Sodium Fluoride → Oleoyl-CoA

HRID 1481958ノート

反応式

HRID 1481958 の反応式
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]

参加物

7

反応物 6

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

生成物 1

HCID 5497111Oleoyl-CoA

実験手順

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.

後処理

  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

出典と根拠

ORDレコード
ord-241171ba139f4f6cbabad722ac846dfe
出典データセット
uspto-grants-2016_08
特許番号
US09410168B2

反応ノート

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