Agriculture Reference
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chloroform phase containing all the lipids, including the unreacted radiolabeled substrate
and the reaction products was removed, the chloroform evaporated in a stream of nitrogen,
and the concentrated lipid fraction subjected to thin-layer chromatography. The plates were
developed to separate the polar lipids such as the phospholipids, and the neutral lipids such
as diacylglycerols and free fatty acids. By quantifying the radiolabel in the defined lipid
classes (as confirmed by authentic standards), the amount of the substrate remaining and
the products formed were determined. Such experiments confirmed that the phospholipid
is broken down immediately to yield PA via removal of the polar head group (choline,
ethanolamine, etc.). PA rarely accumulated in the assay mixture, but appeared to be rapidly
broken down to diacylglycerols through the action of phosphatidate phosphatase. Diacyl-
glycerols were further catabolized to free fatty acids by the action of lipolytic acyl hydrolase
(LAH). LAH does not have positional specificity and can remove fatty acids from either the
sn
-1 or
sn
-2 position. If the fatty acids have a 1,4-pentadiene structure (as in linoleic and
linolenic acids), they may be acted upon by lipoxygenase, yielding hydroperoxide prod-
ucts as well as smaller-chain fatty acids and aldehydes. Accumulation of such degradation
products in the membrane causes destabilization through the formation of gel-phase lipid,
lipid microvesicles, and nonbilayer lipid structures.
The catabolism of radiolabeled phosphatidylcholine by broccoli microsomal membranes
is shown in Fig. 9.1. The broccoli heads were stored at 10
◦
C, and microsomal membranes
100
80
0
60
2
4
40
8
8
6
4
6
8
6
4
2
20
2
6
2
4
8
0
6
0
2
4
0
0
8
0
PC PA DG FFA FAOOH
Fig. 9.1
Changes in the formation of phospholipid degradation products by microsomal membranes from florets
isolated at various stages during storage of broccoli. The broccoli heads were stored in air at 10
◦
C and a relative
humidity of 75-85%. Phospholipid degradation activity was measured by incubating the isolated membranes with
radiolabeled 18:0/20:4 phosphatidylcholine and quantifying the levels radiolabeled degradation products after 1.5
h of incubation. The values are expressed as percentage of recovered radioactivity. The numbers above the bars
refer to days of postharvest storage. PC, phosphatidylcholine; PA, phosphatidic acid; DG, diacylglycerol; FFA,
free (unesterified) fatty acids; FAOOH, peroxidized free fatty acids. (Reproduced with permission from Deschene
et al., 1991.)
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