPI(C210A), in L. major impaired differentiation from the procyclic promastigote to the infective metacyclic promastigote. Susceptibility to a natural l-pGlu-modified antimicrobial peptide, gomesin, was tested using the different cell lines, which were all equally susceptible. Whilst PPI is widespread through the eukaryotic kingdom, this study now suggests that the enzyme is not essential for normal eukaryotic cell function. However, PPI could be involved in regulating the action of l-pGlu-modified peptides required for differentiation of L. major.

Occurrence of the free and Peptide forms of pyroglutamic acid in plasma from the portal blood of rats that had ingested a wheat gluten hydrolysate containing pyroglutamyl peptides.

In order to determine pyroglutamic acid levels in plasma, we developed a method based on precolumn derivatization of the carboxyl group of pyroglutamic acid with 2-nitrophenylhydrazine. Eight-week-old male SD strain rats were administered 200 mg of an acidic peptide fraction obtained from a commercial wheat gluten hydrolysate containing 0.63 mmol/g pyroglutamyl peptide. After administration, significant amounts of free pyroglutamic acid were observed in the ethanol-soluble fraction of the plasma from the portal vein. In addition, pyroglutamate aminopeptidase digestion of the ethanol-soluble fraction liberated significant amounts of pyroglutamic acid, which indicated the presence of the pyroglutamyl peptide. The presence of the pyroglutamyl peptide in the plasma was further confirmed by size exclusion chromatography. The levels of free and peptide forms of pyroglutamic acid increased significantly and reached a maximum (approximately 40 nmol/mL) at 15 and 30 min after administration, respectively.

Structure-activity studies with high-affinity inhibitors of pyroglutamyl-peptidase II.

Inhibitors of PPII (pyroglutamyl-peptidase II) (EC 3.4.19.6) have potential applications as investigative and therapeutic agents. The rational design of inhibitors is hindered, however, by the lack of an experimental structure for PPII. Previous studies have demonstrated that replacement of histidine in TRH (thyrotropin-releasing hormone) with asparagine produces a competitive PPII inhibitor (Ki 17.5 microM). To gain further insight into which functional groups are significant for inhibitory activity, we investigated the effects on inhibition of structural modifications to Glp-Asn-ProNH2 (pyroglutamyl-asparaginyl-prolineamide). Synthesis and kinetic analysis of a diverse series of carboxamide and C-terminally extended Glp-Asn-ProNH2 analogues were undertaken. Extensive quantitative structure-activity relationships were generated, which indicated that key functionalities in the basic molecular structure of the inhibitors combine in a unique way to cause PPII inhibition. Data from kinetic and molecular modelling studies suggest that hydrogen bonding between the asparagine side chain and PPII may provide a basis for the inhibitory properties of the asparagine-containing peptides. Prolineamide appeared to be important for interaction with the S2' subsite, but some modifications were tolerated. Extension of Glp-Asn-ProNH2 with hydrophobic amino acids at the C-terminus led to a novel set of PPII inhibitors active in vitro at nanomolar concentrations. Such inhibitors were shown to enhance recovery of TRH released from rat brain slices. Glp-Asn-Pro-Tyr-Trp-Trp-7-amido-4-methylcoumarin displayed a Ki of 1 nM, making it the most potent competitive PPII inhibitor described to date. PPII inhibitors with this level of potency should find application in exploring the biological functions of TRH and PPII, and potentially provide a basis for development of novel therapeutics.

The amino acid sequence of pancreatic spasmolytic polypeptide.

The sequence of porcine pancreatic spasmolytic polypeptide has been established by a variety of techniques including manual as well as automatic sequencing of fragments resulting from the cleavage of reduced and S-carboxymethylated pancreatic spasmolytic polypeptide with trypsin, chymotrypsin, clostripain, cyanogen bromide and formic acid. The N- and C-terminal sequences were established using pyroglutamate amino-peptidase and carboxypeptidase A, respectively. Pancreatic spasmolytic polypeptide contains 106 amino acid residues in a single chain with seven S-S bridges and a pyroglutamyl blocked N-terminal. The alignment of the sequences representing amino acids 14-49 and 63-98 shows pair-wise identical amino acid residues in 18 out of 36 positions, indicating that these two "domains" have been derived from a common gene.

The unusually slow relaxation kinetics of the folding-unfolding of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus.

In order to understand the thermodynamic and kinetic basis of the intrinsic stability of proteins from hyperthermophiles, the folding-unfolding reactions of cysteine-free pyrrolidone carboxyl peptidase (Cys142/188Ser) (PCP-0SH) from Pyrococcus furiosus were examined using circular dichroism (CD) and differential scanning calorimetry (DSC) at pH 2.3, where PCP-0SH exists in monomeric form. DSC showed a strong dependence of the shape and position of the unfolding profiles on the scan rate, suggesting the stability of PCP-0SH under kinetic control. On DSC timescales, even at a scan rate of 1 deg. C/hour, heat denaturation of PCP-0SH was non-equilibrium. However, over a long period of incubation of the heat-denatured PCP-0SH at pre-transition temperatures, it refolded completely, indicating reversibility with very slow relaxation kinetics. The rates of refolding of the heat-denatured PCP-0SH determined from the time-resolved DSC and CD spectroscopic progress curves were found to be similar within experimental error, confirming the mechanism of refolding to be a two-state process. The equilibrium established with a relaxation time of 5080 seconds (at t(m)=46.5 degrees C), which is unusually higher than the relaxation times observed for mesophilic and hyperthermophilic proteins. The long relaxation time may lead to the apparent irreversibility of an unfolding process occurring on the DSC experiment timescale. The refolding rate (9.8 x 10(-5) s(-1)) peaked near the t(m) (=46.5 degrees C), whereas the stability profile reached maxima (11.8 kJ mol(-1)) at 17 degrees C. The results clearly indicate the unusual mode of protein destabilization via a drastic decrease in the rate of folding at low pH and still maintaining a high activation energy barrier (284 kJ mol(-1)) for unfolding, which provides an effective kinetic advantage to unusually stable proteins from hyperthermophiles.

Subcellular distribution of membrane-bound aminopeptidases in the human and rat brain.

We evaluated the subcellular distribution of four membrane-bound aminopeptidases in the human and rat brain cortex. The particulate enzymes under study--puromycin-sensitive aminopeptidase (PSA), aminopeptidase N (APN), pyroglutamyl-peptidase I (PG I) and aspartyl-aminopeptidase (Asp-AP)--were fluorometrically measured using beta-naphthylamide derivatives. Membrane-bound aminopeptidase activity was found in all the studied subcellular fractions (myelinic, synaptosomal, mitochondrial, microsomal and nuclear fractions), although not homogenously. Human PSA showed highest activity in the microsomal fraction. APN was significantly higher in the nuclear fraction of both species, while PG I showed highest activity in the synaptosomal and myelinic fractions of the human and rat brain. The present results suggest that in addition to inactivating neuropeptides at the synaptic cleft, these enzymes may participate in other physiological processes. Moreover, these peptidases may play specific roles depending on their activity levels at the different subcellular structures where they are localized.