Biology Reference
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can flip in. At t
X the vesicles are then exposed to another non-permeable primary amine
reactive reagent, IAI. IAI then reacts with the PE and PS that flipped from the inner leaflet to
the outer leaflet during the time interval X. After this, the non-reacted IAI is washed away
from the vesicles and the lipids are extracted, separated, and quantified (see Chapter 13).
From the measured amount of PE, PS, PE-TNBS, PS-TNBS, PE-IAI, and PS-IAI, the rate of
flip-flop for PE and PS can be estimated.
¼
Phospholipid Exchange Protein Method to Determine Flip-Flop Rates
Phospholipid exchange proteins (PLEPs, also called lipid transfer proteins, or TPs), offer
a versatile approach to measure lipid asymmetry and flip-flop. PLEPs were first discovered
by Wirtz and Zilversmit at Cornell University in 1968
[31]
. These are a ubiquitous family of
low-molecular weight proteins whose function is to shuttle various lipids from membrane to
membrane
[32]
. Since many PLEPs exhibit absolute specificity for a particular phospholipid,
they do not have to be totally purified but can be employed as a crude cellular mixture. The
experiments can be constructed in almost limitless combinations to address lipid asymmetry
and flip-flop. A simple example to determine the inherent flip-flop rate for PC in a protein-
free lipid vesicle is depicted in
Figure 9.20
. Large unilamellar lipid vesicles are made from
32
P-PC (the method is discussed in more detail in Chapter 13). To the LUVs are added mito-
chondria that provide a large excess of un-labeled (mitochondrial) PC. Mitochondria are
chosen since they are easily obtained and are much bigger and heavier than the
32
P-PC
LUVs. A simple low-speed centrifugation is sufficient to completely separate the LUVs
and mitochondria. To start the experiment, a source of crude PLEP
(PC)
specific for PC is
added. The mixture is incubated until all of the radio-labeled outer leaflet
32
P-PC (black
head group in
Figure 9.20
) is exchanged for the non-labeled mitochondrial PC (white head
group in
Figure 9.20
). The large excess of mitochondrial PC assures that all of the initial
LUV exoleaflet
32
P-PC is replaced by non-radiolabeled mitochondrial PC. The now radiola-
beled mitochondria and PLEP
(PC)
are then removed, leaving the LUVs with a radiolabeled
32
P-PC inner leaflet and a non-radiolabeled mitochondrial PC outer leaflet. The vesicles are
allowed to incubate for a period of time t
X,
the vesicles are exposed to new mitochondria and PLEP
(PC)
. Any
32
P-PC that has flipped
from the inner leaflet to the outer leaflet during the time
¼
X during which flip-flop can occur. At time
¼
X will now appear in the mito-
chondria that can be centrifuged and counted. The method is sensitive enough to detect
only a few flipped
32
P-PC lipids. This number (PCs flipped as followed by counts over
time
¼
¼
X) is compared to the total counts in the vesicles where only the inner leaflet was
radio-labeled. This experiment is then repeated for several longer incubations. From this
an extrapolated half-life for PC flip-flop can be obtained.
Inherent phospholipid flip-flop rates, as determined in protein-free model lipid bilayer
membranes, are very slow, often with half-lives in the days-to-weeks range
[27]
. In sharp
contrast, James Hamilton
[33]
has shown that the flip-flop rates for free fatty acids are very
fast, in the seconds-to-milliseconds range. Therefore it appears that moving the phospholipid
polar head group into the hydrocarbon interior must be the major impediment to flip-flop.
Supporting this is the very rapid flip-flop rate for diacylglycerol whose polar head group is
only a simple uncharged alcohol. Indeed, Horman and Pownall
[34]
showed that at pH 7.4
flip-flop rates increased in the order PC
<
PG
<
PA
<
PE, where the rate for PE was at least
