Postharvest Biology and Technology of Fruits, Vegetables, and Flowers

PHOSPHOLIPASE D, MEMBRANE DETERIORATION, AND SENESCENCE 205
Strawberry is a very acidic fruit, and the organic acids that cause this acidity are sequestered
in the vacuole. Thus, it was of interest to study the pH responses of PLD in strawberry fruit
(Yuan et al., 2005). The mitochondrial PLD was optimally activated at pH values of 5.5 and
6.5. The microsomal PLD also showed dual optimal values of activation at pH values of
5 and 7, respectively. Similar pH activation profiles have been observed in tomato microsomal
membrane (K. Tiwari, unpublished).
9.3.2.2 Kinetic analysis of PLD
PLDs belong to the class of phosphohydrolases with a broad substrate specificity, and can
catalyze hydrolysis of head groups from phospholipids such as phosphatidylcholine, phosphatidylethanolamine,
phosphatidylinositol, and phosphatidylglycerol. Under nonstimulated
physiological conditions, a considerable proportion of PLD may occur in the cytosol.
PLD is ineffective in the cytoplasm and does not exert its activity unless bound to the
membrane. Moreover, in a membrane-bound state, PLD is likely to encounter a variety
of phospholipids, toward which, it may show substrate preferences based on head group
and unsaturation of acyl chains (Brown et al., 1990; Paliyath et al., 1995; Pappan et al.,
1998). Thus, in the true sense, absolute kinetic parameters of PLD cannot be deciphered unless
the PLD molecule becomes membrane-localized as observed under in vivo conditions.
Nevertheless, analysis of substrate–velocity relationships under in vitro conditions may
provide insights into the kinetic properties of PLD localized in different compartments.
To study these aspects, mitochondrial and microsomal membranes were incubated with
varying concentrations of DPPC prepared from a mixture of unlabeled and radiolabeled
DPPC (1 nmol PC/3.7 kBq). Enzyme activities of both mitochondrial and microsomal PLD
followed Michaelis–Menten kinetics. PLD activity in the microsomal fraction showed a
linear increase with increasing DPPC concentration ranging from 0 to 200 μmol attaining
a maximal V max value of >300 nmol/mg protein per 15 min. When DPPC concentration
was increased above 200 μM, PLD activity decreased (Fig. 9.7, top panel). By contrast,
PLD activity in the mitochondrial fraction was considerably lower, showed only a minor
increase in activity with increasing substrate concentration (Fig. 9.7, top panel), and
reached a maximal velocity of 50 nmol/mg protein per 15 min between 200 and 250 μM
substrate concentration, sixfold lower than the maximal activity exhibited by microsomal
PLD. These results suggested that inherent differences might exist in the kinetic properties
of mitochondrial and microsomal PLD.
Transformation of the substrate–velocity data through a Lineweaver–Burke plot provided
further understanding of the kinetic properties of PLD (Fig. 9.7, bottom panel).
Analysis of the 1/v versus 1/[PC] plots was typically linear; the 1/v intercepts (1/V max )
and the X-axis intercept (1/[PC] or [−1/K m ], −0.00878 for mitochondrial PLD, −0.00361
for microsomal PLD) showing differences in kinetic properties between mitochondrial and
microsomal PLD. The kinetic parameters obtained from the Lineweaver–Burke plots are
given in Table 9.1. The V max of microsomal PLD (44.44 nmol/mg protein per minute) was
about 12-fold higher than that of the mitochondrial PLD (3.75 nmol/mg protein per minute).
K m values for the mitochondrial PLD and microsomal PLD were 114 and 277 μM, respectively.
The specific constant (V max /K m ) of microsomal PLD was fivefold higher than that of
mitochondrial PLD, suggesting that PLD in microsomal membranes has a higher catalytic
activity than that of PLD in mitochondria.