NO dehydrogenase (NO-DH) 에 관련된 실험 정리

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Mycobacterium sp. strain JC1 을 대상으로 NO-DH activity 가 존재하는지 확인하기 위해 enzyme assay 와 activity staining 을 실행 (Fig. 1)다른 mycobacteria (Mycobacterium tuberculosis [Fig. 2], Mycobacterium vaccae [Fig. 3]) 를 대상으로 CO-DH 와 NO-DH enzyme assay 를 실행 . CO-DH enzyme activity 와 비교했을 경우 , NO-DH activity 는 상당히 약하기 때문에 NO-DH enzyme assay 만을 분리하여 비교 (Fig. 4). NO-DH activity staining 은 실시했으나 결과를 얻을 수 없었음 .Mycobacterium sp. strain JC1 을 대상으로 한 activity staining 에서 NO-DH 의 활성을 나타내는 band 가 CO-DH 의 band 의 위치가 비슷하기 때문에 (Fig. 1) CO-DH 를 순화하여 NO-DH enzyme assay 를 실행 (Fig. 5). NO-DH activity staining 은 실시했으나 결과를 얻을 수 없었음 .CO-DH 가 NO-DH 의 활성을 가지고 있다고 생각되었기 때문에 CO-DH가 NO 에 의해 (SNP 사용 ) induction 되는지 여부를 확인 . 적절한 SNP의 농도를 결정하기 위해 SNP 농도에 따른 growth curve 를 측정 (Fig. 6). 측정결과 5 mM SNP 를 사용하여 , CO (Fig. 7), methanol (Fig. 8), glucose (Fig. 9) 를 기질로 사용했을 경우 , SNP 에 의한 CO-DH 의 induction여부를 확인 .

SNP 를 가지고 induction 한 결과 , CO-DH 가 induction 되는 형태를 보이기 때문에 , CO-DH enzyme assay, NO-DH enzyme assay (Table 1), Western blot 을 통해 다시 확인 (Fig. 10)CO-DH 가 NO 에 의해 induction 되는 것이 Mycobacterium sp. strain JC1 에 서 만 의 특 성 인 지 다 른 carboxydobacteria 에 서 도 공 통 적 인 형상인지 확인하기 위해 mycobacterial carboxydobacteria 와 non-mycobacterial carboxydobacteria 를 대상으로 SNP 에 의한 CO-DH induction여부를 확인(Fig. 11 and table 2). Mycobacterial carboxydobacteria 의 경우는 현재 진행 중입니다 .Oligotropha carboxidovorans OM5 에 서 SNP 를 가 지 고 induction 한 결과 , CO-DH 가 induction 되는 형태를 보이지 않았기 때문에 , Western blot 을 통해 다시 확인 (Fig. 12)

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Figure 1. CO-DH and NO-DH activity staining of Mycobacterium sp. strain JC1; lane 1 and 3, crude extract of Mycobacterium sp. strain JC1 grown in 7H9 supplement with CO; lane 2 and 4, crude extract of Mycobacterium sp. strain JC1 grown in SMB supplement with CO. A, CO-DH activity staining; B, NO-DH activity staining.

※ NO-DH specific activity : 1.48 CO-DH specific activity : 6.02

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CO-DH activity band NO-DH activity band

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NO-DH and CO-DH enzyme assay (MTB)

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with CO + enzymewith NO + enzymewith NO+ No enzyme

Figure 2. NO-DH and CO-DH enzyme assay of M. tuberculosis. CO-DH specific activity : 9.87 NO-DH specific activity : 1.99

Figure 3. NO-DH and CO-DH enzyme assay of M. vaccae. CO-DH specific activity : 17.45 CO-DH specific activity : 0.55

NO-DH and CO-DH enzyme assay (M. vaccae)

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NO-DH enzyme assay

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Figure 4. NO-DH enzyme assay of MTB and M. vaccae. NO-DH specific activity of MTB and M. vaccae is 1.85 and 3.06, respectively.

NO-DH enzyme assay

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Figure 5. NO-DH enzyme activity of purified CO-DH of Mycobacterium sp. strain JC1. NO-DH specific activity is 32.6

With NO + No enzyme

With NO + purified CO-DH

Figure 6. Growth curve of Mycobacterium sp. Strain JC1 grown in 7H9 supplemented with CO at various concentration of SNP

CO-DH activity band

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A : CBB staining; B : CO-DH activity stainingLane 1-3 : No SNP 0, 20, 40 minLane 4-6 : 5 mM SNP 0, 20, 40 min

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Figure 7. Induction of CO-DH by SNP. Crude extract was prepared from Mycobacterium sp. strain JC1 grown in SMB supplement with CO. Each 17 ㎍ of crude extract was loaded.

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A : CBB staining; B : CO-DH activity stainingLane 1-3 : No SNP 0, 20, 40 minLane 4-6 : 5 mM SNP 0, 20, 40 min

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Figure 8. Induction of CO-DH by SNP. Crude extract was prepared from Mycobacterium sp. strain JC1 grown in SMB supplement with methanol. Each 17 ㎍ of crude extract was loaded.

CO-DH activity band

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A : CBB staining; B : CO-DH activity stainingLane 1-3 : No SNP 0, 20, 40 minLane 4-6 : 5 mM SNP 0, 20, 40 min

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Figure 9. Induction of CO-DH by SNP. Crude extract was prepared from Mycobacterium sp. strain JC1 grown in SMB supplement with glucose. Each 18 ㎍ of crude extract was loaded.

CO-DH activity band

Table 1. CO dehydrogenase and NO dehydrogenase enzyme assay using crude extracts of Mycobacterium sp. strain JC1

CO DH activity

NO-DH activity

No SNPa With SNP

0 20 40 (min) 0 20 40 (min)

aSpecific activity : nmol of INT reduced per mg of protein per minute.

5.68 5.67 6.03 5.68 8.54 0.00

1.47 1.43 1.49 1.47 2.05 0.00

0 20 40 (min)

Figure 10. Western immunoblot analysis of CO-DH. Lane 1-3, crude extract of Mycobacterium sp. strain JC1 harvested after adding 5 mM SNP 0, 20, 40 minutes, respectively. Blots were incubated with antibody raised against Mycobacterium sp. strain JC1 CO-DH medium subunit. Each 90 ㎍ of crude extract was loaded.

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A : CBB staining; B : CO-DH activity stainingLane 1-3 : No SNP 0, 20, 40 minLane 4-6 : 5 mM SNP 0, 20, 40 min

Figure 11. Induction of CO-DH by SNP. Crude extract was prepared from Oligotropha carboxidovorans OM5 grown in SMB supplement with CO. Each 3 ㎍ of crude extract was loaded.

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Table 2. CO dehydrogenase and NO dehydrogenase enzyme assay using crude extracts of Oligotropha carboxidovorans OM5

CO DH activity

NO-DH activity

No SNPa With SNP

0 20 40 (min) 0 20 40 (min)

aSpecific activity : nmol of INT reduced per mg of protein per minute.

17.6 17.0 14.5 17.6 7.6 7.8

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Figure 12. Western immunoblot analysis of CO-DH. Lane 1-3, crude extract of Oligotropha carboxidovorans OM5 harvested after adding CO 0, 20, 40 minutes, respectively; Lane 4-6, crude extract of Oligotropha carboxidovorans OM5 harvested after adding CO and 5 mM SNP 0, 20, 40 minutes, respectively. Blots were incubated with antibody raised against Hydrogenophaga pseudoflava CO-DH. Each 15 ㎍ of crude extract was loaded.