• Title/Summary/Keyword: Adenosine deaminase

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Studies on the Adenosine Deaminase Gene from Nocardioides sp. J-326TK (Nocardioides sp. J-326TK의 Adenosine Deaminase Gene에 관한 연구)

  • 전홍기;백형석;정춘식
    • Journal of Life Science
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    • v.8 no.6
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    • pp.673-680
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    • 1998
  • Adenosine deaminase gene from Nocardioides sp. J-326TK was cloned by polymerase chain reaction using primers (PI, PII and PIII) constructed from the highly conserved amino acid sequences among Escherichia coli, mouse and human. A PCR product of about 800bp, as expected from the sequence of E. coli adenosine deaminase gene, was obtained from Nocardioides sp. J-326TK chromosomal DNA double-digested with EcoRI and Pst I. DNA sequencing of the PCR product after cloning into pT7Blue T-vector shows 99.5% and 98.9% homologies in nucleotide and amino acid sequences, respectively, with the E. coli adenosine deaminase whereas 59.5% and 46.8% homologies with the human adenosine deaminase, indicating the evolutionarily relationship of these organisms.

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Actinomycins에 의한 Adenosine Deaminase의 억제

  • 김경자;조성진
    • Microbiology and Biotechnology Letters
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    • v.24 no.3
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    • pp.380-383
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    • 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.

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Studies on the Adenosine Deaminase Inhibitor Producing Actinomycetes J144-K (방선균 J-144K가 생산하는 Adenosine Deaminase Inhibitor에 관한 연구)

  • Jun, Hong-Ki;Kim, Sam-Woong;Jo, Young-Bae;Yeeh, Yeehn
    • Journal of Life Science
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    • v.6 no.2
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    • pp.120-128
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    • 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.

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Identification of Adenosine Deaminase Inhibitor-producing Bacterium Isolated from Soil

  • SHIN, YONG KOOK;YONG-HA PARK;JAE-DONG LEE;HONG-KI JUN
    • Journal of Microbiology and Biotechnology
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    • v.7 no.1
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    • pp.32-36
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    • 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.

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Purification and Characterization of Adenosine deaminase from Aspergillus oryzae (Aspergillus oryzae에서 Adenosine Deaminase의 정제와 특성)

  • Choi, Hye-Seon
    • Korean Journal of Microbiology
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    • v.31 no.1
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    • pp.54-62
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    • 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.

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Visible and Fast Assay System for Tobacco Transformant Introduced with Adenosine Deaminase Marker Gene (Adenosine Deaminase 표지유전자로 형질전환된 연초의 신속한 Assay 방법)

  • 양덕춘;김용환;임학태;방극수;배창휴
    • Korean Journal of Plant Tissue Culture
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    • v.28 no.3
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    • pp.165-171
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    • 2001
  • New visible and fast assay system have been developed for tobacco transformant introduced with adenosine deaminase (ADA) marker gene, which converts cytotoxic adenosine analogues to non-toxic inosine analogues and ammonia. Ammonia was changed to blue color in the solution of phenol-nitoprusside and alkaline-hypochlorite. It was possible to detect activity of ADA visibly on the holes of 96 well plate using tiny explant of transgenic tobacco leaves within 1 hour incubation time. As substrates of ADA enzyme from transgenic plant on the plate, a number of adenosine analogues such as 9-D-arabinofuranosyl adenine, cordycepin, 2'-deoxyadenosine, adenosine and xylofuranosyl adenine were possible for detection of ADA activity. Optimal condition of substrate for ADA enzyme was each 10 mM and pH 7.5 in adenosine solution. Especially, transgenic plant did not convert adenosine to inosine and ammonia in the presence of ADA inhibitor deoxycoformycin, which means that ammonia produced from transgenic plant is due to expression of ADA gene. Now, we show that this detection system can be easily, sensitively, fast and cheaply as well as visibly assayed in vitro as GUS gene system with very small size of transformant explant.

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Isolation and Characterization of Actinomycetes Producing Extracellular Adenosine Deaminase Inhibitor (세포외 Adenosine Deaminase Inhibitor를 생산하는 방선균의 분리 및 특성)

  • Kim, Kyoung-Ja;Park, Kui-Lea
    • YAKHAK HOEJI
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    • v.38 no.3
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    • pp.274-280
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    • 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.

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Enzymatic Properties of Intracellular Adenosine Deaminase from Nocardioides sp. J-326TK

  • Hong-Ki Jun;Tae-Sook Kim
    • Journal of Life Science
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    • v.9 no.1
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    • pp.64-68
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    • 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.

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Adenosine Deaminase Gene: Possible Selectable Marker for Tobacco Transformation (연초의 형질전환을 위한 새로운 표지유전자로서 Mouse Adenosine Deaminase 유전자의 이용가능성)

  • 양덕춘;한성수;윤의수
    • Korean Journal of Plant Tissue Culture
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    • v.22 no.4
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    • pp.235-240
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    • 1995
  • The development of selectable markers for transformation has been a major factor in the successful genetic manipulation of plant. We established a new selectable marker system for tobacco transformation using chimeric adenosine deaminase (ADA) gene, which confers resistance to cytotoxic adenosine analogues, 9-$\beta$-D-arabinofuranosyl adenine(Ara-A) and cordycepin. The transformants with the chimeric ADA gene in tobacco grew in the presence of normally lethal level of cytotoxic adenosine analogues, 100 $\mu$M Ara-A and 50 $\mu$M cordycepin. We successfully distinguished transformed shoot from non-transformed shoot on the same selectable media with cytotoxic adenosine analogues. In this selectable media, we were able to select seeds with/ without ADA gene from transgenic tobacco seeds. Theses results show that the mammalian ADA gene may serve as a new selectable marker for tobacco transformation.

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Expression of Mouse Adenosine Deaminase Gene in Transgenic Tobacco (Nicotiana tabacum L.) (형질전환 연초(Nicotiana tabacum L.)의 Mouse Adenosine Deaminase 유전자 발현)

  • 양덕춘;박지창;최광태;이정명
    • Korean Journal of Plant Tissue Culture
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    • v.22 no.4
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    • pp.195-200
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    • 1995
  • The mammalian adenosine deaminase(ADA) gene was stably expressed in transgenic tobacco plane. The chimeric ADA gene 35S/35S/AMV/ADA/Tnos, has been constructed. This chimeric gene was introduced into the binary vector pRD400, which was thereafter mobilized into Agrobacterium tumefaiens strain MP90 harboring disarmed Ti-plasmid. The resulting strains were used to transform Nicofiana tabacum L. using the leaf disc. Incorporation of the chimeric gene into plant were confirmed by PCR and Northern blot analyses. Immunoblot analysis showed that ADA protein was successfully synthesized in the transgenic tobacco plants.

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