• 한국동물학회지
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• 제22권4호
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• pp.141-152
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• 1979

고양이 백혈병 바이러스에서 reverse transcriptase를 분리하여 생화학적 및 면역학적 연구를 하였다. 분자량은 72,000이고, DNA polymerase와 RNase H의 활성은 0.05-1 mM $M_n^2+$와 50-80 mM KCl에서 가장 좋았다. DNA polymerase와 RNase H는 같은 단백질 분자에 있으며, chymotrypsin 처리로서 RNase H를 쪼개낼 수 있으며, 이 RNase H도 reverse transcriptase의 항체에 의해서 활성이 거의 억제 된다. Reverse transcriptase의 항체 결합위치와 활성을 내는 위치는 다른 것 같다.

• Journal of Animal Science and Technology
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• 제59권9호
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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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• 제4권1호
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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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• 제3권2호
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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.

• 농업과학연구
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• 제21권1호
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• pp.11-21
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• 1994

RNase를 강하게 분비하는 균을 젓갈류서 분리하여 호염성 Micrococcus sp.로 동정하였다. 선정균은 NaCl을 2M 함유하는 pH 7.0의 CM배지에서 $35^{\circ}C$로 2일간 배양할 때 생육이 가장 양호하였으며 yeast extract 2.0%, casamino acid 1.5%, NaCl 2.0M, pH 8.5의 배지에서 $35^{\circ}C$로 2일간 배양할 때 효소 생산이 최대였다. 조효소액을 유안 염석과 이온교환 크로마토그라피, gel 여과 등으로 정제한 결과 비활성이 98.8 units/mg, 정제도는 39.4배, 수율은 0.9% 이었다. 정제 효소의 작용 최적 pH는 8.0, 온도는 $55^{\circ}C$이었으며 RNA를 기질로 한 Km값은 5mg/ml이었다. NaCI농도에 따른 효소활성은 무첨가조건에서 가장 높았고 1.25M에서 활성이 소설되었다가 다시 증가되어 2.5M에서는 무첨가구의 45% 활성을 보였다.

• 한국미생물·생명공학회지
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• 제8권1호
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• pp.1-8
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• 1980

쌀막걸리 제국중의 핵산관련물질 및 핵산분해효소의 소장을 시험하고 이들 조효소의 효소학적 성질에 대하여 실험한 결과는 다음과 같다. 1) 제국중 산하용성인은 약간 증가하였으나 총인은 그다지 변화가 없었다. 2) 제국중 AMP, IMP등은 약간 증가하였으나 ADP, ATP는 점차 감소하였다. 3) 시간의 경과에 따라 핵산분해효소의 활성은 증가하였다. 4) 국으로 부터 추출한 조효소액에서의 최적 pH는 대체로 RNase가 pH4.0~5.0, PDase와 PMase는 pH 4.0 이었다. 5) RNase와 PMase는 PH 4.0~5.0 부근에서 안정하였고 PDase는 pH4.0에서 대체로 안정하였다. 6) RNase, PDase, PMase의 최적온도는 모두 50~55$^{\circ}C$ 범위였다. 7) 세가지 효소의 열안정성은 RNase>PDase> PMase의 순이었고 특히 PMase는 열안정성이 낮아 7$0^{\circ}C$ 10분간 처리로서 거의 실활되었다. 8) C $u^{++}$, $Zn^{++}$은 RNase의 활성을 저해하였고, C $u^{++}$, NaF, $Na_2$HP $O_4$는 PDase, C $u^{++}$ NaF는 PMase의 활성을 저해하였다.

• 한국환경농학회지
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• 제22권4호
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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.

• 한국식품과학회지
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• 제31권2호
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• pp.465-474
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• 1999

식용 단백질 생산 균주인 S. cerevisiae ATCC 7754에 돌연변이를 유도하고, 핵산 함량이 높으면서도 성장 속도가 빠른 고핵산 축적 균주를 선별하였고 배양 특성을 조사하였다. Saccharomyces cerevisiae ATCC 7754는 pH $3{\sim}4$에서 최대 활성을 보이는 산성 RNase와 pH 9에서 최대활성을 보이는 알칼리성 RNase를 가지고 있는 것이 확인되었으며 산성 RNase의 활성이 훨씬 높았다. Saccharomyces cerevisiae ATCC 7754의 RNase는 금속염에 의하여 영향을 받는데 산성 RNase의 경우는 $0.08\;M\;HgCl_2$에 의하여 활성이 크게 저하되었고, 알칼리성 RNase경우는 2.0 M NaCl이나 KCl에 의하여 활성이 크게 저하되었다. 반면 알칼리성 RNase는 $0.05\;M\;CaCl_2$, $0.02\;M\;ZnSO_4$, $0.008\;M\;HgCl_{2}$에 의하여 활성이 증가되었다. Ethylmethane sulfonate, 자외선, 감마선을 이용한 돌연변이를 통하여 핵산 축적능이 뛰어난 KCl 감수성 균주를 선발하고 YPD 배지에서 배양하면서 건조 균체량과 리보핵산 함량을 측정하여 건조 균체 단위 무게당 리보핵산의 함량이 가장 높은 B24 균주를 고핵산 변이주로 선택하였다. B24 균주는 모균주와 비슷한 증식 특성을 나타내었는데, pH $4.5{\sim}5.5$에서 성장이 잘 되었고 리보핵산 축적은 pH 4.5와 5.0에서 가장 잘 일어났으며, 또한 탄소원으로 당밀을 사용한 경우가 세포 내에 리보핵산이 가장 많이 축적되었고 균의 생육도 빠름을 알 수 있었다. 발효기를 이용한 유가식 배양에서는 배양 초기에 균체내 리보핵산 함량이 급격히 증가하고 이후 정지기까지 서서히 감소하여 최종적으로 리보핵산 함량이 균체 건물량의 19.8%(w/w)로 모균주의 16.1% (w/w)에 비해 우수하였으며, 이때 최대 69.6 g/L(건물량)의 균체를 수득하였다.

• 한국식품과학회지
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• 제15권3호
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• pp.245-251
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• 1983

쌀막걸리 담금중의 핵산분해효소들의 소장과 이효소들을 DEAE-cellulose column chromatography에 의해 분리정제하고 일반적인 성질을 검토함은 물론 핵산관련물질의 변화를 조사한 결과는 다음과 같았다. 1. 담금일수가 경과함에 따라 RNase는 담금 3 일까지 다소 증가하였다가 그후 점차 감소하였으며 PDase 및 PMase는 초기부터 활성이 감소하였다. 2. RNase의 최적pH는 5.0부근이었고, PDase, PMase는 6.0부근이었으며 pH안정성은 대체로 $pH6.0{\sim}7.0$부근이었다. 3. RNase 및 PDase의 최적온도는 $55{\sim}60^{\circ}C$이고, PMase는 50부근이었으며 열안정성은 RNase는 $100^{\circ}C$ PDase는 $80^{\circ}C$에서 10분간반응으로 각각 80%, 90%실활되었으며 PMase는 $70^{\circ}C$에서 10분간 반응으로 거의 샐활되었다. 4. RNase는 $CU^{++},\;Zn{++}$, PMase는 $10^{-3}M\;Na_{2}HPO_{4}$처리로서 약 30%의 효소활성이 저해되었다. 5. 술덧담금 4일째까지 IMP는 증가하였으며 UMP, GMP, AMP는 감소하는 경향을 보였다.

• Animal cells and systems
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• 제5권3호
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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.