Indiana University-Purdue University Indianapolis (IUPUI) / Cellular membranes contain a staggering diversity of lipids. The lipids are heterogeneously
distr
ibuted to create regions, or domains, whose physical properties differ from the bulk
membrane and play an essential role in modulating the function of resident proteins. Many
basic questions pertaining to the formation of these lateral assemblies remain. T
his research
employs model membranes of well
-
defined composition to focus on the potential role of
polyunsaturated fatty acids (PUFAs) and their interaction with cholesterol (chol) in restructuring
the membrane environment. Omega
-
3 (n
-
3) PUFAs are the main
bioactive components of fish
oil, whose consumption alleviates a variety of health problems by a molecular mechanism that is
unclear. We hypothesize that the incorporation of PUFAs into membrane lipids and the effect
they have on molecular organization may be, in part, responsible. Chol is a major constituent in
the plasma membrane of mammals. It determines the arrangement and collective properties of
neighboring lipids, driving the formation of domains via differential affinity for different lipids
. T
he m
olecular organization of 1
-[
2
H
31
]palmitoyl
-2-
eicosapentaenoylphosphatidylcholine (PEPC
-
d
31
) and 1
-[
2
H
31
]palmitoyl
-2-
docosahexaenoylphosphatidylcholine (PDPC
-d
31
) in membran
es with
sphingomyelin (SM) and chol (1:1:1 mol) was compared
by solid
-
state
2
H NMR spectroscopy.
Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are the two major n
-
3 PUFAs found in
fish oil, while PEPC
-d
31
and PDPC
-d
31
are phospholipids containing the respective PUFAs
at the
sn
-
2 position and a perdeuterated palmitic acid a
t the sn
-
1 position
.
Analysis of s
pectra
recorded as a function of temperature indicate
s
that in both cases, formation of PUFA
-
rich (less
ordered) and SM
-
rich (more ordered) domains occurred. A surprisingly substantial proportion of
PUFA was found to infil
trate the more ordered domain. There was almost twice as much DHA
(65%) as EPA (30%)
. The implication is
that n
-
3 PUFA
s
can incorporate
into lipid rafts, which
are
domains
enriched in SM and chol in the plasma membrane,
and
potentially
disrupt the activity of signaling proteins that reside therein. DHA, furthermore, may be the more potent component
of fish oil.
PUFA
-
chol interactions were also examined through affinity measurements. A novel method
utilizing electron paramagnetic resonance (EPR) was develope
d, to monitor
the partitioning of a
spin
-
labeled
analog
of chol
, 3β
-
doxyl
-
5α
-
cholestane (chlstn), between large unilamellar vesicles
(LUVs) and met
hyl
-
β
-
cyclodextrin (mβCD). The EPR spectra for
chlstn in the two environments
are distinguishable due to the substantial differences in tumbling rates
, allowing
the
population
distribution
ratio to
be determined by spectral simulation. Advantages of this approach include
speed of implementation and a
vo
idance of potential
artifact
s associated with
physical
separation of LUV and mβCD
. Additionally, in a check of the method, t
he relative partition
coefficients between lipids measured for the spin label analog agree with values obtained for
chol by isothermal titration calorimetry (ITC). Results from LUV with different composition
confirmed
a hierarchy of
decreased
sterol affinity for phospholipids with increasing
acyl chain
unsaturation
, PDPC possessing half the affinity of the corresponding monounsaturated
phospholipid.
Taken together, the results of
these studies
on model membranes demonstrate the potential for
PUFA
-
driven alteration of the architecture of biomembranes, a mechanism through which
human health may be impacted.
| Identifer | oai:union.ndltd.org:IUPUI/oai:scholarworks.iupui.edu:1805/5067 |
| Date | January 2013 |
| Creators | Williams, Justin A. |
| Contributors | Wassall, Stephen R., Decca, Ricardo, Petrache, Horia, Zhu, Fangqiang, Todd, Brian A. |
| Source Sets | Indiana University-Purdue University Indianapolis |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
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