Biology Reference
In-Depth Information
O
O
O
CH
3
P
O
CH
O
C
N
H
C
HO
(CH
2
)
2
O
-
O
S
O
FMMP
CH
3
NO
2
SO
3
--
NO
2
TNBS
NO
2
NH
EAI
CH
2
CH
3
O
C
H
3
C
NH
IAI
CH
2
CH
2
SO
3
--
O
C
H
3
C
FIGURE 9.9
Types of primary amine tags used to label PE and PS in membrane lipid asymmetry studies: FMMP
(formylmethionylsulphone methyl phosphate, Bretscher's Reagent); TNBS (trinitrobenzene sulfonate); EAI (ethyl-
acetimidate); and IAI (isethionylacetimidate).
the two reagents is that IAI is charged and cannot penetrate the membrane and so only labels
outer leaflet lipids. In contrast, EAI is uncharged and so can readily cross membranes,
labeling both inner and outer leaflet lipids.
Enzymatic Modification
PE and PS both have a highly reactive primary amine on their polar head group. Therefore
they lend themselves well to covalent attachment by a number of reagents. However the
remaining membrane lipids do not possess such a handle and so other methods had to be
sought. Chemical methods do exist that can modify the non-primary amine head groups,
but these methods are much harsher and usually result in membrane destruction. Enzymatic
alteration of a polar head group can replace chemical modification. Enzymes have a tremen-
dous advantage in being highly specific for the substrate lipid. The large size of an enzyme
also makes it impossible to cross an intact membrane. Therefore, the enzyme will only alter
the outer leaflet lipid. While a variety of enzymes have been employed, only three will be dis-
cussed here: phospholipase D; sphingomyelinase; and cholesterol oxidase.
