• 생명과학회지
• /
• 제8권6호
• /
• pp.673-680
• /
• 1998

Nocardioides sp. J-326TK의 adenosine deaminase gene을 분리하기 위하여 genomic DNA를 제한효소로 무작위적으로 절단하여 pBluscript KS에 ligation시켰다. 또한 hu-man과 mouse, E. cali 등의 adenosine deaminase gene의 보존적인 부위를 primer로 합성을 하여 PCR reaction을 행하였다. Genomic DNA를 cloning시킨 pKSN60은 5kb정도의 DNA를 포함하고 있으며 sourthern hybridization 등의 여러 확인 실험을 통하여 adenosine deaminase gene을 포함하고 있다는 알았다. PCR product를 cloning시켜 형성된 recombinant plasmid를 PCR reaction의 primer로서 pTBN20를 sequencing을 행하였다. 그 결과를 다른 ade-nosine deaminase gene의 서열과 비교를 하였는데 미생물인 E. coli와는 nucleotide sequence는 99.5%, amino acid sequence는 98.9%의 homology를 나타내고 human과는 각각 59.5%, 46.8%의 homolosy를 나타내었다.

• 한국미생물·생명공학회지
• /
• 제24권3호
• /
• pp.380-383
• /
• 1996

Adenosine deaminase inhibitor was extracted from culture broth of Streptomyces sp. strain V-8 with ethylacetate. The ethylacetate extract showed the characteristic UV absorption spectrum of actinomycins at 440-450 nm. The ethylacetate extract was compared with respect to inhibitory behavior against adenosine deaminase from calf intestinal mucosa with actinomycin D, -C complex and actinomycin V. The Ki values for actnomycin D, -C complex, and actinomycin V against adenosine deaminase were determined to be 9.9 $\times$ 10$^{-6}$ M, 9.6 $\times$ 10$^{-6}$ M and 9.3 $\times$ 10$^{-6}$ M, respectively. The Ki value for the ethylacetate extract of culture broth against adenosine deaminase was determined to be 5.7 $\times$ 10$^{-6}$ M. The kinetic parameters of actinomycin D, -C complex, -V and ethylacetate extract of culture broth for adenosine deaminase were as follows:I$_{50}$ = 1.5 $\times$ 10$^{-5}$ M (actinomycin D), 2.7 $\times$ 10$^{-5}$ M (actinomycin C complex), 3.5 $\times$ 10$^{-5}$ M (actinomycin V), 8.9 $\times$ 10$^{-6}$ M (ethylacetate extract of culture broth). The adenosine deaminase was inhibited noncompetitively by ethylacetate extract of culture broth as well as by actinomycin D, -C complex and actinomycin V.

• 생명과학회지
• /
• 제6권2호
• /
• pp.120-128
• /
• 1996

In the screening of actinomycetes culture filtrate for inhibitor of adenosine deaminase, a novel inhibitor was found in a cultured broth of strain J-144K. The optimum conditions for the adenosine deaminase inhibitor production from the isolated strain J-144K were evaluated. This strain showed the maximum yield of adenosine deaminase inhibitor when grown at pH 7.0 and 30$\circ$C for 60 hours in the medium of 1.0% dextrose, 0.5% yeast extract, 0.5% peptone and 0.1% KH$_{2}$PO$_{4}$ under the aerobic condition. Through the activated charcoal extraction, methanol fractionation, Dowex 50 H$^{+}$ X-8 ion exchange column chromatography, Dowex CI$^{-}$ X-8 ion exchange column chromatography, and Sephadex G-15 gel filtration procedures, this inhibitor was purified with three materials.

• Journal of Microbiology and Biotechnology
• /
• 제7권1호
• /
• pp.32-36
• /
• 1997

An adenosine deaminase inhibitor-producing bacterium was isolated from soil. An isolate exhibiting high adenosine deaminase inhibitory activity, was designated J-89, and classified as a strain of Bacillus subtilis on the basis of its morphological, phenotypic characteristics, the menaquinone content and cellular fatty acid composition. To confirm the taxonomic position of the strain we need more information such as DNA-DNA homology and other chemotaxonomic characteristics. In this paper we provisionally named strain J-89 as Bacillus sp. J-89 pending further chemotaxonomic study and analysis of adenosine deaminase inhibitor.

• 미생물학회지
• /
• 제31권1호
• /
• pp.54-62
• /
• 1993

Intracellular adenosine deaminase (ADA) from Aspergillus oryzae was purified using ammonium sulfate fractionation, a DEAE-Sephadex A-50 anion exchange chromatography, an ultrafiltration using a PM 10 membrane and two times of Sephadex G-100 gel filtration chromatography. The enzyme was purified 151 fold with a 9% recovery. Purified enzyme gave a single protein band with a molecular weight of 105,000 delton. The enzyme was reasonably stable. The enzyme activity was kept even after 1 hr incubation at 55.deg.C, but decreased significantly at 60.deg.C. The pH optimum was found to be from 6.5 to 7.5. Among tested compounds, the substrate activity was found with adenosine, adenine arainofuranoside, formymcin A, 2'-deoxyadenosine, 3'-deoxyadenosine, 2', 3'-isopropylidene adenosine, 2,6-diaminopurine deoxyriboside, .betha.-nicotinamide adenine dinucleotide (reduced form), 6-chloropurine riboside, 2'-adenine monophosphate (AMP), 3'-AMP and 5'-AMP. The values of Km of adenosine and 2'-deoxyadenosine were calculated to be 500 and .$710\mu$m, respectively. ADA was sensitivite to $Zn^{2+}$, $^Cu{2+}$ and $Fe^{3+}$, p-chloromercuribenzoate and mersalyl acid inactivated the enzyme. The activity of enzyme was not changed when ADA was incubated with dithiothreititol, 2-mercaptoethanol, N-ethylmaleimide, iodoacetic acid and iodoacetamide.

• 식물조직배양학회지
• /
• 제28권3호
• /
• pp.165-171
• /
• 2001

Adenosine deaminase 유전자를 연초의 형질전환용 표지유전자로 활용할 때 형질전환체 여부를 매우 빠르고 눈으로 직접 색깔을 확인할 수 있는 새로운 방법이 개발되었다. ADA 효소는 독성인 adenosine 유도체를 비독성인 inosine 유도체와 암모니아로 변환시키는데, 이때 형성된 암모니아를 phenol-nitoprusside와 alkaline-hypochlorite 용액을 이용하여 청색으로 변환시켜 96 well plate상에서 1시간 내에 형질전환체 여부를 쉽게 확인할 수 있게 되었다. ADA효소의 substrate로서 9-D-arabinofuranosyl adenine, cordycepin, 2'-deoxyadenosine, adenosine and xylofuranosyl adenine이 모두 가능하였으며, substrate 용액의 최적조건은 adenosine 10 mM과 pH 7.5이었다. 특히 형질전환체는 ADA효소의 inhibitor인 deoxycoformycin이 함유되어 있는 용액 속에서는 adenosine을 inosine과 암모니아로 변환시키지 못해 색깔의 변화가 없었는데, 이는 형질전환체에서 색깔의 변화는 ADA효소의 작용 때문에 일어나는 것을 의미한다. 따라서 본 연구결과는 ADA 표지유전자가 도입된 형질전환체의 확인에 있어서 GUS gene system과 같이 눈으로 직접 확인할 수 있을 뿐만 아니라 매우 작은 크기의 형질전환체 절편으로 쉽고, 빠르면, 값싸게 확인할 수 있게 되었다.

• 약학회지
• /
• 제38권3호
• /
• pp.274-280
• /
• 1994

A strain of actinomycetes producing extracellular adenosine deaminase inhibitor, strain V-8, was isolated from soil. Strain V-8 was gam positive and its cell wall chemotype was decided as cell wall chemotype I from analysis of diaminopimelic acid isomers and sugar pattern. This strain had a wide range of sugar utilization as carbon sources. The optimal pH and temperature for growth were $6.8{\sim}7.0$ and $28{\sim}30^{\circ}C$, respectively. From the morphological, chemotaxonomical characteristics and analysis of various physiological characteristics, the strain V-8 was identified Streptomyces sp.

• Journal of Life Science
• /
• 제9권1호
• /
• pp.64-68
• /
• 1999

The properties of purified intracellular adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) of Nocardioides sp. J-326TK isolated from soil have been studied. The enzyme deaminated adenosine and 2`-deoxyadenosine and the respective {TEX}$K_{M}${/TEX} values were 4.0×{TEX}$10^{-4}${/TEX} M and 5.0× {TEX}$10^{-4}${/TEX} M, but the enzyme was not active on 8-bromoadenosine, 6-methylaminopurine riboside, ATP, ADP, 2`-AMP, 3`-AMP, 5`-AMP, dAMP, cAMP, NAD, FAD, NADP and adenine. The enzyme activity was strongly inhibited by the addition of {TEX}$Hg^{2+}${/TEX} and {TEX}$Ag^{+}${/TEX}, {TEX}$Cu^{2+}${/TEX}, {TEX}$Co^{2+}${/TEX} and {TEX}$Mn^{2+}${/TEX} also inhibited the activity but much less extent. The effect of alkyl reagents, metal chelating reagents and certain other compounds on the enzyme activity were also examined. No reagent activated the enzyme. On the contrary, the enzyme reaction was slightly inhibited by o-phenanthroline and 6-benzyladenosine.

• 식물조직배양학회지
• /
• 제22권4호
• /
• pp.235-240
• /
• 1995

식물세포의 형질전환을 위한 새로운 표지유전자의 개발은 식물유전공학의 중요한 관건이 되고 있다. 본 실험은 독성 adenosine 유도체인 9-$\beta$-D-arabinofuranosyl adenine (Ara-A)과 cordycepin등에 저항을 나타내는 adenosine deaminase (ADA) 유전자를 새로운 식물세포의 형질전환용 표지유전자로 사용코자 수행하였다. 정상식물체에서는 치사하는 농도인 Ara-A 100 $\mu$M과 cordycepin 50 $\mu$M이 함유된 선발배지에서 ADA 유전자에 의하여 형질전환된 연초식물체는 생존이 가능하였으며 성공적으로 형질전환체를 선발할 수 있었다. 또한 형질전환된 연초식물체에서 획득한 종자도 동일한 선발배지에서 ADA 유전자가 유전된 종자와 유전되지 않은 종자를 쉽게 구별할 수 있었다. 이런 결과는 동물유전자인ADA 유전자를 연초조직의 새로운 형질전환용 표지유전자로 사용할 수 있음을 시사한다.

• 식물조직배양학회지
• /
• 제22권4호
• /
• pp.195-200
• /
• 1995

동물유전자인 mouse adenosine deaminase (ADA)유전자가 안정적으로 연초 형질전환체에서 발현되었다. ADA cDNA 을 연초에서 강력히 발현시키기 위해서 35S/35S/AMV promoter을 부착시켰으며 식물세포에 도입을 위해서 binary vector인 pRD400을 이용하였다. 연초의 형질전환은 Tri- parental mating에 의해서 도입된 ADA 유전자 함유 binary vector와 disarmed Ti-plasmid을 함유하고 있는 Agrobacterium tmefacience MP9O을 사용하였다. 동시배양은 연초의 잎 disc을 이용해서 kanamycin이 첨가된 배지로부터 직접 shoots을 선발하여 형질전환체로 유도하였다. 형질전환 체에 ADA 유전자의 삽입여부는 PCR을 이용하였으며, 실험결과 ADA 유전자를 확인할 수 있었다. 또한 도입된 mouse ADA 유전자로부터 mRNA 및 단백질 합성여부를 각각 northern blot 및 immunoblot 분석한 결과 형질전환체 에서는 공히 확인되었다.