A. Abousalham et al., STUDY OF FATTY-ACID SPECIFICITY OF SUNFLOWER PHOSPHOLIPASE-D USING DETERGENT PHOSPHOLIPID MICELLES/, European journal of biochemistry, 248(2), 1997, pp. 374-379
The fatty acid specificity of phospholipase D purified from germinatin
g sunflower seeds was studied using mixed micelles with variable deter
gent/phospholipid ratios. The main advantage of this approach is that
since the substrate is integrated in the detergent micelles, compariso
ns can be made between the kinetic constants of a wide range of phosph
atidylcholine (PtdCho) compounds with various fatty acid contents, Pho
spholipase D is subject to interfacial activation as it is most active
on water-insoluble substrates. It is not active on sphingomyelin and
only slightly on lysophosphatidylcholine. By fitting the curves based
on the experimental kinetic data, the interfacial dissociation constan
t of phospholipase B, the maximum hydrolysis rate V-m and the kinetic
constant K-m(B) were determined with the micellar substrate. The speci
ficity of various substrates was examined by comparing the V-m/K-m(B)
values, and it was noted that sunflower phospholipase D is most active
on medium-chain fatty PtdCho compounds, With long-chain natural phosp
holipids, the specificity of phospholipase D was slightly dependent an
the: level of fatty acid unsaturation. The purr: enzyme was able to h
ydrolyse the sunflower phospholipids present in mixed detergent micell
es but not the phospholipids integrated in the natural sunflower oil b
ody structure. We concluded, however, that during the germination of s
unflower seeds, phospholipase D might he involved in the degradation o
f oil bodies, since other factors present in crude seed extracts may m
ake phospholipids accessible to the enzyme.