NO dehydrogenase (NO-DH) 에 관련된 실험 정리

1. NO dehydrogenase (NO-DH)에 관련된 실험 정리 1. 2. 3. 4. Mycobacterium sp. strain JC1을 대상으로 NO-DH activity가 존재하는지 확인하기 위해 enzyme assay와 activity staining 을 실행 (Fig. 1) 다른 mycobacteria (Mycobacterium tuberculosis [Fig. 2], Mycobacterium vaccae [Fig. 3])를 대상으로 CO-DH와 NO-DH enzyme assay를 실행. CO-DH enzyme activity와 비교했을 경우, NO-DH activity는 상당히 약하기 때문에 NO-DH enzyme assay만을 분리하여 비교 (Fig. 4). NO-DH activity staining은 실시했으나 결과를 얻을 수 없었음. Mycobacterium sp. strain JC1을 대상으로 한 activity staining에서 NO-DH의 활성을 나타내는 band가 CO-DH의 band의 위치가 비슷하기 때문에 (Fig. 1) CO-DH를 순화하여 NO-DH enzyme assay를 실행 (Fig. 5). NO-DH activity staining은 실시했으나 결과를 얻을 수 없었음. CO-DH가 NO-DH의 활성을 가지고 있다고 생각되었기 때문에 CO-DH가 NO에 의해 (SNP 사용) induction되는지 여부를 확인. 적절한 SNP의 농도를 결정하기 위해 SNP농도에 따른 growth curve를 측정 (Fig. 6). 측정결과 5 mM SNP를 사용하여, CO (Fig. 7), methanol (Fig. 8), glucose (Fig. 9) 를 기질로 사용했을 경우, SNP에 의한 CO-DH의 induction여부를 확인.

2. 5. 6. 7. SNP를 가지고 induction한 결과, CO-DH가 induction되는 형태를 보이기 때문에, CO-DH enzyme assay, NO-DH enzyme assay (Table 1), Western blot을 통해 다시 확인(Fig. 10) CO-DH가 NO에 의해 induction되는 것이 Mycobacterium sp. strain JC1 에서만의 특성인지 다른 carboxydobacteria에서도 공통적인 형상인지 확인하기 위해 mycobacterial carboxydobacteria와 non-mycobacterial carboxydobacteria를 대상으로 SNP에 의한 CO-DH induction여부를 확인 (Fig. 11 and table 2). Mycobacterial carboxydobacteria의 경우는 현재 진행 중입니다. Oligotropha carboxidovorans OM5에서 SNP를 가지고 induction한 결과, CO-DH가 induction되는 형태를 보이지 않았기 때문에, Western blot을 통해 다시 확인(Fig. 12)

3. A B 1 2 3 4 CO-DH activity band NO-DH activity band Figure 1. CO-DH and NO-DH activity staining of Mycobacterium sp. strain JC1; lane 1 and 3, crude extract of Mycobacterium sp. strain JC1grown in 7H9 supplement with CO; lane 2 and 4, crude extract of Mycobacterium sp. strain JC1grown 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

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

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

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

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

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

9. B A 1 2 3 4 5 6 1 2 3 4 5 6 CO-DH activity band A : CBB staining; B : CO-DH activity staining Lane 1-3 : No SNP 0, 20, 40 min Lane 4-6 : 5 mM SNP 0, 20, 40 min 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.

10. B A 1 2 3 4 5 6 1 2 3 4 5 6 CO-DH activity band A : CBB staining; B : CO-DH activity staining Lane 1-3 : No SNP 0, 20, 40 min Lane 4-6 : 5 mM SNP 0, 20, 40 min 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.

11. B A 1 2 3 4 5 6 1 2 3 4 5 6 CO-DH activity band A : CBB staining; B : CO-DH activity staining Lane 1-3 : No SNP 0, 20, 40 min Lane 4-6 : 5 mM SNP 0, 20, 40 min 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.

12. Table 1. CO dehydrogenase and NO dehydrogenase enzyme assay using crude extracts of Mycobacterium sp. strain JC1 No SNPa With SNP 0 20 40 (min) 0 20 40 (min) CO DH activity 5.68 5.67 6.03 5.68 8.54 0.00 NO-DH activity 1.47 1.43 1.49 1.47 2.05 0.00 aSpecific activity :nmol of INT reduced per mg of protein per minute.

13. 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.

14. 1 2 3 4 5 6 1 2 3 4 5 6 A B A : CBB staining; B : CO-DH activity staining Lane 1-3 : No SNP 0, 20, 40 min Lane 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.

15. Table 2. CO dehydrogenase and NO dehydrogenase enzyme assay using crude extracts of Oligotropha carboxidovorans OM5 No SNPa With SNP 0 20 40 (min) 0 20 40 (min) CO DH activity 17.6 17.0 14.5 17.6 7.6 7.8 NO-DH activity 0.0 0.0 0.0 0.0 0.0 0.0 aSpecific activity :nmol of INT reduced per mg of protein per minute.

16. SM 1 2 3 4 5 6 kDa 250 150 100 75 50 37 25 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.