Inhibitory selectivity of canecystatin: a recombinant cysteine peptidase inhibitor from sugarcane

The cDNA of a cystein peptidase inhibitor was isolated from sugarcane and expressed in Escherichia coli. The protein, named canecystatin, has previously been shown to exert antifungal activity on the filamentous fungus Trichoderma reesei. Herein, the inhibitory specificity of canecystatin was further characterized. It inhibits the cysteine peptidases from plant source papain (Ki =3.3nM) and baupain (Ki=2.1x10(-8)M), but no inhibitory effect was observed on ficin or bromelain. Canecystatin also inhibits lysosomal cysteine peptidases such as human cathepsin B (Ki=125nM), cathepsin K (Ki=0.76nM), cathepsin L (Ki=0.6nM), and cathepsin V (Ki=1.0nM), but not the aspartyl peptidase cathepsin D. The activity of serine peptidases such as trypsin, chymotrypsin, pancreatic, and neutrophil elastases, and human plasma kallikrein is not affected by the inhibitor, nor is the activity of the metallopeptidases angiotensin converting enzyme and neutral endopeptidase. This is the first report of inhibitory activity of a sugarcane cystatin on cysteine peptidases.     

Secretion of serine peptidase by a clinical strain of Candida albicans: influence of growth conditions and cleavage of human serum proteins and extracellular matrix components

Candida albicans expresses a vast number of hydrolytic enzymes, playing roles in several phases of yeast-host interactions. Here, we identified two novel extracellular peptidase classes in C. albicans. Using gelatin-sodium dodecyl sulfate polyacrylamide gel electrophoresis two gelatinolytic activities were detected at physiological pH: a 60-kDa metallopeptidase, completely blocked by 1,10-phenanthroline, and a 50-kDa serine peptidase inhibited by phenylmethylsulfonyl fluoride. In an effort to establish a probable functional implication for these novel peptidase classes, we demonstrated that the 50-kDa secretory serine peptidase was active over a broad pH range (5.0-7.2) and was capable to hydrolyze some soluble human serum proteins and extracellular matrix components. Conversely, when this isolate was grown in yeast carbon base supplemented with bovine serum albumin, a secretory aspartyl peptidase activity was measured, instead of metallo- and serine peptidases, suggesting that distinct medium composition induces different expression of released peptidases in C. albicans. Additionally, we showed by quantitative proteolytic measurement, flow cytometry and immunoblotting assays that the brain heart infusion medium might repress the Sap1-3 production. Collectively, our results showed for the first time the capability of an extracellular proteolytic enzyme other than aspartic-type peptidases to cleave a broad spectrum of relevant host proteinaceous substrates by the human pathogen C. albicans.     

Heterogeneous production of metallo-type peptidases in parasites belonging to the family Trypanosomatidae

Proteolytic enzymes play a central role in the physiology of all living organisms, participating in several metabolic pathways and in different phases of parasite-host interactions. We have identified cell-associated peptidase activities in 33 distinct flagellates, including representatives of almost all known trypanosomatid genera parasitizing insects (Herpetomonas, Crithidia, Leishmania, Trypanosoma, Leptomonas, Phytomonas, Blastocrithidia and Endotrypanum) as well as the biflagellate kinetoplastid Bodo, by using SDS-PAGE containing gelatin as co-polymerized substrate and proteolytic inhibitors. Under the alkaline pH (9.0) conditions employed, all the flagellates presented at least one peptidase, with the exception of Crithidia acanthocephali and Phytomonas serpens, which did not display any detectable proteolytic enzyme activity. All the proteolytic activities were completely inhibited by 1,10-phenanthroline, a zinc-chelating agent, putatively identifying these activities as metallo-type peptidases. EDTA and EGTA, two other metallopeptidase inhibitors, E-64 (a cysteine peptidase inhibitor), pepstatin A (an aspartyl peptidase inhibitor) and PMSF (a serine peptidase inhibitor) did not interfere with the metallopeptidase activities detected in the studied trypanosomatids. Conversely, Bodo-derived peptidases were resistant to 1,10-phenanthroline and only partially inhibited by EDTA, showing a distinct inhibition profile. Together, our data demonstrated great heterogeneity of expression of metallopeptidases in a wide range of parasites belonging to the family Trypanosomatidae.     

The peptidases from fungi and viruses

Fungi and viruses encode a variety of peptidases having a plethora of functions. Many fungal peptidases are extracellular and are likely used to degrade proteins in their environment. Viral peptidases are processing enzymes, intimately involved in the virus infectious cycle. The viral RNA genome is translated by the host-cell machinery into a large polyprotein that is cleaved by the viral peptidases into mature capsid proteins, non-structural proteins and enzymes. I review the structure and catalytic mechanism of scytalidoglutamic peptidase isolated from the wood-destroying fungus Scytalidium lignicolum. This enzyme has a unique beta-sandwich fold and a novel catalytic mechanism based on a glutamate, a glutamine and a nucleophilic water molecule. Hepatitis A virus (HAV) 3C peptidase was the first structure identified for a viral 3C enzyme that exhibited the three-dimensional fold of the chymotrypsin family of serine peptidases but had a cysteine sulfur atom instead of the serine oxygen as the nucleophile. The structure of HAV 3C was unusual in that the Asp residue expected as the third member of the catalytic triad did not interact with the general base His. The present structure is of a beta-lactone-inhibited version of HAV 3C that has a restored catalytic triad.     

A 25-kDa Serine Peptidase with Keratinolytic Activity Secreted by <Emphasis Type="Italic">Coccidioides immitis</Emphasis>

Coccidioides immitis is the causative agent of coccidioidomycosis, a systemic mycosis that attacks humans and a wide variety of animals. In the present study, we showed that the C. immitis mycelial form is able to release proteolytic enzyme into the extracellular environment. Under chemically defined growth conditions, mycelia secreted seven distinct polypeptides ranging from 15 to 65 kDa and an extracellular peptidase of 25 kDa. This enzyme had its activity fully inhibited by phenylmethylsulphonyl fluoride, a serine peptidase inhibitor. Conversely, metallo, cysteine, and aspartyl peptidase inhibitors did not alter the 25-kDa enzyme behavior. This extracellular serine peptidase was able to degrade keratin, a fibrous protein that composes human epidermis. Additionally, this peptidase cleaved different protein substrates, including gelatin, casein, hemoglobin, and albumin. Curiously, an 18-kDa serine peptidase activity was evidenced solely when casein was used as the co-polymerized protein substrate into the gel. The existence of different secreted peptidases could be advantageous for the adaptation of C. immitis to distinct environments during its complex life cycle.     

Influence of parasite encoded inhibitors of serine peptidases in early infection of macrophages with Leishmania major

Ecotin is a potent inhibitor of family S1A serine peptidases, enzymes lacking in the protozoan parasite Leishmania major . Nevertheless, L. major has three ecotin-like genes, termed inhibitor of serine peptidase (ISP). ISP1 is expressed in vector-borne procyclic and metacyclic promastigotes, whereas ISP2 is also expressed in the mammalian amastigote stage. Recombinant ISP2 inhibited neutrophil elastase, trypsin and chymotrypsin with K _is between 7.7 and 83 nM. L. major ISP2–ISP3 double null mutants (Δ isp 2/3) were created. These grew normally as promastigotes, but were internalized by macrophages more efficiently than wild-type parasites due to the upregulation of phagocytosis by a mechanism dependent on serine peptidase activity. Δ isp 2/3 promastigotes transformed to amastigotes, but failed to divide for 48 h. Intracellular multiplication of Δ isp 2/3 was similar to wild-type parasites when serine peptidase inhibitors were present, suggesting that defective intracellular growth results from the lack of serine peptidase inhibition during promastigote uptake. Δ isp 2/3 mutants were more infective than wild-type parasites to BALB/c mice at the early stages of infection, but became equivalent as the infection progressed. These data support the hypothesis that ISPs of L. major target host serine peptidases and influence the early stages of infection of the mammalian host.     

Identification of the amino acid residues essential for proteolytic activity in an archaeal signal peptide peptidase

Signal peptide peptidases (SPPs) are enzymes involved in the initial degradation of signal peptides after they are released from the precursor proteins by signal peptidases. In contrast to the eukaryotic enzymes that are aspartate peptidases, the catalytic mechanisms of prokaryotic SPPs had not been known. In this study on the SPP from the hyperthermophilic archaeon Thermococcus kodakaraensis (SppA(Tk)), we have identified amino acid residues that are essential for the peptidase activity of the enzyme. DeltaN54SppA(Tk), a truncated protein without the N-terminal 54 residues and putative transmembrane domain, exhibits high peptidase activity, and was used as the wild-type protein. Sixteen residues, highly conserved among archaeal SPP homologue sequences, were selected and replaced by alanine residues. The mutations S162A and K214A were found to abolish peptidase activity of the protein, whereas all other mutant proteins displayed activity to various extents. The results indicated the function of Ser(162) as the nucleophilic serine and that of Lys(214) as the general base, comprising a Ser/Lys catalytic dyad in SppA(Tk). Kinetic analyses indicated that Ser(184), His(191) Lys(209), Asp(215), and Arg(221) supported peptidase activity. Intriguingly, a large number of mutations led to an increase in activity levels of the enzyme. In particular, mutations in Ser(128) and Tyr(165) not only increased activity levels but also broadened the substrate specificity of SppA(Tk), suggesting that these residues may be present to prevent the enzyme from cleaving unintended peptide/protein substrates in the cell. A detailed alignment of prokaryotic SPP sequences strongly suggested that the majority of archaeal enzymes, along with the bacterial enzyme from Bacillus subtilis, adopt the same catalytic mechanism for peptide hydrolysis.     

Insulin-like growth factor-I in wound healing of rat skin

Endothelin-1 is involved in physiology and pathophysiology of the alimentary tract. The peptide modulates blood flow in the gastrointestinal microvasculature and regulates contractility of smooth muscles and, when present in excess, may be an important factor contributing to pathogenesis of various forms of mucosal injury and peristaltic disorders. Mechanisms that regulate endothelin concentration in the gastrointestinal tissues are unknown. Therefore, the aim of our study was to identify and characterize endothelin inactivating peptidases in the rat gastrointestinal mucosa and smooth muscle cells. We have found three high affinity and efficient endothelin-1 inactivating peptidases. The acidic (pH optimum 5.5), membrane-bound, thiorphan- (ED(50) 1.2+/-0.2 nM) and phosphoramidon (ED(50) 150+/-25 pM) sensitive, endothelin-1 inactivating peptidase (K(M) 0.12+/-0.03 microM) was present in the mucosal cells of duodenum and small intestine. The enzyme exhibited high molecular weight (>100 kDa) and characteristics similar to that of the rat and human kidney, acidic metalloendopeptidase that was recently described. Two forms of the unique, low molecular weight (100>MW>30 kDa), alkaline (pH optimum 8.5), specific (K(M) 0.5+/-0.2 microM), thiorphan- and phosphoramidon insensitive, 1,10 phenanthroline inhibitable (ED(50) 0.65+/-0.20 mM, mean+/-S.E.M.) endothelin-1 inactivating peptidase were present exclusively in the duodenal mucosal cells; soluble form in cytosol and membrane-bound form exhibiting an abundance ratio 5:1, respectively. Mucosa of the stomach and large intestine, and gastrointestinal smooth muscle cells do not contain the specific endothelin-1 inactivating peptidases. The enzymes may play a crucial role in regulation of endothelin concentration in the gastrointestinal tissues. Whether impairment of activity of the mucosal endothelin inactivating peptidases, resulting in the increase of concentration of endothelin peptides in gastrointestinal tissues, occurs in various pathological conditions is actually studied in our laboratory.     

Expression, purification and characterization of recombinant human serine proteinase inhibitor Kazal-type 6 (SPINK6) in Pichia pastoris

Human serine proteinase inhibitor Kazal-type 6 (SPINK6) belongs to the medically important SPINK family. Malfunctions of SPINK members are linked to many diseases, including pancreatitis, skin barrier defects, and cancer. SPINK6 has been shown to selectively inhibit Kallikrein-related peptidases (KLKs) in human skin. As a SPINK protein, it contains a typical Kazal domain, which requires three intramolecular disulfide bonds for correct folding and activity. Preparation of functional protein is a prerequisite for studying this important human factor. Here, we report the successful generation of tagless SPINK6 using a yeast expression system. The recombinant protein was secreted and purified by cation exchange and size-exclusion chromatography. The protein identity was confirmed by MALDI-TOF MS and N-terminal sequencing. Pichia pastoris-derived recombinant human SPINK6 (rhSPINK6) showed higher inhibitory activity against Kallikrein-related peptidase 14 (KLK14) (K(i)=0.16 nM) than previously reported Escherichia coli-derived rhSPINK6 (K(i)=0.5 nM). This protein also exhibited moderate inhibition of bovine trypsin (K(i)=33 nM), while previous E. coli-derived rhSPINK6 did not. The results indicate that P. pastoris is a better system to generate active rhSPINK6, warranting further studies on this medically important SPINK family candidate.     

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.     

Analysis of the interaction between the aspartic peptidase inhibitor SQAPI and aspartic peptidases using surface plasmon resonance

Aspartic peptidase inhibitors, which are themselves proteins, are strong inhibitors (small inhibition constants) of some aspartic peptidases but not others. However, there have been no studies of the kinetics of the interaction between a proteinaceous aspartic peptidase inhibitor and aspartic peptidases. This paper describes an analysis of rate constants for the interaction between recombinant squash aspartic peptidase inhibitor (rSQAPI) and a panel of aspartic peptidases that have a range of inhibition constants for SQAPI. Purified rSQAPI completely inhibits pepsin at a 1:1 molar ratio of pepsin to rSQAPI monomer (inhibition constant 1 nM). The interaction of pepsin with immobilized rSQAPI, at pH values between 3.0 and 6.0, was monitored using surface plasmon resonance. Binding of pepsin to rSQAPI was slow (association rate constants ca 10(4)M (-1)s(-1)), but rSQAPI was an effective pepsin inhibitor because dissociation of the rSQAPI-pepsin complex was much slower (dissociation rate constants ca 10(-4)s(-1)), especially at low pH values. Similar results were obtained with a His-tagged rSQAPI. Strong inhibition (inhibition constant 3 nM) of one isoform (rSap4) of the family of Candida albicans-secreted aspartic peptidases was, as with pepsin, characterized by slow binding of rSap4 and slower dissociation of the rSap4-inhibitor complex. In contrast, weaker inhibition of the Glomerella cingulata-secreted aspartic peptidase (inhibition constant 7 nM) and the C. albicans rSap1 and Sap2 isoenzymes (inhibition constants 25 and 400 nM, respectively) was, in each case, characterized by a larger dissociation rate constant.     

Peptidases of the rumen bacterium, Prevotella ruminicola

Prevotella (formerly Bacteroides) ruminicola is a numerous rumen bacterium which plays a significant role in the metabolism of proteins and peptides in the rumen. Measurement of the hydrolysis of synthetic aminopeptidase substrates by sonicated extracts and whole cells of different species of rumen bacteria indicated that P. ruminicola had the greatest range and specific activity of dipeptidyl peptidases among the species tested.Streptococcus bovis hydrolysed some dipeptidyl peptidase substrates to a lesser extent, and several species broke down Ala2-p-nitroanilide, including Ruminobacter amylophilus, Ruminococcus spp. and Veillonella parvula. Dipeptidyl peptidases, which cleave dipeptides from the amino-terminus of longer peptides, were much more active than aminopeptidases removing single amino acids in P. ruminicola. Ion-exchange chromatography of sonicated extracts of P. ruminicola M384 revealed at least four distinct activities: one hydrolysed Ala2-p-nitroanilide, ValAla-p-nitroanilide, Ala4and Ala5; another was an O2-sensitive activity hydrolysing GlyArg-4-methoxynapthylamide, ArgArg-4-methoxynaphthylamide, Gly5 and ValGlySerGlu, similar to dipeptidyl peptidase type I DPP-1); a third hydrolysed GlyPro-p-nitroanilide and GlyPro-4-methoxynapthylamide and was similar to dipeptidyl peptidase type IV XDPP-4); a fourth broke down LysAla-4-methoxynaphthylamide. All of the enzymes, and particularly those active against Ala2-p-nitroanilide and GlyPro-p-nitroanilide, were inhibited by serine protease inhibitors, and all except DPP-4 were inhibited by EDTA. Both DPP-1 and the enzyme hydrolysing LysAla-4-methoxynaphthylamide were inhibited strongly by iodoacetate. DPP-4 was inhibited completely by diprotin A. Competitive inhibition experiments suggested that DPP-1 was less important than the other enzymes in the breakdown of peptide mixtures.     

Prediction of peptidase category based on functional domain composition

Peptidases play pivotal regulatory roles in conception, birth, digestion, growth, maturation, aging, and death of all organisms. These regulatory roles include activation, synthesis and turnover of proteins. In the proteomics era, computational methods to identify peptidases and catalog the peptidases into six different major classes-aspartic peptidases, cysteine peptidases, glutamic peptidases, metallo peptidases, serine peptidases and threonine peptidases can give an instant glance at the biological functions of a newly identified protein. In this contribution, by combining the nearest neighbor algorithm and the functional domain composition, we introduce both an automatic peptidase identifier and an automatic peptidase classier. The successful identification and classification rates are 93.7% and 96.5% for our peptidase identifier and peptidase classifier, respectively. Free online peptidase identifier and peptidase classifier are provided on our Web page http://pcal.biosino.org/protease_classification.html.     

New families of carboxyl peptidases: serine-carboxyl peptidases and glutamic peptidases

Peptidases or proteinases are now classified into seven families based on the nature of the catalytic residues [MEROPS-the peptidase database (http://merops.sanger.ac.uk/)]. They are aspartic- (first described in 1993), cysteine- (1993), serine- (1993) metallo- (1993), threonine- (1997), glutamic- (2004) and asparagine-peptidase (2010). By using an S-PI (pepstatin Ac) as a probe, a new subfamily of serine peptidase, serine-carboxyl peptidase (sedolisin) was discovered in 2001. In addition, the sixth family of peptidase, glutamic peptidase (eqolisin) was also discovered in 2004. The former peptidase is widely distributed in nature from archea to mammals, including humans. One of these enzymes is related to a human fatal hereditable disease, Batten disease. In contrast, the distribution of the latter peptidases is limited, with most of them found in human or plant pathogenic fungi. One such enzyme was isolated from a fungal infection in an HIV-infected patient. In this review, the background of the findings, and crystal structures, catalytic mechanisms, substrates specificities and distribution of the new peptidase families are described.     

Enzymic inactivation of neurotensin by hypothalamic and brain extracts of the rat.

A sensitive and specific radioimmunoassay has been developed to study inactivation of neurotensin by hypothalamic and brain peptidases. Degrading activity of peptidases from both hypothalamus and brain seems to have similar activity. These peptidases are temperature- and time- dependent. Brain and hypothalamic enzymes of particulate fractions can be differentiated on the basis of the pH effects; brain peptidase(s) has (have) maximal activity at pH 7.4 and hypothalamic peptidase(s) displaying a maximal activity at pH 5.8. Kidneys and liver extracts contain enzyme(s) degrading neurotensin.     

Comparison of two pig intestinal brush border peptidases with the corresponding renal enzymes.

Intestinal dipeptidyl peptidase IV and gamma-glutamyltransferase were compared to the corresponding kidney enzymes with respect to immunological and electrophoretic properties. The influences of selected effectors on the two enzymes were also studied. The two kidney peptidases exhibited the reaction of total identity with the corresponding intestinal enzymes in immunodiffusion. Furthermore, the intestinal dipeptidyl peptidase IV and gamma-glutamyl transferase showed the same inhibition patterns as the corresponding kidney enzymes and the acceptor specificity of the intestinal gamma-glutamyl-transferase was found to be identical to that of the kidney enzyme. The electrophoretic mobilities of dipeptidyl peptidase IV from the two organs differed greatly. The difference was almost abolished by treatment with neuraminidase, suggesting that the variation in mobility was due to different contents of sialic acid. It is suggested that the intestinal brush border peptidases, dipeptidyl peptidase IV and gamma-glutamyltransferase, are closely related to the corresponding enzymes obtained from the kidney.     

Aspartate-specific peptidases in Salmonella typhimurium: mutants deficient in peptidase E.

The only dipeptide found to serve as a leucine source for a Salmonella strain lacking peptidases N, A, B, D, P, and Q was alpha-L-aspartyl-L-leucine. A peptidase (peptidase E) that specifically hydrolyzes Asp-X peptides was identified and partially purified from cell extracts. The enzyme (molecular weight, 35,000) is inactive toward dipeptides with N-terminal asparagine or glutamic acid. Mutants (pepE) lacking this enzyme were isolated by screening extracts for loss of the activity. Genetic mapping placed the pepE locus at 91.5 map units and established the gene order metA pepE zja-861::Tn5 malB. Duplications of the pepE locus showed a gene dosage effect on levels of peptidase E, suggesting that pepE is the structural gene for this enzyme. Mutations in pepE resulted in the loss of the ability to grow on Asp-Pro as a proline source but did not affect utilization of other dipeptides with N-terminal aspartic acid. Loss of peptidase E did not cause a detectable impairment in protein degradation. Two other peptidases present in cell extracts of mutants lacking peptidases N, A, B, D, P, Q, and E also hydrolyze many Asp-X dipeptides.     

The 1.8 A crystal structure of a proteinase K-like enzyme from a psychrotroph Serratia species

Proteins from organisms living in extreme conditions are of particular interest because of their potential for being templates for redesign of enzymes both in biotechnological and other industries. The crystal structure of a proteinase K-like enzyme from a psychrotroph Serratia species has been solved to 1.8 A. The structure has been compared with the structures of proteinase K from Tritirachium album Limber and Vibrio sp. PA44 in order to reveal structural explanations for differences in biophysical properties. The Serratia peptidase shares around 40 and 64% identity with the Tritirachium and Vibrio peptidases, respectively. The fold of the three enzymes is essentially identical, with minor exceptions in surface loops. One calcium binding site is found in the Serratia peptidase, in contrast to the Tritirachium and Vibrio peptidases which have two and three, respectively. A disulfide bridge close to the S2 site in the Serratia and Vibrio peptidases, an extensive hydrogen bond network in a tight loop close to the substrate binding site in the Serratia peptidase and different amino acid sequences in the S4 sites are expected to cause different substrate specificity in the three enzymes. The more negative surface potential of the Serratia peptidase, along with a disulfide bridge close to the S2 binding site of a substrate, is also expected to contribute to the overall lower binding affinity observed for the Serratia peptidase. Clear electron density for a tripeptide, probably a proteolysis product, was found in the S' sites of the substrate binding cleft.     

Degradation of Neurotensin by Rat Brain Synaptic Membranes: Involvement of a Thermolysin-Like Metalloendopeptidase (Enkephalinase), Angiotensin-Converting Enzyme, and Other Unidentified Peptidases

Neurotensin was inactivated by membrane-bound and soluble degrading activities present in purified preparations of rat brain synaptic membranes. Degradation products were identified by HPLC and amino acid analysis. The major points of cleavage of neurotensin were the Arg8-Arg9, Pro10-Tyr11, and Tyr11-Ile12 peptide bonds with the membrane-bound activity and the Arg8-Arg9 and Pro10-Tyr11 bonds with the soluble activity. Several lines of evidence indicated that the cleavage of the Arg8-Arg9 bond by the membrane-bound activity resulted mainly from the conversion of neurotensin1-10 to neurotensin1-8 by a dipeptidyl carboxypeptidase. In particular, captopril inhibited this cleavage with an IC50 (5.7 nM) close to its K1 (7 nM) for angiotensin-converting enzyme. Thiorphan inhibited the cleavage at the Tyr11-Ile12 bond by the membrane-bound activity with an IC50 (17 nM) similar to its K1 (4.7 nM) for enkephalinase. Both cleavages were inhibited by 1,10-phenanthroline. These and other data suggested that angiotensin-converting enzyme and a thermolysin-like metalloendopeptidase (enkephalinase) were the membrane-bound peptidases responsible for cleavages at the Arg8-Arg9 and Tyr11-Ile12 bonds, respectively. In contrast, captopril had no effect on the cleavage at the Arg8-Arg9 bond by the soluble activity, indicating that the enzyme responsible for this cleavage was different from angiotensin-converting enzyme. The cleavage at the Pro10-Tyr11 bond by both the membrane-bound and the soluble activities appeared to be catalyzed by an endopeptidase different from known brain proline endopeptidases. The possibility is discussed that the enzymes described here participate in physiological mechanisms of neurotensin inactivation at the synaptic level.