2.3.4 Enzyme activity measurement in Exp. 3
The methods for enzyme activity of starch synthesis were slightly
modified from those described by Nakamura,Yuki, Park, & Ohya, 1989 and
Zinselmeier et al. 1995. Four grains from each treatment were sampled at
DAP 10, 25 d after vitro culture in Exp. 3, then frozen in liquid
nitrogen for 2 min and stored at -80℃ for enzyme activity measurements.
The extract contained the following: 50 mM Hepes; 2 mM
MgCl2·6H2O; 3% (V/V)
(CH2OH)2; 1 mM DTT; 1 mM
EDTA-2 Na; 1 N NaCl.
ADP Glc pyrophosphorylase (EC 2.7.7.27) reaction system: 50 mM
Hepes-NaOH (pH 7.2) 100 μL; 10 mM
MgCl2·6H2O 50 μL; 15 mM DTT 100 μL; 1.5
mM ADPG 100 μL; 20 mM PPi 100 μL; Extract 200 μL.
Starch synthase (EC 2.4.1.21) reaction system: 50 mM Hepes-NaOH (pH7.2)
50 μL; 15 mM DTT 50 μL; 1.5 mM ADPG 50 μL; 1 mg Amylopectin; Extract 200
μL.
Sucrose invertase (EC 3.2.1.26) reaction system:
CH3COOH-CH3COONa buffer (pH 4.8) 200 μL;
100 mM Sucrose 200 μL; Extract 100 μL.
Sucrose synthase (EC 2.4.1.13) reaction system: 50 mM Hepes-NaOH (pH7.2)
100 μL; 10 mM UDP 100 μL; 10 mM
MgCl2·6H2O 50 μL; 100 mM Sucrose 150 μL;
Extract 100 μL.