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DOI: 10.1055/s-2002-35649
© Georg Thieme Verlag Stuttgart · New York
Pananotin, a Potent Antifungal Protein from Roots of the Traditional Chinese Medicinal Herb Panax notoginseng
Abstract
The roots of the sanchi ginseng, Panax notoginseng, were extracted with an aqueous buffer. The extract was chromatographed on a CM-cellulose column to remove extraneous unadsorbed proteins. The adsorbed fraction was dialyzed and chromatographed on Affi-gel blue gel. The adsorbed fraction was again collected, dialyzed and applied on a column of Mono S. The second peak was dialyzed and chromatographed on an FPLC-gel filtration Superdex 75 column. An antifungal protein with an N-terminal sequence similar to those of chitinases was isolated from the first peak which had a molecular mass of 35 kDa. The sequence was distinctive in that the third and ninth highly conserved N-terminal residues (C and G) were replaced by H and M, respectively. The protein inhibited mycelial growth in Coprinus comatus, Physalospora piricola, Botrytis cinerea, and Fusarium oxysporum with an IC50 of 100 nM, 1 μM, 630 nM and 560 nM, respectively. It inhibited cell-free translation with an IC50 of 630 nM. Its antifungal and translation-inhibitory activities were more potent than those of previously reported antifungal proteins. It inhibited human immunodeficiency virus-1 reverse transcriptase by 35.8 % at 12.6 μM and 24.7 % at 1.26 μM.
#Introduction
Panax notoginseng is renowned for its beneficial effects on the cardiovascular system [1]. The total saponins of P. notoginseng can improve the scopolamine-induced amnesia learning and memory deficit in rats [2]. Morever, it exhibits anti-carcinogenic [3] and immunostimulating [4] activities.
No information is available in the literature regarding the existence of antifungal proteins in Chinese medicinal materials. It is reported herein that an antifungal protein, possessing an N-terminal amino acid sequence displaying similarities to those of chitinases, can be isolated from P. notoginseng roots. Antifungal proteins are a family of defense proteins which share with lectins and ribosome inactivating proteins the attribute of protection against pathogens and other invasive organisms [5].
#Materials and Methods
The roots of Panax notoginseng (Burk.) F. H. Chen (sanchi ginseng) (1.25 kg) from a local vendor were authenticated by Professor Shiuying Hu, Honorary Professor of Chinese Medicine, The Chinese University of Hong Kong. A voucher has been deposited at the Museum of Chinese Medicine, Institute of Chinese Medicine, The Chinese University of Hong Kong (no. 618). The roots were extracted (4 mL/g) in 10 mM NH4OAc (pH 4.6). After centrifugation the supernatant was loaded on a column of CM-cellulose (5.5 × 25 cm). Following removal of unadsorbed proteins (CM1) with the same buffer, adsorbed proteins (CM2) were desorbed using 50 mM NH4OAc (pH 7). CM2 was dialyzed prior to affinity chromatography on an Affi-gel blue gel column (2.5 × 10 cm) in 10 mM Tris-HCl buffer (pH 7.2). After elution of unadsorbed proteins (BG1), adsorbed proteins (BG2) were eluted with 1.5 M NaCl in 10 mM Tris-HCl buffer (pH 7.2). BG2 was dialyzed and then subjected to ion exchange chromatography on a 1-mL Mono S column (Amersham Biosciences) in 10 mM Tri-HC1 (pH 6.8) by fast protein liquid chromatography. Unadsorbed proteins were eluted with the buffer while the purified antifungal protein was eluted with a linear NaCl concentration gradient (0 - 0.25 M) in the buffer. The protein was then applied to an FPLC-Superdex 75 HR 10/30 column (Amersham Biosciences) which had been calibrated with molecular mass marker proteins. The eluting buffer was 200 mM NH4OAc (pH 7.0).
The chromatographic fractions were monitored for antifungal activity using 90 × 15 mm petri dishes containing 10 ml of potato dextrose agar [8]. After the mycelial colony had developed on the agar, at a distance of 1 cm away from the rim of the colony were placed sterile blank paper disks (0.625 cm in diameter). The samples were dissolved and applied to the disks. The dishes were incubated at 27 °C until mycelial growth had enveloped peripheral disks containing the control and had formed crescents of inhibition around the paper disks with samples possessing antifungal activity. The pathogenic fungal species used included Coprinus comatus, Physalospora piricola, Botrytis cinerea, Mycosphaerella arachidicola and Fusarium oxysporum.
For a quantitative assay [9], five doses of the antifungal protein (2000 nM, 200 nM, 20 nM, 2 nM and 0.2 nM) were added separately to six aliquots each containing 1 ml potato dextrose agar at 45 °C, mixed rapidly, and poured into six separate 3.3-cm petri dishes. After the agar had cooled down, a small amount of mycelia, the same amount to each dish, was inoculated. Buffer only, without antifungal protein, served as a negative control. The leguminous antifungal protein dolichin [10] was used as a positive control. After incubation at 23 °C for 72 h, the area of the mycelial colony was measured and the inhibition of fungal growth determined. IC50 is the concentration of the antifungal protein causing reduction of the area of the mycelial colony to 50 %.
The assay for translation-inhibiting activity using a cell-free reticulocyte lysate system was conducted as previously described [11]. Rabbit reticulocyte lysate was prepared from the blood of rabbits rendered anemic by phenylhydrazine injections. The test sample (10 μL) was added to 10 μl of radioactive mixture (500 mM KCl, 5 mM MgCl2, 130 mM phosphocreatine and 1 μCi [4,5 - 3 H] leucine) and 30 μL working rabbit reticulocyte lysate containing 0.1 μM hemin and 5 μL creatine kinase. Incubation proceeded at 37 °C for 30 min before addition of 330 μL 1 M NaOH and 1.2 % H2O2. Further incubation for 10 min allowed decolorization and tRNA digestion. An equal volume of the reaction mixture was then added to 40 % trichloroacetic acid with 2 % casein hydrolyzate in a 96-well plate to precipitate radioactively labeled protein. The precipitate was collected on a glass fiber Whatman GF/A filter, washed and dried with absolute alcohol passing through a cell harvester attached to a vacuum pump. The filter was suspended in scintillant and counted in an LS6500 Beckman liquid scintillation counter. The ribosome inactivating protein velutin [12] was used as a positive control.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): This was conducted according to the method of Laemmli and Favre [6]. After electrophoresis the gel was stained with Coomassie Brilliant Blue. The molecular mass of pananotin was determined by comparison of its electrophoretic mobility with those of molecular mass marker proteins from Amersham Biosciences.
N-Terminal amino acid sequence analysis: The N-terminal amino acid sequence of pananotin was analyzed by means of automated Edman degradation. Microsequencing was carried out using a Hewlett Packard 1000A protein sequencing equipped with an HPLC system [7].
The assay for HIV reverse transcriptase inhibitory activity was carried out as previously described [13], [14] using a non radioactive kit from Boehringer Mannheim (Germany). The assay takes advantage of the ability of reverse transcriptase to synthesize DNA, starting from the template/primer hybrid poly(A) · oligo (dT) 15. In place of radio-labeled nucleotides, digoxigenin- and biotin-labeled nucleotides in an optimized ratio are incorporated into one and the same DNA molecule, which is freshly synthesized by the reverse transcriptase (RT). The detection and quantification of synthesized DNA as a parameter for RT activity follows a sandwich ELISA protocol: Biotin-labeled DNA binds to the surface of microtiter plate modules that have been precoated with streptavidin. In the next step, an antibody to digoxigenin, conjugated to peroxidase (anti-DIG-POD), binds to the digoxigenin-labeled DNA. In the final step, the peroxidase substrate is added. The peroxidase enzyme catalyzes the cleavage of the substrate, producing a colored reaction product. The absorbance of the samples at 405 nm can be determined using a microtiter place (ELISA) reader and is directly correlated to the level of RT activity. A fixed amount ( 4 - 6 ng) of recombinant HIV-1 reverse transcriptase was used. The inhibitory activity of pananotin was calculated as percent inhibition as compared to a control without the protein. Dolichin [10] and mungin [15] were used as positive and negative controls respectively.
#Results and Discussion
Antifungal activity was adsorbed on CM-cellulose in 10 mM NH4OAc at pH 4.6 and was eluted by 50 mM NH4OAc at pH 7 and collected as fraction CM2 (Fig. [1]). After dialysis, the activity was subsequently adsorbed on Affi-gel blue gel in 10 mM Tris-HCl (pH 7.2), and desorbed by inclusion of 1.5 M NaCl in the eluent and collected as fraction BG2 (Fig. [2]). Following dialysis, the activity was adsorbed on Mono S in 10 mM Tris-HCl (pH 6.8), and desorbed by application of a linear NaCl concentration gradient in the buffer (Fig. [3]). The adsorbed peak MS3 from Mono S exhibited antifungal activity. It yielded two peaks, SD1 and SD2, with a molecular mass of 35 kDa and 15 kDa respectively in gel filtration on Superdex 75 (Fig. [4]). The first peak (SD1) showed a single band with a molecular mass of 35 kDa in SDS-PAGE (Fig. [5]). The N-terminal sequence of this purified antifungal protein, designated pananotin, resembled those of chitinases (Table [1]). Pananotin was obtained with a yield of 800 μg from 1.25 kg P. notoginseng roots. The yields of the crude extract, and fractions CM2, BG2, MS2 and SD1 were respectively 2000 mg, 208 mg, 110 mg, 1.5 mg and 0.8 mg. Pananotin exerted antifungal activity against Coprinus comatus, Physalospora piricola, Botrytis cinerea, and Fusarium oxysporum (Fig. [6]) but not against Mycosphaerella arachidicola. The IC50 of its antifungal activity was 100 nM toward C. comatus, 1 μM toward P. piricola, 630 nM toward B. cinerea and 560 nM toward F. oxysporum. Pananotin inhibited HIV-1 reverse transcriptase to a certain extent. It caused (35.8 ± 5.2) % inhibition at 12.6 μM and (24.7 ± 1.8) % inhibition at 1.26 μM (mean ± SD, n = 3). Pananotin inhibited translation with an IC50 of 630 nM. The N-terminal sequence of pinanotin was EQHGKQAGMALCPNG. It was similar to those of other chitinases (Table [1]).
P. notoginseng antifungal protein (i. e., pananotin) can be distinguished in many ways from quinqueginsin and panaxagin, proteins with antifungal activity purified from American ginseng (P. quinquefolia) roots and Chinese ginseng (P. ginseng) roots [13], [14]. Pananotin is characterized by a chitinase-like N-terminal sequence whereas quinqueginsin and panaxagin possess an N-terminal sequence sharing some similarities with those of ribosome-inactivating proteins and ribonucleases. Pananotin is single-chained and possesses a lower molecular mass (35 kDa) while quinqueginsin and panaxagin possess a higher molecular mass (52 kDa) and are composed of two subunits [13], [14]. While quinqueginsin and panaxagin demonstrate ribonuclease activity, pananotin is devoid of such activity. Pananotin, panaxagin and quinqueginsin are all capable of inhibiting HIV-1 reverse transcriptase. It is noteworthy that while the N-terminal sequences of quinqueginsin and panaxagin do not possess cysteine residues, that of pananotin is characterized by the presence of cysteine residue. The N-terminal sequence of pananotin is remarkably similar to those of chitinases. Its molecular mass is also similar to those of chitinases [10], [16], [17]. Nevertheless, the highly conserved C and G residues at positions 3 and 9 in its N-terminal sequence are replaced by H and M. The first replacement is also encountered in chive (Allium tuberosum) chitinase without an adverse effect on antifungal activity.
The chromatographic behavior of pananotin on ion exchangers (CM-cellulose, Mono S) and the affinity chromatographic media Affi-gel blue gel is similar to that exhibited by other antifungal proteins. Antifungal proteins from leguminous species such as Dolichos lablab [10], Phaseolus mungo [15], Pisum sativum var. macrocarpon [16] and Vigna unguiculata [17], and non-leguminous species such as Ginkgo biloba [18], Panax ginseng [13], Panax quinquefolia [14] and Allium tuberosum [9] are adsorbed on Affi-gel blue gel, CM-ion exchangers and Mono S, just like pananotin.
Not all chitinases possess antifungal activity [19]. Only a few fungi are susceptible to chitinases alone, while many fungi are sensitive to a combination of chitinases and β-1,3-glucanase [20], [21] or another protein or compound that changes the membrane structure or permeability [22].
Pananotin exerts antifungal action against a variety of fungal species, albeit with different potencies. C. comatus is the most sensitive to pananotin, B. cinerea and F. oxysporum show intermediate sensitivity while P. piricola manifests an even lower sensitivity. The antifungal and translation-inhibitory activities of pananotin are more potent than chitinase-like antifungal proteins from field bean [10], cowpea [17] and chive [9].
Fig. 1 Ion exchange chromatography of a crude extract of Panax notoginseng on a CM-cellulose column (5.5 × 25 cm). Eluent: 10 mM NH4OAc (pH 4.6). The eluent was changed to 50 mM NH4OAc (pH 7) when the elution volume was 8 L.
Fig. 2 Affi-gel blue gel chromatography of the fraction of crude P. notoginseng extract adsorbed on CM-cellulose. Eluent: 10 mM Tris-HCl (pH 7.2). The eluent was changed to 1.5 M NaCl in 10 mM Tris-HCl (pH 7.2) when the elution volume was 400 mL.
Fig. 3 Ion exchange chromatography on Mono S. The fraction adsorbed on Affi-gel blue gel was applied to the FPLC Mono S column in 10 mM NaOAc (pH 6.8). After elution of unadsorbed protein the column was eluted with a linear NaCl concentration gradient, as indicated by the slanting line across the chromatogram, to desorb the adsorbed protein.
Fig. 4 FPLC-gel filtration of adsorbed protein peak MS3 from Mono S on Superdex 75 in 200 mM NH4OAc (pH 7). The first peak represents pananotin, the purified antifungal protein. The first and second peaks possess a molecular mass of 35 kDa and 15 kDa, respectively.
Fig. 5 Sodium dedecyl sulfate-polyacrylamide gel electrophoresis of pananotin. Lane 1 represents pananotin. Lane two shows Pharmacia molecular mass markers, from top to downward: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa) and α-lactalbumin (14.4 kDa).
Fig. 6 Antifungal activity of pananotin towards (6.1) Botrytis cinerea (6.2) Coprinus comatus (6.3) Fusarium oxysporum (6.4) Physalospora piricola (A) 100 mM NaOAc (pH 5.5) as negative control (B) 40 μg pananotin in 100 mM NaOAc (pH 5.5) and (C) 8 μg pananotin in 100 mM NaOAc (pH 5.5).
| Residues No. |
Residue No. |
% Identity | ||
| Panax notoginseng | 1 | EQHGKQAGMALCPNG | 15 | 100 |
| Theobroma cacao | 22 | EQCGRQAGGALCPGG | 36 | 73 |
| Persea americana | 26 | EQCGRQAGGALCPGG | 40 | 73 |
| Arabis blepharophylla | 16 | EQCGRQAGGALCPGG | 30 | 73 |
| Medicago sativa | 24 | EQCGKQAGGALCPGG | 39 | 80 |
| Brassica napus | 20 | EQCGRQAGGALCPNG | 35 | 80 |
| Hevea braciliensis | 1 | EQCGRQAGGKLCPNN | 15 | 67 |
| Oryza sativa | 19 | EQCGSQAGGALCPNC | 33 | 73 |
| Ulmus americana | 22 | EQCGSQAGGAVCPVG | 37 | 67 |
| Arabis parishii | 21 | EQCGRQAGGALCPNG | 35 | 80 |
| Phaseolus vulgaris | 1 | EQCGRQAGGALQPGG | 15 | 67 |
| Allium tuberosum | 1 | EQHGSQAGGALHPGX | 15 | 60 |
| Residues identical to corresponding residues in the Panax notoginseng protein are underlined. | ||||
References
- 1 Kwan C Y. Vascular effects of selected antihypertensive drugs derived from traditional herbs. Clin Exp Pharmacol. Physiol Suppl.. 1995; S29 7-9
- 2 Hsieh M T, Peng W H, Wu C R, Wang W H. The ameliorating effects of the cognitive-enhancing Chinese herbs on scopolamine-induced amnesia in rats. Phytother. Res.. 2000; 14 375-7
- 3 Konoshima T, Takasaki M, Tokuda H. Anti-carcinogenic activity of the roots of Panax notoginseng. Biol. Pharm. Bull.. 1999; 22 1150-2
- 4 Gao H, Wang F, Lien E J, Trousdale M D. Immunostimulating polysaccharides from Panax notoginseng. Pharm. Res.. 1996; 13 1196-200
- 5 Graham L S, Sticklan M B. Plant chitinases. Can. J. Bot.. 1994; 72 1057-83
- 6 Laemmli U K, Favre M. Maturation of the head of bacterophage T4. J. Mol. Biol.. 1973; 80 575-99
- 7 Ng T B, Lam Y W. Isolation of a novel agglutinin with complex carbohydrate binding specificity from fresh fruiting bodies of the edible mushroom Lyophyllum shimeiji. Biochem. Biophys. Res. Commun.. 2002; 290 563-8
- 8 Ye X Y, Wang H X, Ng T B. First chromatographic isolation of an antifungal thaumatin-like protein from French bean legumes and demonstration of its antifungal activity. Biochem. Biophys. Res. Commun.. 1999; 263 130-4
- 9 Lam Y W, Wang H X, Ng T B. A robust cysteine-deficient chitinase-like antifungal protein from inner shoots of the edible chive Allium tuberosum. Biochem. Biophys. Res. Commun.. 2000; 279 74-80
- 10 Lam S K, Ng T B. First simultaneous isolation of a ribosome inactivating protein and an antifungal protein from a mushroom (Lyophyllum shimeiji) together with evidence for synergism of their antifungal effects. Arch. Biochem. Biophys.. 2001; 393 271-80
- 11 Ye X Y, Wang H X, Ng T B. Dolichin, a new chtiniase-like antifungal protein isolated from field beans (Dolichos lablab). Biochem. Biophys. Res. Commun.. 2000a; 269 155-9
- 12 Lam S SL, Wang H X, Ng T B. Purification and characterization of novel ribosome inactivating proteins, alpha- and beta-pisavins, from seeds of the garden pea Pisum sativum. Biochem. Biophys. Res. Commun.. 1998; 253 135-42
- 13 Wang H X, Ng T B. Isolation and characterization of velutin, a novel low-molecular-weight ribosome-inactivating protein from winter mushroom (Flammulina velutipes) fruiting bodies. Life Sci. 2001; 68 2151-8
- 14 Ng T B, Wang H X. Panaxagin, a new protein from Chinese ginseng possesses anti-fungal, anti-viral, translation-inhibiting and ribonuclease activities. Life Sci.. 2001; 68 739-49
- 15 Wang H X, Ng T B. Quinqueginsin, a novel protein with anti-human immunodeficiency virus, antifungal, ribonuclease and cell-free translation-inhibitory activities from Amercian ginseng roots. Biochem. Biophys. Res. Commun.. 2000a; 269 203-8
- 16 Ye X Y, Ng T B. Mungin, a novel cyclophilin-like antifungal protein from the mung bean. Biochem. Biophys. Res. Commun.. 2000; 273 1111-5
- 17 Ye X Y, Wang H X, Ng T B. Sativin. A novel antifungal miraculin-like protein isolated from legumes of the sugar snap Pisum sativum var. macrocarpon . Life Sci.. 2000b; 67 775-81
- 18 Ye X Y, Wang H X, Ng T B. Structurally dissimilar proteins with antiviral and antifungal potency from cowpea (Vigna unguiculata) seeds. Life Sci.. 2000c; 67 3199-208
- 19 Wang H X, Ng T B. Ginkbilobin, a novel antifungal protein from Ginkgo biloba seeds with sequence similarity to embryo-abundant protein. Biochem. Biophys. Res. Commun.. 2000b; 279 407-10
- 20 Iseli B, Boller T, Neuhaus J M. The N-terminal cysteine-rich domain of tobacco class I chitinase is essential for chitin binding but not for catalytic or antifungal activity. Plant Physiol.. 1993; 103 221-6
- 21 Mauch F, Mauch-Mani B, Boller T. Antifungal hydrolases in pea tissue, inhibition of fungal growth by combination of chitinases and β-1,3-glucanase. Plant Physiol. 1988; 88 936-41
- 22 Melchers L S, Sela-Buurlage M B, Vloemans S A, Woloshuk C P, Van Roekel J S, Pen J, van den Elzen P J, Cornelissen B J. Extracellular targeting of the vacuolar tobacco proteins AP24, chitinase and beta-1,3-glucanase in transgenic plants. Plant Mol. Biol.. 1993; 21 583-93
- 23 Lorito M, Peterbauer C, Hayes C K, Harman G E. Synergistic interaction between fungal cell wall degrading enzymes and different antifungal compounds enhances inhibition of spore germination. Microbiology. 1994; 140 623-9
T. B. Ng
Department of Biochemistry
Faculty of Medicine
The Chinese University of Hong Kong
Shatin, Hong Kong
China
Fax: +852-2603-5123
Phone: +852-2609-6875
References
- 1 Kwan C Y. Vascular effects of selected antihypertensive drugs derived from traditional herbs. Clin Exp Pharmacol. Physiol Suppl.. 1995; S29 7-9
- 2 Hsieh M T, Peng W H, Wu C R, Wang W H. The ameliorating effects of the cognitive-enhancing Chinese herbs on scopolamine-induced amnesia in rats. Phytother. Res.. 2000; 14 375-7
- 3 Konoshima T, Takasaki M, Tokuda H. Anti-carcinogenic activity of the roots of Panax notoginseng. Biol. Pharm. Bull.. 1999; 22 1150-2
- 4 Gao H, Wang F, Lien E J, Trousdale M D. Immunostimulating polysaccharides from Panax notoginseng. Pharm. Res.. 1996; 13 1196-200
- 5 Graham L S, Sticklan M B. Plant chitinases. Can. J. Bot.. 1994; 72 1057-83
- 6 Laemmli U K, Favre M. Maturation of the head of bacterophage T4. J. Mol. Biol.. 1973; 80 575-99
- 7 Ng T B, Lam Y W. Isolation of a novel agglutinin with complex carbohydrate binding specificity from fresh fruiting bodies of the edible mushroom Lyophyllum shimeiji. Biochem. Biophys. Res. Commun.. 2002; 290 563-8
- 8 Ye X Y, Wang H X, Ng T B. First chromatographic isolation of an antifungal thaumatin-like protein from French bean legumes and demonstration of its antifungal activity. Biochem. Biophys. Res. Commun.. 1999; 263 130-4
- 9 Lam Y W, Wang H X, Ng T B. A robust cysteine-deficient chitinase-like antifungal protein from inner shoots of the edible chive Allium tuberosum. Biochem. Biophys. Res. Commun.. 2000; 279 74-80
- 10 Lam S K, Ng T B. First simultaneous isolation of a ribosome inactivating protein and an antifungal protein from a mushroom (Lyophyllum shimeiji) together with evidence for synergism of their antifungal effects. Arch. Biochem. Biophys.. 2001; 393 271-80
- 11 Ye X Y, Wang H X, Ng T B. Dolichin, a new chtiniase-like antifungal protein isolated from field beans (Dolichos lablab). Biochem. Biophys. Res. Commun.. 2000a; 269 155-9
- 12 Lam S SL, Wang H X, Ng T B. Purification and characterization of novel ribosome inactivating proteins, alpha- and beta-pisavins, from seeds of the garden pea Pisum sativum. Biochem. Biophys. Res. Commun.. 1998; 253 135-42
- 13 Wang H X, Ng T B. Isolation and characterization of velutin, a novel low-molecular-weight ribosome-inactivating protein from winter mushroom (Flammulina velutipes) fruiting bodies. Life Sci. 2001; 68 2151-8
- 14 Ng T B, Wang H X. Panaxagin, a new protein from Chinese ginseng possesses anti-fungal, anti-viral, translation-inhibiting and ribonuclease activities. Life Sci.. 2001; 68 739-49
- 15 Wang H X, Ng T B. Quinqueginsin, a novel protein with anti-human immunodeficiency virus, antifungal, ribonuclease and cell-free translation-inhibitory activities from Amercian ginseng roots. Biochem. Biophys. Res. Commun.. 2000a; 269 203-8
- 16 Ye X Y, Ng T B. Mungin, a novel cyclophilin-like antifungal protein from the mung bean. Biochem. Biophys. Res. Commun.. 2000; 273 1111-5
- 17 Ye X Y, Wang H X, Ng T B. Sativin. A novel antifungal miraculin-like protein isolated from legumes of the sugar snap Pisum sativum var. macrocarpon . Life Sci.. 2000b; 67 775-81
- 18 Ye X Y, Wang H X, Ng T B. Structurally dissimilar proteins with antiviral and antifungal potency from cowpea (Vigna unguiculata) seeds. Life Sci.. 2000c; 67 3199-208
- 19 Wang H X, Ng T B. Ginkbilobin, a novel antifungal protein from Ginkgo biloba seeds with sequence similarity to embryo-abundant protein. Biochem. Biophys. Res. Commun.. 2000b; 279 407-10
- 20 Iseli B, Boller T, Neuhaus J M. The N-terminal cysteine-rich domain of tobacco class I chitinase is essential for chitin binding but not for catalytic or antifungal activity. Plant Physiol.. 1993; 103 221-6
- 21 Mauch F, Mauch-Mani B, Boller T. Antifungal hydrolases in pea tissue, inhibition of fungal growth by combination of chitinases and β-1,3-glucanase. Plant Physiol. 1988; 88 936-41
- 22 Melchers L S, Sela-Buurlage M B, Vloemans S A, Woloshuk C P, Van Roekel J S, Pen J, van den Elzen P J, Cornelissen B J. Extracellular targeting of the vacuolar tobacco proteins AP24, chitinase and beta-1,3-glucanase in transgenic plants. Plant Mol. Biol.. 1993; 21 583-93
- 23 Lorito M, Peterbauer C, Hayes C K, Harman G E. Synergistic interaction between fungal cell wall degrading enzymes and different antifungal compounds enhances inhibition of spore germination. Microbiology. 1994; 140 623-9
T. B. Ng
Department of Biochemistry
Faculty of Medicine
The Chinese University of Hong Kong
Shatin, Hong Kong
China
Fax: +852-2603-5123
Phone: +852-2609-6875
Fig. 1 Ion exchange chromatography of a crude extract of Panax notoginseng on a CM-cellulose column (5.5 × 25 cm). Eluent: 10 mM NH4OAc (pH 4.6). The eluent was changed to 50 mM NH4OAc (pH 7) when the elution volume was 8 L.
Fig. 2 Affi-gel blue gel chromatography of the fraction of crude P. notoginseng extract adsorbed on CM-cellulose. Eluent: 10 mM Tris-HCl (pH 7.2). The eluent was changed to 1.5 M NaCl in 10 mM Tris-HCl (pH 7.2) when the elution volume was 400 mL.
Fig. 3 Ion exchange chromatography on Mono S. The fraction adsorbed on Affi-gel blue gel was applied to the FPLC Mono S column in 10 mM NaOAc (pH 6.8). After elution of unadsorbed protein the column was eluted with a linear NaCl concentration gradient, as indicated by the slanting line across the chromatogram, to desorb the adsorbed protein.
Fig. 4 FPLC-gel filtration of adsorbed protein peak MS3 from Mono S on Superdex 75 in 200 mM NH4OAc (pH 7). The first peak represents pananotin, the purified antifungal protein. The first and second peaks possess a molecular mass of 35 kDa and 15 kDa, respectively.
Fig. 5 Sodium dedecyl sulfate-polyacrylamide gel electrophoresis of pananotin. Lane 1 represents pananotin. Lane two shows Pharmacia molecular mass markers, from top to downward: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa) and α-lactalbumin (14.4 kDa).
Fig. 6 Antifungal activity of pananotin towards (6.1) Botrytis cinerea (6.2) Coprinus comatus (6.3) Fusarium oxysporum (6.4) Physalospora piricola (A) 100 mM NaOAc (pH 5.5) as negative control (B) 40 μg pananotin in 100 mM NaOAc (pH 5.5) and (C) 8 μg pananotin in 100 mM NaOAc (pH 5.5).