Purification and characterization of a new ribonuclease from fruiting bodies of the oyster mushroom Pleurotus ostreatus.

A ribonuclease (RNase), possessing an N-terminal sequence disparate from those of ribonucleases from other mushrooms and previously isolated Pleuotus ostreatus RNases, was purified from the fruiting bodies of the edible mushroom Pleurotus ostreatus. The N-terminal sequence of Pleurotus ostreatus RNase did not manifest homology even to a previously reported RNase from the same mushroom. The ribonuclease was adsorbed on CM-Sepharose and Mono S. It exhibited a molecular mass of 12 kDa in both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and gel filtration on Superdex 75. The ribonuclease displayed an activity of 11490 U/mg on yeast tRNA. The highest ribonuclease activity was exhibited toward poly U, followed by poly A and poly C. No activity was shown toward poly G. The optimal pH for its activity was 7 and the optimal temperature was 55 degrees C. It inhibited cell-free translation in a rabbit reticulocyte lysate with an IC50 of 240 nM.     

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.     

Purification and characterization of a potent homodimeric guanine-specific ribonuclease from fresh mushroom (Pleurotus tuber-regium) sclerotia

From the fresh sclerotia of the mushroom Pleurotus tuber-regium, a potent homodimeric ribonuclease exhibiting a molecular weight of 29 kDa in FPLC-gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was isolated. The protein was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel, CM-cellulose and Mono S. It manifested strong ribonucleolytic activity toward Poly G, slight activity toward Poly U and Poly A, and minimal activity toward Poly C. Its optimal pH was determined to be 6.5 when yeast transfer RNA was used as substrate. Its ribonucleolytic activity was resistant to heating at 100 degrees C for 30 min but was inhibited by a number of salts. The protein inhibited cell-free translation in a rabbit reticulocyte lysate with an IC50 of 0.09 nM. Three out of the four amino acid residues at the active site (positions 38-41) of P. ostreatus ribonuclease, YNNF, were also found at positions 17-20 in the P. tuber-regum RNase. However, unlike P. ostreatus RNase, no cysteine residues were detected in the N-terminal sequence.     

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.     

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.     

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.     

Characterization of a pathogenesis-related class 10 protein (PR-10) from Astragalus mongholicus with ribonuclease activity.

A pathogenesis-related (PR) class 10 protein (designated AmPR-10) was first isolated from the Chinese medicinal material Astragalus mongholicus using a combination of affinity chromatography on Zn-chelate Agarose 4B, ion exchange chromatography on QAE Sephadex A-25 and gel filtration on Sephadex G50. The purified AmPR-10 showed a single band with a molecular mass of 17.2kDa in SDS-PAGE. The molecular mass of intact AmPR-10 was determined to be 32.8kDa by gel filtration. Thus, AmPR-10 is a dimeric protein composed of two identical subunits. AmPR-10 was a glycoprotein detected by periodic acid-Schiff (PAS) staining and its neutral carbohydrate content was 13.7%. The carbohydrate was mainly composed of 73.0% (w/w) arabinose, 15.0% (w/w) glucose and 4.8% (w/w) fructose on the basis of high-performance anion exchange chromatography (HPAEC) analysis. Its N-terminal sequence of 15 amino acid residues was determined as GVISFNEETISTVAP, and showed significant sequence homology to some pathogenesis-related (PR) class 10 proteins. This sequence had 80% identity with the PR-10 protein LlPR10.1C from Lupinus luteus (yellow lupine) followed by 73.3% identity with the PR-10 protein PR10.2 from Medicago sativa (alfalfa), suggesting it is a new member of PR-10 proteins. AmPR-10 exhibited ribonuclease (RNase) activity as do some other PR-10 proteins. The optimal pH and temperature for RNase activity were pH 6.0 and 60 degrees C, respectively. The RNase activity was stable within pH 5.0-11.0. It was stable up to 60 degrees C at pH 6.0. The purification and characterization of AmPR-10 in this investigation furnish additional data to the relatively scanty literature pertaining to Astragali radix proteins.     

Partial Purification and Properties of Tea Leaf Ribonuclease

Four fractions with ribonuclease activity have been isolated from tea leaves by DEAE-cellulose column chromatography and designated as RNase Tf-1, RNase Tf-2, RNase Tf-3 and RNase Tf-4. The bigger fractions of both RNase Tf-3 and RNase Tf-4 have been partially purified by Sephadex G-100 column chromatography.

RNase Tf-3 and RNase Tf-4 were respectively found to have their optimum pH at 4.75 and 4.9 and molecular weights of approximately 13,000 and 16,000, as determined by gel filtration. Both enzymes were inhibited by Cu²⁺ and Hg²⁺, and inactivated by heating at over 50°C. By addition of yeast RNA to the two enzymes, however, their thermostabilities increased. The activities of the enzymes were stable in a pH range of 4.5 to 6.5. Like other plant RNases, RNase Tf-3 and RNase Tf-4 appeared to have no preference for base in RNA.     

Human leukotriene C4 synthase expression in dimethyl sulfoxide-differentiated U937 cells.

Leukotriene C4 (LTC4) synthase was highly expressed in the human U937 monoblast leukemia cell line when differentiated into monocyte/macrophage-like cells by growth in the presence of dimethyl sulfoxide. The specific activity of LTC4 synthase in differentiated cells (399.0 +/- 84.1 pmol of LTC4 formed.min-1.mg-1) was markedly higher (10-fold; p less than 0.001) than in undifferentiated U937 cells (39.9 +/- 16.7 pmol of LTC4 formed.min-1.mg-1) or freshly isolated blood monocytes (21.5 +/- 4.8 pmol of LTC4 formed.min-1.mg-1). The increase in LTC4 synthase activity following dimethyl sulfoxide-induced differentiation was substantially higher than the increase observed for other proteins involved in leukotriene biosynthesis. LTC4 synthase activity was unaffected in U937 cells differentiated by growth in the presence of phorbol 12-myristate 13-acetate. The HL-60 myeloblast leukemia cell line expressed higher LTC4 synthase levels when differentiated into either neutrophil-like or macrophage-like cells by growth in the presence of dimethyl sulfoxide or phorbol 12-myristate 13-acetate (respectively), but reached a specific activity comparable only to undifferentiated U937 cells. Human LTC4 synthase was found to be a unique membrane-bound enzymatic activity completely distinct from alpha, mu, pi, theta, and microsomal glutathione S-transferases, as determined by differential detergent solubilization, chromatographic separation, substrate specificity, and Western blot analysis. An 18-kDa polypeptide was specifically labeled in membranes from dimethyl sulfoxide-differentiated U937 cells using azido 125I-LTC4, a photoaffinity probe based on the product of the LTC4 synthase-catalyzed reaction. Photolabeling of the 18-kDa polypeptide was specifically competed for by LTC4 (greater than 50% at 0.1 microM) but not by 100,000-fold higher concentrations of reduced glutathione (10 mM). Elevation of both the level of the specifically photolabeled 18-kDa polypeptide and of LTC4 synthase specific activity occurred concomitantly with dimethyl sulfoxide differentiation of U937 cells. We conclude that differentiation of U937 cells into monocyte/macrophage-like cells by growth in the presence of dimethyl sulfoxide results in high levels of expression of LTC4 synthase activity. Human LTC4 synthase is a unique enzyme with a high degree of specificity for LTA4 and may therefore be dedicated exclusively to the formation of LTC4 in vivo. An 18-kDa membrane polypeptide, specifically labeled by a photoaffinity derivative of LTC4, is a candidate for being either LTC4 synthase or a subunit thereof.     

Activation of acid ribonuclease from bovine brain by aluminum.

An acid ribonuclease (optimum pH 6.0) has been purified from bovine brain in a five-step procedure. The preparation appeared homogeneous on SDS-polyacrylamide gel electrophoresis. The molecular size of the acid ribonuclease is 70 kDa and it is a dimeric protein with a subunit molecular size of 35 kDa. The acid RNase was activated by aluminum at low concentration. Preincubation of the acid RNase with 10 microM increased the specific activity of the enzyme 2.3-fold at acid pH, while the effect of aluminum was much weaker at alkaline pH under otherwise the same conditions. A stoichiometry of 1: 1 for the binding aluminum to brain acid RNase was estimated. None of the enzyme-bound aluminum was dissociated by dialysis against 50 mM HEPES, pH 7.0 at 4 degrees C for 24 h. Citrate, EDTA, NaF, and apotransferrin abolished the effects of aluminum on the enzyme. Ribonucleic acid also protected the enzyme against the activation caused by aluminum. These results suggest that accumulation of aluminum in brain may change the regulation of ribonucleic acid metabolism.     

Preparation and characterisation of ribonuclease from human hypertrophic prostate gland (RNAase P2).

An endo-type, cyclising, 3'-phosphate-forming rebonuclease was purified to homogeneity from a water/Tween 80 extract of human hypertrophic prostate gland. The enzyme is acid- and heat- resistant and is optimally active at pH 7.0, 0.1 M NaCl. Molecular weight determined by gel filtration on Sephadex G-75 and sucrose density gradient centrifugation gave a mean value of 15 000. The prostatic ribonuclease is inhibited by Cu2+, bromoacetate and photooxidation in the presence of methylene blue. Other divalent ions, EDTA and p-chloromercuribenzoate have no influence on the enzymic activity. Prostatic RNase resembles RNase A in that it preferentially cleaves linkages in RNA after pyrimidine nucleotides to produce oligonucleotides terminated in cyclic 2',3' phosphate. The enzyme is inactive with poly(A) - poly(U) as substrate. Poly(U) is hydrolyzed four times as fast as poly(C), and 1.2 times as fast as RNA.     

Human alpha-N-acetylglucosaminidase. 1. Purification and properties.

alpha-N-Acetylglucosaminidase was purified from human urine to a state of apparent homogeneity. alpha-N-Acetylglucosaminidase is a glycoprrotein with an extensive charge heterogeneity. The molecular weight determined by gel filtration is 307000. Polycarylamide gel electrophoresis in the absence and presence of sodium dodecyl sulfate indicates molecular weight heterogeneity of isocharged forms of the purified enzyme. The enzyme has a pH optimum of 4.5 +/- 0.3 and KM and V values of 0.14-0.74 mM, and 1.04-3.68 mumol mg-1 min-1 for three aryl 2-acetamido-2-deoxy-alpha-D-glucosides and UDP-N-acetylglucosamine. Heparan sulfate, heparin and dermatan sulfate are competitive inhibitors. The enzyme is inhibited by Hg2+ and Cu2+. --SH-protective reagents and thiol reagents have no effect on the enzyme activity. Heating at 65 degrees C and pH values below 5 inactivate the enzyme rapidly.     

Purification from normal human plasma and biochemical characterization of a ribonuclease specific for poly(C) and poly(U)

A new specific ribonuclease from normal human plasma has been purified to homogeneity, following a five-step purification protocol that included DEAE-Sepharose, CM-Sepharose, and Heparin-Sepharose chromatographies. The purified enzyme was found to be glycosylated and appeared as a single 25-kDa band on a SDS polyacrylamide gel. This RNase is poly(C) preferential, degrading poly(U) at a lower rate. Activity of this RNase toward cleavage of native substrates such as ribosomal RNA was also detected. The human plasma ribonuclease is a thermolabile molecule, exhibiting maximum activity at pH 6.5. Comparison between other known plasma RNases and the human plasma ribonuclease described here indicated a variety of differences in their biochemical and catalytic properties.     

Studies on extracellular ribonucleases of Ustilago sphaerogena. Purification and properties

1. Four ribonucleases were isolated from culture media of Ustilago sphaerogena. They were designated ribonucleases U(1), U(2), U(3) and U(4). 2. They were purified about 1600-, 3700-, 1100- and 16-fold respectively. 3. It was shown by gel filtration that ribonucleases U(1), U(2) and U(3) have molecular weights about 10000 like ribonuclease T(1), and that ribonuclease U(4) is much larger. 4. Ribonucleases U(1), U(2) and U(3) are thermostable, but ribonuclease U(4) is not. 5. The pH optimum of ribonucleases U(1) and U(4) is pH8.0-8.5, and that of ribonucleases U(2) and U(3) is pH4.5.     

Cytotoxicity of polyspermine-ribonuclease A and polyspermine-dimeric ribonuclease A

Polyspermine-ribonuclease A (PS-RNase A) and polyspermine-dimeric ribonuclease A (PS-dimeric RNase A) were prepared by cross-linking ribonuclease A or its covalently linked dimer to polyspermine (PS) using dimethyl suberimidate. The two RNase A derivatives were tested for a possible antitumor action. The in vitro and in vivo cytotoxic activity of PS-RNase A, although strong, is not higher than that known for free polyspermine. PS-dimeric RNase A, which was characterized by mass spectroscopy, titration of free amine groups, and enzymatic assays, proved instead to be a definitely more efficient antitumor agent, both in vitro and in vivo. This result could tentatively be explained in view of the importance of positive charges for ribonuclease activity, considering the higher basicity of PS-dimeric RNase A compared to that of PS-(monomeric)RNase A. It must be also taken into account that the dimeric RNase A moiety of PS-dimeric RNase A could evade the cytoplasmic ribonuclease inhibitor, which instead could trap the monomeric RNase A moiety of the other derivative. The two RNase A derivatives degrade poly(A).poly(U) under conditions where native RNase A is inactive. The results of this work demonstrate once again the importance of positive charges for the functions of mammalian pancreatic type ribonucleases in general, in particular for RNase A derivatives, and the potential therapeutic use of the ribonuclease A derivatives.     

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.     

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.