Purification and Characterization of a Novel Peptidase (IImes) from Mesquite (Prosopis velutina) Pollen
1998; Elsevier BV; Volume: 273; Issue: 27 Linguagem: Inglês
10.1074/jbc.273.27.16771
ISSN1083-351X
Autores Tópico(s)Medicinal plant effects and applications
ResumoAlthough the mesquite plant (Prosopis velutina) is not as widely distributed as some other allergenic species, its pollen can induce serious pollinosis in areas where it is localized. We previously isolated and characterized a peptidase from mesquite pollen with trypsin-like specificity (peptidase Imes) (Matheson, N., Schmidt, J., and Travis, J. (1995)Am. J. Respir. Cell Mol. Biol. 12, 441–448). Now we have characterized a second enzyme with specificity for hydrophobic residues (mesquite pollen peptidase IImes). This enzyme has a molecular mass near 92 kDa and activity that was not affected by reducing or chelating agents but was inhibited by specific synthetic serine proteinase inhibitors and the aminopeptidase inhibitor bestatin. However, it was not inhibited by human plasma proteinase inhibitors, nor did it inactivate any of those tested. The enzyme possessed amidolytic activity against p-nitroanilide substrates most effectively after alanine residues and also displayed aminopeptidase activity against non-p-nitroanilide peptides with a preference for phenylalanine. This specificity for hydrophobic amino acid residues was corroborated by inhibition studies with chloromethyl ketone and organophosphonate inhibitors. More interesting from a physiological point of view is that the bioactive peptides, angiotensins I and II and vasoactive intestinal peptide, were also hydrolyzed rapidly, indicating an ability of peptidase IImes to act also as an oligopeptidase. Because these bioactive peptides play a role in the inflammatory responses in allergic asthma, our data suggest that the purified mesquite pollen peptidase IImes may be involved in the degradation of neuro- and vasoactive peptides during pollen-initiated allergic reactions. Although the mesquite plant (Prosopis velutina) is not as widely distributed as some other allergenic species, its pollen can induce serious pollinosis in areas where it is localized. We previously isolated and characterized a peptidase from mesquite pollen with trypsin-like specificity (peptidase Imes) (Matheson, N., Schmidt, J., and Travis, J. (1995)Am. J. Respir. Cell Mol. Biol. 12, 441–448). Now we have characterized a second enzyme with specificity for hydrophobic residues (mesquite pollen peptidase IImes). This enzyme has a molecular mass near 92 kDa and activity that was not affected by reducing or chelating agents but was inhibited by specific synthetic serine proteinase inhibitors and the aminopeptidase inhibitor bestatin. However, it was not inhibited by human plasma proteinase inhibitors, nor did it inactivate any of those tested. The enzyme possessed amidolytic activity against p-nitroanilide substrates most effectively after alanine residues and also displayed aminopeptidase activity against non-p-nitroanilide peptides with a preference for phenylalanine. This specificity for hydrophobic amino acid residues was corroborated by inhibition studies with chloromethyl ketone and organophosphonate inhibitors. More interesting from a physiological point of view is that the bioactive peptides, angiotensins I and II and vasoactive intestinal peptide, were also hydrolyzed rapidly, indicating an ability of peptidase IImes to act also as an oligopeptidase. Because these bioactive peptides play a role in the inflammatory responses in allergic asthma, our data suggest that the purified mesquite pollen peptidase IImes may be involved in the degradation of neuro- and vasoactive peptides during pollen-initiated allergic reactions. Asthma is an allergic inflammation of the lungs which can occur after allergen sensitization. Such inflammatory responses are normally meant to defend against invading organisms or particulates or to effect tissue repair and are thus beneficial; however, in asthma, the response becomes exaggerated (perhaps because of a hereditary predisposition (1DeMonchy J.G.R. Kauffman H.F. Venge P. Koeter G.H. Jansen H.M. Sluiter H.J. DeVries K. Am. Rev. Respir. Dis. 1985; 131: 373-376PubMed Google Scholar)), leading to adverse effects on the airways (2Barnes P.J. Middleton Jr., E. Reed C.E. Ellis E.F. Adkinson Jr., N.F. Yuninger J.W. Busse W.W. Allergy: Principles and Practice. 4th Ed. I. Mosby-Year Book, New York1993: 243-266Google Scholar). Macrophages phagocytize the allergens introduced to the lungs by exposure to various environmental irritants such as dust, pollutants, and pollen, and process them to smaller fragments. As antigen-presenting cells, they then activate T-cells (3Babbit B. Allen P.M. Matsudeda G. Haber E. Unanue E.R. Nature. 1985; 317: 359-361Crossref PubMed Scopus (936) Google Scholar, 4Ashwell J.D. Schwartz R.H. Nature. 1986; 320: 176-178Crossref PubMed Scopus (55) Google Scholar) to stimulate B-cells to produce IgE. This immunoglobulin, when bound to a specific allergen, in turn, stimulates and activates several alveolar cell types to produce the many mediators of inflammation: histamine, prostaglandins, leukotrienes, cytokines, neutral proteases, active oxygen species, and chemoattractants (5Holgate S.T. Robinson C. Church M.K. Middleton Jr., E. Reed C.E. Ellis E.F. Adkinson Jr., N.F. Yuninger J.W. Busse W.W. Allergy: Principles and Practice. 4th Ed. I. Mosby-Year Book, Inc., 1993: 267-279Google Scholar). The interaction of these mediators leads to the pathology of asthma, including bronchoconstriction, hypertrophy of airway smooth muscle, vasodilation, submucosal edema, and mucus hypersecretion (6Barnes P.J. J. Allergy Clin. Immunol. 1989; 83: 1013-1026Abstract Full Text PDF PubMed Scopus (415) Google Scholar). Also, the mucociliary apparatus becomes dysfunctional, reducing the clearance of inhaled particulates. Epithelial cells lining the airways are shed during this inflammatory response, removing a protective barrier (2Barnes P.J. Middleton Jr., E. Reed C.E. Ellis E.F. Adkinson Jr., N.F. Yuninger J.W. Busse W.W. Allergy: Principles and Practice. 4th Ed. I. Mosby-Year Book, New York1993: 243-266Google Scholar) and are also a source of neutral endopeptidase (which normally degrades various bronchoconstrictor peptides (7Frossard N. Rhoden K.J. Barnes P.J. J. Pharmacol. Exp. Ther. 1989; 248: 292-298PubMed Google Scholar)) while exposing nerve endings (8Barnes P.J. The Lancet. 1986; 1: 242-244Abstract PubMed Scopus (590) Google Scholar) that secrete neuropeptides such as vasoactive intestinal peptide (VIP) 1The abbreviations used are: VIP, vasoactive intestinal peptide; pNA, p-nitroanilide; Suc, succinyl; TPCK, tosyl-l-phenylalanine chloromethyl ketone; TLCK,Nα -p-tosyl-l-lysine chloromethyl ketone; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; HIV, human immunodeficiency virus; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; FPLC, fast protein liquid chromatography; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HPLC, high performance liquid chromatography. and substance P, and vasoactive peptides (e.g. angiotensin II). VIP, a neurotransmitter of the nonadrenergic inhibitory system (9Casale T.B. Am. J. Rhinol. 1988; 2: 121-127Crossref Google Scholar), has an anti-inflammatory effect inhibiting lymphocyte proliferation and interleukin-2 release and is also a potent bronchodilator (10Stanisz A.M. Scicchitano R. Bienenstock J. Ann. N. Y. Acad. Sci. 1988; 527: 478-485Crossref PubMed Scopus (25) Google Scholar). Substance P, a neurotransmitter of the nonadrenergic excitatory system (11Barnes P.J. J. Allergy Clin. Immunol. 1987; 79: 285-295Abstract Full Text PDF PubMed Scopus (64) Google Scholar), in contrast, has a proinflammatory effect, increasing vascular permeability and bronchoconstriction, causing macrophages to release proinflammatory substances, and enhancing phagocytosis by neutrophils and macrophages (12Hartung H.-P. Wolters K. Toyka K.V. J. Immunol. 1986; 136: 3856-3863PubMed Google Scholar). Angiotensin II is a strong vasoconstricting agent (13DeBono E. Lee G.de J. Mottram F.R. Pickering G.W. Brown J.J. Keen H. Peart W.S. Sanderson P.H. Clin. Sci. 1963; 25: 123-157PubMed Google Scholar). Pollen is one of the major initiators of allergic asthma. This gamete contains proteins (allergens) that are solubilized in the airway mucus and proceed to induce an immunological response. However, other proteins are also released, of which several have proven to be oligopeptidases (14Matheson N. Schmidt J. Travis J. Am. J. Respir. Cell Mol. Biol. 1995; 12: 441-448Crossref PubMed Scopus (22) Google Scholar, 15Bagarozzi Jr., D.A. Pike R. Potempa J. Travis J. J. Biol. Chem. 1996; 271: 26227-26232Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 16Bagarozzi Jr., D.A. Potempa J. Travis J. Am. J. Respir. Cell Mol. Biol. 1998; 18: 363-369Crossref PubMed Scopus (40) Google Scholar). Because these latter enzymes appear to be members of a family of pollen oligopeptidases with varying specificities for peptide hydrolysis, we propose to name them, at least temporarily, as: peptidases Imes and Irag(trypsin-like specificity from both mesquite and ragweed pollens), peptidase IIrag (chymotrypsin-like specificity from ragweed pollen), and, as described in this report, peptidase IImes(hydrophobic amino acid specificity from mesquite pollen), an enzyme that rapidly degrades VIP, angiotensin II, and its precursor, angiotensin I. We suggest that through exo- and oligopeptidase activity, pollen may have the capability for participation in the inflammatory processes in allergic asthma by mechanisms other than those involving its immunological component. H-Val-pNA, H-Leu-pNA,N-Suc-Ala-Ala-Pro-Phe-pNA,N-Suc-Ala-Ala-Pro-Leu-pNA,N-Suc-Ala-Ala-Val-Ala-pNA,N-Suc-Ala-Ala-Ala-pNA,N-Suc-Phe-pNA, benzoyl-dl-Arg-pNA, TPCK, TLCK, iodoacetamide, bestatin ([(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-l-leucine), angiotensins I and II, VIP, atrial natriuretic peptide, bradykinin, substance P, neurotensin, Phe-Gly-Leu-Met (substance P fragment) (peptide 1), Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Arg (active fragment of myelin basic protein) (peptide 2), Ala-Ser-Thr-Thr-Thr-Asn Tyr-Thr (peptide T = HIV inhibitor) (peptide 3), and Leu-Pro-Pro-Ser-Arg (lymphocyte-activating pentapeptide from the Fc region of human IgG1) (peptide 4) were obtained from Sigma. H-Ala-pNA, H-Ala-Ala-pNA, H-Ala-Ala-Ala-pNA, Ac-Ala-pNA, Ac-Ala-Ala-pNA, H-Phe-pNA, H-Ile-pNA, H-Ala-Phe-pNA, H-Glu-Ala-pNA,N-Suc-Ala-Phe-Pro-Phe-pNA, Suc-Ala-Ala-Pro-Ala-pNA, benzyloxycarbonyl-Ala-Ala-Leu-pNA, and benzoyl-Tyr-pNA were from Bachem. Diisopropyl fluorophosphate and 3,4-dichloroisocoumarin were obtained from Calbiochem, and AEBSF and EDTA were from Boehringer Mannheim. The mesquite pollen was a kind gift from Dr. Justin O. Schmidt (Carl Hayden Bee Research Center, Tuscon, AZ). All chloromethyl ketone (except TPCK and TLCK) and organophosphonate inhibitors were kindly provided by Dr. James Powers (Georgia Institute of Technology, Atlanta). Mesquite pollen (100 g) was extracted by stirring in 400 ml of 0.02 m Bis-Tris, pH 6.5, 5 mm CaCl2 (buffer A) overnight at 4 °C. Purification of the enzyme was performed using exactly the procedures described previously (14Matheson N. Schmidt J. Travis J. Am. J. Respir. Cell Mol. Biol. 1995; 12: 441-448Crossref PubMed Scopus (22) Google Scholar) with ammonium sulfate fractionation, acid precipitation of contaminants, and Cibacron blue-Sepharose, DEAE-Sephacel, and phenyl-Sepharose chromatography. The active eluate from the phenyl-Sepharose column was dialyzed overnight at 4 °C against buffer A with two changes and concentrated to 20 ml using an Amicon P-30 membrane. The final step of purification involved the application of the dialyzed and concentrated enzyme solution to a Mono Q FPLC column (Amersham Pharmacia Biotech) equilibrated with buffer A. The column was washed with buffer A for 5 min, followed by a 0–0.05 m NaCl gradient for 5 min, then a 0.05–0.15m NaCl gradient for 50 min during which the enzyme activity was eluted. The native conformation of the enzyme was obtained by polyacrylamide gel electrophoresis using a Tris-HCl/Tricine buffer system (17Shagger H. Von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10505) Google Scholar) omitting SDS. The molecular weight of the purified enzyme (peptidase IImes) was determined by both SDS-polyacrylamide gel electrophoresis using a Tris-HCl/Tricine buffer system (17Shagger H. Von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10505) Google Scholar) with or without reducing conditions and by gel filtration on a Sephadex G-150 column (2.2 × 90 cm). For routine assays during purification, pH optimum determination, temperature effects, and the effects of inhibitors, the activity of peptidase IImes was only measured spectrophotometrically at 405 nm with H-Ala-pNA (1 mm, final concentration) in either 0.2 or 1.0 ml of 0.1m Tris-HCl, pH 8.0, 0.15% dimethyl sulfoxide at 25 °C. In inhibitor studies, the enzyme was incubated with inhibitors for 15 min at 25 °C before the substrate (H-Ala-pNA) was added. Amidolytic activity of several substrates (1 mm, final concentration) was determined in 0.2 ml of the same buffer and temperature as above. Protein concentration was determined by the bicinchoninic acid-Cu(II) sulfate procedure with bovine serum albumin as the standard (18Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18713) Google Scholar). Peptidase IImes (1.06 nmol) was denatured by boiling in 1% SDS followed by incubation with 0.017 nmol of high molecular weight Arg-gingipain from Porphyromonas gingivalis (19Chen Z. Potempa J. Polanowski A. Wikstrom M. Travis J. J. Biol. Chem. 1992; 267: 18896-18901Abstract Full Text PDF PubMed Google Scholar) in 0.2 ml of 0.02 m Tris-HCl, pH 7.6, and 1 mm fresh cysteine overnight at 37 °C. After SDS-polyacrylamide gel electrophoresis of the digest and electroelution to a polyvinylidene difluoride membrane, sequence analysis was performed with an Applied Biosystems Procise Protein sequencer using the program designed by the manufacturer. For specificity studies, the purified enzyme (35.3–106.0 nm) was incubated with several bioactive peptides (20.0–64.0 μm) at enzyme:substrate molar ratios of 1:400–1:600 in 0.1 mTris-HCl, pH 8.0, at 37 °C. For studies with peptides with NH2-terminal residues of phenylalanine, alanine, and leucine, the purified enzyme (58.7–78.0 nm) was incubated with each of the substrates (64.3–143.2 μm) at enzyme:substrate molar ratios of 1:1,000–1:8,000 in the same buffer and temperature as above. Aliquots of 35 μl were removed at various times and added to 2 μl of 20% trifluoroacetic acid to stop the reaction. Each reaction mixture was subjected to high performance liquid chromatography (HPLC) using an Ultrasphere ODS reverse phase column (4.6 × 25.0 cm, 5 μm) (Beckman Instruments) and a linear gradient from 0.1% trifluoroacetic acid to 0.08% trifluoroacetic acid containing 80% acetonitrile over a 30-min period (1 ml/min). Peptides were detected at 220 nm. The same reaction mixtures were analyzed for amino acid composition by mass spectrometry. Some of the samples were examined by matrix-assisted laser desorption ionization. The matrix (2 μl of a saturated solution of α-cyano-4-hydroxycinnamic acid in a 50:50 mixture of water:acetonitrile with 0.1% trifluoroacetic acid) was placed on the target with approximately 0.5 μl of sample. The samples were then analyzed with a Bruker Reflex time-of-flight mass spectrometer (Billerica, MA) using matrix-assisted laser desorption ionization in linear mode, 100 shots averaged with mass range scanned from 0 to 1,500 m/z for bradykinin to 0–3,600m/z for VIP. Some samples were analyzed by liquid chromatography-mass spectrometry using a PE-Sciex API I (atmospheric pressure ionization) plus mass spectrometer coupled with an Applied Biosystems 140 B solvent delivery system and an ABI 759A absorbance detector. The sample (20 μl) was injected onto an Asahipak ODP C18 column (1 × 250 mm, 5 μm, 200 A) (Keystone Scientific Inc., Bellefonte, PA). The gradient used was from 0 to 100% B over 60 min at a flow rate of 40 μl/min. Solvent A was 0.1% trifluoroacetic acid in water, and solvent B was 90% acetonitrile and 10% water with 0.1% trifluoroacetic acid. The mass spectrometer was scanned from 50 to 500 U using a 0.2-U step and a 1.5-ms dwell time. The UV was monitored at 214 nm, and the signal was amplified 50 times by an Omni Amp II A (Omega Engineering Inc., Stamford, CT). The results of the mass spectrometry were in the form of a chromatographic trace with each peak having a number representing the mass of a specific fragment. Each number was entered into the computer program, Peptidemap 2.2, together with the sequence of the peptide being studied. The output obtained indicated the sequence of the peptide fragment matching the mass of the peak and was an indication of cleavage products from peptidase IImes proteolysis. V max and K m values for amino acid p-NAs were determined using substrate concentrations ranging from 18.75 to 250 μm with the final concentrations of enzyme from 2.0 to 19.1 nm in 0.1m Tris-HCl, pH 8.0, 0.125% dimethyl sulfoxide at 25 °C. Values for the bioactive peptides were measured with substrate concentrations ranging from 10 to 82 μm, with the final concentration of enzyme from 2.2 to 25.4 nm in 0.1m Tris-HCl, pH 8.0, 5 mm CaCl2 at 25 °C with peptidase Imes and 37 °C with peptidase IImes. Aliquots of 35 μl were removed at various times and added to 2 μl of 20% trifluoroacetic acid to stop the reaction. Each sample was subjected to HPLC as described above. The increase in the peak area of the product with time was used to determine the rate of peptide cleavage. V max andK m values were determined by using Hyperbolic Regression Analysis. 2The Hyperbolic Regression Analysis program, written by J. S. Easterby (University of Liverpool, U. K.), was obtained through shareware. Peptidase IImes was readily liberated from the pollen grains by gentle stirring with buffer at 4 °C, with 50% of the activity being released by 2.5 h, and maximum activity at 6 h (data not shown). However, because the enzyme was very stable, extraction was usually performed overnight as a matter of convenience. As shown in Table I, several steps were required to purify peptidase IImes, with the scheme utilized being essentially equivalent to that performed for the isolation of peptidase Imes (14Matheson N. Schmidt J. Travis J. Am. J. Respir. Cell Mol. Biol. 1995; 12: 441-448Crossref PubMed Scopus (22) Google Scholar). Although a single enzyme activity directed toward hydrolysis of H-Ala-pNA was obtained during all procedures up to the Mono Q FPLC step, three activities separated during this final chromatographic procedure. However, all forms exhibited identical specific activities against either H-Ala-pNA or H-Leu-pNA, all were 92 kDa, and all were inhibited by TPCK, 3,4-dichloroisocoumarin, AEBSF, or the aminopeptidase inhibitor bestatin. A native polyacrylamide gel revealed a single, diffuse, unresolvable band (data not shown). Because of these identical properties, we assumed that the various forms were isozymes of each other, pooled them together, and utilized the combined enzyme in the studies described below.Table IPurification of mesquite pollen peptidase IImesFractionation stepTotal activityaBased on enzymatic activity usingl-Ala-pNA where 1 unit = nmol of pNA released/min.Total proteinSpecific activityPurificationYieldunitsmgunits/mg-fold%Crude extract81,70010,2008.01100(NH4)2SO4, 30–60%75,8005,52013.7293pH 4.5 supernatant67,8001,45046.7683Cibacron blue-Sepharose36,40037497.31245DEAE-Sephacel34,600122284.03542Phenyl-Sepharose24,3007.43280.041030Mono Q FPLC19,7000.7227,400.03,42024Results are based on 100 g of pollen.a Based on enzymatic activity usingl-Ala-pNA where 1 unit = nmol of pNA released/min. Open table in a new tab Results are based on 100 g of pollen. As in the case of peptidase I mes, peptidase IImes was also stable for at least several months at −20 °C, although frequent freezing and thawing caused some loss. However, in comparison, Ca2+ was not required either for stability or activity. Treatment of the purified enzyme with SDS followed by gel electrophoresis revealed a major band with a molecular mass of 92 kDa and some very faint minor bands (Fig. 1). The molecular mass of the major band agreed very well with that determined by Sephadex G-150 gel filtration of active enzyme (96 kDa). Unfortunately, no amino-terminal sequence could be found, indicating that this enzyme has a blocked amino terminus. Utilizing the amidolytic activity assays with H-Ala-pNA, it was found that the enzyme had a broad pH optimum from pH 7.5 to 9.5 and was stable for at least 48 h at pH 8.0 and 25° or 37 °C. Peptidase IImes activity was tested with several amino acid and peptide p-NAs (Table II). H-Ala-pNA was the preferred substrate by far (and thus was used in general assays), with the next best being H-Ala-Ala-pNA. Longer peptides were even less effective as substrates. An NH2-terminal blocking group nearly or completely abolished activity, with Suc-Ala-Ala-Ala-pNA, Suc-Phe-pNA, Ac-Ala-pNA, and Ac-Ala-Ala-pNA not acting as substrates at all; however, there was substantial activity against the corresponding non-succinylated or non-acetylated p-NAs. Ac-Ala-pNA and Ac-Ala-Ala-pNA, in fact, acted as inhibitors at 10 and 20 times the concentration of the substrate, H-Ala-pNA. These results indicate that the amidolytic activity of peptidase IImes requires a free amino group at the NH2 terminus of a substrate, whereas a blocked NH2 terminus can create a competitive inhibitor. It is possible that the enzyme may be sequentially removing the NH2-terminal amino acid or cleaving internally in the peptide pNA substrates since, as shown below utilizing non-pNA peptide substrates, both aminopeptidase and oligopeptidase activity could be detected.Table IIAmidolytic activity of mesquite pollen peptidase IImesSubstrateActivitynmol pNA released/μg/minH-Ala-pNA26.24H-Ala-Ala-pNA3.51H-Phe-pNA1.89H-Val-pNA0.95H-Ala-Ala-Ala-pNA0.79H-Leu-pNA0.63H-Ile-pNA0.53H-Ala-Phe-pNA0.45H-Glu-Ala-pNA0.34Suc-Met-Val-Pro-Phe-pNA0.07Suc-Ala-Phe-Pro-Phe-pNA0.04Suc-Ala-Ala-Pro-Phe-pNA0.03Suc-Ala-Ala-Pro-Leu-pNA0.03Suc-Ala-Ala-Pro-Ala-pNA0.02Suc-Ala-Ala-Val-Ala-pNA0Suc-Ala-Ala-Ala-pNA0Z-Ala-Ala-Leu-pNAaZ, benzyloxycarbonyl.0Suc-Phe-pNA0Bz-Tyr-pNAbBz, benzoyl.0Bz-Arg-pNA0The assay was performed at 25 °C in 0.1 M Tris-HCl, pH 8.0, 0.15% Me2SO with 1 mM, final concentration, of the substrates above. The enzyme to substrate ratio was 1:100,000 for H-Ala-pNA and 1:25,000 for the others.a Z, benzyloxycarbonyl.b Bz, benzoyl. Open table in a new tab The assay was performed at 25 °C in 0.1 M Tris-HCl, pH 8.0, 0.15% Me2SO with 1 mM, final concentration, of the substrates above. The enzyme to substrate ratio was 1:100,000 for H-Ala-pNA and 1:25,000 for the others. The demonstration of exo- and oligopeptidase activity of peptidase IImes against both bioactive and randomly selected peptides is given in Table III. In peptides chosen because they contained unblocked phenylalanine, leucine, or alanine residues at the NH2 terminus, hydrolysis at the amino terminus occurred at low E:S molar ratios: 1:8,000 for enzyme:FGLM, 1:2,000 for LPPSR, and 1:1,000 for both FSWGAEGQR and ASTTTNYT. Internal residues of alanine and leucine were untouched at these short times of incubation and low E:S ratios. The enzyme was particularly effective in cleaving after NH2-terminal phenylalanine residues, especially in the tetrapeptide, FGLM. Hydrolysis after either the NH2-terminal leucine or alanine residues was much slower (Fig. 2). Thus, peptidase IImes exhibited aminopeptidase activity. It is puzzling, however, why phenylalanine should be preferred rather than alanine, as was seen with the pNA substrates.Table IIISpecificity of cleavage of bioactive peptides by mesquite pollen peptidase IImesPeptideSequence ↓Angiotensin ID-R-V-Y-I-H-P-F-H-L ↓Angiotensin IID-R-V-Y-I-H-P-F ↓ ↓ ↓ ↓ ↓Vasoactive intestinal peptideH-S-D-A-V-F-T-D-N-Y-T-R-L-R-K-Q-M-A-V- ↓ ↓K-K-Y-L-N-S-I-L-N ↓Atrial natriuretic peptideS-L-R-R-S-S-C-F-G-G-R-M-D-R-I-G-A-Q-S-G-↓ ↓L-G-C-N-S-F-R-Y ↓BradykininR-P-P-G-F-S-P-F-R ↓ ↓Substance PR-P-K-P-Q-Q-F-F-G-L-M ↓ ↓NeurotensinE-L-Y-E-N-K-P-R-R-P-Y-I-L↓Peptide 1F-G-L-M↓Peptide 2F-S-W-G-A-E-G-Q-R↓Peptide 3A-S-T-T-T-N-Y-T↓Peptide 4L-P-P-S-R Open table in a new tab Non-pNA bioactive peptides of 8–28 amino acids were excellent substrates at E:S molar ratios of 1:400 to 1:600 (Table III). Angiotensins I and II were cleaved relatively rapidly (Fig. 3) with complete hydrolysis by 50 and 100 min, respectively, at these very low ratios. VIP was fragmented somewhat more slowly, 40% being cleaved by 90 min; atrial natriuretic peptide, bradykinin, substance P, and neurotensin were only slowly degraded. These results indicate that peptidase IImes also has oligopeptidase activity. This is not a novel concept because multiple reports indicate that many purified enzymes have both aminopeptidase and oligopeptidase activity. These include cathepsin H (20Barrett A.J. Kirschke H. Methods Enzymol. 1981; 80: 535-561Crossref PubMed Scopus (1729) Google Scholar). In addition, many peptidyldipeptidases, including cathepsin B (21McKay M.J. Offerman M.K. Barrett A.J. Bond J.S. Biochemistry. 1983; 213: 467-471Crossref Scopus (32) Google Scholar), also have oligopeptidase activity. In all cases, this has been demonstrated with peptide substrates rather than with proteins, a result that is paralleled in this study. The hydrolysis of all bioactive peptides occurred exclusively and internally after isoleucine, leucine, phenylalanine, alanine, and methionine residues (most rapidly after isoleucine) in the substrates tested but not after every such residue in every peptide. Six of the peptides had one to three cleavages, but VIP was cleaved at seven sites. Hydrolysis after methionine residues also occurred in the dipeptides Met-Phe and Met-Tyr, which are usually used as internal standards in determining kinetic constants by HPLC. Also, a small amount of inhibition (20%) of the hydrolysis of the bioactive peptides occurred when Met-Phe or Met-Tyr were present. No preference for either hydrophilic or hydrophobic residues in either the P1′ or P2 position was obvious. (The amino acid residues in substrates are numbered as P3, P2, P1, etc. toward the NH2 terminus from the cleavage site and P1′, P2′, P3′, etc. toward the COOH terminus (22Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4783) Google Scholar).) Although phenylalanine or alanine was the favored NH2-terminal residue for aminopeptidase activity and isoleucine for the oligopeptidase activity, cleavage was exhibited in both cases after all three residues. Significantly, no NH2-terminal amino acid cleavage occurred with the bioactive peptides tested because none had a suitable hydrophobic residue in that position, supporting our contention of a single enzyme with two activities of defined specificities. As with mesquite pollen peptidase Imes, peptidase IImes only very slowly hydrolyzed proteins, such as azocasein and blue hide powder (a matter of days), whereas the plasma serpins, α1-proteinase inhibitor and α1-antichymotrypsin, were not hydrolyzed despite the known susceptibility to proteolytic attack within their respective reactive site loops. These results differ from data obtained recently with a chymotrypsin-like peptidase from ragweed pollen which rapidly inactivated human α1-proteinase inhibitor (15Bagarozzi Jr., D.A. Pike R. Potempa J. Travis J. J. Biol. Chem. 1996; 271: 26227-26232Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Peptidase IImes was not inhibited by cysteine or metalloproteinase inhibitors (TableIV) or by the specific serpins α1-proteinase inhibitor and α1-antichymotrypsin. It was, however, inactivated by lowM r serine proteinase inhibitors such as diisopropyl fluorophosphate, AEBSF, and 3,4-dichloroisocoumarin, although at rather high concentrations. As expected, TPCK was a good inhibitor, by virtue of the specificity of the enzyme toward phenylalanine residues, whereas TLCK was not inhibitory. In support of the exopeptidase activity exhibited by peptidase IImes, the aminopeptidase inhibitor bestatin was very effective in reducing enzyme activity on synthetic substrates. However, both 1,10-phenanthroline (a metal chelator) and 4,7-phenanthroline (a non-chelator) inhibited as well. The non-chelating analog can bind nonspecifically to the active site of some enzymes (23Barrett A.J. Methods Enzymol. 1994; 244: 11Google Scholar), which indicates that no metal ion is involved in the enzymatic activity.Table IVEffect of class-specific inhibitors on the amidolytic activity of mesquite pollen peptidase IImesInhibitorClassConcentrationResidual activity%EDTAMetallo5 mm1001,10-PhenanthrolineMetallo1 mm444,7-PhenanthrolineNonspecific1 mm51IodoacetamideCysteine10 mm100TLCKCysteine/serine trypsin-like1 mm100DFPaDFP, diisopropyl fluorophosphate.Serine0.2 mm562 mm0AEBSFCysteine/serine1 mm683,4-DCICSerine0.5 mm57BestatinAminopeptidase15 μm44120 μm10TPCKCysteine/serine25 μm43chymotrypsin-likeResults are for a 15-min incubation at 25 °C in 0.1 mTris-HCl, pH 8.0, with 1 mm H-Ala-pNA as substrate.a DFP, diisopropyl fluorophosphate. Open table in a new tab Results are for a 15-min incubation at 25 °C in 0.1 mTris-HCl, pH 8.0, with 1 mm H-Ala-pNA as substrate. In support of the specificity of peptidase IImes toward substrates with P1 hydrophobic
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