tina sui
(Tina Sui)
#1
Because of the increasing interest in using PLDs as catalysts for the synthesis of
new phospholipids (D’Arrigo and Servi, 1997; Servi, 1999), or in connection with
the biological importance of PLD, several methods for the determination of PLD
activity with different detection methods have been published in the recent past.
These are based on head group release by hydrolysis of [^3 H]-methylcholine labeled
PC, on transphosphatidylation with [^3 H]-acyl-PC or [^32 P]-PC, and on the change in
the amount of charged particles during the course of reaction (conductivity measure-
ments) (for a review of their advantages and drawbacks, see Morris et al., 1997).
Becker et al. (1997) developed a flow injection analysis (FIA) where the choline
produced in presence of PC and PLD is oxidized by choline oxidase (EC
1.1.3.17). The simultaneously released hydrogen peroxide is monitored by chemi-
luminescence detection (see below). The viability of this method has been demon-
strated by the characterization of PLDs from three different microbial sources with
respect to their kinetic data. The detection limit for choline is given with 75 pmol
min–1.
As already pointed out for PLA 2 the activity of PLD also depends strongly on
parameters such as co-factor concentration or the ratio of surfactants (SDS, Triton
X) to substrate concentration – in other words, the physical and chemical properties
of the interface. Furthermore, it should be considered that many PLD-catalyzed
syntheses are carried out in organic solvents which had been taken into account
by Aurich et al. (1999), who presented a simple, sensitive, and exact method for
the determination of PLD activity in emulsion systems with transphosphatidylation
of PC in presence of 1-butanol and PC dissolved in dichloromethane, and sodium
acetate buffer containing the PLD (Streptomycessp.). For the detection of products
arising from both the hydrolysis, as well as the transphosphatidylation reaction,
HPTLC proved to be a most suitable method.
The majority of the well-known analytical methods can be applied for the deter-
mination of PLD activity, and also for the analysis of phospholipid concentrations.
However, a major disadvantage of all these procedures is wasting of the often very
expensive enzymes applied. This problem can be overcome by using the involved
biocatalysts in immobilized state, as described by Masoom et al. (1990). Controlled
pore glass (Fluka) derivatized with 3-aminopropyl triethoxysilane was used for the
covalent binding of enzymes through glutaraldehyde as spacer. The authors estab-
lished two different flow injection systems. The first utilized a column containing
immobilized PLC that was connected with a column filled with alkaline phosphatase
(EC 3.1.3.1) and choline oxidase co-immobilized on the SiO 2 support. The second
approach made use of immobilized PLD and choline oxidase in two different col-
umns. The amount of H 2 O 2 developed through the action of choline oxidase was
determined amperometrically. The detection limits for PC in phospholipid mixtures
were 1 nmol and 10 nmol, respectively and the time required for one quantitation
including calibration with standard PC-solutions is 15 min.
Yaqoob et al. (1997) developed a flow injection procedure for the determination of
glycerol-3-phosphate (GP) and glycerolphosphorylcholine (GPC). A reliable method
for measuring GPC concentrations is important in connection with clinical investi-
gations involving epididymal secretion. In their study, the authors reported a flow
injection analysis with chemiluminescence detection. Based on the following
scheme:
13.4 Immobilization of phospholipases 285