反应详情

Gal-1-Phospate uridyltransferase (UT): Two separate methods were used to measure UT activity in light of the near absence of activity in melon fruit. A continuous coupled enzyme assay modified from Elsevier and Fridovich-Keil (1996), J Biol Chem 271: 32002-32007, was carried out in a 0.5 ml reaction mixture containing 50 mM HEPES-NaOH (pH 7.8), 5 mM MgCl2, 0.5 mM DTT, 10 mM Gal-1-P, 1 mM NAD, 10 μM gl-1,6 bis P, 1 U G6PDH (from Leuconostoc), 2 U PGM, and enzyme sample. The reaction was initiated by 4 mM UDP-Glc and monitored for 10 min at 37° C. The amount of Glc-1-P produced was expressed as the amount of enzyme necessary to produce 1 μmol Glc-1-P per min at 37° C. Alternatively, a two-step end point assay, modified from Main et al. (1983), Physiol Plant 59: 387-392, was used for determining UT activity. The reaction buffer contained 25 mM HEPES-NaOH (pH 7.5), 1 mM EDTA, 5 mM MgCl2, 0.5 mM DTT with addition of 10 mM Gal-1-P and 10 μl of partially purified enzyme sample in a 100 μl reaction mixture. The reaction was initiated by 4 mM UDP-Glc and stopped after 10 min by boiling for 2 min and the mixture was cooled on ice. For the measurement of the Glc-1-P product 400 μl consisting of 50 mM HEPES-NaOH pH (7.8) 5 mM MgCl2, 1 mM NAD, 10 μM gl-1,6 bis P, 2 U PGM and 1 U G6PDH. After 40 min incubation at 30° C. , absorbance of NADH product was recorded at 340 nm.

1. customGal-1-Phospate uridyltransferase (UT): Two separate methods
2. customwas carried out in a 0.5 ml reaction mixture