Postharvest Biology and Technology of Fruits, Vegetables, and Flowers

206 POSTHARVEST BIOLOGY & TECHNOLOGY OF FRUITS, VEGETABLES, & FLOWERS
Table 9.1 Kinetic parameters of PLD toward
dipalmitoylphosphatidylcholine
Mitochondrial PLD Microsomal PLD
V max (nmol/mg protein min) 3.75 44.44
K m (μM) 114 277
V max /K m 0.033 0.160
Choline released (nmol/mg
protein)
Mitochondrial membrane
350
Microsomal membrane
300
250
200
150
100
50
0
0 100 200 300 400 500 600
Substrate concentration ( M)
1/[V]
0.12
0.1
0.08
0.06
0.04
0.02
y = 2.0255x + 0.0178
y = 0.415x + 0.0015
Mitochondrial membrane
Microsomal membrane
0
0.0000 0.0100 0.0200 0.0300 0.0400 0.0500
1/[PC]
Fig. 9.7 Substrate–velocity plot (top panel) of PLD activity in mitochondrial and microsomal membrane fractions
of strawberry fruit. Phospholipase D activity was measured by the release of radiolabeled choline from 16:0/16:0
phosphatidylcholine (L 3 -phosphatidyl (N-methyl- 3 H) choline, 1,2-dipalmitoyl) in a 1-mL reaction mixture resulting
from a 15-min reaction period. The substrate was prepared by mixing cold PC and radiolabeled PC in a ratio
of 1 nmol/3.7 kBq, also containing 0.1% Triton x-100 and 50-mM Hepes, pH 7.0 (final). Appropriate volumes
of the substrate were added to the reaction mixtures to give the desired concentrations. A separate blank without
the enzyme was made for each concentration of the substrate used to correct for the enzyme activity. The values
are mean ±SE from three separate experiments. Lineweaver–Burk plot of the data is given in the bottom panel
without taking the plateau region. (Reproduced with permission from Yuan et al., 2005.)