A membrane-bound metallo-endopeptidase from rat kidney. Characteristics of its hydrolysis of peptide hormones and neuropeptides.

A membrane-bound metallo-endopeptidase that hydrolyzes human parathyroid hormone (1-84) and reduced hen egg lysozyme between hydrophilic amino acid residues was isolated from rat kidney [Yamaguchi et al. (1991) Eur. J. Biochem. 200, 563-571]. In this study, the hydrolyses of various peptide hormones and neuropeptides by the metallo-endopeptidase were examined using an automated gas-phase protein sequencer. The purified enzyme hydrolyzed the oxidized insulin B chain and substance P most rapidly, followed by big endothelin 1, neurotensin, angiotensin 1, endothelin 1, rat alpha-atrial natriuretic peptide and bradykinin, in this order. The enzyme mainly cleaved these peptides at bonds involving a hydrophilic amino acid residue. However, it cleaved bonds between less hydrophilic amino acid pairs in several short peptides, e.g. at the His5-Leu6 bond in oxidized insulin B chain, the Ile28-Val29 bond in big endothelin-1 and the Ile5-His6 and Phe8-His9 bonds in angiotensin 1. The enzyme cleavage sites of oxidized insulin B chain and angiotensin 1 were different from the reported sites cleaved by meprin and by endopeptidase 2, respectively. Kinetic determination of bradykinin hydrolysis by the purified enzyme yielded values of Km = 18.1 microM and kcat = 0.473 s-1, giving a ratio of kcat/Km = 2.62 x 10(4) s-1.M-1. The Km value was about 20-fold lower than that reported for meprin and endopeptidase 2. These results indicate that the membrane-bound metallo-endopeptidase from rat kidney is distinguished from meprin and endopeptidase 2 in its substrate specificity and is not parathyroid hormone specific, but has potential capacities to inactivate various biologically active peptide hormones and neuropeptides in vivo.     

Specific cleavages of arginyl peptide bonds at basic amino acid pairs by a serine proteinase from the microsomal membranes of rat liver

The specificity of action of a serine proteinase from the microsomal membranes of rat liver was investigated at pH 7.5 and 37 degrees C using various peptides as substrates. HPLC analyses of the peptides produced followed by their amino acid analyses have revealed that the enzyme is a unique endopeptidase specifically cleaving arginyl peptide bonds at paired basic amino acid residues. Thus, the enzyme is suggested to be a kind of processing proteinase involved in the conversion of proproteins to their mature forms. Indeed, the enzyme cleaved specifically the NH2-terminal 20-residue peptide of proalbumin at the Arg-Arg sequence.     

Serratia Protease

The substrate specificity of Serratia protease was determined using various synthetic substrates. The enzyme did not participate in the hydrolysis of di- and tri-peptides except benzoylglycylleucinamide which was split at a limited rate into hippuric acid and leucinamide. The enzyme action on larger peptides was also studied. The enzyme cleaved the gly-leu bond in eledoisin related peptide and the gly-phe bond in bradykinin. The enzyme split oxidized insulin B-chain at twelve different peptide bonds.     

Purification and characterization of a novel neurotensin-degrading peptidase from rat brain synaptic membranes

A peptidase that cleaved neurotensin at the Pro10-Tyr11 peptide bond, leading to the formation of neurotensin-(1-10) and neurotensin-(11-13), was purified nearly to homogeneity from rat brain synaptic membranes. The enzyme appeared to be monomeric with a molecular weight of about 70,000-75,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography filtration. Isoelectrofocusing indicated a pI of 5.9-6. The purified peptidase could be classified as a neutral metallopeptidase with respect to its sensitivity to pH and metal chelators. Thiol-blocking agents and acidic and serine protease inhibitors had no effect. Studies with specific peptidase inhibitors clearly indicated that the purified enzyme was distinct from enzymes capable of cleaving neurotensin at the Pro10-Tyr11 bond such as proline endopeptidase and endopeptidase 24-11. The enzyme was also distinct from other neurotensin-degrading peptidases such as angiotensin-converting enzyme and a recently purified rat brain soluble metalloendopeptidase. The peptidase displayed a high affinity for neurotensin (Km = 2.6 microM). Studies on its specificity revealed that neurotensin-(9-13) was the shortest neurotensin partial sequence that was able to fully inhibit [3H]neurotensin degradation. Shortening the C-terminal end of the neurotensin molecule as well as substitutions in positions 8, 9, and 11 by D-amino acids strongly decreased the inhibitory potency of neurotensin. Among 20 natural peptides, only angiotensin I and the neurotensin-related peptides (xenopsin and neuromedin N) were found as potent as unlabeled neurotensin.     

Gamma-glutamyl hydrolase conjugase). Purification and properties of the bovine hepatic enzyme.

Bovine hepatic gamma-glutamyl hydrolase (conjugase) has been purified to homogeneity. A feature of the purification procedure was the use of high affinity macromolecular polyanion enzyme inhibitors which formed tight complexes with the enzyme altering its solubility, gel filtration, and ion exchange properties. The enzyme, which cleaves the gamma-glutamyl bonds of pteroylpolyglutamates, has a molecular weight of 108,000. It is a glycoprotein with an acid pH optimum, properties consistent with its lysosomal localization. Zinc is essential for enzyme stability. The presence of highly reactive sulfhydryl groups was evident from the extreme sensitivity to oxidizing agents and organomercurials. Very little thermal denaturation occurs below 65 degrees, but the enzyme is extremely sensitive to 0uffer anions, in keeping with the polyanionic nature of the substrate. In order to study the mechanism of action of the enzyme, a wide range of pteroylpolyglutamates, N-t-Boc polyglutamates and free polyglutamates were synthesized containing L-[U-14C]glutamic acid residues in different positions. Two pteroyltriglutamate derivatives were also synthesized in which an alpha bond replaced one of the two available gamma bonds. Time course studies of the products of the action of conjugase on these various substrates enabled us to draw the following conclusions about the enzyme: (a) peptide bond cleavage occurred only at gamma-glutamyl bonds and the presence of a COOH-terminal gamma bond was essential for enzyme action; (b) bond cleavage occurred with equal facility at internal points of the peptide chain and the enzyme should therefore be more appropriately classified as an acid hydrolase; (c) longer chain gamma-glutamyl peptides were preferentially attacked by the enzyme, the cleavage of diglutamyl peptides being extremely slow; and (d) cleavage of gamma bonds was independent of the NH2-terminal pteroyl moiety. Studies with polyanions such as the glycosaminoglycans and dextran sulfate supported the concept that the polyanion structure of the substrate was a major factor in substrate-active site interaction.     

The metabolism of neuropeptides. The hydrolysis of peptides, including enkephalins, tachykinins and their analogues, by endopeptidase-24.11.

Endopeptidase-24.11 (EC 3.4.24.11), purified to homogeneity from pig kidney, was shown to hydrolyse a wide range of neuropeptides, including enkephalins, tachykinins, bradykinin, neurotensin, luliberin and cholecystokinin. The sites of hydrolysis of peptides were identified, indicating that the primary specificity is consistent with hydrolysis occurring at bonds involving the amino group of hydrophobic amino acid residues. Of the substrates tested, the amidated peptide substance P is hydrolysed the most efficiently (Km = 31.9 microM; kcat. = 5062 min-1). A free alpha-carboxy group at the C-terminus of a peptide substrate is therefore not essential for efficient hydrolysis by the endopeptidase. A large variation in kcat./Km values was observed among the peptide substrates studied, a finding that reflects a significant influence of amino acid residues, remote from the scissile bond, on the efficiency of hydrolysis. These subsite interactions between peptide substrate and enzyme thus confer some degree of functional specificity on the endopeptidase. The inhibition of endopeptidase-24.11 by several compounds was compared with that of pig kidney peptidyldipeptidase A (EC 3.4.15.1). Of the inhibitors examined, only N-[1(R,S)-carboxy-2-phenylethyl]-Phe-p-aminobenzoate inhibited endopeptidase-24.11 but not peptidyldipeptidase. Captopril (D-3-mercapto-2-methylpropanoyl-L-proline), Teprotide (pGlu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro) and MK422 [N-[(S)-1-carboxy-3-phenylpropyl]-L-Ala-L-Pro] were highly selective as inhibitors of peptidyldipeptidase. Although not wholly specific, phosphoramidon was a more potent inhibitor of endopeptidase-24.11 than were any of the synthetic compounds tested.     

Functional characterization of the Mycobacterium tuberculosis zinc metallopeptidase Zmp1 and identification of potential substrates

Abstract Zinc metallopeptidases of bacterial pathogens are widely distributed virulence factors and represent promising pharmacological targets. In this work, we have characterized Zmp1, a zinc metallopeptidase identified as a virulence factor of Mycobacterium tuberculosis and belonging to the neprilysin (NEP; M13) family, whose X-ray structure has been recently solved. Interestingly, this enzyme shows an optimum activity toward a fluorogenic substrate at moderately acidic pH values (i.e., 6.3), which corresponds to those reported for the Mtb phagosome where this enzyme should exert its pathological activity. Substrate specificity of Zmp1 was investigated by screening a peptide library. Several sequences derived from biologically relevant proteins were identified as possible substrates, including the neuropeptides bradykinin, neurotensin, and neuropeptide FF. Further, subsequences of other small bioactive peptides were found among most frequently cleaved sites, e.g., apelin-13 and substance P. We determined the specific cleavage site within neuropeptides by mass spectrometry, observing that hydrophobic amino acids, mainly phenylalanine and isoleucine, are overrepresented at position P1'. In addition, the enzymatic mechanism of Zmp1 toward these neuropeptides has been characterized, displaying some differences with respect to the synthetic fluorogenic substrate and indicating that the enzyme adapts its enzymatic action to different substrates.     

Purification, identification and characterisation of seprase from bovine serum

The study and identification for the first time of a soluble form of a seprase activity from bovine serum is presented. To date, this activity has only been reported to be an integral membrane protease but has been known to shed from its membrane. The activity was purified 30,197-fold to homogeneity, using a combination of column chromatographies, from bovine serum. Inhibition by DFP, resulting in an IC(50) of 100:nM confirms classification as a serine protease. The protease after separation and visualisation by native PAGE was subjected to tryptic digestion and the subsequent peptides sequenced. Each peptide sequenced was found to be present in the primary structure of seprase/fibroblast activation protein (FAP), a serine gelatinase specific for proline-containing peptides and macromolecules. Substrate specificity studies using kinetic, RP-HPLC and LC-MS analysis of synthetic peptides suggest that this peptidase has an extended substrate-binding region in addition to the primary specificity site S(1). This analysis revealed at least five subsites to be involved in enzyme-substrate binding, with the smallest peptide cleaved being a tetrapeptide. A proline residue in position P(1) was absolutely necessary therefore showing high primary substrate specificity for the Pro-X bond, while a preference for a hydrophobic residue at the C-terminal end of the scissile bond (P'(1)) was evident. The enzyme also showed complete insensitivity to the prolyl oligopeptidase specific inhibitors, JTP-4819, Fmoc-Ala-pyrrCN and Z-Phe-Pro-BT. To date, no physiological substrate has clearly been defined for this protease but its ability to effectively degrade gelatin suggests a candidate protein substrate in vivo and a possible role in extracellular matrix protein degradation.     

Modulation by neurotensin and neuromedin N of adherence and chemotaxis capacity of murine lymphocytes.

The action of neurotensin and related peptides has not been yet studied on lymphocytes, although there are studies indicating the stimulative action of neurotensin, a peptide first isolated from bovine hypothalamus, on different functions of phagocytic immune cells. The present study demonstrates that neurotensin and a related peptide, neuromedin N, increased significantly the adherence and chemotaxis capacity of murine peritoneal lymphocytes, when they were incubated in the presence of neuropeptide concentrations between 10(-9) M and 10(-12) M. With respect to their adherence capacity, neuromedin N showed a slightly higher stimulation than neurotensin at a shorter time. However, both neuropeptides stimulated the chemotaxis capacity in a similar percentage. The study of the action mechanisms of these neuropeptides showed that intracellular cAMP levels were not modified by neurotensin or neuromedin N, but using an extracellular calcium chelator, EGTA (1 mM), and a blocker of calcium channels in endoplasmic reticulum, ryanodine (0.5 mM), we observed that neurotensin and neuromedin N could produce their effects through an augmentation of the intracellular Ca2+ concentration. As adherence and chemotaxis are initial processes of immune response, the results show that both neuropeptides may be physiological modulators of the lymphocyte function.     

Multicatalytic, High-Mr Endopeptidase from Postmortem Human Brain

The main high molecular weight (650K) multicatalytic endopeptidase has been purified from postmortem human cerebral cortex. As in other tissues and species, this enzyme is composed of several subunits of 24-31K and has three distinct catalytic activities, as shown by the hydrolysis of the fluorogenic tripeptide substrates glutaryl-Gly-Gly-Phe-7-amido-4-methylcoumarin, benzyloxycarboxyl-Gly-Gly-Arg-7-amido-4-methylcoumarin, and benzyloxycarboxyl-Leu-Leu-Glu-2-naphthylamide with hydrophobic (Phe), basic (Arg), and acidic (Glu) residues in the P1 position, respectively. These activities are distinguishable by their differential sensitivity to peptidase inhibitors. The enzyme hydrolysed neuropeptides at pH 7.4 at multiple sites with widely differing rates, ranging from 113 nmol/min/mg for substance-P, down to 2 nmol/min/mg for bradykinin. The enzyme also had proteinase activity as shown by the hydrolysis of casein. For the hydrolysis of the Tyr5-Gly6 bond in luteinizing hormone-releasing hormone, the Km was 0.95 mM and the specificity constant (kcat/Km) was 4.7 X 10(3) M-1 s-1. The bond specificity of the enzyme at neutral pH was determined by identifying the degradation products of 15 naturally occurring peptide sequences. The bonds most susceptible to hydrolysis had a hydrophobic residue at P1 and either a small (e.g., -Gly or -NH2) or hydrophobic residue at P'1. Hydrolysis of -Glu-X bonds (most notably in neuropeptide Y) and the Arg6-Arg7 bond in dynorphin peptides was also seen. Thus the three activities identified with fluorogenic substrates appear to be expressed against oligopeptides.     

Post-proline cleaving enzyme (prolyl endopeptidase) from bovine brain

A post-proline cleaving enzyme [prolyl endopeptidase, EC 3.4.21.26] was purified about 3,700-fold from an extract of bovine brain by a series of column chromatographies on DEAE-Sephadex, hydroxyapatite and PCMB-T-Sepharose, and gel filtration on Sephadex G-200 using N-carbobenzoxy-Gly-Pro-beta-naphthylamide (Z-Gly-Pro-2-NNap), thyrotropin releasing hormone (TRH) and oxytocin as substrates. The purified enzyme appeared homogeneous as judged by disc gel and SDS gel electrophoreses. The enzyme was most active at pH 7.5 and 7.2 with Z-Gly-Pro-2-NNap and TRH, respectively, and hydrolyzed peptide bonds involving Pro-X (X=amino acid, peptide, ester and amide) bonds of synthetic substrates, oxytocin, vasopressin, neurotensin, substance P, tuftsin, bradykinin, and insulin B chain. However, the enzyme was inert toward collagen, gelatin, and casein. The enzyme was completely inactivated by diisopropylphosphorofluoridate (DFP), Z-Gly-Pro-chloromethyl ketone and p-chloromercuribenzoate (PCMB), while it was not inhibited by phenylmethane sulfonylfluoride (PMSF) or metal chelators. Determination of the amino acid composition revealed that the enzyme contained 25 half-cystines. Modification of three cysteine residues of the enzyme by PCMB led to complete inactivation. The isoelectric point of the enzyme was 4.8, and the molecular weight was estimated to be 76,000 by ultracentrifugal analysis and 75,000-74,000 by both gel filtration and sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme is present as a monomer. These results indicate that the post-proline cleaving enzyme from bovine brain is very similar to those previously purified from lamb brain and kidney in its enzymatic properties, substrate specificity and physicochemical properties, in sharp contrast with the results obtained by Tate, who reported that the bovine brain prolyl endopeptidase was inert toward oxytocin, vasopressin and bradykinin.     

Proteases from human immunodeficiency virus and avian myeloblastosis virus show distinct specificities in hydrolysis of multidomain protein substrates.

The virally encoded proteases from human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) have been compared relative to their ability to hydrolyze a variant of the three-domain Pseudomonas exotoxin, PE66. This exotoxin derivative, missing domain I and referred to as LysPE40, is made up of a 13-kilodalton NH2-terminal translocation domain II connected by a segment of 40 amino acids to enzyme domain III of the toxin, a 23-kilodalton ADP-ribosyltransferase. HIV protease hydrolyzes two peptide bonds in LysPE40, a Leu-Leu bond in the interdomain region and a Leu-Ala bond in a nonstructured region three residues in from the NH2-terminus. Neither of these sites is cleaved by the AMV enzyme; hydrolysis occurs, instead, at an Asp-Val bond in another part of the interdomain segment and at a Leu-Thr bond in the NH2-terminal region of domain II. Synthetic peptides corresponding to these cleavage sites are hydrolyzed by the individual proteases with the same specificity displayed toward the protein substrate. Peptide substrates for one protease are neither substrates nor competitive inhibitors for the other. A potent inhibitor of HIV type 1 protease was more than 3 orders of magnitude less active toward the AMV enzyme. These results suggest that although the crystallographic models of Rous sarcoma virus protease (an enzyme nearly identical to the AMV enzyme) and HIV type 1 protease show a high degree of similarity, there exist structural differences between these retroviral proteases that are clearly reflected by their kinetic properties.     

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.     

Reactivity of Lys(NH2)-containing peptides toward endopeptidases.

Lys(NH2)-containing peptides were subjected to various proteolytic enzymes which were selected for their well-documented specificity for arginyl and/or lysyl peptide bonds. Lys(NH2)-containing peptides were cleaved more rapidly by clostripain than the corresponding lysyl peptides. On the other hand, they proved to be resistant to Achromobacter protease I hydrolysis. The modified peptides synthesized in this study were more stable than the arginyl and lysyl analogues when incubated with trypsin or thrombin. The same tendency was observed when Lys(NH2)-containing peptides were incubated in diluted human serum, suggesting that the replacement of Arg or Lys by Lys(NH2) could be used to increase the stability of peptides in vivo.     

Synthetic peptide models for the redox-active disulfide loop of glutaredoxin. Conformational studies

Two cyclic peptide disulfides (Sequence: see text). (X = L-Tyr or L-Phe) have been synthesized as models for the 14-membered redox-active disulfide loop of glutaredoxin. 1H NMR studies at 270 MHz in chloroform solutions establish a type I beta-turn conformation for the Pro-X segment in both peptides, stabilized by a 4----1 hydrogen bond between the Cys(1) CO and Cys(4) NH groups. Nuclear Overhauser effects establish that the aromatic ring in the X = Phe peptide is oriented over the central peptide unit. In dimethyl sulfoxide solutions two conformational species are observed in slow exchange on the NMR time scale, for both peptides. These are assigned to type I and type II beta-turn structures with -Pro-Tyr(Phe)- as the corner residues. The structural assignments are based on correlation of NMR parameters with model 14-membered cyclic cystine peptides with Pro-X spacers. Circular dichroism studies based on the -S-S- n-omega* transition suggest a structural change in the disulfide bridge with changing solvent polarity, establishing conformational coupling between the peptide backbone and the disulfide linkage in these systems.     

Substrate recognition of isopeptidase: specific cleavage of the epsilon-(alpha-glycyl)lysine linkage in ubiquitin-protein conjugates

The structural chromatin protein A24 (uH2A) is a conjugate of histone H2A and a non-histone protein, ubiquitin. Eukaryotic cells contain an enzyme, generically termed isopeptidase, which can cleave A24 stoichiometrically into H2A and ubiquitin in vitro. Isopeptidase, free of proteinase activity, has been partially purified from calf thymus by ion-exchange chromatography, gel filtration and affinity chromatography, and analyzed for its substate specificity. There are three major types of isopeptide bonds besides the epsilon-(alpha-glycyl)lysine bond between H2A and ubiquitin; namely, the disulfide bridge, the aldol and aldimide bonds and the epsilon-(gamma-glutamyl)lysine crosslink. Under conditions where A24 was completely cleaved into H2A and ubiquitin, none of these naturally occurring isopeptide bonds was cleaved by isopeptidase. Furthermore, the bonds formed in vitro by transglutaminase reaction between casein and putrescine, through the gamma-NH2 of glutamine residue and the NH2 of putrescine, were not cleaved by the enzyme. The enzyme also failed to cleave the glycyl-lysyl and other orthodox peptide linkages within proteins. Among various proteins examined, the substrates for isopeptidase reaction were confined to conjugates between ubiquitin and other proteins, formed through epsilon-(alpha-glycyl)lysine bonds. Since ubiquitin released by isopeptidase is re-usable for an ATP-dependent conjugation with other proteins, its carboxyl terminal -Gly-Gly-COOH most likely is preserved intact, and is not blocked. These results suggest that isopeptidase specifically recognizes and cleaves the epsilon-(alpha-glycyl)lysine bond. A possible biological significance of this enzyme is discussed.     

Selective cleavage of peptide bonds by cathepsins L and B from rat liver

The selective cleavage of peptide bonds by cathepsin L from rat liver was examined with a hexapeptide, luteinizing hormone releasing hormone, neurotensin and oxidized insulin A chain as model substrates. The specificity of cathepsin L was compared with that of cathepsin B. Cathepsin L cleaved peptide bonds that have a hydrophobic amino acid, such as Phe, Leu, Val, and Trp or Tyr, in position P2. A polar amino acid, such as Tyr, Ser, Gly, Glu, Asp, Gln, or Asn, in position P1. enhanced the susceptibility of the peptide bond to cathepsin L, though the importance of the amino acid residue in position P1' was not as great as that of the amino acid in position P2 for the action of cathepsin L. These results suggest that, in contrast to cathepsin B, cathepsin L shows very clear specificity.     

Treatment of an encephalitogenic peptide from guinea pig myelin basic protein with alpha-protease and thermolysin. Isolation of fragments and determination of cleavage sites.

Two peptic fragments (residues 37-88 and 43-88) of guinea pig myelin basic protein which are capable of inducing experimental allergic encephalomyelitis in Lewis rats were cleaved to shorter fragments with alpha-protease (Crotalus atrox proteinase, EC 3.4.24.1) and thermolysin (EC 3.4.24.4). The fragments were isolated, purified, and identified by amino acid composition and NH2- and COOH-terminal residues. The time courses of the reactions, monitored by thin layer electrophoresis of the digests, showed that alpha-protease cleaves peptide (43-88) initially at the Pro(71)-Gln(72) bond, and that the product peptides are subsequently attacked at the Arg(63) -Thr(64), Ser(74)-Gln(75), Arg(78)-Ser(79), and Ser(76)-Gln(80) bonds. No significant cleavages occurred at the -Leu, -Val, and -Ala bonds. These results are in striking contrast to those obtained previously by others workers with other peptide substrates, where selective cleavage at hydrophobic residues occurred. Thermolysin was found to attack peptide (37-88) at the Phe(42)-Phe(43) bond very rapidly; the product peptides were subsequently attacked at the His(60)-Ala(61), Ser(38)-Ile(39)-Tyr(67)-Gly(68), and Pro(84)-Val(85) bonds. These cleavages are compatible with the known specificity of this enzyme. Several of the fragments prepared with these two enzymes, peptides (43-71), (61-88), (75-88), and (72-84) have been used in other studies to locate the encephalitogenic site in the parent peptic peptide.     

Inhibition of Rabbit Brain Prolyl Endopeptidase by N-Benzyloxycarbonyl-Prolyl-Prolinal, a Transition State Aldehyde Inhibitor

Prolyl endopeptidase cleaves peptide bonds on the carboxyl side of proline residues within a peptide chain. The enzyme readily degrades a number of neuropeptides including substance P, neurotensin, thyrotropin-releasing hormone, and luteinizing hormone-releasing hormone. The finding that the enzyme is inhibited by benzyloxycarbonyl-prolyl-proline, with a Ki of 50 microM, prompted the synthesis of benzyloxycarbonyl-prolyl-prolinal as a potential transition state analog inhibitor. Rabbit brain prolyl endopeptidase was purified to homogeneity for these studies. The aldehyde was found to be a remarkably potent inhibitor of prolyl endopeptidase with a Ki of 14 nM. This Ki is more than 3000 times lower than that of the corresponding acid or alcohol. By analogy with other transition state inhibitors, it can be assumed that binding of the prolinal residue to the S1 subsite and the formation of a hemiacetal with the active serine of the enzyme greatly contribute to the potency of inhibition. The specificity of the inhibitor is indicated by the finding that a variety of proteases were not affected at concentrations 150 times greater than the Ki for prolyl endopeptidase. The data indicate that benzyloxycarbonyl-prolyl-prolinal is a specific and potent inhibitor of prolyl endopeptidase and that consequently it should be of value in in vivo studies on the physiological role of the enzyme.     

Peripheral inactivation of neurotensin. Isolation and characterization of a metallopeptidase from rat ileum

A peptidase that inactivated neurotensin by cleaving the peptide at the Pro10-Tyr11 bond, generating the biologically inactive fragments neurotensin(1-10) and neurotensin(11-13) was purified from whole rat ileum homogenate. The purified enzyme behaved as a 70-75-kDa monomer as determined by SDS-PAGE analysis in reducing or non-reducing conditions and gel permeation on Ultrogel AcA34. The peptidase was insensitive to thiol-blocking agents and acidic and serine protease inhibitors but could be strongly inhibited by 1,10-phenanthroline, EDTA, dithiothreitol and heavy metal ions such as zinc, copper and cobalt. Zinc was the only divalent cation able potently to reactivate the apoenzyme. This enzyme could be distinguished from endopeptidases EC 3.4.24.15 and EC 3.4.24.11, angiotensin-converting enzyme, proline endopeptidase, aminopeptidase and pyroglutamyl-peptide hydrolase since it was not affected by micromolar concentrations of their specific inhibitors. The peptidase displayed a high affinity for neurotensin (1.6 microM). Studies concerning the specificity of the enzyme towards the sequence of neurotensin established the following. (a) Neurotensin(9-13) was the shortest partial sequence that fully inhibited tritiated neurotensin degradation; shortening the C-terminal part of the neurotensin molecule led to inactive fragments. (b) Amidation of the C-terminal end of the peptide did not prevent the recognition by the peptidase. (c) There existed a strong stereospecificity of the peptidase for the residues in positions 8, 9 and 11 of the neurotensin molecule. (d) Pro-Xaa dipeptides (where Xaa represented aromatic or hydrophobic residues) were the most potent inhibitors of tritiated neurotensin degradation while all the Xaa-Pro dipeptides tested were totally ineffective. (e) The neurotensin-related peptides: neuromedin N, xenopsin and [Lys8-Asn9]neurotensin(8-13), as well as angiotensins I and II and dynorphins(1-8) and (1-13) were as potent as neurotensin in inhibiting [3H]neurotensin hydrolysis.