Udp-Galactose → N-Acetyl-D-Glucosamine

HRID 429740

반응식

HRID 429740 반응식
[CH3:1][C:2]([NH:4][C@H:5]1[CH:10]([OH:11])[O:9][C@H:8]([CH2:12][OH:13])[C@@H:7]([O:14][C@@H]2O[C@H](CO)[C@H](O)[C@H](O)[C@H]2O)[C@@H:6]1[OH:26])=[O:3].C1C(=O)NC(=O)N([C@@H]2O[C@H](COP(OP(O[C@H]3O[C@H](CO)[C@H](O)[C@H](O)[C@H]3O)(O)=O)(O)=O)[C@@H](O)[C@H]2O)C=1>[As](C)(C)(=O)[O-]>[OH:11][CH:10]1[O:9][C@H:8]([CH2:12][OH:13])[C@@H:7]([OH:14])[C@H:6]([OH:26])[C@H:5]1[NH:4][C:2]([CH3:1])=[O:3]

참가 물질

4

반응물 2

HCID 18068Udp-Galactose2회HCID 9800166beta-D-galactosyl-(1->4)-N-acetyl-D-glucosamine

생성물 1

HCID 439174N-Acetyl-D-Glucosamine

시약·촉매·용매 1

HCID 167250Cacodylate용매

실험 절차

Initial velocities of the enzyme reaction were determined by measuring the rate of LacNAc formation with a slight modification of the assay by Pierce et al. [Pierce et al., Anal. Biochem., 102:441 (1980)]. All the reactions were carried out in 100 mM cacodylate buffer (pH 7.5) with fixed concentrations of Mn2+ (9.3 mM) and UDP-Gal (0.1 mM; 58.5 cpm/pmol of UDP-14C-Gal) in 100 mL of solution. The reaction was initiated by the addition of GalT (0.05 U, 120 mg protein; from Sigma) and permitted to stand at 20° C. for 30 minutes. Nonspecific hydrolysis of UDP-Gal was measured by the control reaction in the absence of GalT. The reaction was stopped by passing through a column of QAE-Sephadex (700 mL), and eluted by gentle air pressure to remove the unreacted UDP-Gal. The reaction vial was rinsed twice with 400 mL of water each and passed through the resin column. The filtrates were collected and directly transferred into a scintillation vial. The scintillation fluid was added to the vial, and then radioactivity was counted by a liquid scintillation counter. The data were analyzed by a double reciprocal plot to obtain Km (1.5 mM for GlcNAc) [a value of 1.3±1 mM was reported in Palcic et al., Carbohydr. Res., 159:315 (1987)] and Ki (0.46±0.06 mM) for UDP. Similarly, IC50 value of UDP for GalT was determined using different concentration of UDP.

후처리

  1. customInitial velocities of the enzyme reaction
  2. additionThe reaction was initiated by the addition of GalT (0.05 U, 120 mg protein; from Sigma)
  3. customwas measured by the control reaction in the absence of GalT
  4. washeluted by gentle air pressure
  5. customto remove the unreacted UDP-Gal
  6. customThe reaction
  7. washvial was rinsed twice with 400 mL of water each and
  8. customThe filtrates were collected
  9. additionThe scintillation fluid was added to the vial, and then radioactivity

출처 및 근거

ORD 기록
ord-05aae5b58f2242e8b57f42f1e8ec4a6d
출처 데이터셋
uspto-grants-2001_11
특허 번호
US06319695B1

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