Reacción #2143504

ord-30555b9d73864a10998d1042d0b4c156

Ecuación de reacción

CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O[C@@H]2O[C@H](CO)[C@H](O)[C@H](O)[C@H]2O)[C@@H]1O
LacNAc
O=c1ccn([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)O[C@H]3O[C@H](CO)[C@H](O)[C@H](O)[C@H]3O)[C@@H](O)[C@H]2O)c(=O)[nH]1
UDP-Gal
O=c1ccn([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)O[C@H]3O[C@H](CO)[C@H](O)[C@H](O)[C@H]3O)[C@@H](O)[C@H]2O)c(=O)[nH]1
UDP-Gal
→
CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O
GlcNAc

Reactantes

—
QAE-Sephadex
CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O[C@@H]2O[C@H](CO)[C@H](O)[C@H](O)[C@H]2O)[C@@H]1O
LacNAc
O=c1ccn([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)O[C@H]3O[C@H](CO)[C@H](O)[C@H](O)[C@H]3O)[C@@H](O)[C@H]2O)c(=O)[nH]1
UDP-Gal
O=c1ccn([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)O[C@H]3O[C@H](CO)[C@H](O)[C@H](O)[C@H]3O)[C@@H](O)[C@H]2O)c(=O)[nH]1
UDP-Gal

Reactivos

Ninguno

Disolventes

—
solution
C[As](C)(=O)[O-]
cacodylate

Condiciones de reacción

Condiciones detalladas
See reaction.notes.procedure_details.

Tratamiento posterior

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

Procedimiento

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.

Fuente

DOI: 10.6084/m9.figshare.5104873.v1Patente: US07335500B2uspto-grants-2008_02