CHARACTERIZATION OF A REACTIVE OXYGEN SPECIES-RESISTANT HIGH YIELDING MUTANT R3-1 OF PISUM SATIVUM CV. ‘ALASKA’ WITH HIGH TEMPERATURERESISTANT PHENOTYPE - Pisum Sativum - page 61

to be used as a soluble fraction was aliquated to 0.1 mL and stored at -80 ℃ in
the dark. The resulting pellet was resuspended in 2.5 mL of resuspension
medium (0.25 M sucrose, 4 mM KNO 3 , 5 mM potassium phosphate buffer, pH
7.8, 120 mM N-methyl- D -glucamine, 0.01 mM leupeptine, 0.01 mM
pepstatine and 0.5 mM PMSF). The mixture was 10 times homogenized by a
small Teflon-glass homogenizer, and 0.1 mL aliquots were frozen at -80 ℃ in
the dark. Protein concentration was determined using BSA as a standard,
according to the method of Bradford (1976).
Phosphorylation of Proteins by [ γ - 32 P]ATP
The standard reaction mixture (20 µL) contained 4 µL of five-fold
concentrated reaction mixture [0.1 M PIPES (pH6.4), 0.5 mM
ethylenediaminetetraacetate (EDTA), 0.5 M NaCl, 7.5 mM MgCl 2 , 1 mM
PMSF and 3.6x10 8 Bq [ γ - 32 P]ATP], 2 µL of 1 % triton X-100, 4 µL of
resuspension medium containing 0.1 M NaCl, and 10 µL of the soluble faction
(5 µg) or the crude membrane (microsomal) fraction. After incubation for 15
sec on ice, the reaction was terminated by adding 12 L of six-fold
concentrated SDS sample buffer containing 10% SDS, 0.125 M TrisHCl, pH
6.8, 2 mM DTT, 10% glycerol and 0.004% bromophenol blue (BPB). The
radiolabeled proteins were then fractionated by SDS-PAGE slab gels of 12.5%
polyacrylamide essentially as described by Laemmli (1970). The proteins in
the gels were transferred to a nitrocellulose filter (pore size, 0.2m; Schleicher
and Schuell, Dassel, Germany) by electroblotting, and the radioactivity on the
filter was measured by exposure to a Kodak X-Omat AR film (Boston, MA,
USA).
Estimation of Chlorophyll Concentration of Leaf
The relative concentration of chlorophyll in a leaf was measured by SPAD
photometer (SPAD-502, Konica Minolta).