Endopeptidase Substrates Research Articles

Major increase in endopeptidase activity of human cathepsin B upon removal of occluding loop contacts.

The main feature distinguishing cathepsin B from other cysteine proteases of the papain family is the presence of a large insertion loop, termed the occluding loop, which occupies the S' subsites of the enzyme. The loop is held in place mainly by two contacts with the rest of the enzyme, involving residues His110 and Arg116 on the loop that form salt bridges with Asp22 and Asp224, respectively. The influence of this loop on the endopeptidase activity of cathepsin B has been investigated using site-directed mutagenesis and internally quenched fluorogenic (IQF) substrates. Wild-type cathepsin B displays poor activity against the substrates Abz-AFRSAAQ-EDDnp and Abz-QVVAGA-EDDnp as compared to cathepsin L and papain. Appreciable increases in kcat/KM were observed for cathepsin B containing the single mutations D22A, H110A, R116A, and D224A. The highest activity however is observed for mutants where both loop to enzyme contacts are disrupted. For the triple-mutant D22A/H110A/R116A, an optimum kcat/KM value of 12 x 10(5) M-1 s-1 was obtained for hydrolysis of Abz-AFRSAAQ-EDDnp, which corresponds to a 600-fold increase relative to wild-type cathepsin B and approaches the level of activity observed with cathepsin L or papain. By comparison, the mutations have little effect on the hydrolysis of Cbz-FR-MCA. The influence of the mutations on the pH dependency of activity also indicates that the complexity of pH activity profiles normally observed for cathepsin B is related to the presence of the occluding loop. The major increase in endopeptidase activity is attributed to an increase in loop "flexibility" and suggests that the occluding loop might move when an endopeptidase substrate binds to the enzyme. The possible contribution of these interactions in regulating endopeptidase activity and the implications for cathepsin B activity in physiological or pathological conditions are discussed.

A Plant Chloroplast Glutamyl Proteinase.

A glutamyl proteinase was partially purified from Percoll gradient-purified spinach (Spinacia oleracea) chloroplast preparations and appeared to be predominantly localized in the chloroplast stroma. The enzyme degraded casein, but of the 11 synthetic endopeptidase substrates tested, only benzyloxycarbonyl-leucine-leucine-glutamic acid-[beta]-napthylamide was hydrolyzed at measurable rates. In addition, the enzyme cleaved the oxidized [beta]-chain of insulin after a glutamic acid residue. There was no evidence that native ribulose-1,5-bisphosphate carboxylase/oxygenase was cleaved by this proteinase. The apparent Km for benzyloxycarbonyl-leucine-leucine-glutamic acid-[beta]NA at the pH optimum of 8.0 was about 1 mM. Cl-ions were required for both activity and stability. Of the proteinase inhibitors covering all four classes of the endopeptidases, only 4-(2-aminoethyl)-benzenesulfonyl-fluoride HCl and L-1-chloro-3-[4-tosylamido]-4-phenyl-2-butanone significantly inhibited the proteinase. The partially purified enzyme had a molecular weight of about 350,000 to 380,000, based on size-exclusion chromatography. The enzyme has both similar and distinctive properties to those of the bacterial glutamyl proteinases. To our knowledge, this is the first description of a plant glutamyl proteinase found predominantly or exclusively in the chloroplast.

Taenia saginata Oncosphere Excretory/Secretory Peptidases

To identify oncosphere excretory/secretory peptidases, Taenia saginata adult worms were collected from 3 patients. Eggs were hatched and activated in vitro and oncospheres cultured in vitro. The culture medium from the oncospheres was assayed with peptide substrates coupled to 7-amino-4-trifluoromethyl coumarin (AFC), and free AFC was detected fluorometrically. The endopeptidase substrates Z-Phe-Arg-AFC and Z-Arg-AFC as well as the aminopeptidase substrate Arg-AFC were hydrolyzed when incubated with spent media from the oncospheres compared to control culture medium removed prior to incubation. Hydrolysis of Z-Phe-Arg-AFC was inhibited 78% by preincubation of the medium with the serine proteinase inhibitor Phenylmethylsulfonyl fluoride. Endopeptidase activity was partially enhanced in the presence of exogenous thiols and partially inhibited with the cysteine proteinase inhibitor E-64, suggesting the presence of both serine and cysteine endopeptidases. No significant inhibition was noted with pepstatin or phenanthroline. The peptidase activities detected with Z-Phe-Arg-AFC and Arg-AFC were separated by gel-filtration fast protein liquid chromatography and eluted at volumes corresponding to molecular weights of 18 and 30 kDa, respectively. These data demonstrate that T. saginata oncospheres produce excretory/secretory peptidases, including serine and cysteine endopeptidases and an aminopeptidase. These enzymes may play a role in invasion of the intestinal mucosa of the intermediate host.

Identification and properties of a neuropeptide-degrading endopeptidase (neprilysin) ofAscaris suummuscle

We have previously identified in membranes of the locomotory muscle of Ascaris suum a phosphoramidon-sensitive endopeptidase which hydrolyses the neuropeptide AF1 (Lys-Asn-Glu-Phe-Ile-Arg-Phe-NH2) by cleavage of the Glu3-Phe4 bond (Sajid & Isaac, 1994). We have determined the properties of this neuropeptide-degrading enzyme of A. suum muscle using AKH-1 (pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2) and [D-Ala2, Leu5]enkephalin as convenient endopeptidase substrates. Phosphoramidon, thiorphan and SQ 28603, potent inhibitors of mammalian neprilysin (neutral endopeptidase, endopeptidase 24.11), inhibited the endopeptidase activity towards AKH-I with IC50 values of 0.13 microM, 22 microM and 6.3 microM, respectively. Two other neprilysin inhibitors (SCH 32615 and SCH 39370) and the bivalent metal ion chelators, EDTA (1 mM) and 1, 10 bis-phenanthroline (1 mM) failed to inhibit the nematode enzyme. The endopeptidase had a neutral pH optimum and a significant proportion (45%) of the enzyme activity partitioned into the detergent-rich phase of Triton X-114, indicating that the enzyme is an integral membrane protein. The muscle enzyme also attacked [D-Ala2, Leu5]enkephalin cleaving the Gly3-Phe4 bond and this hydrolytic activity was inhibited by phosphoramidon and thiorphan (IC50, 0.28 microM and 15.8 microM, respectively) but not by EDTA and 1, 10 bis-phenanthroline. The phosphoramidon-sensitive endopeptidase activity was detected on intact muscle cells prepared by collagenase treatment of the body wall musculature, indicating that endopeptidase is accessible to peptide molecules that interact with the cell surface.

Molecular Cloning and Expression of Rat Brain Endopeptidase 3.4.24.16

We have isolated by immunological screening of a lambda ZAPII cDNA library constructed from rat brain mRNAs a cDNA clone encoding endopeptidase 3.4.24.16. The longest open reading frame encodes a 704-amino acid protein with a theoretical molecular mass of 80,202 daltons and bears the consensus sequence of the zinc metalloprotease family. The sequence exhibits a 60.2% homology with those of another zinc metallopeptidase, endopeptidase 3.4.24.15. Northern blot analysis reveals two mRNA species of about 3 and 5 kilobases in rat brain, ileum, kidney, and testis. We have transiently transfected COS-7 cells with pcDNA3 containing the cloned cDNA and established the overexpression of a 70-75-kDa immunoreactive protein. This protein hydrolyzes QFS, a quenched fluorimetric substrate of endopeptidase 3.4.24.16, and cleaves neurotensin at a single peptide bond, leading to the formation of neurotensin (1-10) and neurotensin (11-13). QFS and neurotensin hydrolysis are potently inhibited by the selective endopeptidase 3.4.24.16 dipeptide blocker Pro-Ile and by dithiothreitol, while the enzymatic activity remains unaffected by phosphoramidon and captopril, the specific inhibitors of endopeptidase 3.4.24.11 and angiotensin-converting enzyme, respectively. Altogether, these physicochemical, biochemical, and immunological properties unambiguously identify endopeptidase 3.4.24.16 as the protein encoded by the isolated cDNA clone.

Stability of neurotensin and acetylneurotensin 8–13 in brush-border membrane, cytosol, and homogenate of rat small intestine

The stability of neurotensin and acetylneurotensin 8–13 in small intestinal brush-border membrane, cytosol, and homogenate was studied. Proteolytic degradation of both compounds was pH dependent. At pH 7.5, rapid degradation was observed in brush-border membrane, cytosol, and homogenate. At pH 4.5, both compounds were stable in brush-border membrane, but were slowly degraded in mucosal homogenate of the proximal intestine. In general, acetylneurotensin 8–13 was more stable than neurotensin. Degradation of both compounds by 27 000 × g pellets, rich in brush-border membrane, was highest among the subcellular fractions. Studies using enzyme inhibitors suggested that both compounds were degraded by brush-border endopeptidase-24.11 and angiotensin-converting enzyme (ACE), and that endopeptidase-24.11 was the major enzyme degrading acetylneurotensin 8–13. At pH 4.5, degradation in homogenates was mainly due to serine proteases. In cytosol, degradation of both compounds was inhibited by a specific substrate of prolyl endopeptidase (EC 3.4.21.26) which is also called post-proline cleaving enzyme, an enzyme cleaving at the carboxyl end of proline. In summary, inhibitor studies suggest that both compounds are degraded at pH 7.5 by endopeptidase-24.11 and ACE in brush-border membrane, by activities of prolyl endopeptidase in cytosol, and at pH 4.5 by serine protease(s) in homogenate.

Structural requirements of bioactive peptides for interaction with endopeptidase 22.19

A series of biologically active peptides and related compounds (opioid peptides, neurotensin, and bradykinin) were used as substrates or competitive inhibitors to study the structural requirements for peptide interaction with endopeptidase 22.19. The kinetics of hydrolysis of these peptides indicated that, in contrast to other proteases, the substrate specificity of endopeptidase 22.19 is not determined by the amino acids flanking the sensitive bonds of the substrates. The competition between bioactive peptide analogues and the quenched fluorescence substrate of endopeptidase 22.19 indicated that their length and their flexibility may be the dominant factors to explain their binding specificities. These peculiar features of endopeptidase 22.19 may be of importance to understand the physiological processes of conversion and inactivation of biologically active peptides.

Inhibition of Neutral Endopeptidase Augments Anaphylactic Constriction of Guinea Pig Tracheal Smooth Muscle

To determine whether tachykinins participate in antigen-induced constriction of tracheal smooth muscle, we examined the effects of a neutral endopeptidase inhibitor, phosphoramidon, the tachykinin antagonist (D-Pro4, D-Trp7,9,10)-substance P(4-11), and capsaicin-induced tachykinin depletion on the responses to antigen in tracheal rings from ovalbumin-sensitized guinea pigs. In these preparations, the antigen (ovalbumin, 0.1 microgram/ml) produced reproducible and durable constriction of tracheal smooth muscle. Incubation with phosphoramidon (10 min, 10 microM) prior to antigen challenge significantly augmented the magnitude of ovalbumin-induced constriction by 22% after 30 min and by 31% after 45 min. The addition of phosphoramidon at the plateau level of antigen-induced constriction produced a similar, significant increase in the magnitude of the constriction. Following incubation with tachykinin antagonist (D-Pro4,D-Trp7,9,10)-substance P(4-11) (5 microM), the contractile response of the tracheal rings to the antigen was not altered. Furthermore, the addition of phosphoramidon (10 microM) did not significantly affect this contraction. Similarly, neither tachykinin antagonist nor phosphoramidon altered the ovalbumin-induced constriction of the tracheal rings from capsaicin-treated guinea pigs. Based on these findings, we hypothesize that tachykinins or similar broncho-constricting neutral endopeptidase substrates were released from tachykinin-containing nerve endings during immediate hypersensitivity reaction in airways, manifesting a modest and delayed constrictive effect. Following alteration of endopeptidase activity, these substances could modulate the anaphylactic constriction of the airway smooth muscle.

Detection and Preliminary Characterization of Taenia solium Metacestode Proteases

The metacestode of Taenia solium persists for years in the human central nervous system. As proteolytic enzymes play an important role in the survival of tissues helminths, we examined extracts of T. solium metacestodes for proteolytic activity using 9 synthetic peptide substrates and 3 proteins (hemoglobin, albumin, and immunoglobulin G). The proteolytic enzymes were classified based on their inhibitor profiles. At neutral pH, aminopeptidase(arginine-7-amino-4-trifluoromethylcoumarin) and endopeptidase(benzyloxy-carbonyl-glycine-glycine-arginine-7-amino-4- trifluoromethylcoumarin) substrates were cleaved. Hydrolysis of both substrates was inhibited by chelating agents, which inhibit metalloproteases. Peak activity with both substrates eluted in gel filtration fractions corresponding to a molecular weight of about 104 kDa. Cysteine protease activity was identified, which cleaved benzyloxy-carbonyl-phenylalanine-arginine-7-amino- 4-trifluoromethylcoumarin (Z-Phe-Arg-AFC) and hemoglobin. Cleavage of Z-Phe-Arg-AFC was maximal at acid pH, was stimulated by thiols, and was inhibited by leupeptin and Ep459. Peak cysteine protease activity eluted in gel filtration fractions corresponding to a molecular weight of 32 kDa. Aspartic protease activity was identified by specific inhibition with pepstatin of acid digestion of hemoglobin and immunoglobulin G. Immunoglobulin digestion occurred at acid pH, with preferential degradation of the heavy chain. Upon gel filtration chromatography, the aspartic protease activity eluted as a broad peak with maximal activity at about 90 kDa. No serine protease activity was detected. None of the parasite enzymes digested albumin. Proteolytic enzymes of T. solium may be important for parasite survival in the intermediate host, by providing nutrients and digesting host immune molecules.

Fluorimetric assay of the neurotensin-degrading metalloendopeptidase, endopeptidase 24.16

Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp (Mcc = 3-carboxy-7-methoxycoumarin; Dnp = dinitrophenyl), a quenched fluorimetric substrate originally designed as a probe to measure Pz-peptidase (also called endopeptidase 24.15), was examined as a putative substrate of the neurotensin-degrading neutral metalloendopeptidase, endopeptidase 24.16. During the purification of endopeptidase 24.16 the neurotensin(1-10) and neurotensin(11-13) formation due to this enzyme was coeluted with Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp-hydrolysing activity. By both fluorimetric and h.p.l.c. analyses, we observed that the latter activity was dose-dependently and completely abolished by neurotensin with an IC50 value (2.6 microM) that closely corresponds to the affinity of purified endopeptidase 24.16 for neurotensin (Km = 2.5 microM). Furthermore, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp hydrolysis was inhibited by a series of dipeptides with a rank of order of potencies that parallels that observed in competition experiments of tritiated neurotensin hydrolysis by brain and intestinal endopeptidase 24.16. Altogether, these data clearly demonstrate that, in addition to Pz-peptidase, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp also behaves as a substrate of endopeptidase 24.16, with a Km of about 26 microM. In addition, we show that, even in crude membrane preparations, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp behaves as a useful tool to monitor and accurately quantify endopeptidase 24.16.

Hydrolysis of thymic humoral factor γ2 by neutral endopeptidase (EC 3.4.24.11)

A search for the natural substrates for neutral endopeptidase (NEP; EC 3.4.24.11) in the immune system led to investigation of the enzyme's action on thymic humoral factor gamma 2 (THF). The ectoenzyme rapidly and efficiently hydrolyses the Lys6-Phe7 bond of the octapeptide. The site of cleavage was confirmed by h.p.l.c. analysis, amino acid analysis and sequence determination of the products. Phosphoramidon (3.6 microM), a potent inhibitor of the enzyme, prevents this cleavage even during prolonged incubation. The high efficiency of hydrolysis of THF by NEP is similar to that reported for [Leu5]enkephalin, and the dipeptide Phe-Leu is the C-terminal product in the hydrolysis of both peptides. The presence of NEP, reportedly identified as the common acute lymphoblastic leukaemia antigen (CALLA), in bone-marrow cells and other cells of the immune system raises the possibility that it may play a role in modulating the activity of peptides such as THF.

Neutral metalloendopeptidase associated with the smooth muscle cells of pregnant rat uterus

The pregnant rat uterus contains a membrane-bound metalloendopeptidase that is biochemically and immunologically similar to kidney enkephalinase (E.C.3.4.24.11). The uterus enzyme readily cleaved specific neutral endopeptidase substrates and oxytocin as well as the synthetic elastase substrate, Suc(Ala)3-pNA, yet did not digest native elastin. Using specific inhibitors, the uterus endopeptidase was identified as a metallopeptidase and not a serine protease, having an absolute requirement for zinc and perhaps calcium for maximal activity. The uterus endopeptidase cross-reacted with polyclonal antiserum to kidney microvillar endopeptidase and a monoclonal antibody to common acute lymphocytic leukemia antigen. Immunohistochemical localization of the enzyme in a 17 day pregnant uterus indicated that the enzyme was localized on the smooth muscle bundles of the myometrium and the endometrial epithelium. Total enzyme activity was 25 times higher in the late-term pregnant uterus (17th day of pregnancy) than in the nonpregnant uterus. Enzyme levels dropped rapidly prior to parturition and within 4 days after delivery the enzyme activity had returned to control levels. Inhibition of NEP in uterine strips with phosphoramidon resulted in a marked potentiation of oxytocin-induced contractions. Our results suggest that the uterine endopeptidase may have an important role in regulating uterine smooth muscle cell contraction during the later stages of pregnancy through its action on oxytocin and perhaps other biologically active peptides.

The hydrolysis of endothelins by neutral endopeptidase 24.11 (enkephalinase).

Endothelins 1-3 are a family of 21-amino acid peptides whose structure consists of two rings formed by intra-chain disulfide bonds and a linear "COOH-terminal tail." These peptides were originally described on the basis of their potent vasoconstrictor activity. The hydrolytic inactivation of endothelin action has recently been implicated to be attributed, at least in part, to the enzyme neutral endopeptidase 24.11 (Scicli, A. G., Vijayaraghavan, J., Hersh, L., and Carretero, O. (1989) Hypertension 14, 353). The kinetic properties and mode of hydrolysis of the endothelins by this enzyme are reported in this study. The Km for endothelins 1 and 3 hydrolysis is approximately 2 microM while endothelin2 exhibits a 5-fold higher Km. Endothelins 1 and 2 exhibit similar Vmax values while endothelin3 is hydrolyzed considerably more slowly. The initial cleavage site in endothelin1 is at the Ser5-Leu6 bond located within one of the cyclic structures. Thermolysin, a bacterial neutral endopeptidase with a similar substrate specificity to neutral endopeptidase 24.11 initially cleaves endothelin1 between His16-Leu17 which lies within the COOH-terminal linear "tail" portion of the molecule. The cleavage of endothelins 2 and 3 by neutral endopeptidase 24.11 differs from that observed with endothelin1 in that cleavage of these endothelins occurs at Asp18-Ile19 within the linear COOH-terminal tail structure. These results demonstrate that the endothelins are good substrates for neutral endopeptidase 24.11 and suggest that their mode of cleavage is dependent upon both amino acid sequence as well as peptide conformation.

Effect of electron withdrawing substituents on substrate hydrolysis by and inhibition of rat neutral endopeptidase 24.11 (enkephalinase) and thermolysin

A series of N-acylphenylalanylglycine dipeptides were synthesized and examined as substrates for neutral endopeptidase 24.11 (NEP) and thermolysin. Those N-acyl dipeptides containing an N-acyl group derived from an acid whose p K a is below 3.5 were considerably more reactive with both enzymes than those peptides containing an N-acyl group derived from an acid whose p K a is above 4. The data are interpreted to suggest that electron withdrawal at the scissile bond increases k cat for both NEP and thermolysin. The pH dependence for inhibition by the dipeptides Phe-Ala, Phe-Gly, and Leu-Ala showed binding dependent upon the basic form of an enzyme residue with a p K a of 7 for NEP and a p K a of 6 for thermolysin. In the case of thermolysin this p K a was decreased to 5.3 in the enzymeinhibitor complex. When examined as alternate substrate inhibitors of NEP, N-acyl dipeptides showed three distinct profiles for the dependence of K i on pH. With N-trifluoroacetyl-Phe-Gly as inhibitor, binding is dependent upon the basic form of an enzyme residue with a p K a value of 6.2. N-methoxyacetyl-Phe-Gly inhibition appears pH independent, while N-acetyl-Phe-Gly inhibition is dependent upon the acidic form of an enzyme residue with a p K a of approximately 7. All inhibitions of thermolysin by N-acyl dipeptides exhibit a dependence on the acidic form of an enzyme residue with a p K a of 5.3 to 5.8. These results suggest that with NEP, binding interactions at the active site involve one or more histidine residues while with thermolysin binding involves an active site glutamic acid residue.

A novel leupeptin-sensitive serine endopeptidase present in normal and malignant rat mammary tissues

N-Methyl-N-nitrosourea (MNU)-induced rat mammary adenocarcinomas contain high levels of a novel leupeptin-sensitive serine endopeptidase. Its properties apparently differ from those of other similar endopeptidases reported to be present in various normal and malignant mammalian tissues. The same leupeptinsensitive serine endopeptidase was also detected in normal rat mammary tissues, but at levels approximately 20 times lower than those in MNU-induced mammary tumors. This enzyme, which is a trypsin-like serine endopeptidase, preferentially hydrolyzes various synthetic endopeptidase substrates at the carboxyl side of an arginyl residue. It has an apparent Mr of approximately 160,000 and a Stokes radius of 49 A, as determined by gel filtration. Its isoelectric points range from 4.5 to 4.8, and it has a pH optimum of approximately 7.0. The enzyme is stable from pH 4.0 to 7.0, but is extremely unstable above pH 7.0. Besides leupeptin, its activity is inhibited by antipain, aprotinin, N alpha-p-tosyl-L-lysine chloromethyl ketone and phenylmethylsulfonyl fluoride, but is not inhibited by soybean trypsin inhibitor. Many other potential inhibitors or activators such as 2-mercaptoethanol, p-hydroxymercuribenzoic acid and EDTA have no effect on its activity. The enzyme is adsorbed to p-aminobenzamidine agarose affinity beads at pH 6.5 and elutes at pH 4.0.

Proteolytic activity of oral streptococci

Streptococcus mutans and Streptococcus sobrinus were the least proteolytic of 8 species of oral streptococci while Streptococcus oralis and Streptococcus sanguis were the most proteolytic. Degradation of FITC-BCA was significantly correlated with the hydrolysis of synthetic endopeptidase substrates. As S. oralis strains proliferate in dental plaque in the absence of dietary food their success, in vivo, might be due partially to their greater proteolytic activity compared to other oral streptococci.

Proinsulin endopeptidase substrate specificities defined by site-directed mutagenesis of proinsulin

Two endopeptidases are involved in the conversion of proinsulin; a type I activity directed at the B chain, Arg31,Arg32, C-peptide junction, and type II which cleaves the C-peptide, Lys64,Arg65, A chain junction. To define further the substrate specificities of these enzymes, a series of mutant preproinsulin cDNAs were generated by site-directed and deletion mutagenesis. These were inserted into pT7 plasmids and capped cRNA transcripts synthesized, that were then microinjected into Xenopus oocytes. Oocytes were biosynthetically radiolabeled with [3H]leucine and the secreted peptides (greater than 95% present as unprocessed proinsulins) then incubated with types I and II endopeptidase activities prepared from isolated insulinoma secretory granules. The reaction products were analyzed by high performance liquid chromatography. Des-38-62-proinsulin, in which all but six amino acids of C-peptide were deleted was not processed by either enzyme. The mutant Lys64,Arg65 to Thr64,Arg65 was not cleaved by the type II enzyme but was still a substrate for the type I enzyme. The mutant Arg31,Arg32 to Arg31,Gly32 correspondingly was not cleaved by the type I enzyme; however, in this case it was not attacked by the type II enzyme. These results indicate that not only is the presence of a dibasic sequence essential, but also that the secondary structure of the protein is important in determining whether the prohormone is susceptible to proteolytic processing.

The inhibition of proinsulin-processing endopeptidase activities by active-site-directed peptides.

Inhibitor studies were performed on the two endopeptidase activities involved in proinsulin conversion in isolated insulin secretory granules [Davidson, Rhodes & Hutton (1988) Nature (London) 333, 93-96]. The active-site-directed peptides L-alanyl-L-arginyl-L-arginylmethyldimethylsulphonium and L-alanyl-L-lysyl-L-arginylmethyldimethylsulphonium inhibited these activities in accordance with the observed cleavage pattern, suggesting that the primary amino acid sequence of the dibasic site was an important determinant of the endopeptidase substrate specificities.

Porcine muscle prolyl endopeptidase and its endogenous substrates.

Prolyl endopeptidase [EC 3.4.21.26] was purified 4,675-fold with a yield of 26.3% from porcine muscle. The purified enzyme was shown to be very similar to the liver enzyme with respect to its molecular weight (72,000-74,000), antigenicity, substrate specificity, and susceptibility to protease inhibitors. Among several bioactive peptides, angiotensins I, II, and III had the lowest Km of 0.6 to 3 microM with the lowest kcat of 0.19 to 0.85 s-1, while thyrotropin-releasing hormone had the highest Km of 98 microM with the highest kcat of 14.4 s-1. Interestingly, mastoparan was hydrolyzed at alanyl bonds, but insulin was only slightly hydrolyzed and glucagon was not hydrolyzed although the latter two peptides contain prolyl and/or alanyl bonds. Muscle prolyl endopeptidase failed to hydrolyze proteins with high molecular weight such as albumin, immunoglobulin G, elastin, collagen, and muscle soluble and insoluble proteins. However, 8 of 14 peptides with molecular weights lower than 3,000, which were isolated from muscle extract, were digested by this enzyme, and they were proved to contain prolyl and/or alanyl residues in their molecules. The data suggest that they are probable endogenous substrates for prolyl endopeptidase.

Rapid degradation of D- and L-succinimide-containing peptides by a post-proline endopeptidase from human erythrocytes.

We have been interested in the metabolic fate of proteins containing aspartyl succinimide (Asu) residues. These residues can be derived from the spontaneous rearrangement of Asp and Asn residues and from the spontaneous demethylation of enzymatically methylated L-isoAsp and D-Asp residues. Incubation of the synthetic hexapeptide N-Ac-Val-Tyr-Pro-Asu-Gly-Ala with the cytosolic fraction of human erythrocytes resulted in rapid cleavage of the prolyl-aspartyl succinimide bond producing the tripeptide N-Ac-Val-Tyr-Pro. The rate of this reaction is equal for both L- and D-Asu-containing peptides and is 10-fold greater than the rate of cleavage of a corresponding peptide containing a normal Pro-Asp linkage. When the aspartyl succinimide ring was replaced with an isoaspartyl residue, the cleavage rate was about 5 times that of the normal Pro-Asp peptide. The tripeptide-producing activity copurified on DEAE-cellulose chromatography with an activity that cleaves N-carbobenzoxy-Gly-Pro-4-methylcoumarin-7-amide, a post-proline endopeptidase substrate. These two activities were both inhibited by an antiserum to rat brain post-proline endopeptidase, and it appears that they are catalyzed by the same enzyme. This enzyme has a molecular weight of approximately 80,000 and is covalently labeled and inhibited by [3H]diisopropyl fluorophosphate. The facile cleavage of the succinimide- and isoaspartyl-containing peptides by this post-proline endopeptidase suggests that it may play a role in the metabolism of peptides containing altered aspartyl residues.