Biology Reference
In-Depth Information
Phospholipase D (specific for PC, PLD (PC) ) can be used to measure the asymmetry of PC.
The enzyme catalyzes the reaction:
PLD ð PC Þ PA
PC
!
þ
Choline
Since the enzyme cannot cross the membrane, it only converts outer leaflet PC to PA. It does
not affect the inner leaflet PC. The PC and PA content is determined before and after hydrolysis
by PLD (PC) . The decrease in PC equals the increase in PA, and this represents the outer leaflet
PC content. From this measurement the membrane asymmetry of PC can be determined.
In a similar fashion, sphingomyelinase (SMase) can be used to measure the membrane
asymmetry of SM. The enzyme catalyzes the following reaction:
SMase
SM
ceramide
þ
phosphorylcholine
!
As with PLD (PC) , only the outer leaflet SM is hydrolyzed and SM and ceramide content is
determined before and after enzyme treatment. The decrease in SM equals the increase in
ceramide, and this represents the outer leaflet SM content. From this measurement the
membrane asymmetry of SM can be determined.
Some other types of phospholipases are less useful since they produce products that desta-
bilize the membrane. For example phospholipase A 1 or A 2 produce lysophospholipids and
free fatty acids, both of which adversely affect membrane structure. Phospholipase C
produces the non-lamellar phase-preferring compound diacylglycerol (Chapter 5).
Cholesterol is not hydrolysable and so its asymmetry must be determined using a very
different method. Cholesterol also presents an additional problem since its inherent rate of
trans-membrane diffusion (flip-flop) is so much faster than phospholipids. Accurate measure-
ment of cholesterol asymmetry is difficult and no method is without its flaws. One common
method employs the use of cyclodextrin (Chapter 10) [11] . A less commonly used method is
outlined in Figure 9.10 . It presents a theoretically easy UV method that would be available to
CHOLESTEROL
HO
inner
leaflet
CHOLESTANONE
outer
leaflet
O
TIME
FIGURE 9.10 Conversion of cholesterol to cholestanone by cholesterol oxidase. The initial rapid increase in
absorbance is due to oxidation of outer leaflet cholesterol to UV light-absorbing cholestanone. The slower oxidation
occurs after inner leaflet cholesterol has flipped to the outer leaflet. Cholesterol asymmetry can be determined from
the ratio of inner to outer leaflet cholesterol.
 
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