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.     

Formation of Hydrogen Peroxide by a Polyvinyl Alcohol Degrading Enzyme

A method for purification and crystallization, and some properties of ribonuclease from Aspergillus sp. [EC 2.7.7.17] (RNase L) are reported. The purification procedure consisted of six steps, including acetone precipitation, column chromatographies on Duolite A–2 and DEAE-cellulose, repeated chromatography on DEAE-Sephadex A–50 column and affinity chromatography on 5’-AMP-Sepharose 4B column. Crystallization was performed by the dialysis against ammonium sulfate solution at 60% saturation.

The crystalline enzyme was shown to be homogeneous by polyacrylamide disc electrophoresis and ultracentrifugation. Svedberg value of the crystalline enzyme was 4.2. The enzyme was the most active at pH 3.5 and 60~65°C, and it was inhibited markedly with Fe.³⁺

RNase L has no absolute base specificity, and produces four kinds of 3’-mononucleotides from yeast RNA. However, the susceptibility of four nucleotide residues to RNase L increases in the order; G < A < C < U and is quite different from those of RNase T₂, RNase M and RNase R.     

Toxic Metabolites of an Unidentified Filamentous Fungus Isolated from Zinnia Leaves

Two ribonucleases (RNases) designated RNase I and RNase II were found in Euphausia superba and isolated by (NH₄)₂SO₄ fractionation, 2 cycles of CM-cellulose chromatography and gel filtration on Sephadex G-100. This procedure resulted in a 2,116-fold purification of RNase I and a 130-fold purification of RNase II. The molecular weight of both purified enzymes was estimated by gel filtration to be 31,500. The isoelectric points were 6.0 (RNase I) and 7.0 (RNase II). Each enzyme hydrolyzed poly A-U, poly U but did not degrade poly G, poly C and DNA. Both enzymes were classified as endonuclease from the hydrolysis product of yeast RNA and poly A. The enzymes were located mainly in the cardiac and pyloric portion of the stomach.     

Isolation and partial characterization of catalytic antibodies with oligonuclease activity from bovine colostrum

Catalytic antibodies (abzymes) which hydrolyze RNA and DNA were isolated from bovine colostrum by sequential chromatography on Protein A Sepharose, denaturated DNA-cellulose, Mono Q, and gel permeation chromatography on Superose 12 at pH 2.3 after acidic shock. Metachromatic agar containing toluidine blue and yeast RNA was used to measure RNase activity. Electrophoresis in agarose showed DNase activity on plasmid DNA from Escherichia coli and DNA from calf thymus in fractions from all 4 purification steps. Gel permeation chromatography showed that the abzymes hydrolysed both a single-stranded polyadenylic acid (Poly A) and single-stranded polycitidylic acid (Poly C), while partially purified RNase from the colostrum hydrolysed Poly (C), but not Poly (A). Electrophoresis of purified abzymes under denaturing conditions showed protein bands of molecular mass corresponding to heavy and light chains of IgG. The abzymes immunoreacted with anti-bovine IgG. The RNase activity of the purified abzymes represented 0.022% of total RNase activity in the colostrum; acid shock and gel filtration at low pH reduced the specific RNase activity of abzymes 3.6-fold. The RNase activity of abzymes at pH 6.6 was reduced by 90% by heat treatment at 75 degrees C for 52 min.     

Purification and properties of beta-galactosidase from fungus, Curvularia inaequalis]

Highly purfied beta-galactosidase from fungus Curvularia inaequalis cultural fluid with a specific activity of 50 units per mg of protein was obtained by 2-fold purification of the enzyme, using chromatography on DEAE-cellulose and on hydroxylapatite. The enzyme was found to hydrolyze o-nitrophenyl-beta-D-galactopyranoside (pH optimum of 3.7--4.5) and lactose (pH optimum 3.9--5.3). The isoelectric point was observed at pH 4.4 the temperature optimum was 60 degrees C. The molecular weight (115 000--126 000) and the amino acid composition of the enzyme were determined. Km values for o-nitrophenyl-beta-D-galactopyranoside and lactose were 0.55-10(-3) M and 4.5-10(-3) M respectively. Disc-electrophoresis in polyacrylamide gel revealed a single band with a specific activity. The homogeneity of the enzyme was found in ultracentrifuge.     

Tertiary structure of RNase Pch1 predicted from the model structure of RNase Ms and the crystal structure of RNase T1

In this paper we predict the structure of RNase Pch1 as modelled from the previously predicted structure of RNase Ms and the crystal structure of RNase T1 in the complex with 2GMP. The predicted structures and their initial energy minimized structural RNase T1 template are compared. The predicted structures of RNase Pch1 show, independent of their prediction form RNase Ms or T1, a higher structural similarity to RNase T1 than to RNase Ms, in agreement with higher sequence similarity and specificity — RNaes T1 and Pchl are specific for guanine whereas RNase Ms is base-unspecific with preference for guanine. Offprint requests to: W Saenger     

Purification and some properties of an extracellular ribonuclease with antiviral activity against tobacco mosaic virus from <Emphasis Type="Italic">Bacillus cereus</Emphasis>

A new ribonuclease (RNase) with tobacco mosaic virus inhibition was isolated and purified from Bacillus cereus ZH14 through ammonium sulfate precipitation, ultrafiltration, ion-exchange chromatography of DEAE-Sephadex A-50 column, and gel chromatography of Sephacryl S-200HR column. The enzyme was purified approximately 134-fold with a recovery of 9.2%. The RNase had an MW of 75.6 kDa in SDS-PAGE, which differed from RNases reported previously. The inhibitory activity of the RNase in the purification process against tobacco mosaic virus was tested, and the percentage inhibition of the purified RNase (48 U/ml) reached 90%. The protein could tolerate 90°C and pH 4.0.     

Extracellular RNAase Pch2 of Penicillium chrysogenum 152A: purification, specificity and physico-chemical properties

Acid RNAase Pch2 was isolated from a filtrate of the cultural fluid of the fungus Penicillium chrysogenum 152A and purified to homogeneity. An analysis of RNAase Pch2 action on RNA and synthetic substrates showed that the enzyme can be attributed to non-specific true ribonucleases (ribonucleate-3'-oligo-nucleotide hydrolase, EC 3.1.4.23). The maximal effect of the enzyme on RNA is observe at pH 4.5 and 55 degree. The RNAase Pch2 is not activated by bivalent metal ions, p-chloromercurybenzoate or beta-mercaptoethanol and is reversibly inactivated by 8 M urea. The enzyme molecule consists of 332 amino acid residues; its molecular weight is 36160, the isoelectric point lies at 5.2.     

Extracellular lipase of Pseudomonas sp. strain ATCC 21808: purification, characterization, crystallization, and preliminary X-ray diffraction data.

A procedure for the purification of a very hydrophobic lipase from Pseudomonas sp. strain ATCC 21808 was elaborated by avoiding the use of long-chain detergents in view of subsequent crystallization of the enzyme. The purification procedure included chromatography on Q-Sepharose in the presence of n-octyl-beta-D-glucopyranoside, Ca2+ precipitation of fatty acids, and Octyl-Sepharose chromatography. The enzyme was purified 260-fold to a yield of 35% and a specific activity of 3,300 U/mg. The molecular weight was determined as 35,000; a polyacrylamide gel under nondenaturing conditions revealed a band at 110,000, and the isoelectric point proved to be at 4.5 to 4.6. The lipase crystallized with different salts and ethylene glycol polymers in the presence of n-octyl-beta-D-glucopyranoside and one alkyloligooxyethylene compound (CxEy) in the range from C5E2 to C8E4. The crystals diffract to a resolution of about 0.25 nm. Precession photographs revealed that they belong to space group C2 with lattice constants of a = 9.27 nm, b = 4.74 nm, c = 8.65 nm, and beta = 122.3 degrees, indicating a cell content of one molecule per asymmetric unit of the crystal. In hydrolysis of triglycerides, the lipase showed substrate specificity for saturated fatty acids from C6 to C12 and unsaturated long-chain fatty acids. Monoglycerides were hydrolyzed very slowly. The N-terminal sequence is identical to that of the lipase from Pseudomonas cepacia. Treatment with diethyl-p-nitrophenylphosphate affected the activities toward triolein and p-nitrophenylacetate to the same extent and with the same velocity.     

A 3'-deoxymononucleotide-producing nuclease from the marine sponge Verongia aerophoba. I. Purification.

A four-step procedure for purification of a nuclease from the keratinous sponge Verongia aerophoba is described. The extracted material is lyophilized, acidified, and subjected to chromatography on Sephadex, hydroxyapatite, and phosphocellulose. The nuclease is purified about 1,000-fold from the crude extract and approximately 1,600-fold from concomitant acid RNase. Phosphodiesterase is lost after chromatography on Sephadex. The purified enzyme solution contains one single activity as determined by polyacrylamide gel electrophoresis.     

A rapid and simple method for the purification of rat liver RNase inhibitor

A rapid and simple method for the purification of rat liver alkaline RNase inhibitor from a 105,000 g supernatant is reported. It involves protein precipitations by (NH₄)₂SO₄ and chromatography on carboxymethyl cellulose-RNase column. The purification procedure gives a 1020-fold increase in specific activity with a yield of 32%. This purified inhibitor can be stored for 5 weeks without any loss in activity.     

Novel extracellular ribonuclease from Bacillus intermedius--binase II: purification and some properties of the enzyme

The recombinant enzyme binase II was isolated from the culture liquid of Bacillus subtilis 3922 transformed with the pJF28 plasmid bearing the birB gene. The procedure of the enzyme purification included precipitation by polyethylene glycol with subsequent chromatography on DEAE-cellulose, heparin-Sepharose, and Toyopearl TSK-gel. The enzyme was purified 142-fold yielding a preparation with specific activity 1633 U/mg. The molecular weight of binase II is 30 kD. The enzyme is activated by Mg²⁺ and virtually completely inhibited by EDTA. The pH optimum for the reaction of RNA hydrolysis is 8.5. The properties of the enzyme are close to those of RNase Bsn from B. subtilis. The character of cleaving of synthetic single- and double-stranded polyribonucleotides by binase II suggests that the enzyme binds the substrate in the helix conformation, and its catalytic mechanism is close to that of RNase VI from cobra venom.     

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 novel method for isolation and concentration of ribonuclease T1 from Taka-Diastase

A novel method for isolation and concentration of RNase T1 from Taka-Diastase is developed. It is a combination method of bentonite adsorption with dialysis desorption. In the present method, RNase T1 can be concentrated about ten-fold, the recovery of total activity was greater than 95%, and specific activity was raised 8-10 folds. Further purification with ammonium sulfate precipitation and chromatography on DEAE-cellulose and DEAE-Sephadex yields a RNase T1 which contains no pMase. pDase nor RNase T2 activities and a 750 fold increase in specific activity. Our method is more simple, rapid, and efficient than previous methods.     

Purification and physicochemical characterization of murine T cell replacing factor (TRF)

Murine T cell replacing factor (TRF) was purified from a cellfree supernatant of a T cell hybridoma (B151K12) that constitutively produces TRF. Two assay systems for TRF activity were employed: 1) induction of anti-DNP IgG PFC responses in cultures of splenic B cells from DNP-KLH-primed BALB/c mice, and 2) induction of IgM PFC in chronic B cell leukemic cells (BCL1). The purification scheme consisted of ammonium sulfate precipitation, DEAE-cellulose chromatography, Blue-Sepharose chromatography, hydroxylapatite chromatography, gel permeation with fast protein liquid chromatography (FPLC), and disc polyacrylamide gel electrophoresis. Overall, TRF was purified approximately 34,000-fold with a maximum 3.8% recovery of activity, and the specific activity of the purified TRF was approximately 9.6 X 10(4) U/mg. The TRF that is active in these systems is distinct from the other lymphokines such as IL 1, IL 2, BCGFI (now known as BSFp1), and gamma-interferon. The TRF is extremely hydrophobic, with an apparent m.w. of 50,000 to 60,000 on gel permeation chromatography and 18,000 on SDS-PAGE under reducing conditions. Highly purified B151-TRF abrogated the activity by treatment with trypsin but not with RNase. Moreover, it bound to lima bean agglutinin-Sepharose specific for N-acetylgalactosamine residues, indicating that B151-TRF is a glycosylated glycoprotein containing N-acetylgalactosamine residues. The role of N-acetylgalactosamine residues on TRF activity was additionally substantiated by the fact that the addition of appropriate amounts of N-acetylgalactosamine in the assay systems for TRF preferentially induced a profound suppression for TRF-mediated PFC responses.     

Crystallization and properties of acylphosphatase from porcine skeletal muscle

A crystalline acylphosphatase has been obtained from porcine skeletal muscle. The purification procedure consists of extraction with water, phosphocellulose column chromatography, CM-cellulose column chromatography, and crystallization. The enzyme was homogeneous by polyacrylamide gel electrophoresis. A high yield (39%) of the pure enzyme was attained by the use of buffers containing 10 mM 2-mercaptoethanol to prevent dimerization of the enzyme in the purification process. Activity assay in the presence of bovine serum albumin showed a high specific activity of the enzyme (about 7,000 mumol/min/mg at 25 degrees C with benzoyl phosphate as substrate). The molecular weight was determined to be 11,100 by sedimentation equilibrium. The amino acid composition of the enzyme was determined. The amino-terminus of the enzyme was blocked and the carboxyl-terminal residue was tyrosine.     

A rapid method for the isolation of coagulation factor XII from human plasma

Zinc chelate affinity chromatography was used to develop a rapid, three-step procedure to isolate coagulation factor XII from human plasma. The first step was ammonium sulphate fractionction which gave a 2-fold purification and 90% recovery in the 25–50% saturation fraction. The second step was zinc chelate affinity chromatography which gave a 240-fold purification and 67.5% recovery. The third step was zinc chelate affinity chromatography again, but with the application of a pH gradient. The overall recovery of zymogen factor XII was 21.7% and the total purification was 1992-fold. The purified factor XII had an apparent molecular weight of 77 600 as determined by SDS-polyacrylamide gel electrophoresis and a specific activity of 50 units/mg on a clotting assay.     

Purification, stabilization, and characterization of rat hepatic triglyceride lipase

Hepatic triglyceride lipase (H-TGL) was purified to near homogeneity from heparin-containing rat liver perfusates with the following column chromatography steps: heparin-Sepharose affinity chromatography, anion-exchange chromatography on DEAE-Sephacel, and gel filtration on Ultrogel AcA 34. A final specific activity of 45,000 μmol fatty acid/mg/h was obtained with an overall 31% recovery of catalytic activity. The heparin-Sepharose step resulted in a 20-fold purification, while the DEAE and gel filtration steps led to further purification with complete recovery of activity. An extensive survey of various detergents as potential stabilizers of H-TGL activity led to the selection of Triton N-101 for use in the column buffers of the DEAE and gel filtration steps. Relative to initial H-TGL activity upon dilution in buffer without detergent, recoveries between 90 and 100% were consistently obtained with Triton N-101-containing buffers following a 24-h incubation at 20°C. In contrast after a 24-h incubation at 20°C those control samples lacking detergent were at least 95% inactivated. The highly purified H-TGL exhibited a single major band by sodium dodecyl sulfate-electrophoresis. The use of DEAE chromatography and stabilization of H-TGL with Triton N-101 are the improvements in purification that resulted in an 8-fold enhancement in specific activity relative to the highest previous report of purification from rat liver perfusates.     

A rapid and efficient new method of purification of glutamate dehydrogenase by affinity chromatography on GTP-sepharose

The very high affinity for GTP of glutamate dehydrogenase was used to purify this enzyme by affinity chromatography. After periodic acid oxidation, GTP was covalently bound to an activated Sepharose. When crude mitochondrial extracts were applied on a column of this GTP-Sepharose, glutamate dehydrogenase was retained with very few other proteins. Glutamate dehydrogenase from rat liver was eluted with a KCl gradient with only one contaminating protein. From a pig heart mitochondrial extract the enzyme was purified 300-fold in one step. A chromatography on hydroxyapatite was sufficient to achieve the purification. This very simple technique avoids the long and troublesome crystallization steps generally involved in glutamate dehydrogenase purification.     

Purification of pharmaceutical-grade plasmid DNA by anion-exchange chromatography in an RNase-free process

Anion-exchange is the most popular chromatography technique in plasmid DNA purification. However, poor resolution of plasmid DNA from RNA often results in the addition of bovine-derived ribonuclease (RNase) A to degrade RNA impurities which raises regulatory concerns for the production of pharmaceutical-grade plasmid DNA. Low capacity for plasmid of most commercial media is another issue affecting the suitability of anion-exchange chromatography for large-scale processing. This study reports the use of anion-exchange chromatography to remove RNA in an RNase-free plasmid purification process. Resolution was achieved through careful selection of adsorbent and operating conditions as well as RNA reduction steps before chromatography. Dynamic capacity for plasmid was significantly increased (to 3.0mg/ml) so that it is now possible to envisage the large-scale manufacturing of therapeutic-grade plasmid DNA in the absence of added RNase using anion-exchange chromatography as a polishing step.