Sodium Fluoride → Oleoyl-CoA

HRID 1481958노트

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HRID 1481958 반응식
C(O)C(N)(CO)CO.Cl.P([O:22][CH2:23][C@H:24]1O[C@@H:27](N2C3N=CN=C(N)C=3N=C2)[C@H:26](O)[C@@H:25]1O)(OP(OP(O)(O)=O)(O)=O)(=O)O.C(N(CC(O)=O)CC(O)=O)CN(CC(O)=O)CC(O)=O.[F-].[Na+].CCC(CO[C:70](C(N(CC[NH+](C)C)C)=O)([C:77]1[CH:82]=[CH:81][CH:80]=[CH:79][CH:78]=1)[C:71]1[CH:76]=[CH:75][CH:74]=[CH:73][CH:72]=1)CC.[Cl-].[C@@H:93]1([N:131]2[C:140]3[N:139]=[CH:138][N:137]=[C:135]([NH2:136])[C:134]=3[N:133]=[CH:132]2)[O:130][C@H:102]([CH2:103][O:104][P:105]([O:108][P:109]([O:112][CH2:113][C:114]([C@H:117]([C:119]([NH:121][CH2:122][CH2:123][C:124]([NH:126][CH2:127][CH2:128][SH:129])=[O:125])=[O:120])[OH:118])([CH3:116])[CH3:115])([OH:111])=[O:110])([OH:107])=[O:106])[C@@H:96]([O:97][P:98]([OH:101])([OH:100])=[O:99])[C@H:94]1[OH:95]>>[C:23]([S:129][CH2:128][CH2:127][NH:126][C:124](=[O:125])[CH2:123][CH2:122][NH:121][C:119](=[O:120])[C@H:117]([OH:118])[C:114]([CH3:116])([CH3:115])[CH2:113][O:112][P:109]([OH:111])(=[O:110])[O:108][P:105]([OH:107])(=[O:106])[O:104][CH2:103][C@H:102]1[O:130][C@@H:93]([N:131]2[C:140]3[N:139]=[CH:138][N:137]=[C:135]([NH2:136])[C:134]=3[N:133]=[CH:132]2)[C@H:94]([OH:95])[C@@H:96]1[O:97][P:98]([OH:101])([OH:100])=[O:99])(=[O:22])[CH2:24][CH2:25][CH2:26][CH2:27][CH2:78][CH2:79][CH2:80]/[CH:81]=[CH:82]\[CH2:77][CH2:70][CH2:71][CH2:76][CH2:75][CH2:74][CH2:73][CH3:72]

참가 물질

7

반응물 6

HCID 5235Sodium FluorideHCID 59575'-AtpHCID 6049Ethylenediaminetetraacetic AcidHCID 19153X 100HCID 87642Coenzyme AHCID 93573Tromethamine hydrochloride

생성물 1

HCID 5497111Oleoyl-CoA

실험 절차

For acyl-CoA synthetase activity, the method of, for example, Kameda et al. (J. Biol. Chem. 256:5702, 1981) can be used by growing cells to mid-log phase in NSM with antibiotics as required, harvesting cells by centrifugation, washing twice with NSM, suspending the cells to a density of 1.2×109 cells/mL in 10 mM Tris-HCl, pH 7.5, and then lysing by three cycles of sonication on ice. Reaction mixtures are prepared, in a total volume of 0.5 ml, to include 200 mM Tris-HCl, pH 7.5, 2.5 mM ATP, 8 mM MgCl, 2 mM EDTA, 20 mM NaF, 0.1% Triton® X-100, 10 pM [3H]oleate, 0.5 mM coenzyme A, and cell extract. The enzyme reactions are initiated with the addition of coenzyme A, incubated at 35° C. for 10 minutes, and terminated by the addition of 2.5 ml isopropyl alcohol:n-heptane:1M H2SO4 (40:10:1). The radioactive oleic acid is removed by organic extraction using n-heptane, while oleoyl-CoA formed during the reaction remains in the aqueous fraction to be quantified by scintillation counting. Protein concentrations in the enzyme extracts are determined using the Bradford assay with bovine serum albumin as a standard.

후처리

  1. washwashing twice with NSM
  2. custompH 7.5, and then lysing by three cycles of sonication on ice
  3. customReaction mixtures
  4. customare prepared, in a total volume of 0.5 ml
  5. extractionextract
  6. additionThe enzyme reactions are initiated with the addition of coenzyme A
  7. customterminated by the addition of 2.5 ml isopropyl alcohol
  8. customThe radioactive oleic acid is removed by organic extraction

출처 및 근거

ORD 기록
ord-241171ba139f4f6cbabad722ac846dfe
출처 데이터셋
uspto-grants-2016_08
특허 번호
US09410168B2

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