Functional Characteristics of a Phospholipase A2Inhibitor from Notechis ater Serum
2000; Elsevier BV; Volume: 275; Issue: 2 Linguagem: Inglês
10.1074/jbc.275.2.983
ISSN1083-351X
AutoresPeter G. Hains, Kah-Leong Sung, Albert Tseng, Kevin W. Broady,
Tópico(s)Vitamin K Research Studies
ResumoA phospholipase A2 inhibitor has been purified from the serum of Notechis ater using DEAE-Sephacel chromatography. The inhibitor was found to be composed of two protein subunits (α and β) that form the intact complex of approximately 110 kDa. The α-chain is a 30-kDa glycoprotein and the β-chain a nonglycosylated, 25-kDa protein. N-terminal sequence analysis reveals a high level of homology to other snake phospholipase A2 inhibitors. The inhibitor was shown to be extremely pH and temperature stable. The inhibitor was tested against a wide variety of phospholipase A2 enzymes and inhibited the enzymatic activity of all phospholipase A2 enzymes tested, binding with micromole to nanomole affinity. Furthermore, the inhibitor was compared with the Eli-Lilly compound LY311727 and found to have a higher affinity for human secretory nonpancreatic phospholipase A2 than this chemical inhibitor. The role of the carbohydrate moiety was investigated and found not to affect thein vitro function of the inhibitor. A phospholipase A2 inhibitor has been purified from the serum of Notechis ater using DEAE-Sephacel chromatography. The inhibitor was found to be composed of two protein subunits (α and β) that form the intact complex of approximately 110 kDa. The α-chain is a 30-kDa glycoprotein and the β-chain a nonglycosylated, 25-kDa protein. N-terminal sequence analysis reveals a high level of homology to other snake phospholipase A2 inhibitors. The inhibitor was shown to be extremely pH and temperature stable. The inhibitor was tested against a wide variety of phospholipase A2 enzymes and inhibited the enzymatic activity of all phospholipase A2 enzymes tested, binding with micromole to nanomole affinity. Furthermore, the inhibitor was compared with the Eli-Lilly compound LY311727 and found to have a higher affinity for human secretory nonpancreatic phospholipase A2 than this chemical inhibitor. The role of the carbohydrate moiety was investigated and found not to affect thein vitro function of the inhibitor. phospholipase A2 enzyme 1-hexadecanoyl-2-(1-prendecanoyl)-sn-glycero-3-phosphocholine 1-hexadecanoyl-2-(1-prendecanoyl)-sn-glycero-3-phosphoglycerol, ammonium salt human secretory type II PLA2 PLA2 inhibitor N. ater inhibitor semi-purified preparation reverse phase high pressure liquid chromatography For many years research on PLA21 enzymes has centred on snake venom PLA2s, largely because of the fact that PLA2 enzymes are extremely abundant in snake venoms and are easily purified. PLA2 enzymes have also been identified and purified from bovine, porcine, and human pancreas (1.Fleer E.A. Verheij H.M. De Haas G.H. Eur. J. Biochem. 1978; 82: 261-269Crossref PubMed Scopus (124) Google Scholar, 2.Verheij H.M. Westerman J. Sternby B. de Haas G.H. Biochim. Biophys. Acta. 1983; 747: 93-99Crossref PubMed Scopus (75) Google Scholar, 3.Puijk W.C. Verheij H.M. de Haas G.H. Biochim. Biophys. Acta. 1977; 492: 254-259Crossref PubMed Scopus (94) Google Scholar) and in human synovial fluid aspirates from rheumatoid and osteoarthritis patients (4.Parks T.P. Lukas S. Hoffman A.F. Adv. Exp. Med. Biol. 1990; 275: 55-81Crossref PubMed Scopus (12) Google Scholar, 5.Seilhamer J.J. Plant S. Pruzanski W. Schilling J. Stefanski E. Vadas P. Johnson L.K. J. Biochem. (Tokyo). 1989; 106: 38-42Crossref PubMed Scopus (49) Google Scholar). PLA2 enzymes have been grouped according to structural and sequence characteristics. Pancreatic PLA2 enzymes belong to group I, whereas the human secretory nonpancreatic PLA2 is a group II enzyme. PLA2 enzymes from snake venom belong to group I or II (6.Davidson F.F. Dennis E.A. J. Mol. Evol. 1990; 31: 228-238Crossref PubMed Scopus (292) Google Scholar, 7.Dennis E.A. J. Biol. Chem. 1994; 269: 13057-13060Abstract Full Text PDF PubMed Google Scholar, 8.Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (754) Google Scholar). Recently PLA2 enzymes have been implicated as playing a role in a range of diseases including rheumatoid and osteoarthritis, asthma, acute pancreatitis, psoriasis, multiple organ failure, septic shock, and adult respiratory distress syndrome (9.Vadas P. Pruzanski W. Lab. Invest. 1986; 55: 391-404PubMed Google Scholar, 10.Vadas P. Browning J. Edelson J. Pruzanski W. J. Lipid Med. 1993; 8: 1-30PubMed Google Scholar, 11.Michaels R.M. Chang Z.L. Beezhold D.H. Clin. Exp. Rheum. 1994; 12: 643-647PubMed Google Scholar). Several review articles (9.Vadas P. Pruzanski W. Lab. Invest. 1986; 55: 391-404PubMed Google Scholar, 10.Vadas P. Browning J. Edelson J. Pruzanski W. J. Lipid Med. 1993; 8: 1-30PubMed Google Scholar, 12.Mukherjee A.B. Miele L. Pattabiraman N. Biochem. Pharmacol. 1994; 48: 1-10Crossref PubMed Scopus (188) Google Scholar, 13.Pruzanski W. Vadas P. Immunol. Today. 1991; 12: 143-146Abstract Full Text PDF PubMed Scopus (397) Google Scholar, 14.Chang J. Musser J.H. McGregor H. Biochem. Pharmacol. 1987; 36: 2429-2436Crossref PubMed Scopus (305) Google Scholar) have examined the physiological and potential disease role of hsPLA2-II. Of major importance are the reaction by-products of PLA2 activity, a free fatty acid and a lysophospholipid. The fatty acid is often arachidonic acid, one of the main constituents of the cell membrane in several tissues (15.Katsuki H. Okuda S. Prog. Neurobiol. 1995; 46: 607-636Crossref PubMed Scopus (245) Google Scholar, 16.Buhl W.J. Eisenlohr L.M. Preuss I. Gehring U. Biochem. J. 1995; 311: 147-153Crossref PubMed Scopus (15) Google Scholar, 17.Barrington P.L. Soons K.R. Rosenberg P. Toxicon. 1986; 24: 1107-1116Crossref PubMed Scopus (13) Google Scholar). Arachidonic acid is the precursor for inflammatory mediators such as thromboxanes, leukotrienes, and prostaglandins (18.Flower R.J. Blackwell G.J. Biochem. Pharmacol. 1976; 25: 285-291Crossref PubMed Scopus (522) Google Scholar, 19.Sommers C.D. Bobbitt J.L. Bemis K.G. Snyder D.W. Eur. J. Pharmacol. 1992; 216: 87-96Crossref PubMed Scopus (33) Google Scholar). Lysophospholipids are the precursor of platelet activating factor (20.Mukherjee A.B. Cordella-Miele E. Miele L. DNA Cell Biol. 1992; 11: 233-243Crossref PubMed Scopus (54) Google Scholar). The suggestion that PLA2 enzymes play a role in diseases process has intensified research on PLA2 inhibitors. Many investigators have purified and characterized PLIs from numerous sources including plant, fungi, and bacteria (21.Cuellar M.J. Giner R.M. Recio M.C. Just M.J. Manez S. Rios J.L. J. Nat. Prod. 1996; 59: 977-979Crossref PubMed Scopus (60) Google Scholar, 22.Hyodo S. Fujita K.I. Kasuya O. Takahashi I. Uzawa J. Koshino H. Tetrahedron. 1995; 51: 6717-6724Crossref Scopus (16) Google Scholar, 23.Matsumoto K. Tanaka K. Matsutani S. Sakazaki R. Hinoo H. Uotani N. Tanimoto T. Kawamura Y. Nakamoto S. Yoshida T. J. Antibiotics. 1995; 48: 106-112Crossref PubMed Scopus (29) Google Scholar, 24.Gil B. Ferrandiz M.L. Sanz M.J. Terencio M.C. Ubeda A. Rovirosa J. San-Martin A. Alcaraz M.J. Paya M. Life Sci. 1995; 57: PL25-PL30Crossref PubMed Scopus (36) Google Scholar, 25.Jain M.K., Yu, B.Z. Rogers J.M. Smith A.E. Boger E.T.A. Ostrander R.L. Rheingold A.L. Phytochem. 1995; 39: 537-547Crossref PubMed Scopus (89) Google Scholar, 26.Lindahl M. Tagesson C. Inflammation. 1997; 21: 347-356Crossref PubMed Scopus (129) Google Scholar, 27.Fujii S. Tahara Y. Toyomoto M. Hada S. Nishimura H. Inoue S. Ikeda K. Inagaki Y. Katsumura S. Samejima Y. Omori-Satoh T. Takasaki C. Hayashi K. Biochem. J. 1995; 308: 297-304Crossref PubMed Scopus (14) Google Scholar, 28.Ortiz A.R. Pisabarro M.T. Gago F. J. Med. Chem. 1993; 36: 1866-1879Crossref PubMed Scopus (36) Google Scholar). A large research effort has gone into the design of synthetic compounds, such as the Eli-Lilly compound LY311727, which specifically inhibits hsPLA2-II (29.Schevitz R.W. Bach N.J. Carlson D.G. Chirgadze N.Y. Clawson D.K. Dillard R.D. Draheim S.E. Hartley L.W. Jones N.D. Mihelich E.D. Olkowski J.L. Snyder D.W. Sommers C. Wery J.P. Nat. Struct. Biol. 1995; 2: 458-465Crossref PubMed Scopus (233) Google Scholar). Additionally, a number of PLIs have been purified from the serum of elapid and crotalid snakes. These PLIs have all been shown to be homologous or heterologous complexes that interact with PLA2 enzymes to inhibit enzymatic activity (30.Ohkura N. Inoue S. Ikeda K. Hayashi K. Biochem. Biophys. Res. Commun. 1994; 200: 784-788Crossref PubMed Scopus (34) Google Scholar, 31.Ohkura N. Inoue S. Ikeda K. Hayashi K. Biochem. Biophys. Res. Commun. 1994; 204: 1212-1218Crossref PubMed Scopus (56) Google Scholar, 32.Perales J. Villela C. Domont G.B. Choumet V. Saliou B. Moussatche H. Bon C. Faure G. Eur. J. Biochem. 1995; 227: 19-26Crossref PubMed Scopus (60) Google Scholar, 33.Fortes-Dias C.L. Lin Y. Ewell J. Diniz C.R. Liu T.Y. J. Biol. Chem. 1994; 269: 15646-15651Abstract Full Text PDF PubMed Google Scholar, 34.Fortes-Dias C.L. Fonseca B.C.B. Kovcha E. Diniz C.R. Toxicon. 1991; 29: 997-1008Crossref PubMed Scopus (40) Google Scholar, 35.Kihara H. J. Biochem. (Tokyo). 1976; 80: 341-349Crossref PubMed Scopus (30) Google Scholar, 36.Inoue S. Kogaki H. Ikeda K. Samejima Y. Omori-Satoh T. J. Biol. Chem. 1991; 266: 1001-1007Abstract Full Text PDF PubMed Google Scholar, 37.Kogaki H. Inoue S. Ikeda K. Samejima Y. Omori-Satoh T. Hamaguchi K. J. Biochem. (Tokyo). 1989; 106: 966-971Crossref PubMed Scopus (50) Google Scholar, 38.Nobuhisa I. Inamasu S. Nakai M. Tatsui A. Mimori T. Ogawa T. Shimohigashi Y. Fukumaki Y. Hattori S. Kihara H. Ohno M. Eur. J. Biochem. 1997; 249: 838-845Crossref PubMed Scopus (45) Google Scholar, 39.Ohkura N. Ohkura H. Inoue S. Ikeda K. Hayashi K. Biochem. J. 1997; 325: 527-531Crossref PubMed Scopus (75) Google Scholar, 40.Ohkura N. Inoue S. Ikeda K. Hayashi K. J. Biochem. (Tokyo). 1993; 113: 413-419Crossref PubMed Scopus (52) Google Scholar, 41.Lizano S. Lomonte B. Fox J.W. Gutierrez J.M. Biochem. J. 1997; 326: 853-859Crossref PubMed Scopus (62) Google Scholar, 42.Ohkura N. Kitahara Y. Inoue S. Ikeda K. Hayashi K. J. Biochem. (Tokyo). 1999; 125: 375-382Crossref PubMed Scopus (28) Google Scholar, 43.Okumura K. Ohkura N. Inoue S. Ikeda K. Hayashi K. J. Biol. Chem. 1998; 273: 19469-19475Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). We now report the purification and functional characterization of a PLI from Notechis ater (Australian tiger snake) serum. The inhibitor termed, N. ater inhibitor (NAI) has been shown to inhibit the enzymatic activity of all PLA2 enzymes that it was tested against. Additionally, the affinity of the interaction was in the nanomole to micromole range dependent on the PLA2examined. Notexin from Notechis scutatus (Common tiger snake) and taipoxin from Oxyuranus scutellatus(Australian taipan) were purchased from Venom Supplies (Tanunda, South Australia). Bee venom PLA2 (Apis millifera), bovine pancreatic PLA2 (Bos taurus), andCrotalus atrox PLA2 (Western diamondback rattlesnake) were purchased from Sigma. Recombinant human secretory human type II PLA2 was generously donated by the Garvan Institute (Darlinghurst, New South Wales, Australia). The PLA2 inhibitor LY311727 was generously donated by Eli-Lilly and Co. (Indianapolis, Indiana). Pooled N. ater serum was obtained from World Tiger Snake Farm (Launceston, Tasmania). It should be noted that the serum was pooled from two very similar subspecies ofN. ater, namely N. ater serventyi (Chappell Island tiger snake) and N. ater humphreysi (Tasmanian tiger snake). NAI (which elutes in the 0.5 m peak) was purified from whole N. aterserum using a DEAE-Sephacel (Amersham Pharmacia Biotech) column (1.5 × 15 cm). Serum (10 mg of total protein) was loaded onto the column that had been equilibrated in 0.01 m ammonium acetate, pH 7.0, at a flow rate of 0.5 ml/min. Proteins were eluted with an increasing concentration of 0.1, 0.25, 0.5, and 1.0m ammonium acetate, pH 7.0, at a flow rate of 0.5 ml/min. The eluant was monitored at 280 nm (ISCO, UA-6 UV-visible Detector), and fractions were collected (ISCO, Retriever II). The 0.5m peak (referred to as the semi-purified preparation or SPP) was used for functional characterization of NAI. The α- and β-chains of NAI were purified on a Amersham Pharmacia Biotech smart system fitted with a Amersham Pharmacia Biotech Sephasil C8 5 μm (2.1 × 100 mm) column. The column was equilibrated in 0.1% (v/v) trifluoroacetic acid in water (buffer A) at a flow rate of 100 μl/min. The SPP collected form the DEAE-Sephacel column was loaded onto the C8 column and eluted with a gradient of 0.08% (v/v) trifluoroacetic acid, 80% (v/v) acetonitrile in water (buffer B). The percentage of buffer B was increased as follows: 0–2 min 0%, 2–10 min 0–55%, 10–50 min 55–70%, and 50–60 min 70–100%. The eluant was monitored at 214 and 280 nm. Software controlled peak fractionation was used to collect peaks with the integrated fraction collector. Following reduction and alkylation the proteins were N-terminally sequenced with a Perkin-Elmer Applied Biosystems Procise 494 protein sequencer. The molecular mass of NAI was assessed by size exclusion chromatography according to the method of Andrews (44.Andrews P. Biochem. J. 1965; 96: 595-606Crossref PubMed Scopus (2427) Google Scholar). A Superose 12 HR 10/30 (Amersham Pharmacia Biotech) column was calibrated with; aldolase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and lysozyme (14.4 kDa) at a flow rate of 0.5 ml/min in 0.1 m ammonium acetate, pH 7.0. The SPP was chromatographed under identical conditions, and the native mass of NAI was estimated using the elution volume of the peak containing NAI. Two PLA2assays were utilized, either the spectrofluorometric assay of Radvanyiet al. (45.Radvanyi F. Jordan L. Russo-Marie F. Bon C. Anal. Biochem. 1989; 177: 103-109Crossref PubMed Scopus (235) Google Scholar) or a mixed micelle assay based on the method of Seilhamer et al. (46.Seilhamer J.J. Pruzanski W. Vadas P. Plant S. Miller J.A. Kloss J. Johnson L.K. J. Biol. Chem. 1989; 264: 5335-5338Abstract Full Text PDF PubMed Google Scholar). Assays were carried out as described except that enzymes were dissolved in saline/0.1% (w/v) bovine serum albumin. For the spectrofluorometric assay substrates utilized were either 1-hexadecanoyl-2-(1-prendecanoyl)-sn-glycero-3-phosphocholine (10pPC, Molecular Probes, Inc.) or 1-hexadecanoyl-2-(1-prendecanoyl)-sn-glycero-3-phosphoglycerol, ammonium salt (10pPG, Molecular Probes, Inc.). Phosphatidyl ethanolamine, l-α-1-palmitoyl-2-arachidonyl, [arachidonyl-1-14C] (NEN Life Science Products) (specific activity, 2.035 Gbq/mmol) was used as substrate in the mixed micelle assay. The pH stability was investigated by altering the pH of the solution in which the SPP (10 μg/assay) was dissolved and testing this in the fluorometric PLA2 assay. The assay was performed using N. scutatus venom as the PLA2 source (50 ng/assay with 10pPC as substrate) at room temperature. The pH values tested were 2, 4, 6, 7, 8, 9, 10, and 12. The SPP was made up to the appropriate pH and incubated overnight at 4 °C prior to testing. Temperature stability was assessed in the same manner as the pH stability. Samples were heated or cooled at the appropriate temperature for 30 min and immediately tested in the fluorometric PLA2 assay at room temperature. The temperatures examined were 4, 25, 37, 50, 60, 70, 80, 90, and 100 °C. For both experiments the assay was performed at room temperature. Also, samples were not preincubated with venom because the stability of the PLA2 under varying pH and temperature values could not be assured. All samples were performed in triplicate with appropriate positive and negative controls. Results are expressed as percentage inhibition relative to control values. The SPP was lyophilized to dryness, resuspended in 100 μl of 20 mm sodium phosphate solution, pH 7.2, 50 mm EDTA, 0.1% (w/v) SDS, and digested with 0.8 unit of N-glycosidase F or 5.0 milliunits ofO-glycosidase (Roche Molecular Biochemicals) for 18 h at 37 °C. Following incubation, the SPP was either subjected to SDS-polyacrylamide gel electrophoresis (47.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205492) Google Scholar) under reducing conditions and silver stained (48.Blum H. Beier H. Gross H.J. Electrophoresis. 1987; 8: 93-99Crossref Scopus (3721) Google Scholar) or blotted onto nitrocellulose and carbohydrate detected by the digoxigenin glycan detection system (Roche Molecular Biochemicals). Salts present in the deglycosylation buffer were removed by running samples over a AG11A8/G10 column. The AG11A8 matrix (Bio-Rad) represented 1.5 × 7.5 cm of the column; poured directly on top of this was Sephadex G10 (Amersham Pharmacia Biotech) matrix, representing 1.5 × 6 cm of the column. The column was equilibrated in water at a flow rate of 0.5 ml/min, and samples were eluted under the same conditions. The eluant was monitored at 280 nm (ISCO, UA-6 UV-visible Detector), and fractions were collected. The eluted sample was tested in the fluorometric PLA2 assay for inhibitory activity against N. scutatus venom,Agkistrodon bilineatus venom (Central American Moccasin) and bee venom PLA2. For N. scutatus venom andA. bilineatus venom, 10pPC was used as substrate; 10pPG was used as substrate for bee venom PLA2. All PLA2sources were dissolved in saline/0.1% (w/v) bovine serum albumin.N. scutatus venom was used at 33.3 ng/assay, A. bilineatus venom 500 ng/assay and bee venom PLA2 25 ng/assay, all in a final volume of 10 μl. Native NAI was used as the positive control. Results are expressed as the percentage of inhibition relative to control values. The α- and β-chains were purified by RP-HPLC (see above) after deglycosylation and analyzed by mass spectrometry using a Micromass Platform II single quadrupole instrument. The mass of the reverse phase purified proteins were also measured before deglycosylation. All enzymes except hsPLA2-II and bovine pancreatic PLA2 were tested in the fluorometric assay using 10pPC as substrate. Bovine pancreatic PLA2 and hsPLA2-II were tested in the fluorometric assay using 10pPG as substrate and in the mixed micelle assay. The amount of enzyme added for the fluorometric assay were as follows; notexin, 16.7 ng; taipoxin, 800 ng; C. atrox PLA2, 40 ng; bee venom PLA2, 10 ng; bovine pancreatic PLA2, 6250 ng. For the mixed micelle assay 100 ng of bovine pancreatic PLA2 or 5 ng of hsPLA2-II were used. All enzymes were dissolved in saline/0.1% (w/v) bovine serum albumin, except bovine pancreatic PLA2 and hsPLA2-II, which were dissolved in 20 mm Tris, pH 8.0, and 1 m NaCl, 10 mm Tris, pH 7.5, respectively. The IC50 was determined by preincubating varying concentrations of NAI (10.5 mg/mL) in a constant volume, against a constant amount of enzyme (see above and Refs. 49.Todhunter J.A. Methods Enzymol. 1979; 63: 383-411Crossref PubMed Scopus (38) Google Scholar, 50.Aitafoun M. Mounier C. Heymans F. Binisti C. Bon C. Godfroid J.J. Biochem. Pharmacol. 1996; 51: 737-742Crossref PubMed Scopus (62) Google Scholar, 51.Binisti C. Mounier C. Touboul E. Heymans F. Bon C. Godfroid J.J. J. Lipid. Mediat. Cell. Signal. 1997; 16: 171-187Crossref PubMed Scopus (4) Google Scholar). The concentration of NAI was varied between 30 μm and 70 pm (PLA2- and assay-dependent) until a sigmoidal dose response curve was generated allowing calculation of the IC50 value. For comparison, the IC50 for the Lilly compound LY311727 was determined using the mixed micelle assay with hsPLA2-II. All samples were performed in triplicate. Results were analyzed by nonlinear regression with GraphPad Prism (version 2.01) and expressed as the percentage of inhibition relative to control values. NAI was purified from whole N. ater serum utilizing a DEAE-Sephacel column (Fig.1). All peaks were collected, concentrated, and tested for inhibitory activity against N. scutatus venom in the fluorometric assay. Peaks 3 and 4 inhibited; however, when samples were diluted to a equal protein concentration, inhibition of PLA2 enzymatic activity was only detected in peak 4. The purity of NAI is approximately 90% as judged by SDS-polyacrylamide gel electrophoresis analysis (Fig.2). NAI is composed of two proteins, an α-chain (∼30 kDa) and a β-chain (∼25 kDa). These proteins are noncovalently associated because a similar separation pattern is observed when the gel is run under nonreducing conditions (not shown). The native mass of NAI was estimated at 110 kDa by size exclusion chromatography (not shown). The α- and β-chains can be separated by RP-HPLC (Fig. 3), and it is apparent that isoforms of the α-chain are present. By measuring the area under the curve for the α- and β-chains at 280 nm and correcting for their relative absorbance using extinction co-efficients calculated from the amino acid sequence, the NAI complex is suggested to be composed of two α-chains and one β-chain. The N terminus of the purified chains were sequenced (Table I). Two N termini were present in the peak containing NAIα, demonstrating that isoforms were partially separated under the RP-HPLC conditions utilized (Fig.3).Figure 2SDS-polyacrylamide gel electrophoresis analysis of DEAE-Sephacel separated N. aterserum. Peaks were collected (as shown in Fig. 1) and analyzed by SDS-polyacrylamide gel electrophoresis, and the gel was silver-stained. Lanes 1 and 8, molecular mass markers (sizes as indicated, kDa); lane 2, whole serum;lane 3, peak 1; lane 4, peak 2; lane 5, peak 3; lane 6, peak 4; lane 7, peak 5 from Fig. 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3RP-HPLC purification of NAI α and NAI β. The SPP (∼80 μg) was purified with a Amersham Pharmacia Biotech Sephasil C8 (2.1 × 100 mm) column at a flow rate of 100 μl/min. The column was equilibrated in 0.1% (v/v) trifluoroacetic acid, water, and the proteins were eluted with an increasing concentration of buffer B (0.08% (v/v) trifluoroacetic acid, 80% acetonitrile, water) as indicated on the graph.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IN-terminal sequence of NAIα and NAIβChainN-terminal sequenceα-ChainHSCEICHNFGRDCQSDEAEECASPEDQCGL K ETE Tβ-ChainLECEICIGLGLECNTWTKTCDANQOTCVFollowing RP-HPLC separation and reduction and alkylation, both chains were N-terminally sequenced. Open table in a new tab Following RP-HPLC separation and reduction and alkylation, both chains were N-terminally sequenced. The temperature and pH stability of NAI was investigated by varying the temperature or pH of the solution in which NAI was dissolved and measuring the inhibitory activity of NAI. The temperature stability of NAI is shown in Fig.4. As can be seen, NAI is active following exposure to a wide range of temperatures, with 76 ± 13% inhibitory activity still present after heating at 100 °C for 30 min. NAI was found to retain greater than 89% of its inhibitory activity following exposure to temperatures between 4 and 90 °C. A similar situation was seen for the pH profile of NAI (Fig.5). NAI retained greater than 94% of its inhibitory activity following overnight incubation at pH 4–12; at pH 2 the level of inhibition was 35 ± 7%.Figure 5pH stability of NAI. NAI was incubated in solutions of varying pH overnight and tested in the fluorometric assay for inhibitory activity against N. scutatus venom. Results are represented as the averages ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The glycosylation status of NAIα and NAIβ was investigated with the digoxigenin glycan detection system. After determining that only the α-chain was glycosylated, the type of linkage was analyzed, using enzymes specific for N-linked or O-linked sugars (52.Haselbeck A. Roos A. Hosel W. Topics Biochem. 1992; 10Google Scholar, 53.Haselbeck A. Hosel W. Topics Biochem. 1988; 8: 1-4Google Scholar). All carbohydrate moieties were removed byN-glycosidase F but were not affected byO-glycosidase (not shown). Mass spectrophotometric analysis of the native and deglycosylated α-chain (not shown) indicated mass differences of 2207 and 2864 Da, which are consistent with carbohydrate masses of biantennary sugars (54.Settineri C.A. Burlingame A.L. Chapman J.R. Protein and Peptide Analysis by Mass Spectrometry. 1st ed. 61. Humana Press, Totowa, New Jersey1996: 255-278Google Scholar). After deglycosylation, NAI (in the form of the SPP) was tested for inhibitory activity against wholeN. scutatus venom, A. bilineatus venom, and bee venom PLA2 using the fluorometric assay (Fig.6). It is apparent that deglycosylated NAI is as active on all PLA2 enzymes as untreated control samples. The IC50 values determined (with 95% confidence interval) using the fluorometric assay (Fig. 7) with 10pPC as substrate for notexin, taipoxin, C. atrox PLA2, and bee venom PLA2 enzymes are as follows: 0.13 nm(0.05–0.28 nm), 0.89 nm (0.53–1.5 nm), 2.4 nm (1.4–3.9 nm), and 0.16 nm (0.085–0.31 nm), respectively. The IC50 for bovine pancreatic PLA2 (Fig.8) calculated using the fluorometric assay with 10pPG as substrate (the enzyme is not active on 10pPC) was 32.3 nm (21.8–47.7 nm). Using the mixed micelle assay the IC50 values for bovine pancreatic PLA2 and hsPLA2-II (Fig.9) were 12,050 nm (2,030–71, 490 nm) and 287.3 nm (146.4–563.9 nm), respectively. The IC50 using LY311727 (Fig. 10) on hsPLA2-II in the mixed micelle assay was calculated at 808 nm(551.4–1,184 nm).Figure 8Inhibition curve for NAI versusbovine pancreatic PLA2 enzyme. For experimental details see “Experimental Procedures.” The IC50 value was determined for bovine pancreatic PLA2 using the fluorometric assay with 10pPG as substrate. Results are represented as the averages ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 9Inhibition curves for NAI versusgroup I PLA2 enzymes. For experimental details see “Experimental Procedures.” IC50 values were determined for hsPLA2-II and bovine pancreatic PLA2 using the mixed micelle assay with phosphatidyl ethanolamine as substrate. Results are represented as the averages ± S.D.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 10Inhibition curves for NAI versusLilly LY311727 for hsPLA2-II. For experimental details see “Experimental Procedures.” IC50 values were determined for rhPLA2 using the mixed micelle assay with phosphatidyl ethanolamine as substrate. NAI and Lilly LY311727 were used as inhibitors. Results are represented as the averages ± S.D.View Large Image Figure ViewerDownload Hi-res image Download (PPT) N-terminal sequencing revealed two isoforms of NAIα that could be partially separated under the conditions utilized. These isoforms have high homology to other PLIs, with 67–70% homology to the 30-kDa chain of the PLI from Naja naja kaouthia (Thailand cobra), depending on which sequence was used for comparison. Only one sequence was present for NAIβ, which also shows high homology (84%) to the 25-kDa chain of the PLI purified from N. naja kaouthia (31.Ohkura N. Inoue S. Ikeda K. Hayashi K. Biochem. Biophys. Res. Commun. 1994; 204: 1212-1218Crossref PubMed Scopus (56) Google Scholar). A number of PLIs have been isolated from either elapid or crotalid serum. Many of these PLIs have been sequenced and show homology to two protein families. We propose that PLIs with homology to urokinase plasminogen activator receptor, Ly-6, and CD59 will be classified as Class A PLIs. Those with homology to the carbohydrate recognition domain of C-type lectins will be classified as Class B PLIs, whereas other PLIs will be classified as Class C PLIs. Experiments were designed to investigate the pH stability and thermostability of NAI (Figs. 4 and 5). NAI was found to be extremely pH- and temperature-resistant with greater than 89% inhibition recorded for temperatures between 4 and 90 °C and greater than 94% inhibitory activity retained between pH 4 and 12. These experiments also serve to demonstrate that NAI is an effective inhibitor whether it is added before the PLA2 (IC50 studies) or added after the PLA2 (pH or temperature studies). When NAI was added after the PLA2, inhibition was immediate and as effective as when NAI was preincubated with the PLA2enzyme. Preincubation was used for the IC50 studies because the assay was empirically determined to be more robust when performed in this fashion. Carbohydrate moieties are common on snake PLIs and have been detected or presumptive evidence for their existence has been found on at least one chain for all snake PLIs characterized (30.Ohkura N. Inoue S. Ikeda K. Hayashi K. Biochem. Biophys. Res. Commun. 1994; 200: 784-788Crossref PubMed Scopus (34) Google Scholar, 33.Fortes-Dias C.L. Lin Y. Ewell J. Diniz C.R. Liu T.Y. J. Biol. Chem. 1994; 269: 15646-15651Abstract Full Text PDF PubMed Google Scholar, 34.Fortes-Dias C.L. Fonseca B.C.B. Kovcha E. Diniz C.R. Toxicon. 1991; 29: 997-1008Crossref PubMed Scopus (40) Go
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