Potassium Chloride → Udp-Glucose

HRID 1957572

反応式

HRID 1957572 の反応式
CN/C(/NCCSCC1[O:17][C:16](CN(C)C)=CC=1)=C\[N+]([O-])=O.Cl.C1N(CCS(O)(=O)=O)CC[O:25]C1.[OH2:35].[Mg+2].[Cl-].[Cl-].C1N=C(N)C2N=CN([C@@H:48]3[O:52][C@H:51]([CH2:53][O:54][P:55]([O:58][P:59]([O:62][CH2:63][C@H:64]4[O:68][C@@H:67]([N:69]5[CH:74]=[C:73]([C:75]([NH2:77])=[O:76])CC=[CH:70]5)[C@H:66]([OH:78])[C@@H:65]4[OH:79])([OH:61])=[O:60])([OH:57])=[O:56])[C@@H:50]([OH:80])[C@H:49]3[OH:81])C=2N=1.C(N(CC(O)=O)CC(O)=O)CN(CC(O)=O)CC(O)=O.[Cl-].[K+]>>[CH:73]1[C:75](=[O:76])[NH:77][C:70](=[O:35])[N:69]([C@@H:67]2[O:68][C@H:64]([CH2:63][O:62][P:59]([O:58][P:55]([O:54][C@H:53]3[O:52][C@H:48]([CH2:16][OH:17])[C@@H:49]([OH:81])[C@H:50]([OH:80])[C@H:51]3[OH:25])([OH:57])=[O:56])([OH:61])=[O:60])[C@@H:65]([OH:79])[C@H:66]2[OH:78])[CH:74]=1

参加物

7

反応物 6

HCID 4873Potassium ChlorideHCID 6049Ethylenediaminetetraacetic AcidHCID 439153NadhHCID 3033332Ranitidine Hydrochloride2 回HCID 5360315Magnesium ChlorideHCID 162184172-(N-Morpholino)ethanesulfonic acid hydrate

生成物 1

HCID 8629Udp-Glucose

実験手順

N-acylsphingosine glucosyltransferase (EC 2.4.1.80) activity was measured in rat brain homogenates and mouse macrophage tissue cultured cell (WEHI-3B) homogenates using a method adapted as follows from published conventional procedures [Vunnam and Radin, Chem. & Phys. of Lipids 26, 265-278 (1980); Shukla and Radin, Arch. Biochem. Biophys. 283, 372-378 (1990)]: Dioleoylphosphatidylcholine and cerebroside liposomes containing 200 nmol ceramides Type IV (Sigma) were added to a reaction mixture (100 μl) composed of 40 mM 2-[N-morpholino]ethanesulfonic acid (MES) buffer, pH 6.5, 5 mM MnCl2, 2.5 mM MgCl2, 1 mM NADH and 8 μM UDP-[14C]-glucose (318 mCi/mmol, Amersham International, Amersham, U.K.). After incubation at 37° C. for 1-2 hr. the reaction was stopped by the addition of EDTA (25 mM) and KCl (50 mM). Radiolabelled glycolipids were extracted with 500 μl of chloroform:methanol (2:1 v/v) for 10 min. and the phases separated. The lower phase was washed twice with Folch theoretical upper phase and portions taken for scintillation counting. When imino sugars were tested for inhibitory activity, these were added at appropriate concentrations to homogenates and preincubated for 10 min. before sonication with ceramide containing liposomes. Control reactions were performed with liposomes containing no ceramide to measure the activity of transfer to endogenous acceptors.

後処理

  1. extractionRadiolabelled glycolipids were extracted with 500 μl of chloroform:methanol (2:1 v/v) for 10 min.
  2. customthe phases separated
  3. washThe lower phase was washed twice with Folch theoretical upper phase and portions
  4. additionthese were added at appropriate concentrations to homogenates
  5. custompreincubated for 10 min. before sonication with ceramide containing liposomes
  6. additioncontaining no ceramide

出典と根拠

ORDレコード
ord-08f10c478fec48eb83aa7c73e1a515ac
出典データセット
uspto-grants-1998_07
特許番号
US05786368

関連ノート

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