Fluorigenic Substrates Research Articles

Comparison of Fluorigenic Peptide Substrates PL50, SNAPtide, and BoTest A/E for BoNT/A Detection and Quantification: Exosite Binding Confers High-Assay Sensitivity

Detection and quantification of low doses of botulinum toxin serotype A (BoNT/A) in medicinal preparations require precise and sensitive methods. With mounting pressure from governmental authorities to replace the mouse LD50 assay, interest in alternative methods such as the endopeptidase assay, quantifying the toxin active moiety, is growing. Using internal collision-induced fluorescence quenching, Pharmaleads produced peptides encompassing the SNAP-25 cleavage site: a 17-mer (PL63) and a 48-mer (PL50) reaching the previously identified α-exosite, with PL50 showing higher apparent affinity for BoNT/A. Peptide mapping experiments revealed that this increased affinity is mainly due to a connecting peptide sequence between the N-terminus of PL63 and the α-exosite, identifying a new cooperative exosite on BoNT/A. Other endopeptidase substrates available, including SNAPTide and BoTest A/E, are both based on fluorescent resonance energy transfer (FRET) technology. To compare these assays, their limits of detection and quantification were determined using light chain or 150-kDa BoNT/A. Detection limits of PL50 and BoTest were over 100 times better than those using SNAPtide in standard conditions. Although the BoTest possessed a detection limit around 0.2 pM for either BoNT/A form, its quantification limit (9.7 pM) using purified BoNT/A was 12 times inferior to PL50, estimated at 0.8 pM, suitable for medicinal preparation quantification.

The increased dipeptidyl peptidase‐4 activity is not counteracted by optimized glucose control in type 2 diabetes, but is lower in metformin‐treated patients

Dipeptidyl peptidase (DPP)-4 in responsible for incretin degradation and some observations suggest that DPP-4 activity is increased in type 2 diabetes (T2D). We aimed to assess the effect of T2D and glucose control on DPP-4 activity. In the first set (SET1) of patients, we compared plasma DPP-4 activity between 30 T2D and 20 age- and sex-matched non-diabetic subjects. In the second set (SET2), we measured serum DPP-4 activity in 42 T2D patients before and after a trial of glucose control achieved by add-on basal insulin therapy (NCT00699686). Serum/plasma DPP-4 activity was determined using chromogenic and fluorigenic substrates, as well as several positive and negative controls. In SET1, plasma DPP-4 activity was significantly higher in T2D vs. controls (32.2 ± 1.2 U/l vs. 21.2 ± 1.1 U/l, p < 10(-6)). From a meta-analysis of the literature, we found that T2D is associated with a 33% increase in DPP-4 activity compared to controls. In SET2, serum DPP-4 activity was not lowered by intensified glucose control, despite an average haemoglobin A1c (HbA1c) reduction of 1.5%. In both sets of diabetic patients, the use of metformin was associated with a significantly lower DPP-4 activity, independently of age, sex, body mass index and HbA1c. DPP-4 activity is increased in T2D, but is not lowered by glucose control, suggesting that hyperglycaemia is not a direct determinant of DPP-4 activity. However, metformin may indirectly reduce DPP-4 activity.

Compromising the Unfolded Protein Response Induces Autophagy-Mediated Cell Death in Multiple Myeloma Cells

ObjectiveTo determine whether the Unfolded Protein Response (UPR) sensors (PERK, ATF6 and IRE-1) can be targeted to promote death of Multiple Myeloma (MM) cells.MethodsWe have knocked-down separately each UPR stress sensor in human MM cell lines using RNA interference and followed MM cell death by monitoring the membrane, mitochondrial and nuclear alterations. Involvement of caspases in MM cell death consecutive to UPR sensor knock-down was analyzed by western blotting, measurement of their enzymatic activity using fluorigenic substrates and susceptibility to a pan-caspase inhibitor. Activation of the autophagic process was measured directly by detection of autophagosomes (electronic microscopy), monodansylcadaverine staining, production of the cleaved form of the microtubule-associated protein 1A/1B light chain 3 (LC3) and indirectly by analyzing the impact of pharmacological inhibitors of autophagy such as 3MA and bafilomycin A1.ResultsWe show that extinction of a single UPR stress sensor (PERK) induces a non-apoptotic form of cell death in MM cells that requires autophagy for its execution. We also show that this cytotoxic autophagic process represses the apoptosis program by reducing the cytosolic release of the apoptogenic factors Smac/DIABLO and cytochrome c.InterpretationAltogether our findings suggest that autophagy can contribute to execution of death in mammalian cells that are exposed to mild ER stress. They also suggest that the autophagic process can regulate the intrinsic apoptotic pathway by inhibiting production of death effectors by the mitochondria, thus preventing formation of a functional apoptosome. Altogether these findings give credit to the idea that UPR sensors can be envisaged as therapeutic targets for the treatment of MM.

Probing the S1 specificity pocket of the aminopeptidases that generate antigenic peptides

ERAP1 (endoplasmic reticulum aminopeptidase 1), ERAP2 and IRAP (insulin-regulated aminopeptidase) are three homologous enzymes that play critical roles in the generation of antigenic peptides. These aminopeptidases excise amino acids from N-terminally extended precursors of antigenic peptides in order to generate the correct length epitopes for binding on to MHC class I molecules. The specificity of these peptidases can affect antigenic peptide selection, but has not yet been investigated in detail. In the present study we utilized a collection of 82 fluorigenic substrates to define a detailed selectivity profile for each of the three enzymes and to probe structural and functional features of the S1 (primary specificity) pocket. Molecular modelling of the three S1 pockets reveals substrate-enzyme interactions that are critical determinants for specificity. The substrate selectivity profiles suggest that IRAP largely combines the S1 specificity of ERAP1 and ERAP2, consistent with its proposed biological function. IRAP, however, does not achieve this dual specificity by simply combining structural features of ERAP1 and ERAP2, but rather by an unique amino acid change at position 541. The results of the present study provide insights on antigenic peptide selection and may prove valuable in designing selective inhibitors or activity markers for this class of enzymes.

A continuous fluorigenic assay for the measurement of the activity of endoplasmic reticulum aminopeptidase 1: Competition kinetics as a tool for enzyme specificity investigation

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a recently discovered enzyme that plays critical roles in antigen presentation and the immune response. Unlike other aminopeptidases, ERAP1 displays strong sequence preferences for residues distal to the peptide–substrate’s N terminus. This unusual substrate specificity necessitates the development of new assays that are appropriate for the study of such aminopeptidases. Here we describe a continuous fluorigenic assay suitable for the analysis of the enzymatic properties of ERAP1. In this assay, signal is generated by the excision of an internally quenched N-terminal tryptophan residue from a 10mer peptide by the aminopeptidase, resulting in the enhancement of tryptophan fluorescence in the solution. This method overcomes the limitations of previously used fluorigenic and high-performance liquid chromatography (HPLC)-based assays and is appropriate for small molecule inhibitor screening as well as for rapid substrate specificity analysis by kinetic competition experiments. Such efficient peptidic fluorigenic substrates like the ones described here should greatly simplify specificity analysis and inhibitor discovery for ERAP1 and similar aminopeptidases.

Detection and Quantification of Botulinum Neurotoxin Type A by a Novel Rapid In Vitro Fluorimetric Assay

Botulinum neurotoxin type A (BoNT/A), the most poisonous substance known to humans, is a potential bioterrorism agent. The light-chain protein induces a flaccid paralysis through cleavage of the 25-kDa synaptosome-associated protein (SNAP-25), involved in acetylcholine release at the neuromuscular junction. BoNT/A is widely used as a therapeutic agent and to reduce wrinkles. The toxin is used at very low doses, which have to be accurately quantified. With this aim, internally quenched fluorescent substrates containing the fluorophore/repressor pair pyrenylalanine (Pya)/4-nitrophenylalanine (Nop) were developed. Nop and Pya were, respectively, introduced at positions 197 and 200 of the cleavable fragment (amino acids 187 to 203) of SNAP-25 (with norleucine at position 202 [Nle(202)]), which is acetylated at its N terminus and amidated at its C terminus. Cleavage of this peptide occurred between positions 197 and 198, as in SNAP-25, and was easily quantified by the strong fluorescence emission of the metabolite. To increase the assay sensitivity, the peptide sequence of the previous substrate was lengthened to account for exosite binding to BoNT/A. We synthesized the peptide PL50 (SNAP-25-NH(2) acetylated at positions 156 to 203 [Nop(197), Pya(200), Nle(202)]) and its analogue PL51, in which all methionines were replaced by nonoxidizable Nle. Consistent with a large increase in affinity for BoNT/A, PL50 and PL51 exhibit catalytic efficiencies of 2.6 x 10(6) M(-1) s(-1) and 8.85 x 10(6) M(-1) s(-1), respectively, and behave as the best fluorigenic substrates of BoNT/A reported to date. Under optimized assay conditions, they allow simple quantification of as little as 100 and 60 pg of BoNT/A, respectively, within 2 h with a classical fluorimeter. Calibration of the method against the mouse 50% lethal dose assay unequivocally validates the enzymatic assay.

Non-invasive in vivo optical imaging of the lacZ and luc gene expression in mice

The bacterial lacZ gene encoding for beta-galactosidase (beta-gal) is a common reporter gene used in transgenic mice. Nonetheless, the absence of fluorigenic substrates usable in live animals greatly hampered the non-invasive follow-up of this reporter gene expression. We used far-red fluorescence for imaging beta-Gal expression in live cells in vitro or in vivo. The 9H-(1,3-dichloro-9,9-dimethylacridin- 2-one-7-yl) beta-D-galactopyranoside substrate was used to monitor beta-Gal expression as a reporter of tumor growth, or of the physiological levels of an endogenous gene or of gene transfer in lung. A quantitative evaluation of this method as well as a comparison of its sensitivity with Firefly Luciferase-based bioluminescence was also performed. In vivo measurements showed that 10(3) beta-Gal tumor cells located under the skin were detectable. In deeper organs like lung, as little as 5 ng of beta-Gal or Luciferase enzymes per mg of proteins were measured, confirming that both techniques reached similar sensibilities. Nonetheless, quantitative comparison of beta-Gal levels measured with far-red imaging or with a standardized enzymatic evaluation after killing revealed that the 2D-fluorescent reflectance imaging method is submitted to a color-dependent disparity of the organs and cannot supply quantitative measurements but that a simple correction can be applied.

Fluorimetric determination of activity and isoenzyme composition of N-acetyl-β-D-hexosaminidase in seminal plasma of fertile men and infertile patients with secretory azoospermia

The activity and isoenzyme composition of N-acetyl-beta-D-hexosaminidase (EC.3.2.1.52) in seminal plasma of fertile and infertile men have been evaluated. However, no data are available on the isoenzyme content in seminal plasma from patients with secretory azoospermia. The activity and isoenzyme composition of seminal plasma from 15 normozoospermic controls and 18 patients with secretory azoospermia were determined by fluorimetric methods. 4-Methylumbelliferil-2-acetamido-2-deoxy-beta-D-glucopyranoside and 4-methylumbelliferil-2-acetamido-2-deoxy-beta-D-glucopyranoside-6-sulfate were used as fluorigenic substrates. Receiver-operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic efficiency of the assays. No significant difference was found in total enzyme activity between the two groups, while isoenzyme A activity was significantly lower (p=0.004) and the ratio between total enzyme activity and isoenzyme A activity was significantly higher (p=0.04) in azoospermic patients compared to controls. The diagnostic efficiency of these evaluations was low (< or =75.7%). Our findings show that the isoenzyme composition of N-acetyl-beta-D-hexosaminidase in seminal plasma from patients with secretory azoospermia is significantly different from controls, but this difference does not represent a useful marker of secretory azoospermia. The fluorimetric assays are simple and rapid methods for evaluating the isoenzyme composition.

Aprataxin Is an AMP-Lysine Hydrolase

Ataxia-oculomotor apraxia syndrome 1 (AOA), a neurological disorder characterized by abnormal movements of the head and eye, is associated with mutation of APTX , the gene that encodes aprataxin (a nuclear protein that mediates protein-protein interactions as part of the DNA damage response). Curiously, though, aptx -mutant cells do not show evidence of a major deficit in this response. Noting that APTX mutations associated with AOA involve either a histidine triad (HIT) domain similar to that of Hint (an AMP-lysine hydrolase) or the HIT domain's immediate C-terminal region, Seidle et al. used fluorigenic substrates to show that aprataxin acted as an AMP-lysine and GMP-lysine hydrolase. Aprataxin contains an N-terminal forkhead-associated (FHA) domain and a C-terminal zinc-finger domain, as well as the HIT domain; its AMP-lysine hydrolase activity depended on the Hint active site and also on the presence of the FHA domain, whereas the zinc-finger domain was necessary for protein stability. The authors used SDS-PAGE and Western analysis, together with analysis of biochemical activity against an adenylylated lysine substrate, to show that eight disease-associated mutations were characterized by a major loss of both protein stability and enzymatic activity. A mutation associated with less severe disease displayed a less dramatic loss of protein stability and enzymatic activity. Thus, the authors conclude that it is the loss of the ability of aprataxin to hydrolyze nucleotidylated protein modifications that is associated with the development of AOA. H. F. Seidle, P. Biegnowski, C. Brenner, Disease-associated mutations inactivate AMP-lysine hydrolase activity of aprataxin. J. Biol. Chem. 280 , 20927-20931 (2005). [Abstract] [Full Text]

Disease-associated Mutations Inactivate AMP-Lysine Hydrolase Activity of Aprataxin

Ataxia-oculomotor apraxia syndrome 1 is an early onset cerebellar ataxia that results from loss of function mutations in the APTX gene, encoding Aprataxin, which contains three conserved domains. The forkhead-associated domain of Aprataxin mediates protein-protein interactions with molecules that respond to DNA damage, but the cellular phenotype of the disease does not appear to be consistent with a major loss in DNA damage responses. Disease-associated mutations in Aprataxin target a histidine triad domain that is similar to Hint, a universally conserved AMP-lysine hydrolase, or truncate the protein NH2-terminal to a zinc finger. With novel fluorigenic substrates, we demonstrate that Aprataxin possesses an active-site-dependent AMP-lysine and GMP-lysine hydrolase activity that depends additionally on the zinc finger for protein stability and on the forkhead associated domain for enzymatic activity. Alleles carrying any of eight recessive mutations associated with ataxia and oculomotor apraxia encode proteins with huge losses in protein stability and enzymatic activity, consistent with a null phenotype. The mild presentation allele, APTX-K197Q, associated with ataxia but not oculomotor apraxia, encodes a protein with a mild defect in stability and activity, while enzyme encoded by the atypical presentation allele, APTX-R199H, retained substantial function, consistent with altered and not loss of activity. The data suggest that the essential function of Aprataxin is reversal of nucleotidylylated protein modifications, that all three domains contribute to formation of a stable enzyme, and that the in vitro behavior of cloned APTX alleles can score disease-associated mutations.

Fluorigenic Substrates for the Protease Activities of Botulinum Neurotoxins, Serotypes A, B, and F

Fluorigenic Substrates for the Protease Activities of Botulinum Neurotoxins, Serotypes A, B, and F

Fluorigenic substrates for the protease activities of botulinum neurotoxins, serotypes A, B, and F.

The seven botulinum neurotoxins (BoNTs) are zinc metalloproteases that cleave neuronal proteins involved in neurotransmitter release and are among the most toxic natural products known. High-throughput BoNT assays are needed for use in antibotulinum drug discovery and to characterize BoNT protease activities. Compared to other proteases, BoNTs exhibit unusually stringent substrate requirements with respect to amino acid sequences and polypeptide lengths. Nonetheless, we have devised a strategy for development of fluorigenic BoNT protease assays, based on earlier structure-function studies, that has proven successful for three of the seven serotypes: A, B, and F. In synthetic peptide substrates, the P(1) and P(3)' residues were substituted with 2,4-dinitrophenyl-lysine and S-(N-[4-methyl-7-dimethylamino-coumarin-3-yl]-carboxamidomethyl)-cysteine, respectively. By monitoring the BoNT-catalyzed increase in fluorescence over time, initial hydrolysis rates could be obtained in 1 to 2 min when BoNT concentrations were 60 ng/ml (about 1 nM) or higher. Each BoNT cleaved its fluorigenic substrate at the same location as in the neuronal target protein, and kinetic constants indicated that the substrates were selective and efficient. The fluorigenic assay for BoNT B was used to characterize a new competitive inhibitor of BoNT B protease activity with a K(i) value of 4 micro M. In addition to real-time activity measurements, toxin concentration determinations, and kinetic studies, the BoNT substrates described herein may be directly incorporated into automated high-throughput assay systems to screen large numbers of compounds for potential antibotulinum drugs.

Multiple kinetics of mitochondrial cytochrome c release in drug-induced apoptosis.

We investigated cytochrome c release kinetics in response to three apoptosis-inducing agents (tumor necrosis factor-alpha, staurosporine, and valinomycin) in MCF-7/Casp-3 cells stably transfected with enhanced green fluorescent protein (EGFP)-tagged cytochrome c. All three agents induced significant caspase activation in the cultures determined by monitoring the cleavage of fluorigenic caspase substrates in extracts from drug-treated MCF-7/Casp-3 cells, albeit the valinomycin-induced activation was less pronounced. Time-lapse confocal microscopy showed that tumor necrosis factor-alpha and staurosporine caused rapid, one- or multiple-step release of cytochrome c-EGFP from mitochondria. In contrast, valinomycin-induced cytochrome c-EGFP release occurred slowly over several hours. Unlike staurosporine, the valinomycin-induced cytochrome c release was not associated with translocation of the proapoptotic Bax protein to the mitochondria, and was not accompanied by co-release of the proapoptotic Smac protein. Immunoprecipitation experiments revealed that cytochrome c was also released out of the cell into the extracellular space before loss of plasma membrane integrity. Our data indicate the existence of multiple kinetics of cytochrome c release in drug-induced apoptosis.

Extracellular enzyme activities during slow sand filtration in a water recharge plant

Activities of the extracellular enzymes β-glucosidase and phosphatase and bacterial densities were investigated during the filtration process at several sites in a groundwater recharge plant at the Ruhr river (Hengsen recharge plant in Schwerte, Germany). Low numbers of microorganisms and low levels of activity in this type of habitat, compared to most surface waters, caused methodological problems when determining microbial activity. In this study, fluorigenic model substrates, which enable hydrolytic rates as low as 1 nmol ( L× h) −1 to be measured, were used to determine extracellular enzyme activities. Highest activities were determined in surface water (107 nmol ( L× h) −1 for β-glucosidase and 252 nmol ( L× h) −1 for phosphatase), which decreased during the filtration process in the gravel prefilter and the main sand filter until the end of subsurface flow (1.6 nmol ( L× h) −1 and 6.8 nmol ( L× h) −1, respectively). Similarly, bacterial numbers decreased from 3.4×10 6 to 0.29×10 6 cells mL −1. These data showed that microbial activity within the prefilter and the shallow layers of the sand filter had the greatest impact on water quality. In addition to its involvement in the continuous purification of surface water, the microbial community in the sand filter probably acts as a biological buffer against ephemeral increases in the loads of organic matter and nutrients in the recharge plant.

A new long-wavelength fluorigenic substrate for alkaline phosphatase: synthesis and characterisation

Naphthofluorescein diphosphate has been synthesised from the parent dye, and shown to be an attractive long-wavelength alternative to other fluorigenic substrates for the determination of alkaline phosphatase. Its application to the determination of theophylline, an inhibitor of this enzyme, has been demonstrated. The optimum excitation wavelength of the hydrolysis product naphthofluorescein has been found to depend on the presence of additives such as cyclodextrins and (3-[3-cholamidopropyl]-dimethylamino)-1-propane sulfonate (CHAPS): such effects can be used to raise the excitation wavelength to match the output of a 635 nm diode laser in a simple and sensitive fluorescence detector.

Measurement of Low‐level Extracellular Enzyme Activity in Natural Waters Using Fluorigenic Model Substrates

AbstractActivities of the extracellular enzymes ß‐D‐glucosidase, phosphatase, and leucine aminopeptidase were investigated in unpolluted waters to test the suitability of fluorigenic model substrates for measuring enzyme activities in water samples expected to sustain low levels of activity. The waters tested were surface running waters, shallow groundwaters, and deep groundwaters from water extraction wells. Enzyme activity was detected in all the waters tested, although some of the hydrolytic rates were at the lower limit of current analytical capability. Rates varied between 1.1 and 291 nmol/(L·h) for ß‐D‐glucosidase, between 9.2 and 320 nmol/(L·h) for phosphatase, and between 5.8 and 1560 nmol/(L·h) for leucine aminopeptidase. The method thus proved to be suitable for measuring low levels of extracellular enzyme activity in natural waters, but in any case, it is necessary to adapt the technique to the type of water investigated.

Studies on the activation by ATP of the 26 S proteasome complex from rat skeletal muscle

The 26 S proteasome complex is thought to catalyse the breakdown of ubiquitinated proteins within eukaryotic cells. In addition it has been found that the complex also degrades short-lived proteins such as ornithine decarboxylase in a ubiquitin-independent manner. Both proteolytic processes are paralleled by the hydrolysis of ATP. Here we show that ATP also affects the hydrolytic activity towards fluorigenic peptide substrates by the 26 S proteasome complex from rat skeletal muscle tissue. Low concentrations of ATP (about 25 microM) optimally activate the so-called chymotryptic and tryptic activity by increasing the rate of peptide hydrolysis but not peptidylglutamylpeptide hydrolysis. Activation of the enzyme by ATP is transient but this effect can be enhanced and prolonged by including in the assay an ATP-regenerating system, indicating that ATP is hydrolysed by the 26 S proteasome complex. Although ATP cannot be substituted for by adenosine 5'-[beta,gamma-methylene]triphosphate or AMP, hydrolysis of the phosphoanhydride bond of ATP seems not to be necessary for the activation process of the proteasome complex, a conclusion drawn from the findings that ATP analogues such as adenosine 5'-[beta,gamma-imido]triphosphate, adenosine 5'-O-[gamma-thio]triphosphate, adenosine 5'-O-[beta-thio]-diphosphate and adenosine 5'-[alpha,beta-methylene]triphosphate give the same effect as ATP, and vanadate does not prevent ATP activation. These effects are independent of the presence of Mg2+. Thus, ATP and other nucleotides may act as allosteric activators of peptide-hydrolysing activities of the 26 S proteasome complex as has also been found with the lon protease from Escherichia coli.

Multiple glutathione S-transferases in Galleria mellonella; their detection with Fluorigenic substrates

Multiple glutathione S-transferase (GST) isoenzymes from Galleria mellonella have been purified by use of a combination of BSP affinity chromatography and isoelectric focusing to give catalytically active proteins isoelectric at pI 5.2, 6.9, 7.7 and 8.2. All isoforms were active with 1-chloro-2,4-dinitrobenzene (CDNB), 3-4-dichloro-nitrobenzene (DCNB) and were capable of cleaving β-methyl-umbelliferyl acetate (MUA). The pI 8.2 isoenzyme had by far the greatest activity with respect to CDNB. All four isoforms had activities of approximately the same magnitude with the other two substrates. All isoforms had detectable activity with monobromobimane and this substrate, or β-methyl umbelliferyl acetate could be used to localize the isoenzymes in electrophoresis gels by virtue of the fluorescent products of the reactions.

High-performance liquid chromatographic method for the simultaneous purification of cathepsins B, H and L from human liver

A high-performance liquid chromatographic procedure for the isolation of the three cysteine proteinases, namely cathepsins B, H and L, is described. The method is based on the following four steps. (1) A classical AcA 44 gel permeation separation with a 30–70% ammonium sulphate fraction from the human liver homogenate is used to remove the non-enzymic high-molecular-mass components. (2) Preparative cation-exchange chromatography on a CM-SW TSK column can separate the three proteinases. (3) An anion-exchange step on a semi-preparative DEAE-SW TSK column for the cathepsin H fraction is used to remove a small amount of cathepsins B and L activities. (4) The three separated enzymes are purified on an analytical TSK gel 2000 SW column. The purity of each enzyme is assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and electrofocusing on polyacrylamide gels. To check the activities of the purified proteinases, the kinetic constants [Michaelis constant ( K M) and catalytic constant ( K cat)] and the ratio K cat/ K M against the fluorigenic substrates Arg-NH-Mec, Z-Arg-Arg-NH-Mec and Z-Phe-Arg-NH-Mec after active-site titration using E-64, were determined. Z-Phe-Phe-CNH 2 was also used as a specific inhibitor of cathepsin L. This method requires only 6 g of human liver, and gives a high yield of the three lysosomal cysteine-proteinases: thus, about 150 μg of cathepsin B and 50 μg each of cathepsins L and H are obtained in a single run.

Neuropeptide-hydrolysing activities in synaptosomal fractions from dog ileum myenteric, deep muscular and submucous plexi. Their participation in neurotensin inactivation

The mapping of neuropeptidases in synaptosomal fractions prepared from dog ileum myenteric, deep muscular and submucous plexus was established by means of fluorigenic substrates and specific inhibitors. Endopeptidase 24.11, angiotensin-converting enzyme and aminopeptidases were found in all tissues, the highest amounts being recovered in the submucous preparation. Post-proline dipeptidyl aminopeptidase was obtained in high quantities whatever the tissue source while proline endopeptidase was detected in low amounts and pyroglutamyl-peptide hydrolase was never detectable. The above peptidases were examined for their putative participation in the inactivation of neurotensin by monitoring the effect of specific inhibitors on the formation of the metabolites of labeled neurotensin separated by HPLC. Endopeptidases 24.11, 24.15 and 24.16 were respectively responsible for the formation of neurotensin(1–11), neurotensin(1–8) and neurotensin(1–10) that are devoid of biological activity. The secondary attacks occurring on neurotensin degradation products were the following: cleavage of neurotensin(1–10) into neurotensin(1–8) by angiotensin-converting enzyme; conversion of neurotensin(9–13) into neurotensin(11–13) by post-proline dipeptidyl aminopeptidase; hydrolysis of neurotensin(11–13) into free tyrosine by aminopeptidase(s).