• The Korean Journal of Zoology
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• v.22 no.4
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• pp.141-152
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• 1979

Feline leukemia virus DNA polymerase was purified by ion-exchange and nucleic acid affinity chromatographies. The enzyme consists of a single polypeptide chain of approximately 72, 000 molecular weight as determined by both of a glycerol density gradient centrifugation and SDS-polyacrylamide gel electrophoresis. The preferred divalent cation for DNA synthesis is $Mn^2+$ on a variety of template-primers, and its optimum concentration appears to be significantly lower than reported results of other mammalian type-C viral enzymes. The divalent cation requirement for maximum activity of RNase H is similar to those of DNA polymerase. Both DNA polymerase and RNase H activities appear to reside on the same molecule as demonstrated by the copurification of both activities through various purification steps. An additional RNase H without detectible polymerase activity was generated by a limited chymotrypsin digestion. This RNase H activity was inhibited equally effectively as RNase H in the intact reverse transcriptase by antisera prepared against reverse transcriptase of feline leukemia virus. Neutralization and binding test showed that antibody binding to reverse transcriptase molecule did not completely inhibit the polymerase activity.

• Journal of Animal Science and Technology
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• v.59 no.9
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• pp.20.1-20.9
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• 2017

Background: Ribonuclease (RNase) is one of the few toxic proteins that are present constantly in snake venoms of all types. However, to date this RNase is still poorly studied in comparison not only with other toxic proteins of snake venom, but also with the enzymes of RNase group. The objective of this paper was to investigate some properties of RNase from venom of Vietnam cobra Naja atra. Methods: Kinetic methods and gel filtration chromatography were used to investigate RNase from venom of Vietnam cobra. Results: RNase from venom of Vietnam cobra Naja atra has some characteristic properties. This RNase is a thermostable enzyme and has high conformational stability. This is the only acidic enzyme of the RNase A superfamily exhibiting a high catalytic activity in the pH range of 1-4, with $pH_{opt}=2.58{\pm}0.35$. Its activity is considerably reduced with increasing ionic strength of reaction mixture. Venom proteins are separated by gel filtration into four peaks with ribonucleolytic activity, which is abnormally distributed among the isoforms: only a small part of the RNase activity is present in fractions of proteins with molecular weights of 12-15 kDa and more than 30 kDa, but most of the enzyme activity is detected in fractions of polypeptides, having molecular weights of less than 9 kDa, that is unexpected. Conclusions: RNase from the venom of Vietnam cobra is a unique member of RNase A superfamily according to its acidic optimum pH ($pH_{opt}=2.58{\pm}0.35$) and extremely low molecular weights of its major isoforms (approximately 8.95 kDa for RNase III and 5.93 kDa for RNase IV).

• IMMUNE NETWORK
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• v.4 no.1
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• pp.16-22
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• 2004

Background: To develop a novel treatment strategy for hepatitis B virus infection, a major cause of liver chirosis and cancer, we aimed to make human monoclonal antibodies inhibiting RNase H activity of P protein playing in important role in HBV replication. In this regard, phage display technology was employed and demonstrated as an efficient cloning method for human monoclonal antibody. So this study analysed the usability of human monoclonal antibody as protein based gene therapy. Methods: RNase H of HBV was expressed as fusion protein with maltose binding protein and purified with amylose resin column. Single chain Fv (scFv) phage antibody library was constructed by PCR cloning using total RNAs of PBMC from 50 healthy volunteers. Binders to RNase H were selected with BIAcore 2000 from the constructed library, and purified as soluble antibody fragment. The affinity and sequences of selected antibody fragments were analyzed with BIAcore and ABI automatic sequencer, respectively. And finally RNase H activity inhibiting assay was carried out. Results: Recombinant RNase H expressed in E. coli exhibited an proper enzyme activity. Naive library of $4.46{\times}10^9cfu$ was screened by BIAcore 2000. Two clones, RN41 and RN56, showed affinity of $4.5{\times}10^{-7}M$ and $1.9{\times}10^{-7}M$, respectively. But RNase H inhibiting activity of RN41 was higher than that of RN56. Conclusion: We cloned human monoclonal antibodies inhibiting RNase H activity of P protein of HBV. These antibodies can be expected to be a good candidate for protein-based antiviral therapy by preventing a replication of HBV if they can be expressed intracellularly in HBV-infected hepatocytes.

• Asian-Australasian Journal of Animal Sciences
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• v.3 no.2
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• pp.115-120
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• 1990

Extracellular nuclease(s) in buffalo rumen fluid were purified from strained rumen fluid by a procedure involving Seitz filtration, acetone fractionation and gel filtration on Sephadex G-100. The enzyme resolved into two peaks exhibiting both DNase and RNase activities. The molecular weight of enzyme corresponding to peaks I and II were approximately 30,000 and 12,000 respectively. The properties of enzymes from the two peaks, however, were same. Optimum temperature for both DNase and RNase activities was at $50^{\circ}C$. Whereas DNase activity was stable upto $60^{\circ}C$, RNase activity was stable only up to $50^{\circ}C$. DNase activity recorded two pH optima, one at pH 5.5 and the other at pH 7.0. RNase activity recorded a broad pH optimum between pH 6.0-8.0. pH stability of the enzyme coincided with pH optima for both the activities. DNase activity was stimulated by $Mg^{2+}$ and $Mn^{2+}$ and inhibited by $Fe^{2+}$, $Zn^{2+}$, $Hg^{2+}$ and $Ag^+$. RNase activity was also stimulated by $Mg^{2+}$ and $Mn^{2+}$ and inhibited by $Cu^{2+}$, $Fe^{2+}$, $Zn^{2+}$, $Hg^{2+}$ and $Ag^+$. Reducing agents stimulated both the activities.

• Korean Journal of Agricultural Science
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• v.21 no.1
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• pp.11-21
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• 1994

The halophile, Micrococcus sp. which produces RNase was isolated from salted and fermented food. The optimum growth condition of the Micrococcus sp. in pH 7.0 of complex medium containing 2M NaCl, and at $35^{\circ}C$. Optimum condition for enzyme production by this strain was when it was grown in the CM medium, containing 2% yeast extract, 1.5% casamino acid and 2M NaCl in the initial pH 8.5 for 2 days. The maximal RNase activity was observed at pH 8.0 and $55^{\circ}C$. The Km value for RNA was determined to be 5mg/ml by Lineweaver-Burk plot. The RNase activity in the absence of NaCl was maximum, but it was completely lost by adding of 1.25M NaCl and it was increased above 1.25M to 2.5M NaCl. When 2.5M NaCl was added, the activity of RNase showed 45% of maximum value.

• Microbiology and Biotechnology Letters
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• v.8 no.1
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• pp.1-8
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• 1980

Changes of nucleic acid related substances and their enzymes during rice makkulli koji making were observed and enzymological properties of crude enzymes were examined. The results obtained were as follows : (1) The amounst of acid soluble phosphorus were increased, while no remarkable changes were observed in the component of total phosphorus during koji making. (2) AMP and IMP were increased, while ADP and ATP were decreased gradually in the course of process. (3) Activities of nucleic acid degrading enzymes were increased with the lapse of time. (4) In the crude enzyme solution extracted from rice makkulli koji, the optimal pH of RNase was 4.0~5.0 and those of PDase PNase were 5.0. (5) RNase and PMase were stable at the range of pH 4.0~5.0 and PDase was stable at the pH 4.0. (6) The optimal temperature of RNase was 55$^{\circ}C$, and that of PDase was at the range of 50~55$^{\circ}C$, and 5$0^{\circ}C$ for PMase. (7) Among the three enzymes, the heat stability was in order RNase, PDase and PMase, and especially PMase was so heat labile that it was almost inactivated at 7$0^{\circ}C$ for 10 min. (8) Inhibition by metal ions and other inhibitors was disclosed : C $u^{++}$ and Z $n^{++}$ inhibited the activity of RNase, and C $u^{++}$, NaF and N $a_2$HP $O_4$ inhibited that of PDase, while C $u^{++}$ and NaF inhibited the PMase activity.ctivity.

• Korean Journal of Environmental Agriculture
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• v.22 no.4
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• pp.290-293
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• 2003

To measure an effects of strontium on secretion of ${\alpha}$-amylase and RNase, wheat aleurone layers were isolated after the pre-incubation in a solution with or without 10 mM $SrCl_2$ or $CaCl_2$ for 3 days at $25^{\circ}C$ in the dark under aseptic conditions. The secretion of ${\alpha}$-amylase reached a maximum at 72 h after incubation. $Sr^{2+}$ induced more effectively secretion of ${\alpha}$-amylase than $Ca^{2+}$. The ${\alpha}$-amylase secretions by $Sr^{2+}$ or $Ca^{2+}$ ware about $2 (Ca^{2+})$ to $2.5 (Sr^{2+})$ fold higher than it without divalent ions, When aleurone layers were incubated without divalent ions, however, the ${\alpha}$-amylase was remarkably retained in the tissues. Total ${\alpha}$-amylase synthesis (ie. tissues + media) was slightly lowered by 10mM $SrCl_2$ addition. It seemed that the RNase secretion begins at 18 h after incubation. This meaned that the RNase secretion may process slower than ${\alpha}$-amylasee secretion. $Ca^{2+}$ effect on RNase secretion is stronger than $Sr^{2+}$ unlikely to ${\alpha}$-amylase. The secretion process is likely to be suddenly induced between 72 hand 96 h. These results suggested that the secretion was enhanced after the accumulation in aleurone layers.

• Korean Journal of Food Science and Technology
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• v.31 no.2
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• pp.465-474
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• 1999

RNase activity of Saccharomyces cerevisiae ATCC 7754 was investigated to obtain strains with high ribonucleic acid (RNA) content. The yeast strain contained two RNase activities; an acidic RNase with a optima of pH $3{\sim}4$ and an alkaline RNase with a optima pH 9. The acidic RNase activity was inhibited by $0.08\;M\;HgCl_{2}$ most drastically. The alkaline RNase activity was inhibited by 2.0 M NaCl or KCl, while enhanced by addition of $0.05\;M\;CaCl_{2},\;0.02\;M\;ZnSO_{4},\;or\;0.008\;M\;HgCl_{2}$. Various mutants of Saccharomyces cerevisiae ATCC 7754 were isolated by ethylmethane sulfonate (EMS) treatment or $\gamma$-ray/ultra violet irradiation. Among the mutants that were sensitive to high concentration of KCl which inhibits alkaline RNase, B24 was selected for high RNA content per culture volume. Growth characteristics of the mutant were comparable to those of the mother strain with optimum growth at pH $4.5{\sim}5.5$. The mutant accumulated higher content of RNA than the mother strain when glucose was used as the carbon source. However, both growth rate and total RNA content of the mutant were higher in molasses medium than in glucose medium. RNA content of the mutant increased rapidly during the early stage of growth, and then decreased gradually until the culture reached stationary phase by a fed-batch culture in a 5 L jar fermenter. Maximal cell harvest and the final RNA content using the mutant B24 were 69.6 g/L culture broth and 19.8 g/100 g of the dry cell while those using the mother strain were 68 g/L culture broth and 16.1 g/100 g of dry cell, respectively.

• Korean Journal of Food Science and Technology
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• v.15 no.3
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• pp.245-251
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• 1983

Nucleic acid degrading enzymes (RNase, PDase, PMase) isolated from rice Makgeoly brewing were purified by DEAE-cellulose column technique and their enzymological properties were examined. Changes of nucleotides and their related substances during the brewing were also investigated. The results obtained were as follows: 1. RNase activity was increased in the earlier phase of brewing and then decreased after 3 days brewing, while PDase and PMase activities were decreased with the lapse of time. 2. The optimum pH of RNase was 5.0 and those of PDase and PMase were 6.0. Activities of these three enzymes were almost stable in the range of pH 6.0-7.0. 3. The optimum temperature of RNase and PDase were in the range of $55{\sim}60^{\circ}C$ and that of PMase was about $50^{\circ}C$. When RNase was treated at $100^{\circ}C$ for 10 min., 80% to of activity was lost PDase lost 90% of activity when heated at $70^{\circ}C$ for 10 min, while PMase was completely inactivated at the same condition. 4. $CU^{++},\;Zn{++}$ inhibited the activity of NRase, Activity of PMase was reduced about 30% by adding $10^{-3}M\;Na_{2}HPO_{4}$5. Until 4 day brewing, IMP was increased, while UMP, GMP, AMP were decreased gradually.

• Animal cells and systems
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• v.5 no.3
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• pp.195-198
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• 2001

Hepatitis B virus (HBV) polymerase, which possesses the activities of terminal binding, DNA polymerase, reverse transcriptase and RNaseH, has been shown to accomplish viral DNA replication through a pregenomic intermediate. Because the HBV polymerase has not been purified, the expression of HBV polymerase was examined in an E. coli expression system that is under the regulation of arabinose operon. The expressed individual domain containing terminal binding protein, polymerase, or RNaseH turned out to be insoluble. The activities of those domains were not able to be recovered by denaturation and renaturation using urea or guanidine-HCI. The expressed reverse transcriptase containing the polymerase and RNaseH domains became extensively degraded, whereas the proteolysis was reduced in a Ion- mutant. These results indicate that Lon protease proteolyzes the HBV reverse transcriptase expressed in E. coli.