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A ribonuclease with distinctive features from the wild green-headed mushroom Russulus virescens
BBRC Biochemical and Biophysical Research Communications 312 (2003) 965–968 www.elsevier.com/locate/ybbrc
A ribonuclease with distinctive features from the wild green-headed mushroom Russulus virescens Hexiang Wanga and Tzi Bun Ngb,* a
Department of Microbiology, College of Biological Science, China Agricultural University, and State Key Laboratory of Agrobiotechnology, Beijing, China b Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China Received 17 October 2003
Abstract A ribonuclease with an N-terminal sequence different from those of other ribonucleases has been purified from fruiting bodies of the mushroom Russula virescens. The RNase was adsorbed on DEAE–cellulose and Q-Sepharose in 10 mM Tris–HCl buffer (pH 7.1–7.3) and on CM–Sepharose in 10 mM NH4 OAc buffer (pH 4.6), unlike other mushroom ribonucleases which are unadsorbed on DEAE–cellulose. The RNase demonstrated a molecular mass of 28 kDa in both gel filtration and sodium dodecyl sulfate–polyacrylamide gel electrophoresis. In contrast to other mushroom ribonucleases which are monospecific, it exhibited co-specificity towards poly A and poly C. It demonstrated a pH optimum of 4.5, which is lower than values reported for other mushroom ribonucleases, and a temperature optimum of 60 °C. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Ribonuclease; Mushroom; Purification
Ribonucleases (RNases) belong to an important family of proteins which have been studied intensively. Several Nobel prizes have been awarded to eminent researchers who contributed to the early studies on RNases. RNases isolated from different mammalian tissues, e.g., brain, liver, and pancreas, may have different structures [1–3]. Seminal [4,5] and milk [6] RNases have also been characterized. Other isolated RNases comprise those from submammalian vertebrates, invertebrates, plants, fungi, and bacteria [7–20]. A spectrum of activities in RNases has been enumerated including antiviral [9,10,21], immunomodulatory [5], antitumor [4], angiogenic [6], and antifungal [10,11] activities. Russulus virescens is a wild mushroom of which little research has been conducted. The aim of the present study was to isolate a ribonuclease from this species and to compare its characteristics with those of RNases previously isolated from common edible mushrooms including the straw mushroom Volvariella volvacea [17], the shitake mushroom Lentinus edodes [14], the oyseter * Corresponding author. Fax: +852-2603-5123. E-mail address: [email protected] (T.B. Ng).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.10.201
mushroom Pleurotus ostreatus [15,18], the lung-shaped mushroom Pleurotus pulmonarius [19], the tiger milk mushroom Pleurotus tuber-regium [16], and the mushroom Irpex lacteus [13].
Materials and methods Fresh fruiting bodies of the green-headed mushroom R. virescens (1.5 kg) were purchased from a local market. They were first soaked and homogenized in distilled water. The homogenate was centrifuged at 10,000g for 30 min and the supernatant was removed. Tris–HCl buffer (pH 7.3) was added to the supernatant until the concentration of Tris reached 10 mM. The supernatant was then applied to a column of DEAE–cellulose (Sigma) (5 20 cm). Unadsorbed proteins were eluted as fraction D1 with 10 mM Tris–HCl buffer (pH 7.3). Adsorbed proteins were eluted as fraction D2 by addition of 0.8 M NaCl to the buffer. Fraction D2 was dialyzed against 10 mM NH4 OAc buffer (pH 4.6) before application to a column of CM–Sepharose CL-6B (Amersham Biosciences) (2.5 20 cm). After removal of unadsorbed proteins, adsorbed proteins (peaks CM2 and CM3) were eluted with a linear concentration gradient (0–1 M) of NaCl in the NH4 OAc buffer. Peak CM2 was dialyzed against 10 mM Tris–HCl buffer (pH 7.1) prior to chromatography on a Q-Sepharose column (Amersham Biosciences) (1.5 20 cm). The unadsorbed fraction Q1 was eluted with 10 mM Tris–HCl buffer (pH 7.1). The adsorbed proteins were separated into
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peaks Q2 and Q3 by elution with a linear concentration gradient (0–1 M) of NaCl in 10 mM Tris–HCl buffer (pH 7.1). The most strongly adsorbed peak Q3 was fractionated by FPLC on a Superdex 75 HR 10/30 column (Amersham Biosciences) into two peaks, SU1 and SU2. Molecular mass determination by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and by FPLC-gel filtration. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was carried out in accordance with the procedure of Laemmli and Favre [22], using a 12% resolving gel and a 5% stacking gel. At the end of electrophoresis, the gel was stained with Coomassie brilliant blue. FPLC-gel filtration was carried out using a Superdex 75 HR 10/30 column that had been calibrated with molecular-mass standards (Amersham Biosciences). Analysis of N-terminal amino acid sequence. Amino acid sequence analysis was carried out using an HP G1000A Edman degradation unit and an HP1000 HPLC system [23]. Assay for activity of ribonuclease. The activity of RNase toward yeast tRNA was assayed by determining the generation of acid-soluble, UV-absorbing species with the method of Mock et al. [24]. The RNase was incubated with 200 lg tRNA in 150 lg of 100 mM Mes buffer (pH 6.0) at 37 °C for 1 h. The reaction was terminated by introduction of 350 ll of ice-cold 3.4% perchloric acid. After leaving on ice for 15 min, the sample was centrifuged (15,000g, 15 min) at 4 °C. The OD260 of the supernatant was read after appropriate dilution. One unit of enzymatic activity is defined as the amount of enzyme that brings about an increase in OD260 of one per minute in the acid-soluble fraction per milliliter of reaction mixture under the specified condition. Activity of RNase toward polyhomoribonucleotides. The ribonucleolytic activity of RNase toward polyhomoribonucleotides was determined with a modification of the method of Wang and Ng [16]. Incubation of RNase with 100 lg of poly A, poly C, poly G or poly U in 250 ll of 100 mM sodium acetate (pH 5.0) was carried out at 37 °C for 1 h, prior to addition of 250 ll of ice-cold 1.2 N perchloric acid containing 20 mM lanthanum nitrate to terminate the reaction. After standing on ice for 15 min, the sample was centrifuged at 15,000g for 15 min at 4 °C. The absorbance of the supernatant, after appropriate dilution, was read at 260 nm (in the case of poly A, poly G, and poly U) or at 280 nm (in the case of poly C).
Fig. 1. Ion exchange chromatography on Q-Sepharose. Column dimensions: 1.5 20 cm. Starting buffer: 10 mM Tris–HCl buffer (pH 7.3). Sample: fraction of fruiting body extract adsorbed on DEAE– cellulose and then on CM–Sepharose (i.e., fraction CM2). Dotted slanting line across the right half of the chromatogram indicates the linear NaCl concentration gradient (0–1 M) used to elute adsorbed proteins.
Table 1 RNase activities of chromatographic fractions obtained at various stages of the purification process Chromatographic fraction
RNase activity (U/mg)
D1 D2 D2CM1 D2CM2 D2CM3 D2CM2Q1 D2CM2Q2 D2CM2Q3 D2CM2Q3SU1 (purified RNase) D2CM2Q3SU2
<1 22.6 <1 49.6 5.9 3.4 10.8 124.8 216.9 13.5
Results RNase activity was located in fraction D2 of the fruiting body extract that was adsorbed on DEAE– cellulose. When D2 was chromatographed on CM– Sepharose, RNase activity was found to reside in fraction CM2, the first adsorbed fraction (data not shown). CM2 was separated into a small unadsorbed peak Q1 devoid of RNase activity, and two adsorbed peaks of roughly equal size, Q2 and Q3 (Fig. 1). RNase activity was concentrated in Q3 (Table 1). Q3 was fractionated on Superdex 75 into a larger peak SU1 and a smaller peak SU2 that was eluted later (Fig. 2). The molecular mass of SU1 was 28 kDa as judged by gel filtration (Fig. 2) and SDS–PAGE (Fig. 3). High RNase activity of the enzyme was observed over the pH range 4.0–6.0 and minimal activity over the pH range 7.5–9.0. There was a precipitous fall in activity between pH 6.5 and 7.5 (Fig. 4). Maximal activity of the ribonuclease was observed at 60 °C. The enzyme activity underwent a rapid decline both when the temperature was reduced from 60 °C and when it was raised further (Fig. 5).
Fig. 2. Fast protein liquid chromatography-gel filtration on a Superdex 75 HR 10/30 column. Buffer: 0.2 M NH4 HCO3 (pH 8.5). Flow rate: 0.4 ml/min. Fraction size: 0.8 ml. Sample: fraction Q3. Peak SU1 represented purified RNase.
The activity of R. virescens RNase toward poly A, poly C, poly U, and poly G was, respectively, 125.3, 130.7, 9.1, and 6.2 U/mg. The RNase activities of various chromatographic fractions derived from the fruiting
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Table 2 N-terminal sequence of R. virescens (RV) ribonuclease in comparison with ribonucleases from P. pulmonarius (PP), V. volvacea (VV), L. edodes (LE), I. lacteus (IL), P. ostreatus (PO), and P. tuber-regium (PT) RV: PP: VV: LE: IL:
TDHTLDTMMTHTLRD AISANNERKGVNQQSVQNTYQENDV APYVQLFRPLIQPQVLATFAIANNMAQY ISSGCGTTGALSCSSNAKGTCCFEAPGGLI VNSGCGTSGAESCSNSDDGTCCFEAPGGLL
PO:
ETGVRSCNCAGRSFTGTDVTNAIRSARAGGSGN
PT:
ALTAQDNRVRVGNRIVGNNFNFAAVQAAYY
Discussion Fig. 3. Molecular mass determination by SDS–PAGE. Right lane: molecular mass markers from Amersham Biosciences. From top downward: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa), and lactalbumin (14.4 kDa). Left lane: peak SU1.
Fig. 4. Effect of pH on RNase activity of R. virescens RNase.
Fig. 5. Effect of temperature on RNase activity of R. virescens RNase.
body extract are shown in Table 1. The yield of the RNase was 9 mg from 1.5 kg fruiting bodies. The Nterminal sequence of R. virescens RNase did not resemble those of other mushroom RNases (Table 2).
Russula virescens RNase is unique in that it shows cospecificity toward poly A and poly C. The RNases previously isolated from P. ostreatus [15], P. pulmonarius [19], P. tuber-regium [16], and L. edodes [14] demonstrate specificity toward poly G, poly C, poly G, and poly A, respectively. Pleurotus tuber-regium RNase is much more robust compared with R. virescens RNase. The activity of the former RNase is preserved after exposure to 100 °C for 30 min [16]. An abrupt drop in activity of R. virescens RNase is observed when the ambient temperature is elevated above 70 °C. Its optimal temperature is 60 °C, close to that for P. pulmonarius RNase [19] which also undergoes rapid inactivation when the temperature rises above 60 °C. A pH of 4.5 is optimal for the activity of R. virescens RNase while the optimum pH is 6.5 for P. tuber-regium RNase [16] and 7.0 for P. pulmonarius RNase [19]. The molecular mass of R. virescens RNase (28 kDa) is close to that of P. tuber-regium RNase (29 kDa). However, the former is monomeric while the latter [16] is dimeric. R. virescens RNase is smaller than V. volvacea RNase [17] in molecular mass (42 kDa), but is much larger than P. ostreatus and R. pulmonarius RNases (around 10 kDa) [15,19]. Russulus virescens RNase is adsorbed on both DEAE–cellulose and CM–Sepharose. By contrast, P. tuber-regium RNase [16] and V. volvacea RNase [17] are unadsorbed on DEAE–cellulose and adsorbed on CM–cellulose. The starting buffer used for DEAE– cellulose chromatography is 10 mM Tris–HCl buffer (pH 7.3) for R. virescens RNase and V. volvacea RNase and 10 mM Tris–HCl buffer (pH 7.8) for P. tuber-regium RNase. Hence the adsorption of R. virescens RNase on DEAE–cellulose is a distinctive characteristic. The N-terminal sequence of R. virescens RNase is distinctly different from those of other mushroom RNases. All in all, R. virescens RNase is a unique RNase in many ways.
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Acknowledgments We thank Ms. Fion Yung and Ms. Christine Chung for their excellent secretarial assistance.
References [1] M.P. Sasso, A. Carsana, E. Confalone, C. Cosi, S. Somentino, M. Viola, M. Palmieri, E. Russo, A. Furia, Molecular cloning of the gene encoding the bovine brain ribonuclease and its expression in different regions of the brain, Nucleic Acids Res. 19 (1991) 6469– 6474. [2] J. Hofsteenge, R. Matthies, S.R. Stone, Primary structure of a ribonuclease from porcine liver, a new member of the ribonuclease superfamily, Biochemistry 28 (1989) 9806–9813. [3] M. Irie, R. Nitta, K. Ohgi, Y. Niwata, H. Watanabe, M. Iwama, J.J. Beintema, A. Sanda, Y. Takizawa, Primary structure of a nonsecretory ribonuclease from bovine kidney, J. Biochem. (Tokyo) 104 (1988) 289–296. [4] B.S. Adinolfi, V. Cafaro, G. D’Alessio, D. Di Donato, Full antitumor action of recombinant seminal ribonuclease depends on the removal of its N-terminal methionine, Biochem. Biophys. Res. Commun. 213 (1995) 525–532. [5] J. Matousek, J. Soucek, J. Riha, T.R. Zankel, S.A. Benner, Immunosuppressive activity of angiogenin in comparison with bovine seminal ribonuclease and pancreatic ribonuclease, Comp. Biochem. Physiol. 112B (1995) 235–241. [6] R. Shapiro, B.L. Vallee, Human placental ribonuclease inhibitor abolishes both angiogenic and ribonucleolytic activities of angiogenin, Proc. Natl. Acad. Sci. USA 84 (1987) 2238–2241. [7] Y.D. Liao, A pyrimidine-guanine sequence-specific ribonuclease from Rana catesbiana (bullfrog) oocytes, Nucleic Acids Res. 20 (1992) 1371–1377. [8] P.J. Green, The ribonucleases of higher plants, Annu. Rev. Plant Physiol. Plant Mol. Biol. 45 (1994) 421–445. [9] H.X. Wang, T.B. Ng, Quinqueginsin, a novel protein with antihuman immunodeficiency virus, antifungal, ribonuclease and cellfree translation-inhibitory activities from American ginseng roots, Biochem. Biophys. Res. Commun. 269 (2000) 155–159. [10] T.B. Ng, H.X. Wang, Panaxagin, a new protein from Chinese ginseng possesses antifungal, antiviral, translation-inhibiting and ribonuclease activities, Life Sci. 68 (2001) 739–749. [11] S.K. Lam, T.B. Ng, Isolation of a novel thermolabile heterodimeric ribonuclease with antifungal and antiproliferative activities from roots of the sanchi ginseng Panax notoginseng, Biochem. Biophys. Res. Commun. 285 (2001) 419–423.
[12] P.A. Bariola, P.J. Green, Plant ribonucleases, in: G. D’Alessio, J. Riordan (Eds.), Ribonucleases Structures and Functions, Academic Press, New York, 1997, pp. 163–190. [13] H. Watanabe, F. Hamid, M. Iwami, T. Onda, K. Ohgi, M. Irie, Primary structure of RNase from Irpex lacteus, Biosci. Biotechnol. Biochem. 59 (1995) 2092–2103. [14] H. Kobayashi, N. Inokuchi, T. Koyama, H. Watanabe, M. Iwami, K. Ohgi, M. Irie, Primary structure of the nonspecific and adenylic acid preferential ribonuclease from the fruit bodies of Lentinus edodes, Biosci. Biotechnol. Biochem. 55 (1992) 2003– 2010. [15] N. Nomura, N. Inokuchi, H. Kobayashi, T. Koyama, M. Iwama, K. Ohgi, M. Irie, Purification and primary structure of a new guanylic acid—specific ribonuclease from Pleurotus ostreatus, J. Biochem. (Tokyo) 116 (1994) 26–33. [16] H.X. Wang, T.B. Ng, Purification and characterization of a potent homodimeric guanine-specific ribonuclease from fresh mushroom (Pleurotus tuber-regium) scleotia, Int. J. Biochem. Cell Biol. 33 (2001) 390–483. [17] H.X. Wang, T.B. Ng, Isolation of a new ribonuclease from fresh fruiting bodies of the straw mushroom, Biochem. Biophys. Res. Commun. 264 (1999) 714–718. [18] X.Y. Ye, T.B. Ng, A novel peptide with ribonuclease and translation-inhibitory activity from fruiting bodies of the oyster mushroom Pleurotus ostreatus, J. Peptide Sci. 8 (2002) 235–240. [19] X.Y. Ye, T.B. Ng, A novel and potent ribonuclease from fruiting bodies of the mushroom Pleurotus pulmonarius, Biochem. Biophys. Res. Commun. 293 (2002) 857–861. [20] D.E. Mossakowska, K. Nyberg, A.R. Fersht, Kinetic characterization of the recombinant ribonuclease from Bacillus amyloliquefaciens (barnase) and investigation of key residues in catalysis by site-directed mutagenesis, Biochemistry 28 (1989) 3843– 3850. [21] T.B. Ng, T.L. Lam, T.K. Au, X.Y. Ye, C.C. Wang, Inhibition of human immunodeficiency virus type 1 reverse transcriptase, protease and integrase by bovine milk proteins, Life Sci. 69 (2001) 2217–2223. [22] U.K. Laemmli, M. Favre, Maturation of the head of bacteriophage T4. I. DNA packaging events, J. Mol. Biol. 80 (1970) 575– 599. [23] S.S.L. Lam, H.X. Wang, T.B. Ng, Purification and characterization of novel ribosome inactivating proteins, alpha- and betapisavins, from seeds of the garden pea Pisum sativum, Biochem. Biophys. Res. Commun. 253 (1998) 135–142. [24] J.W.Y. Mock, T.B. Ng, R.N.S. Wong, Q.Z. Yao, H.W. Yeung, W.P. Fong, Demonstration of ribonuclease activity in the plant ribosome-inactivating proteins alpha- and beta-momorcharins, Life Sci. 59 (1996) 1853–1859.
A ribonuclease with distinctive features from the wild green-headed mushroom Russulus virescens Hexiang Wanga and Tzi Bun Ngb,* a
Department of Microbiology, College of Biological Science, China Agricultural University, and State Key Laboratory of Agrobiotechnology, Beijing, China b Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China Received 17 October 2003
Abstract A ribonuclease with an N-terminal sequence different from those of other ribonucleases has been purified from fruiting bodies of the mushroom Russula virescens. The RNase was adsorbed on DEAE–cellulose and Q-Sepharose in 10 mM Tris–HCl buffer (pH 7.1–7.3) and on CM–Sepharose in 10 mM NH4 OAc buffer (pH 4.6), unlike other mushroom ribonucleases which are unadsorbed on DEAE–cellulose. The RNase demonstrated a molecular mass of 28 kDa in both gel filtration and sodium dodecyl sulfate–polyacrylamide gel electrophoresis. In contrast to other mushroom ribonucleases which are monospecific, it exhibited co-specificity towards poly A and poly C. It demonstrated a pH optimum of 4.5, which is lower than values reported for other mushroom ribonucleases, and a temperature optimum of 60 °C. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Ribonuclease; Mushroom; Purification
Ribonucleases (RNases) belong to an important family of proteins which have been studied intensively. Several Nobel prizes have been awarded to eminent researchers who contributed to the early studies on RNases. RNases isolated from different mammalian tissues, e.g., brain, liver, and pancreas, may have different structures [1–3]. Seminal [4,5] and milk [6] RNases have also been characterized. Other isolated RNases comprise those from submammalian vertebrates, invertebrates, plants, fungi, and bacteria [7–20]. A spectrum of activities in RNases has been enumerated including antiviral [9,10,21], immunomodulatory [5], antitumor [4], angiogenic [6], and antifungal [10,11] activities. Russulus virescens is a wild mushroom of which little research has been conducted. The aim of the present study was to isolate a ribonuclease from this species and to compare its characteristics with those of RNases previously isolated from common edible mushrooms including the straw mushroom Volvariella volvacea [17], the shitake mushroom Lentinus edodes [14], the oyseter * Corresponding author. Fax: +852-2603-5123. E-mail address: [email protected] (T.B. Ng).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.10.201
mushroom Pleurotus ostreatus [15,18], the lung-shaped mushroom Pleurotus pulmonarius [19], the tiger milk mushroom Pleurotus tuber-regium [16], and the mushroom Irpex lacteus [13].
Materials and methods Fresh fruiting bodies of the green-headed mushroom R. virescens (1.5 kg) were purchased from a local market. They were first soaked and homogenized in distilled water. The homogenate was centrifuged at 10,000g for 30 min and the supernatant was removed. Tris–HCl buffer (pH 7.3) was added to the supernatant until the concentration of Tris reached 10 mM. The supernatant was then applied to a column of DEAE–cellulose (Sigma) (5 20 cm). Unadsorbed proteins were eluted as fraction D1 with 10 mM Tris–HCl buffer (pH 7.3). Adsorbed proteins were eluted as fraction D2 by addition of 0.8 M NaCl to the buffer. Fraction D2 was dialyzed against 10 mM NH4 OAc buffer (pH 4.6) before application to a column of CM–Sepharose CL-6B (Amersham Biosciences) (2.5 20 cm). After removal of unadsorbed proteins, adsorbed proteins (peaks CM2 and CM3) were eluted with a linear concentration gradient (0–1 M) of NaCl in the NH4 OAc buffer. Peak CM2 was dialyzed against 10 mM Tris–HCl buffer (pH 7.1) prior to chromatography on a Q-Sepharose column (Amersham Biosciences) (1.5 20 cm). The unadsorbed fraction Q1 was eluted with 10 mM Tris–HCl buffer (pH 7.1). The adsorbed proteins were separated into
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peaks Q2 and Q3 by elution with a linear concentration gradient (0–1 M) of NaCl in 10 mM Tris–HCl buffer (pH 7.1). The most strongly adsorbed peak Q3 was fractionated by FPLC on a Superdex 75 HR 10/30 column (Amersham Biosciences) into two peaks, SU1 and SU2. Molecular mass determination by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and by FPLC-gel filtration. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was carried out in accordance with the procedure of Laemmli and Favre [22], using a 12% resolving gel and a 5% stacking gel. At the end of electrophoresis, the gel was stained with Coomassie brilliant blue. FPLC-gel filtration was carried out using a Superdex 75 HR 10/30 column that had been calibrated with molecular-mass standards (Amersham Biosciences). Analysis of N-terminal amino acid sequence. Amino acid sequence analysis was carried out using an HP G1000A Edman degradation unit and an HP1000 HPLC system [23]. Assay for activity of ribonuclease. The activity of RNase toward yeast tRNA was assayed by determining the generation of acid-soluble, UV-absorbing species with the method of Mock et al. [24]. The RNase was incubated with 200 lg tRNA in 150 lg of 100 mM Mes buffer (pH 6.0) at 37 °C for 1 h. The reaction was terminated by introduction of 350 ll of ice-cold 3.4% perchloric acid. After leaving on ice for 15 min, the sample was centrifuged (15,000g, 15 min) at 4 °C. The OD260 of the supernatant was read after appropriate dilution. One unit of enzymatic activity is defined as the amount of enzyme that brings about an increase in OD260 of one per minute in the acid-soluble fraction per milliliter of reaction mixture under the specified condition. Activity of RNase toward polyhomoribonucleotides. The ribonucleolytic activity of RNase toward polyhomoribonucleotides was determined with a modification of the method of Wang and Ng [16]. Incubation of RNase with 100 lg of poly A, poly C, poly G or poly U in 250 ll of 100 mM sodium acetate (pH 5.0) was carried out at 37 °C for 1 h, prior to addition of 250 ll of ice-cold 1.2 N perchloric acid containing 20 mM lanthanum nitrate to terminate the reaction. After standing on ice for 15 min, the sample was centrifuged at 15,000g for 15 min at 4 °C. The absorbance of the supernatant, after appropriate dilution, was read at 260 nm (in the case of poly A, poly G, and poly U) or at 280 nm (in the case of poly C).
Fig. 1. Ion exchange chromatography on Q-Sepharose. Column dimensions: 1.5 20 cm. Starting buffer: 10 mM Tris–HCl buffer (pH 7.3). Sample: fraction of fruiting body extract adsorbed on DEAE– cellulose and then on CM–Sepharose (i.e., fraction CM2). Dotted slanting line across the right half of the chromatogram indicates the linear NaCl concentration gradient (0–1 M) used to elute adsorbed proteins.
Table 1 RNase activities of chromatographic fractions obtained at various stages of the purification process Chromatographic fraction
RNase activity (U/mg)
D1 D2 D2CM1 D2CM2 D2CM3 D2CM2Q1 D2CM2Q2 D2CM2Q3 D2CM2Q3SU1 (purified RNase) D2CM2Q3SU2
<1 22.6 <1 49.6 5.9 3.4 10.8 124.8 216.9 13.5
Results RNase activity was located in fraction D2 of the fruiting body extract that was adsorbed on DEAE– cellulose. When D2 was chromatographed on CM– Sepharose, RNase activity was found to reside in fraction CM2, the first adsorbed fraction (data not shown). CM2 was separated into a small unadsorbed peak Q1 devoid of RNase activity, and two adsorbed peaks of roughly equal size, Q2 and Q3 (Fig. 1). RNase activity was concentrated in Q3 (Table 1). Q3 was fractionated on Superdex 75 into a larger peak SU1 and a smaller peak SU2 that was eluted later (Fig. 2). The molecular mass of SU1 was 28 kDa as judged by gel filtration (Fig. 2) and SDS–PAGE (Fig. 3). High RNase activity of the enzyme was observed over the pH range 4.0–6.0 and minimal activity over the pH range 7.5–9.0. There was a precipitous fall in activity between pH 6.5 and 7.5 (Fig. 4). Maximal activity of the ribonuclease was observed at 60 °C. The enzyme activity underwent a rapid decline both when the temperature was reduced from 60 °C and when it was raised further (Fig. 5).
Fig. 2. Fast protein liquid chromatography-gel filtration on a Superdex 75 HR 10/30 column. Buffer: 0.2 M NH4 HCO3 (pH 8.5). Flow rate: 0.4 ml/min. Fraction size: 0.8 ml. Sample: fraction Q3. Peak SU1 represented purified RNase.
The activity of R. virescens RNase toward poly A, poly C, poly U, and poly G was, respectively, 125.3, 130.7, 9.1, and 6.2 U/mg. The RNase activities of various chromatographic fractions derived from the fruiting
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Table 2 N-terminal sequence of R. virescens (RV) ribonuclease in comparison with ribonucleases from P. pulmonarius (PP), V. volvacea (VV), L. edodes (LE), I. lacteus (IL), P. ostreatus (PO), and P. tuber-regium (PT) RV: PP: VV: LE: IL:
TDHTLDTMMTHTLRD AISANNERKGVNQQSVQNTYQENDV APYVQLFRPLIQPQVLATFAIANNMAQY ISSGCGTTGALSCSSNAKGTCCFEAPGGLI VNSGCGTSGAESCSNSDDGTCCFEAPGGLL
PO:
ETGVRSCNCAGRSFTGTDVTNAIRSARAGGSGN
PT:
ALTAQDNRVRVGNRIVGNNFNFAAVQAAYY
Discussion Fig. 3. Molecular mass determination by SDS–PAGE. Right lane: molecular mass markers from Amersham Biosciences. From top downward: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa), and lactalbumin (14.4 kDa). Left lane: peak SU1.
Fig. 4. Effect of pH on RNase activity of R. virescens RNase.
Fig. 5. Effect of temperature on RNase activity of R. virescens RNase.
body extract are shown in Table 1. The yield of the RNase was 9 mg from 1.5 kg fruiting bodies. The Nterminal sequence of R. virescens RNase did not resemble those of other mushroom RNases (Table 2).
Russula virescens RNase is unique in that it shows cospecificity toward poly A and poly C. The RNases previously isolated from P. ostreatus [15], P. pulmonarius [19], P. tuber-regium [16], and L. edodes [14] demonstrate specificity toward poly G, poly C, poly G, and poly A, respectively. Pleurotus tuber-regium RNase is much more robust compared with R. virescens RNase. The activity of the former RNase is preserved after exposure to 100 °C for 30 min [16]. An abrupt drop in activity of R. virescens RNase is observed when the ambient temperature is elevated above 70 °C. Its optimal temperature is 60 °C, close to that for P. pulmonarius RNase [19] which also undergoes rapid inactivation when the temperature rises above 60 °C. A pH of 4.5 is optimal for the activity of R. virescens RNase while the optimum pH is 6.5 for P. tuber-regium RNase [16] and 7.0 for P. pulmonarius RNase [19]. The molecular mass of R. virescens RNase (28 kDa) is close to that of P. tuber-regium RNase (29 kDa). However, the former is monomeric while the latter [16] is dimeric. R. virescens RNase is smaller than V. volvacea RNase [17] in molecular mass (42 kDa), but is much larger than P. ostreatus and R. pulmonarius RNases (around 10 kDa) [15,19]. Russulus virescens RNase is adsorbed on both DEAE–cellulose and CM–Sepharose. By contrast, P. tuber-regium RNase [16] and V. volvacea RNase [17] are unadsorbed on DEAE–cellulose and adsorbed on CM–cellulose. The starting buffer used for DEAE– cellulose chromatography is 10 mM Tris–HCl buffer (pH 7.3) for R. virescens RNase and V. volvacea RNase and 10 mM Tris–HCl buffer (pH 7.8) for P. tuber-regium RNase. Hence the adsorption of R. virescens RNase on DEAE–cellulose is a distinctive characteristic. The N-terminal sequence of R. virescens RNase is distinctly different from those of other mushroom RNases. All in all, R. virescens RNase is a unique RNase in many ways.
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Acknowledgments We thank Ms. Fion Yung and Ms. Christine Chung for their excellent secretarial assistance.
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