A novel ribonuclease with antiproliferative activity from fresh fruiting bodies of the edible mushroom Hypsizigus marmoreus

An 18-kDa ribonuclease (RNase) with a novel N-terminal sequence was purified from fresh fruiting bodies of the mushroom Hypsizigus marmoreus. The purification protocol comprised ion exchange chromatography on DEAE cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose and Q-Sepharose and gel filtration by fast protein liquid chromatography on Superdex 75. The starting buffer was 10 mM Tris-HCl buffer (pH 7.2), 10 mM Tris-HCl buffer (pH 7.2), 10 mM NH(4)OAc buffer (pH 5), 10 mM NH(4)HCO(3) buffer (pH 9.4) and 200 mM NH(4)HCO(3) (pH 8.5), respectively. Absorbed proteins were desorbed using NaCl added to the starting buffer. A 42-fold purification of the enzyme was achieved. The RNase was unadsorbed on DEAE cellulose, Affi-gel blue gel and CM-cellulose but adsorbed on Q-Sepharose. It exhibited maximal RNase activity at pH 5 and 70 degrees C. Some RNase activity was detectable at 100 degrees C. It demonstrated the highest ribonucleolytic activity (196 U/mg) toward poly C, the next highest activity (126 U/mg) toward poly A, and much weaker activity toward poly U (48 U/mg) and poly G (41 U/mg). The RNase inhibited [(3)H-methyl]-thymidine uptake by leukemia L1210 cells with an IC(50) of 60 microM.     

A ribonuclease from the wild mushroom Boletus griseus

A ribonuclease (RNase) with a molecular mass of 29 kDa and cospecific for poly A and poly U was isolated from fruiting bodies of the mushroom Boletus griseus. Its N-terminal sequence exhibited some similarity to those of RNases from the mushrooms Irpex lacteus and Lentinus edodes. The RNase was adsorbed on diethylaminoethyl-cellulose, Q-Sepharose, and Affi-gel blue gel and was unadsorbed on CM-cellulose. The enzyme exhibited a temperature optimum between 60 and 70°C and a pH optimum at 3.5.     

A homodimeric laccase with unique characteristics from the yellow mushroom Cantharellus cibarius

The aim of the present study was to isolate a laccase from fruiting bodies of the yellow mushroom Cantharellus cibarius. The fruiting body extract was subjected to a purification protocol that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel and Con A-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The laccase was unadsorbed on DEAE-cellulose and Affi-gel blue gel and adsorbed on Con A-Sepharose. The laccase was composed of two identical subunits each with a molecular mass of 46 kDa. The laccase exhibited a temperature-dependent rise in activity over the temperature range 20-50 degrees C. When the temperature was raised above 60 degrees C there was a fall in enzyme activity. The enzyme manifested maximal activity at pH 4. At and above pH 6 there was a dramatic reduction in activity. The unique features of this fruiting body laccase compared with previously reported mycelial laccases include homodimeric nature, a distinctive N-terminal sequence, a higher optimal pH, and adsorption on only ConA-Sepharose among the various chromatographic media tested.     

A novel alkaline protease with antiproliferative activity from fresh fruiting bodies of the toxic wild mushroom Amanita farinosa

A novel protease with a molecular mass of 15 kDa was purified from fresh fruiting bodies of the wild mushroom Amanita farinosa. The purification protocol entailed anion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and SP-Sepharose. It demonstrated a single 15-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and a 15-kDa peak in gel filtration. The optimal pH and optimal temperature of the protease were pH 8.0 and 65 °C, respectively. Proliferation of human hepatoma HepG2 cells was inhibited by the protease with an IC(50) of 25 μM. The protease did not have antifungal or ribonuclease activity.     

Isolation of a ribonuclease from fruiting bodies of the wild mushroom Termitomyces globulus

A ribonuclease, with a molecular mass of 13 kDa and a ubiquitin-like N-terminal sequence, has been isolated from fruiting bodies of the mushroom Termitomyces globulus. The ribonuclease demonstrated ribonucleolytic activity toward poly A, poly C, poly G and poly U, with the activity toward poly A and poly C being much higher than that toward poly G and poly U. The ribonuclease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and CM-Sepharose. The enzyme required a temperature of 70 degrees C for expression of maximal activity. However, the enzyme expressed nearly the same optimal activity over a wide pH range of 5.0-8.0.     

Isolation of a ribonuclease from sanchi ginseng (Panax pseudoginseng) flowers distinct from other ginseng ribonucleases

A single-chained ribonuclease was isolated from the aqueous extract of sanchi ginseng (Panax pseudoginseng) flowers. It exhibited a molecular mass of 23 kDa, an N-terminal sequence with some similarity to other enzymes involved in RNA metabolism but different from known ribonucleases, and considerably higher activity toward poly U than poly C and only slight activity toward poly A and poly G. The purification protocol entailed ion exchange chromatography on diethylaminoethyl (DEAE)-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on carboxymethyl (CM)-cellulose, and gel filtration on Superdex 75. The ribonuclease was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and CM-cellulose. Maximal activity of the ribonuclease was attained at pH 7. On either side of this pH the enzyme activity underwent a drastic decline. The enzyme activity was at its highest at 50 degrees C and dropped to about 20% of the maximal activity when the temperature was decreased to 20 degrees C or elevated to 80 degrees C. The characteristics of sanchi ginseng flower ribonuclease were different from those of the ribonucleases previously purified from sanchi ginseng and Chinese ginseng roots including ribonuclease from Chinese ginseng flowers which are morphologically very similar to sanchi ginseng flowers.     

Isolation of a new ribonuclease from fruiting bodies of the silver plate mushroom Clitocybe maxima

A ribonuclease, with an N-terminal sequence exhibiting some homology to ribonuclease from Pleurotus ostreatus (Family Pleurotaceae), has been purified from fruiting bodies of the silver plate mushroom Clitocybe maxima (Family Tricholomataceae). However, there is little resemblance between the N-terminal sequences of ribonucleases from various Pleurotus species, and a lesser extent of resemblance between ribonucleases from C. maxima and Pleurotus tuber-regium. No structural relationship exists between ribonuclease from C. maxima, and those from Volvariella volvacea, Lentinus edodes and Irpex lacteus. The purification protocol involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-Sepharose, and fast protein liquid chromatography on Superdex 75. The ribonuclease was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and CM-Sepharose. It exhibited a molecular mass of 17.5 kDa in both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It manifested roughly the same ribonucleolytic potency toward poly A and poly G followed by poly U. Its activity toward poly C was, by comparison, meager. The temperature and pH required for its optimal activity were, respectively, 70 degrees C and 6.5-7.0.     

A novel ribonuclease from fruiting bodies of the common edible mushroom Pleurotus eryngii

A ribonuclease with a temperature optimum of about 70 degrees C and a pH optimum of 6.5 was isolated from fruiting bodies of the mushroom Pleurotus eryngii. The ribonuclease was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and S-Sepharose. It possessed a molecular mass of 16 kDa, and exhibited higher ribonucleolytic activity toward poly A and poly G and lower ribonucleolytic activity toward poly C and poly U. Its N-terminal sequence was distinctly different from those of other mushroom ribonucleases, and resembled that of Pleurotus tuber-regium only by 40%. Furthermore, its thermostability characteristics, polyhomoribonucleotide specificity and molecular mass were dissimilar to those of other mushroom ribonucleases.     

Isolation and characterization of a novel lectin from the wild mushroom Xerocomus spadiceus

A lectin was isolated from extracts of fruiting bodies of the mushroom Xerocomus spadiceus using a procedure that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose, and gel filtration by fast protein liquid chromatography on Superdex 75. The lectin was unadsorbed on DEAE-cellulose and Affi-gel blue gel and adsorbed on CM-Sepharose. The lectin was capable of eliciting an approximately four-fold stimulation of mitogenic response in murine splenocytes. The hemagglutinating activity was stable up to 60 degrees C, halved at 70 degrees C, reduced to 25% at 75 degrees C and dwindled to an undetectable level at 80 degrees C. The activity remained unaltered in the presence of various divalent (Ca2+, Mg2+, Zn2+ and Mn2+) chlorides up to a salt concentration of 10 mM except in the case of ZnCl2. FeCl3 up to a concentration of 10 mM did not affect the hemagglutinating activity of the lectin. The hemagglutinating activity of the lectin was doubled in the presence of 5 mM AlCl3 or 10 mM ZnCl2 and quadrupled in the presence of 10 mM AlCl3. The activity was reduced in the presence of HCl and NaOH. Among the large number of carbohydrates tested, only inulin was able to inhibit the hemagglutinating activity of the lectin.     

An alkaline protease from fresh fruiting bodies of the edible mushroom <Emphasis Type="Italic">Pleurotus citrinopileatus</Emphasis>

A protease was purified from fresh fruiting bodies of the edible mushroom Pleurotus citrinopileatus. The isolation procedure included ion exchange chromatography on DEAE-cellulose, CM-cellulose, and Q-Sepharose and fast protein liquid chromatography-gel filtration on Superdex 75. The protease was unadsorbed on DEAE-cellulose and Q-Sepharose, but adsorbed on CM-cellulose. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protease demonstrated a single band with a molecular mass of 28 kDa. The protease showed an optimal pH at 10 and an optimal temperature at 50°C. The activity of the protease was not affected by EDTA, indicating that it is not a metalloprotease. The protease exhibited a higher activity in the presence of K⁺ and Li⁺, but its activity was potently inhibited by Al³⁺, Cu²⁺, and Hg²⁺ ions. It manifested a K _m of 3.44 mg/ml and a V _max of 0.139 mg ml⁻¹ min⁻¹. It was devoid of ribonuclease and antifungal activities.     

Purification and characterization of a ubiquitin-like peptide with macrophage stimulating,antiproliferative and ribonuclease activities from the mushroom Agrocybe cylindracea

A peptide, with a molecular mass of 9.5kDa and demonstrating an N-terminal sequence similar to ubiquitin, was isolated from fruiting bodies of the mushroom Agrocybe cylindracea. The peptide was isolated with a purification protocol involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, FPLC-ion exchange chromatography on Mono S and FPLC-gel filtration on Superdex 75. The peptide was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and Mono S. It showed antiproliferative activity on leukemia cell line (M1) and hepatoma cell line (HepG2), and enhanced nitric oxide production in murine peritoneal macrophages with a potency comparable to that of lipopolysaccharide. A pH of 6.0 was required for optimal RNase activity. Its RNase activity was stable over the temperature range of 0-60 degrees C. It exerted ribonucleolytic activity preferentially on polyC, much lower activity on polyU, and negligible activity on polyA and polyG.     

A ribonuclease with distinctive features from the wild green-headed mushroom Russulus virescens

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 10mM Tris-HCl buffer (pH 7.1-7.3) and on CM-Sepharose in 10mM NH(4)OAc buffer (pH 4.6), unlike other mushroom ribonucleases which are unadsorbed on DEAE-cellulose. The RNase demonstrated a molecular mass of 28kDa 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 degrees C.     

A distinctive ribonuclease from fresh fruiting bodies of the medicinal mushroom Ganoderma lucidum

A ribonuclease with an N-terminal sequence distinct from other mushroom ribonucleases was isolated from fresh fruiting bodies of the medicinal mushroom Ganoderma lucidum. The ribonuclease was adsorbed on DEAE-cellulose and Q-Sepharose, and unadsorbed on CM-Sepharose. It possessed a molecular mass of 42 kDa as judged by gel filtration by fast protein liquid chromatography on Superdex 75 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its molecular mass was similar to that of straw mushroom ribonuclease but much higher compared with those of other mushroom ribonucleases. The ribonuclease was unique among mushroom ribonucleases in that it exhibited the highest potency toward poly(U), followed by poly(A). Its activity toward poly(G) and poly(C) was about one-half of that toward poly(A) and one-quarter of that toward poly(U). A pH of 4.0 and a temperature of 60 degrees C were required for optimal activity of the enzyme. The optimum pH was low compared with those reported for other mushroom ribonucleases.