Partial purification and characterization of a double-stranded RNA-specific nuclease from human placenta

A double-stranded RNA-specific nuclease (ds RNase) has been isolated and partially purified from human placenta by DEAE-cellulose and DNA-cellulose column chromatography. Denatured DNA-cellulose retained most of the single-stranded RNA-specific nuclease (ss RNase) activity, whereas the ds RNase came out in the void volume. N-ethylmaleimide at a concentration of 5 mM, selectively inhibited ds RNase activity by 60% under the conditions in which the ss RNase activity was inhibited to an extent of 7%. The ds RNase was specifically inhibited by Penicillium chrysogenum viral ds RNA and by ethidium bromide. The partially purified ds RNase showed requirements for Mg+ whereas Mn2+ and NH4+ ions were inhibitory. The DEAE-enzyme cleaved 32P-labelled 45S ribosomal precursor RNAs from Yoshida ascites sarcoma cells into species that had similar electrophoretic mobilities as the mature rRNAs.     

Purification and characterization of an extracellular, uracil specific ribonuclease from a Bizionia species isolated from the marine environment of the Sundarbans

The first ribonuclease (RNase) from the Cytophaga-Flavobacterium-Bacteroides phylum, dominant in the marine environment, and also from the first Bizionia species isolated from the tropics was purified and characterized. Extracellular RNase production occurred when the culture medium contained 5-7% (w/v) NaCl. The 53.0 kDa enzyme was purified 29 folds with a recovery of 4% and specific activity of 630unit/mg protein. The pH and temperature optima are 6.5 and 35 degrees C, respectively and the enzyme retains more than half of its activity (relative to optimal assay conditions) after 1h pre-incubation separately with 5% (w/v) NaCl or from pH 5.0 to 8.5 or at 50 degrees C. Dithiothreitol and beta-mercaptoethanol do not inhibit whereas human placental RNase inhibitor protein halves the RNase activity. While Mg(2+), Ba(2+) and Ca(2+) enhanced the enzyme activity, Fe(2+), Cu(2+) and Hg(2+) inactivated it. This RNase degrades uracil containing nucleic acids only. Our isolate could be a novel renewable source of deoxyribonuclease (DNase)--free RNase enzyme.     

Purification, properties, and partial amino acid sequence of chitinase from a marine Alteromonas sp. strain O-7.

Chitinase (EC 3.2.1.14) was isolated from the culture supernatant of a marine bacterium, Alteromonas sp. strain O-7. The enzyme (Chi-A) was purified by anion-exchange chromatography (DEAE-Toyopearl 650 M) and gel filtration (Sephadex G-100). The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of Chi-A were 70 kDa and 3.9, respectively. The optimum pH and temperature of Chi-A were 8.0 and 50 degrees C, respectively. Chi-A was stable in the range of pH 5-10 up to 40 degrees C. Among the main cations, such as Na+, K+, Mg2+, and Ca2+, contained in seawater, Mg2+ stimulated Chi-A activity. N-Bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide inhibited Chi-A activity. The amino-terminal 27 amino acid residues of Chi-A were sequenced. This enzyme showed sequence homology with chitinases from terrestrial bacteria such as Serratia marcescens QMB1466 and Bacillus circulans WL-12.     

Isolation of Ca2+, Mg2+-dependent nuclease from calf thymus chromatin

Ca2+,Mg2+-dependent nuclease was isolated from calf thymus chromatin by stepwise chromatography on DEAE-Sepharose, CM-Sephadex and DNA-Sepharose. The enzyme was purified more than 700-fold. SDS-PAGE electrophoresis revealed one protein band possessing an enzymatic activity. The molecular mass of the nuclease as determined by gel filtration is 25700 Da, that determined by 12% SDS polyacrylamide gel electrophoresis is 28,000 Da. In the presence of various ions the enzyme activity decreases in the following order: (Ca2+ + Mn2+) greater than (Ca2+ + Mg2+) greater than Mn2+; the pH optimum is at 8.0. In media with Mg2+, Ca2+, Co2+ and Zn2+ the nuclease is inactive. Some other properties of the enzyme are described.     

A novel nuclease from mitochondria of rat liver

Alkaline RNase partially purified from rat liver mitochondria hydrolyzes both RNA and denatured DNA. The behaviors of RNase activity of the nuclease are closely similar to those of the DNase activity. The nuclease has a pH optimum between 9.0 and 9.5, and the activity is absolutely dependent on Mg²⁺ and reversibly inhibited by $\text{p}$-hydroxymercuribenzoate.     

酿脓链球菌Cas9核酸酶的重组表达、分离纯化及酶学特性

【背景】Cas9核酸酶是一种RNA引导的核酸内切酶,可与单链向导RNA (single-guide RNA,sgRNA)形成稳定的核糖核蛋白复合物,识别和切割特定的核苷酸片段。由于其具备高灵活性和高效率的特点,目前已经成为基础科学研究领域和临床治疗方法中使用最广泛的基因编辑工具。【目的】为Cas9核酸酶的合理开发和利用提供理论依据。【方法】利用大肠杆菌表达系统表达野生型酿脓链球菌(Streptococcus pyogenes) Cas9核酸酶,经硫酸铵沉淀和镍柱亲和层析两步纯化获得较高纯度表达产物,并对其热稳定性、pH稳定性、金属离子的影响等酶学特性进行研究。【结果】经高密度发酵后,大肠杆菌湿菌重达191.0 g/L。纯化后酿脓链球菌Cas9核酸酶的比酶活达641.29 U/mg,纯化倍数为16.02,收率为46.40%。Cas9核酸酶在25-42°C保温2 h后剩余酶活保持在65%以上,而在45°C保温15 min后全部失活;其在pH 6.0-10.0范围内稳定性较高,剩余酶活大于68%,在pH9.0时稳定性最高;0.5-20.0mmol/L浓度范围内的Mg²⁺...     

Studies on chromatin-associated nuclease from barley leaves

A nuclease associated with the chromatin of barley leaves hasbeen solubilized and purified 20 fold. The purified preparationhydrolyzes native or denatured DNA and RNA, but exhibits nophosphodiesterase or phosphomonoesterase activity. The ratiosof RNase and DNase activities remain essentially constant throughoutall the steps of purification. The two enzyme activities hadpH optimum of 7.0 and showed similar effects of phosphate, Zn++,Mg++ and other metal cations, EDTA, inhibitors, freezing andthawing, heat treatment and precipitation by protamine sulfateand streptomycin sulfate. RNA and DNA were degraded by the enzymein endonucleolytic fashion. (Received January 30, 1971; )     

Calpain II-like proteinase of scallop (Patinopecten yessoensis) striated adductor muscle.

1. A Ca(2+)-dependent cysteine proteinase was purified from scallop striated adductor muscle by ammonium sulfate fractionation and column chromatography on DEAE-cellulose and Sephacryl S-300. 2. The enzyme is of Mr approximately 200,000, composed of two Mr 100,000 subunits. 3. The enzyme is a cysteine proteinase with optimum activity at pH 6.8 and about 18 degrees C. In addition, it requires 1.7 mM Ca2+ for half-maximal activity and more than 10 mM Ca2+ for maximal activity. Thus the enzyme can be classified as calpain II.     

酿酒酵母胞内无机焦磷酸酶的分离纯化及性质

An inorganic pyrophosphatase (EC3.6.1.1) from Saccharomyces cerevisiae was purified to PAGE homogeneity by sonication disruption. (NH4)2SO4 fractionation and DEAE-cellulose colunm chromatography. The optimum pH and temperature of the enzyme were 7.4～7. 8 and 60℃, respectively. The Km was 19.3 mmol / L. The enzyme required Mg2+ as a cofactor for hydrolysis of pyrophosphate and was inhibited by Ca2+, Hg2+, Pb2+, Mn2+.     

Purification and characterization of a mammalian endo-exonuclease.

An endo-exonuclease has been purified from cultured monkey (CV-1) cells. The enzyme which was purified to near homogeneity to be a 65 kDa monomeric protein. The single-strand DNase activity is endonucleolytic and nonprocessive, whereas the double-strand DNase activity is exonucleolytic and processive. The enzyme was also found to have RNase activity using poly-rA as substrate. The pH optimum for ss-DNase is 8 and for ds-DNase it is 7.5. Both DNase activities require a divalent metal ion (Mg2+, Mn2+, Ca2+, Zn2+) for activity and exhibit the same kinetics of heat inactivation. The purified protein binds to and cleaves a synthetic Holliday junction substrate. The overall enzymatic characteristics of the mammalian protein are very similar to the putative recombination endo-exonucleases purified from Neurospora crassa, Aspergillus nidulans and Saccharomyces cerevisiae.     

Purification and thermal characterization of a novel peroxidase from a local chick pea cultivar

A novel peroxidase isolated from a local chick pea (Cicer arietinum L.) cultivar (Balksar 2000) was purified by means of ammonium sulfate precipitation, DEAE-cellulose chromatography and two runs on gel filtration. The purified enzyme has a specific activity of 2045 U/mg with 17 % activity recovery. The molecular mass of the enzyme was estimated to be 39 kDa by SDS-polyacrylamide gel electrophoresis. Optimum pH and temperature of the enzyme were 5.5 and 45 degrees C respectively. The thermal denaturation of local chick pea peroxidase was studied in aqueous solution at temperatures ranging from 45 degrees C to 65 degrees C. The temperature of 50% inactivation of the enzyme was found to be 68 degrees C. The enthalpy (DeltaH*) and free energy (DeltaG*) of thermal denaturation of chick pea peroxidase were 101.4 and 103.4 k J/mol respectively at 65 degrees C.Metals like Zn2+, Mn2+, Hg2+, Co2+ and Al3+ slightly inhibited the peroxidase activity while Ca2+, Mg2+ and Ba2+ have no effect on enzyme activity. The high specific activity and thermal stability make chick pea peroxidase an alternative to horseradish peroxidase (HRP) in various applications.     

Purification and properties of the major nuclease from mitochondria of Saccharomyces cerevisiae

The vast majority of nuclease activity in yeast mitochondria is due to a single polypeptide with an apparent molecular weight of 38,000. The enzyme is located in the mitochondrial inner membrane and requires non-ionic detergents for solubilization and activity. A combination of heparin-agarose and Cibacron blue-agarose chromatography was employed to purify the nuclease to approximately 90% homogeneity. The purified enzyme shows multiple activities: 1) RNase activity on single-stranded, but not double-stranded RNA, 2) endonuclease activity on single- and double-stranded DNA, and 3) a 5'-exonuclease activity on double-stranded DNA. Digestion products with DNA contain 5'-phosphorylated termini. Antibody raised against an analogous enzyme purified from Neurospora crassa (Chow, T. Y. K., and Fraser, M. (1983) J. Biol. Chem. 258, 12010-12018) inhibits and immunoprecipitates the yeast enzyme. This antibody inhibits 90-95% of all nuclease activity present in solubilized mitochondria, indicating that the purified nuclease accounts for the bulk of mitochondrial nucleolytic activity. Analysis of a mutant strain in which the gene for the nuclease has been disrupted supports this conclusion and shows that all detectable DNase activity and most nonspecific RNase activity in the mitochondria is due to this single enzyme.     

从海洋中分离的弧菌QY102褐藻胶裂解酶的纯化和性质研究

从马尾藻(Sargassum)表面分离到一株产生高效胞外褐藻胶裂解酶的海洋弧菌（Vibrio sp.） QY102。以褐藻胶为唯一碳源发酵培养后,发酵液上清通过0.22μm滤膜过滤、DEAESepharose离子交换和Superdex75凝胶过滤得到电泳纯的褐藻胶裂解酶。酶的性质研究表明：其分子量约为28.5kD（SDSPAGE）,反应最适温度为40℃,最适pH为7.1,Ca2+、Mg2+对酶活有促进作用,而Ni2+、Al3+、Zn2+、Ba2+对酶活有抑制作用。该酶的活性明显高于已报道的褐藻胶裂解酶,pH稳定范围广（5～10）,并且对聚甘露糖醛酸的活性高于对聚古罗糖醛酸的活性。     

Identification and purification of a nuclease from Zinnia elegans L.: a potential molecular marker for xylogenesis

A single-strand specific nuclease was identified during a particular stage of a defined cellular differentiation pathway characteristic of xylem development. Using a hormone-inducible system in which cultured mesophyll cells of Zinnia elegans differentiated to xylem cells in synchrony, the enzymatic activity on single-stranded (ss) DNA was highest during the maturation phase of differentiation. Nondifferentiating cells contained little of this activity throughout a similar course of culture. After electrophoresis of extracts from differentiating cells, a 43-kilodalton (kDa) polypeptide was detected by its activity in the gels containing either ssDNA or RNA. Lectins specific for mannose residues on glycoproteins bound to the 43-kDa nuclease and were used to distinguish it from several ribonucleases. The nuclease was purified by a two-step chromatographic procedure: a lectin-affinity column followed by a phosphocellulose column. The purified protein was determined to be a single polypeptide with a relative molecular mass of 43000 by the analysis of its mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration of the native enzyme. A sensitive detection system using biotinylated-concanavalin A and avidin was demonstrated to be specific as a probe for the nuclease protein. An N-terminal amino-acid sequence was derived from 5 pmol of the enzyme. The nuclease was most active on ssDNA at pH 5.5 in the presence of Zn²⁺ and dithiothreitol. The purified preparation hydrolyzed RNA and to a lesser extent, native DNA. It digested closed circular duplex DNA by introducing a single endonucleolytic cleavage followed by random hydrolysis. During the induced pathway of synchronous differentiation in Zinnia the 43-kDa nuclease rapidly increased just prior to the onset of visibly differentiated features, and developed to a maximum level during xylem cell maturation. At this time a similar but slightly smaller nuclease appeared and became dominant as differentiation continued, and subsequently both enzymes decayed. After autolysis, a nuclease of about 37 kDa was found together with the 43-kDa enzyme in the culture medium. Complementing these analyses was the examination of the tissue distribution of the 43-kDa enzyme in Zinnia and other dicotyledonous plants, which also indicated an invivo role of the nuclease in autolysis, the terminal stage of vascular differentiation in plants. The Zinnia nuclease is therefore a potential marker for xylogenesis.Abbreviations Con A Canavalia ensiformis (concanavalin) agglutinin - DNase deoxyribonuclease - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - kDa kilodalton - M_r relative molecular mass - RNase ribonuclease - ss single-stranded - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Extracellular nuclease from Rhizopus stolonifer: purification and characteristics of - single strand preferential - deoxyribonuclease activity

An extracellular nuclease from Rhizopus stolonifer (designated as nuclease Rsn) was purified to homogeneity by chromatography on DEAE-cellulose followed by Blue Sepharose. The M(r) of the purified enzyme determined by native PAGE was 67? omitted?000 and it is a tetramer and each protomer consists of two unidentical subunits of M(r) 21? omitted?000 and 13? omitted?000. It is an acidic protein with a pI of 4.2 and is not a glycoprotein. The purified enzyme showed an obligate requirement of divalent cations like Mg(2+), Mn(2+) and Co(2+) for its activity but is not a metalloprotein. The optimum pH of the enzyme was 7.0 and was not influenced by the type of metal ion used. Although, the optimum temperature of the enzyme for single stranded (ss) DNA hydrolysis in presence of all three metal ions and for double stranded (ds) DNA hydrolysis in presence of Mg(2+) was 40 degrees C, it showed higher optimum temperature (45 degrees C) for dsDNA hydrolysis in presence of Mn(2+) and Co(2+). Nuclease Rsn was inhibited by divalent cations like Zn(2+), Cu(2+) and Hg(2+), inorganic phosphate and pyrophosphate, low concentrations of SDS, guanidine hydrochloride and urea, organic solvents like dimethyl sulphoxide, dimethyl formamide and formamide but not by 3'- or 5'-mononucleotides. The studies on mode and mechanism of action showed that nuclease Rsn is an endonuclease and cleaves dsDNA through a single hit mechanism. The end products of both ssDNA and dsDNA hydrolysis were predominantly oligonucleotides ending in 3'-hydroxyl and 5'-phosphoryl termini. Moreover, the type of metal ion used did not influence the mode and mechanism of action of the enzyme.     

Partial purification and properties of thermostable intracellular amylases from a thermophilic Bacillus sp. AK-2

Intracellular thermostable amylases from a thermophilic Baccilus sp. AK-2 have been isolated and purified. The crude enzyme, having pH optimum at 6.5. and temperature optimum at 68 degrees C was purified by DEAE-cellulose column chromatography. Three separable enzyme fractions having starch hydrolyzing property were eluted by lowering the pH from 8.5 to 7.0. Electrophoretic mobility of these fractions showed a single band. Calcium ion up to a concentration of 20 mM had an activating effect on the three fractions. The optimum temperature for the three fractions (FI, FII and FIII) was 65 degrees C and the pH optimum for each was 6.0, 6.5 and 6.0, respectively. The -SH group in the amylase molecule was essential for enzyme activity. Except for Ca2+, Mg2+, Sr2+ and Mn2+ all other metal ions studied inhibited both alpha and beta-amylase activities. EDTA showed dose dependent non-competitive inhibition. Product formation studies proved FI and FIII to be of the alpha-amylase type and FII of the beta-amylase type. The Km for the substrate (starch) in the presence or absence of EDTA was 0.8 X 10(-3) and 1.13 X 10(-3) g/ml for alpha-amylase and beta-amylase, respectively.     

A novel oligoribonuclease of Escherichia coli. I. Isolation and properties.

A new ribonuclease has been isolated from Escherichia coli. The enzyme is present in the 100,000 times g supernatant fraction and has been purified over 200-fold. Studies of the enzyme reveal that: 1. The enzyme shows a marked preference for oligoribonucleotides; indeed, the reaction rate is inversely proportional to the chain length of the substrate. The enzyme does not attack polynucleotides even at high concentrations of enzyme and has no detectable DNase activity. 2. The enzyme is stimulated strongly by Mn2+, less strongly by Mg2+, and not at all by Ca2+ and monovalent cations. 3. The enzyme is purified free of RNase I, RNase II, RNase III, polynucleotide phosphorylase, and other known ribonucleases of E. coli. The enzyme displays identical properties when isolated from mutants of E. coli that are deficient in the above ribonucleases. 4. The enzyme has a marked thermostability, a point of further distinction from RNase II.     

Purification and characterization of a mitochondrial endonuclease from Drosophila melanogaster embryos.

A mitochondrial endonuclease from Drosophila melanogaster embryos was purified to near homogeneity by successive fractionation with DEAE-cellulose and heparin--avidgel-F, followed by FPLC chromatography on mono S, Superose 12 and a second mono S column. This enzyme digests double-stranded DNA more efficiently than heat-denatured DNA. The endonuclease activity has a molecular mass of 44 kDa, as determined under native conditions using a gel-filtration Superose 12 column. The prominent peptide detected by SDS/polyacrylamide gel electrophoresis likewise has a molecular mass of 44 kDa, suggesting a monomeric protein. The enzyme has an absolute requirement for divalent cations, preferring Mg2+ over Mn2+. No activity could be detected when these cations were replaced by Ca2+ or Zn2+. The pH optimum for this enzyme activity is 6.5-7.4 and its isoelectric point is 4.9. Both single-strand and double-strand breaks are introduced simultaneously into a supercoiled substrate in the presence of MgCl2 or MnCl2. Endonuclease-treated DNA serves as a substrate for DNA polymerase I from Escherichia coli, suggesting that 3'-OH termini are generated during cleavage. The enzyme is free from any detectable DNA exonuclease activity but not from RNase activity. Partial inhibition by antibodies raised against mitochondrial endonucleases derived from bovine heart and Saccharomyces cerevisiae have revealed a potential structural homology between these nucleases.     

Purification and some properties of a specific nuclease which cleaves transfer RNA precursors from the posterior silk gland of Bombyx mori.

A specific endonuclease involved in the processing of tRNA precursors was isolated and partially purified from the posterior silk gland of Bombyx mori, and designated as RNase P.Bmo. This enzyme was shown to catalyze the conversion of 4.5 S precursor RNA to 4.1 S RNA by trimming the 5'-additional segment from the precursor RNA. RNase P.Bmo required divalent cations, Mg2+ or Mn2+. In the presence of these divalent cations, K+ or NH4+ activated the RNase P.Bmo reaction. Optimum pH was observed around 8.0. Ribosomal RNA's and mature tRNA from the silk gland were not cleaved by RNase P.Bmo. A 4.5 S precursor RNA fraction containing formycin, an adenosine analog, was less susceptible to RNase P.Bmo than the normal one. These results indicate that RNase P.Bmo has a high substrate specificity. An additional nuclease(s) was isolated. This activity was assumed to remove the extra 3'-segment of the 4.5 S precursor RNA.     

In vitro replication of tobacco mosaic virus RNA in tobacco callus cultures: solubilization of membrane-bound replicase and partial purification.

A fraction containing membrane-bound tobacco mosaic virus RNA replicase was isolated form tobacco mosaic virus-infected tobacco callus cultures. The replicase activity reached a maximum 60 h after inoculation and then declined. The enzyme activity was insensitive to actinomycin D and DNase. The corresponding fraction from healthy callus contained essentially no activity. The viral RNA synthesis in vitro proceeded linearly for 30 min and required the four nucleotide triphosphates and Mg2+ ions. Mn2+ was a poor substitute for Mg2+. During RNA synthesis the product was at least 70% resistant to RNase in 2X SSC (0.15 M NaCl plus 0.015 M sodium citrate), but completely digested by RNase in 0.1X SSC. Analysis of the product by polns) that appeared to be replicative form and a partially RNase-resistant structure similar to replicative intermediate form. Washing the membrane-bound replicase with Mg2+-deficient buffer solubilized enzyme. The solubulized enzyme was further purified by DEAE-Sephadex column chromatography. The DEAE-purified enzyme was nearly completely dependent upon tobacco mosaic virus RNA for activity. Analysis of the product on a sucrose gradient revealed a double-stranded RNA with sedimentation of 16S and smaller heterogeneous RNase-sensitive products.