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FATTY ACID METABOLISM
Essential fatty acids in membrane: physical properties and function
CHRISTOPHER D. STUBBS* and
ANTHONY D. SMITH?
* Department of Pathology mid Cell Biology, Thomas Jefferson
University, Philadelphia, PA 19107, U.S.A. arid t Departmerit
of’C’hemicalPathology, Utiiversity College arid Middlesex
School of Medicine, Loridori WII’ 6DR, U.K.
The physicochemical properties of membranes are largely
governed by the nature of the fatty acid components. Membrane proteins, e.g. receptors, enzymes, ion channels, etc.,
are highly sensitive to the lipid environment. The critical
features of the fatty acid which govern the physicochemical
properties are the chain length and the position and number
of cis-double bonds. All of the major unsaturated w - 6 and
w - 3 fatty acids are considered to be essential in that a
dietary source is required [ 11. Therefore a great deal of effort
has been directed towards understanding the relationship
between these fatty acid properties and the physicochemical
environment and in turn its effect o n the protein functioning.
The primary function of fatty acyl unsaturation is to provide
a liquid-crystalline or fluid lipid bilayer phase which will
allow the various membrane proteins t o function optimally.
An unresolved question is the nature of the control of membrane protein functioning exerted at the level of fatty acyl
unsaturation. One direct functional role is of course t o provide the precursors of eicosanoids. These aspects have been
extensively reviewed elsewhere and will not be dealt with
here (see the other papers in this Colloquium).The release of
fatty acids by phospholipase A, will also create altered localizcd physical properties which will differ according to the
fatty acid type. In this article the major issue is the relationship beween essential fatty acids and membrane physical
properties (for other reviews, see 12-51).
Membrane proteins are surrounded by the fatty acyl constituents of membrane phospholipids. To what extent is the
function of the protein affected by the nature of the fatty acid
in terms of its chain length and unsaturation? This appears to
vary considerably from protein to protein. For example,
there is strong evidence that Ca?’-ATPase activity is affected
more by chain length and is insensitive to the degree of
unsaturation [6-8). By contrast, Na’,K+-ATPase does
appear to be sensitive to unsaturation 19-1 I]. An important
consideration is that just because the reconstituted enzyme
activity in question is supported equally well by any crystalline phase phospholipid bilayer, it does not necessarily follow
that the enzyme is not subject to more subtle modulation by
changes in unsaturation in the natural membrane. In addition, sensitivity of the enzyme to such changes may be lost in
a reconstituted enzyme. In general, because these types of
questions require complex reconstitution procedures the role
of unsaturation has not been examined in many systems. For
example, while transport processes examined in intact cells
or membrane preparations appear to be very sensitive to
modified unsaturation, as modified by dietary or cell-culture
means (reviewed in [ 12]), little is known of the response o f
particular channel proteins.
The fatty acid composition of cell membranes is highly
susceptible to dietary manipulation. However, it has become
apparent that this manipulation can only occur within very
specific limits; for example, an increase in 2 2 : 6 , w 3 at the
expense of 20:4,w6 [8,131. This leads to the question of
how membrane function may be directly altered by the modified fatty acid composition affecting the physicochemical
environment of the membrane.
Abbreviations used: T,, gel-liquid-crystalline phase transition
temperature; DPH, diphenyl-hexatriene.
There are a number of diverse types o f physical properties
which can be ascribed to a cell membrane and which are subject to modification by alterations in the level o f unsaturation. In addition, the membrane can be subdivided into at
least three distinct areas: the phospholipid head group
region, the central hydrocarbon chain region and the region
adjacent to membrane proteins. The major physicochemical
property of membranes and the most widely studied is
known as ‘membrane fluidity’ [ 14). The term membrane
fluidity is often misunderstood. The reason why investigation
of membrane fluidity is so popular is that it appears to be
relatively simple to measure, and can apparently be dealt
with as a single physical ‘parameter’. I t is o f course not surprising that such a complex dynamic structure as a cell membrane can only be inadequately described by a single physical
parameter. First, it ignores the two basic motional parameters of membranes which can be described as the rate and
order. The order relates to the packing of the fatty acid
chains which below the gel-liquid-crystalline phase transition temperature ( T,) is highly ordered (all-trans configuration), whereas above 7, the phospholipid fatty acyl chains
will contain a number of gauche rotomers that loosen the
fatty acid packing and cause a more disordered structure,
which is the situation in most mammalian membranes. Thc
degree of order and disorder can be obtained from various
types of spectroscopic measurements and a parameter relating to the extent of angular motion described by the fatty acid
chain segment obtained, i.e. its degree of orientational constraint. How fast the fatty acyl chain describes the angular
motion gives the rate of motion.
This rate of motion refers (with respect t o ?H-n.m.r., e.s.r.
and fluorescence anisotropy measurements) t o rotational
motion. Another rate term refers to the lateral motion for
which different physical techniques are used 1141. The term
membrane fluidity is used in general sense t o cover the rate
and order of rotational motion. Probably membrane fluidity
should refer only t o the rate of motion; however, the term has
taken o n wide usage as a semi-empirical term. In general, the
parameters it refers t o are the steady-state fluorescence
anisotropy of membrane fluorophore probes rather than the
time-resolved fluorescence components o f rate and order
[ 141. In this article wc use membrane fluidity only in a
general way to cover both the rate and range o f motion as
embodied in the steady-state fluorescence anisotropy parameter as commonly measured with the bilayer fluorophorc
probe diphenyl-hexatricne (DPH). Another major disadvantage of the use of the term membrane fluidity is that it is by
definition a bulk o r average tcrm which applies to the whole
membrane in question. This has been an attraction in sim ler
organisms and the theory o f homoeoviscous adaptation YlS I
has been invoked to describe the maintenance o f membrane
fluidity at different growth temperature by an altered lcvel of
unsaturation in the phospholipids. However, this rather elegant system does not appear t o translate very well t o more
complex mammalian systems, although the basic philosophy
behind the idea has influenced the field for a number o f
years. Importantly, it does not allow for distinct areas o r
regions of differing physical properties t o exist. Such areas
have been proposed in the past [ 161 and their attraction is
that they allow a membrane to respond t o differing compositional stress from diet or other means by tailoring the properties of specific regions according t o their needs and of
buffering sensitive regions from larger changes. Although
this approach has obvious attractions there is a lack o f convincing evidence.
There are a number of approaches t o the study o f the
effect of differing unsaturation on membrane properties. One
780
is to use dietary or cell culture manipulation methods. Dietary methods have been widely used since the results may be
o f more immediate direct significance, but again can only be
used to obtain modifications within certain limitations. With
cell culture thc limitations are less and to answer specific
questions the cells can often be forced to incorporate higher
levels of a specific test fatty acid than would be possible by
dietary means. The third approach is to study model or
reconstituted systems where the components of interest can
be isolated and a much simplified system can be used consisting only o f the components of interest. The advantage of
the latter approach is that conditions can be much more precisely set to test various aspects. It is also possible to isolate
particular membrane protein components and to examine
these with a precisely defined reconstituted system protein.
Since thc extent of modification of the membrane fatty
acid composition which the cell will allow is limited, it is not
surprising that the physical properties, in terms of bulk or
average membrane fluidity parameters, can also only change
by small amounts. Thus it seems reasonable to conclude that
the reason that the cell will not allow the proportion of saturated fatty acids to change appreciably is because the membrane fluidity would change in an undesirable manner. The
same applies to the incorporation of unsaturated fatty acids
such as 18:l,w6, 18:2,w6, 18:3,w6, 20:4,w6 or of
2 0 : S , w 3 and 22:6,w3. Faced with an apparently overwhelming dietary or cell-culture challenge of large amounts
of these fatty acids, the cell responds by incorporating the
fatty acid within fairly modest specified levels and compensating accordingly by reducing the fatty acid which it most
nearly resembles. Thus, for example, for a dietary elevation
of the highly unsaturated 2 0 : S , w 3 , 22:5,w3 and 22:6,w3,
as found in marine-oil-based food products, the 20:4,w6
will decrease accordingly (e.g. [ 81). Of course, although this
might maintain the physicochemical equilibrium, it has the
potential of changing the type o f eicosanoid formed, allowing
other biological effects to occur. One major question which is
unresolved is the nature o f the compensation mechanism
which appears t o allow sensing o f the membrane physical
properties in some form that can be recognized by the cell
membrane fatty acyl desaturase-elongase modification
system (reviewed in [ 1 71j. We would propose that whatever
the nature o f the system for sensing the membrane physical
properties in the cell membrane, changes to the membrane
fatty acid composition which result in significant overall bulk
changes t o the membrane physical properties cannot occur, a
process that may be termed a passive type of ’homoeoviscous
adaptation’ [ 81.
In order t o address the question o f the effect o f fatty acids
o n membrane physical properties in localized areas such as
domains o r adjacent to proteins, it is important to understand how each typc of unsaturated phospholipid component
behaves o n an individual basis. Using this type of approach
useful basic information on the behaviour of unsaturated
fatty acyl constituents in membranes can be determined.
First, with respect to the 7;. it has been established by
Keough and co-workers [ 18, 191 that while the first and
second cis-double bonds decrease the Tc, further additions
into the sn-2 chain o f a phospholipid eventually reverse the
effect with further double bonds actually increasing T,. The
extreme o f this is 16 : 0 / 2 2:6-phosphatidylcholine where T,
is almost the same as that of 16 :O/ I8 : I-phosphatidylcholine
[20-221. Also, after the first double bond in a phospholipid,
further double bonds have a much diminished effect compared to the first 1241. With the 2 2 : 6 chain it is probably the
16:O sn-1 chain which is actually undergoing the transition
due to the increasing stiffness introduced by the inflexible
carbon-carbon double bonds and also the helical configuration of the 2 2 : 6 chain ( [ 2 ,21, 251 and see reviews dealing
with 2 2 : 6 126, 271). How this chain behaves at the
BIOCHEMICAL SOCIETY TRANSACTIONS
protein-lipid interface compared with less unsaturated
chains is currently a topic of investigation.
In addition to the order and rate of acyl chain motion
there are other physicochemical properties worthy of investigation. For example, the fluorescence lifetime of a membrane
probe such as DPH is highly dependent on its immediate
environment or ‘solvent cage’ experienced while in the
excited state. By analysing the fluorescence decay process as
a distribution of decay rates, the degree of environmental
heterogeneity experienced by the fluorophore can be ascertained [28]. This will vary according t o the depth in the
bilayer, due to differing degrees of water penetration 128,
291, and this is increased for the more unsaturated fatty acids
(C. Ho, B. W. Williams & C. D. Stubbs, unpublished work). A
heterogeneity of decay rates is also caused by membrane
proteins, probably due to a combination of the hydrophobic
amino acid side chains and the adjacent fatty acyl chains [ 3 11.
In this case the role of unsaturation remains to be determined. However, due to the potential of this region in influencing protein conformation and function this should be a
fruitful area of future investigation.
The financial support of PHS grants A A 0 8 0 2 2 and 072 IS and a
grant from the Alcoholic Beverage Medical Research Foundation
are gratefully acknowledged.
I. Vergroesen, A . J . & Crawford, M. (d.)
( I YYO) The Hob, oj’l.ii/.s
in Human Nrt/ri/iori Academic Press, London and New York
2. Stubbs, C. D. & Smith, A . D.( I Y84) Hiochim. Hiophys. Acw
779.89-137
3. Spector, A . A . & York, M. A . ( 1 Y 8 S ) J. Lipid Hes. 26,
1 0 I 5- I 035
4. McMurchie, E. J . ( I Y88) in Physiologicd Kegiilciriorr o/’ Mcvnhrcinr Fluidity, pp. I XY-237.Alan Liss. New York
5. Quinn, P. J., JOO, F.& Vigh, L. ( I YXY) /'rag. Hiophys. M o l . I1iol.
53.7 1-1 03
6. East, J . M.,
Jones, 0.T., Sirnmonds. A . C. & Lee, A . G . (10x4)
J . Hiol. (’hem. 259.8070-807I
7. Gould, G.W., McWhirter, J . M., East. J . M. and Lee, A . G .
( I Y87)Biochem. J. 245.75 1-755
8. Stuhbs, C.D.& Kisielewski, A . ( 1090) />Ipil/.s in the press
9. Bruni, A,. van Dijk, P. W. M. & de Gier, J . ( 1075)Hiochinr. Riophys. AC/U406.3 15-328
10. Brivio-Haughland. K. P., Louis. S. L.. Musch, K., Waldcck, N . &
Williams, M. A . ( I Y76)Hiochim. l1iophy.s. Acw 433. I SO- 163
I I . Galo, M.G., Unates. L. €5. & Farias. K. N. ( I Y8 I ) J . Hiol. (’hem.
256.71 13-71 I4
12. Spector, A . A,, Mathur. S. N.. Kaducc. I., L. & Hyman. 13. -1.
( I Y 8 1 ) I’rog. LipidKes. 10,56-67
13. Conroy, D. C..Stubbs, C. D., Belin, J.. Pryor. C . L. & Smith.
A . D. ( 1986) Biochim. Hiophys. A m 86 1,457-462
14. Stubbs, C.D.( 19x3) I:‘ssriys Biochcwi. 19.I - 3 Y
IS. Sinensky, M. (1974)I’roc. N d . Accicl. S c i . U.S.A. 71.522-525
16. Karnovsky, M. J.. Kleinfeld, A . M.. Hoover, K. L. & Klausner.
R. D. ( I 982) J . C M mi.
94,I -6
17. Quinn, P. J. ( 198 I ) Prog Hiophy. Mol. Hiol. 38. I - I04
I 8. Keough, K. M. W.. Giffin. B. & Kariel. N. ( I YX7)H i o c h i n i . /1iophy.~.AC/O902.I - I0
19. Keough, K. M. W. & Kariel, N. ( 1987)Hiochini. Hiophys. Acrci
902,II-lX
20. DKKSK,
A . J., Dratz, E. A,, Dahlquist, F. W. & Paddy, M. K.
( I98 I ) Hiochemi.s/qj 20.6420-6427
21. Stubbs, C. D., Pryor, C . L., Nie, Y., Taraschi. 1’.F.. Ellingson, J .
& Mendelsohn, R. ( 1986) Hiophys. J. 49.3 13
22. Stubbs, C. D. ( 1089) C’O/~OC/. INSEHM 195. 125- I34
23. Reference deleted
24. Stubbs, C. D.. Kouyama, T.. Kinosita. K.. Jr. & Ikegami. A .
( I YX I ) Hiochrmisrr?/20,4257-4262
25. Applegate. K. R. & Glomset. J . A . ( 1 9 8 6 ) J. I i p i c l Hcs. 27,
658-680
26. Salem, N., Kim, H.-Y. & Yergey, J . A . ( I Y86) in Hralrh EJpc~so j ’
I’olyiirisciritrrtred F(i/rj, Acids iri .Secijbods (Simopoulos. A . P..
Kefer. R. R. bi Martin. R. E.. (eds.). pp. 263-3 17. Academic
Press. London and New York
78 1
FATTY ACID METABOLISM
27. Dratz. E. A. & Deese, A. J. ( 1986) in Health Eflects of I’olyctn.sutctmted /+city Acids in Secrjbods (Simopoulos. A. P.. Kefer.
R. R. & Martin, R. E.. eds.). pp. 263-317, Academic Press,
London and New York
28. Williams. B. W. & Stubbs, C. D. (1988) Biochemistry 27,
7994-7999
29. Stubbs, C . D., Williams, B. W. & Ho, C. ( 1990) I’roc. S I ’ I E
30. Reference deleted
3 1. Willliams, B. W.. Scotto. A. W. & Stubbs. C. D. ( 1990) Hiochemistry29,3248-3255
Received 25 April 1990
1204,448-455
Structural and enzymological properties of cellular phospholipases A,
HENK VAN DEN BOSCH.* ANTON J. AARSMAN,*
RON H. N. VAN SCHAIK? CASPER G. SCHALKWIJK,*
FRED W. NEIJS* and AUGUESTE STURKt
* Ccritrefor Riometnbranes arid Lipid Enzymology, Universiry
of Utrecht, Transitoriiim 3, Padiraluun 8,3.584 CH Utrecht,
71ie Netherlarids. and t Department of Hematology, University
of‘Atnster(lam, Academic Medical Center, Meibergdreef 9,
I105 AZ Atristerdam. The Netherlands
Introdirctiori
Phospholipases A,, which catalyse the hydrolysis of acyl
cstcr bonds at the sn-2-position of naturally occurring
phosphoglycerides, occur widely in nature. Pancreatic juice
and snake venoms are rich sources and detailed information
on the structure and catalytic mechanism of these enzymes is
available [ 11. The enzymes are also found in minute quantities in almost any mammalian cell, often associated with
more than one subcellular organelle, at least in vitro after cell
disruption [2]. A long-standing question has been what the
relationship in structural properties is between the extracellular and cellular enzymes on one hand and between thosc
in the different subcellular organelles on the other hand.
From the point of view o f cell biology there is a general interest in the regulation of cellular phospholipases A, because
the uncontrolled action of the membrane-associated
enzymes might easily destroy biomembrane function and
compartmentalization in eukaryotic cells. The enzymes have
also received much attention because they are implicated in
the release of arachidonate and 1- 0-alkyl-2-hydroxy-snglycero-3-phosphocholine (lyso-platelet-activating factor,
lyso-PAF ), i.e. precursors for bioactive lipids 13, 41. Whether
these precursors are formed by specific phospholipases A?
that exhibit specificities for either the polar headgroup, the
acyl-chain at the sn-2-position or the type of linkage at the
sri-1-position, i.e. an ester or an ether linkage, remains a
rather open question at present. This is largely the result of
two commonly encountered problems. First, cellular
phospholipases A? are extremely low-abundancy proteins
and only few of them have been obtained in sufficient
amounts and purity for detailed substrate specificity studies.
Secondly, the enzymes act at interfaces formed by the waterinsoluble substrates so that the physico-chemical factors that
govern substrate organization have a profound effect on
enzymic activity, as will be illustrated.
It~liierice of substrate organization
oti
phospholipase A
logical as it may be, be reconciled with the well-known
phenomenon that membrane-associated phospholipases A,
can be assayed with exogenously added substrates? Studies
on the substrate selectivity o f the enzyme provided a clue to
this problem. The membrane-associated enzyme showed a
2- to 3-fold preference for phosphatidylethanolaminc over
phosphatidylcholine hydrolysis when acting on endogenous
mitochondrial phospholipids (Fig. 1 ). A comparison o f the
two substrates, when added exogenously, indicated that the
preference for phosphatidylethanolamine hydrolysis became
20- to 30-fold. At first sight one would be inclined t o conclude from this comparison that the enzyme is rather specific
for phosphatidylethanolamine. However, the observed selcctivity does not reflect a true enzyme specificity but appears to
be governed by physicochemical factors. Phosphatidylcholine forms stable bilayer membranes that do not intcract
well with the mitochondrial membrane and, therefore, is
poorly degraded, unless a phosphatidylcholine-exchange
protein is included in the incubation mixture t o insert the
exogenous substrate into the biomembrane having the
enzyme [S].Alternatively, proper contact o f enzyme and substrate can be induced by addition o f detergents. In contrast,
phosphatidylethanolaminc. especially in the presencc of
Ca2+ ions necessary for measurements of phospholipase A2
activity, forms hexagonal structures that become easily associated with the mitochondrial membranes as could be
demonstrated by sucrose-gradient centrifugations 15. I. This
BIOMEMBRANE 6.uI
EXOGENOUS
SUBSTRATE
PC
MEMBRANE
ASSOCIATION
MODEL MEMBRANE
(PE PC = 40160)
PLUS ENZYME
.,A
PE
n ,
RATIO PWPC
20
- 30
READILY
.
3-4
uctivitv
Rat liver mitochondria contain a peripherally membraneassociated phospholipase A? that acts exclusively on substrates in its own membrane [S].How can this observation,
Abbreviations used: lyso-PAF, lyso-platelet activating factor ( 1 O-alkyl-2-hydroxy-s~~-~lycero-3-ph~~sphoch~~line);
PAF, plateletactivating factor.
Vol. 18
Fig. 1. Actrori of mernbrutie-associated mitochondria1 phospholipase A 2 towards endogenous arid exogerioiis substrutes
The large preference for exogenous phosphatidylethanolm i n e (PE) compared with phosphatidylcholine (PC) is
explained by its tendency to form hexagonal structures that
become easily associated with the biomembrane containing
the enzyme.