Agriculture Reference
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9.3.2.3 Activation of PLD by calcium
Stimulation of PLD by calcium may arise through more than one mechanism. Two pos-
sibilities are calcium-dependent membrane association, which increases enzyme substrate
interactions and substrate availability, and direct activation of the enzyme by calcium. PLD
activity in strawberry fruit membranes was also stimulated by calcium (Fig. 9.8, top panel).
Both microsomal and mitochondrial PLD were maximally stimulated (sixfold stimulation
for microsomal membranes, threefold stimulation for mitochondrial membranes) between
1 and 3-mM added calcium. The assay mixture contained 0.2-mM ethylene glycol-bis-
-
aminoethyl ether,
N
,
N
,
N
,
N
tetra-acetic acid (EGTA) that acts as a calcium-buffering agent
and provides precise levels of free calcium, especially at low levels of added calcium. Micro-
somal membranes showed nearly fourfold stimulation between 0.15- and 0.25-mM added
β
Mitochondrial membrane
70.00
60.00
Microsomal membrane
50.00
40.00
30.00
20.00
10.00
0.00
0123
45678
9 0 1
CaCl
2
(mM)
Mitochondrial membrane
70.00
Microsomal membrane
60.00
50.00
40.00
30.00
20.00
10.00
0.00
0
0.2
0.4
0.6
0.8
1
1.2
CaCl
2
(mM)
Fig. 9.8
Effect of increasing calcium on PLD activity in mitochondrial and microsomal membrane of strawberry
fruit. Precise levels of free calcium levels were obtained by including 0.2-mM EGTA in the assay mixture.
Under these conditions, free calcium concentrations for 100, 150, 200, and 250
μ
M added calcium ranged from
≤
M, respectively. PLD 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 lower panel is an expansion of lower concentrations of
calcium shown in the top panel. The values are mean
1, 1, 15, and 40
μ
±
SE from three separate experiments. (Reproduced with
permission from Yuan et al., 2005.)
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