F{byV1

~ ~~~ 01-~11.s;2V'~ Ed,'J-.:-.-

Ph~L' f L, Y-eQ~/q31-

tkA~ ~0- I )-1-(P1t>tfl-~

2

The Lipids of Cell Membranes

The most fundamental structure of the membranes of cells, the lipidbilayer, is formed from membrane lipids.. The membrane lipids generallyhave an amphipathic structure. One end ofthe lipid molecule has a polargroup, such as a phosphate, an amine, and an alcohol. The remainder of themolecule is usually hydrophobic with extensive hydrocarbon character.These lipids are organized in an aqueous environment according to thehydrophobic effect, which often leads to the formation of the lipid bilayer.The formation of lipid bilayers will be discussed in Chapter 4. Here thechemical structures of the lipids will be examined, since it is the chemicalstructure that most fundamentally determines lipid behavior.

I. PHOSPHOLIPIDS

A. Chemical Structure

A good place to begin the story of the lipids is by looking at the bestknown and understood of the membrane lipids, the phospholipids.2 Phos-pholipids derive their name from the phosphate group found in the polarheadgroups of these lipids. The general structure is shown in Fig. 2.1.Two nonpolar hydrocarbon chains are esterified to a glycerol which inturn is esterified to the phosphate in the 3' position. The phosphate, inits turn, is esterified to an alcohol. The name of the alcohol lends a furtherdistinction to the nomenclature of the phospholipid. In the absence ofany alcohol, the phospholipid is called phosphatidic acid. Phosphatidate,which refers to its normal, ionized form, is not found in large quantitiesin cell membranes, as it is largely a biosynthetic intermediate. This phos-pholipid will be known in this book by the abbreviation PA.

If the alcohol esterified to the phosphate is choline, the phospholipidis called phosphatidylcholine (or lecithin in an older nomenclature). Phos-18

I. Phospholipids 19

AlcoholoEb-p=o

o,'CHz

"- CHz

6c=oCHz

(CHz)mCH.

z'- CH6

i C=O2 CHz

(CHz)nCH.

AlcoholsCholine , HO CHz CHz~ (CH.).

HO CHz CHz~.

~'" .

HO CHz -CH

'coo-HO-CHz-CH-CHzOH

OH

Elhonolamine

Serine

Glycerol

~HH~ OH

Fig. 2.1. Generic structure for the phospholipids. The alcohols listed in the lower leftcan be substituted at the position marked alcohol in the upper right structure to form thevarious classes of phospholipids.

Inosllol

phatidylcholine (often abbreviated as PC) is one of the most common ofcell membrane phospholipids.

If the alcohol esterified to the phosphate is ethanolamine, the resultingphospholipid is called phosphatidylethanolamine (or cephalin in an oldernomenclature). Phosphatidylethanolamine (commonly abbreviated as PE)is another common cell membrane lipid. Note that PE contains a freeamino group. This amino group can be stripped of a proton at high pH(9-10) to give an uncharged, primary amine.

Other alcohols may be esterified to the phosphate. These include theL-amino acid serine, and sugars such as glycerol, and inositol. The phos-pholipids are then named, following the example of phosphatidylcholine,according to the corresponding alcohol. This nomenclature produces thenames phosphatidylserine, phosphatidylglycerol, and phosphatidylinosi-tol, respectively (commonly abbreviated as PS, PO, and PI, respectively).

20 2. The Lipids oCCell Membranes

!

!

The phospholipids so far listed include most of the common phospholipidsfound in cell membranes. All of them contain only one phosphate. Diphos-phatidylglycerol (cardiolipin in an older nomenclature), which is found inmitochondrial membranes, is unique among common phospholipids inthat it contains two diester phosphates linked by a glycerol (often abbrevi-ated as DPG). The structures of some of these phospholipids appear inFig. 2.2.

B. Electrical Charge

The chemical structure of the polar headgroup of these phospholipidsdetermines what charge the phospholipid as a whole may carry. Phosphati-dylcholine at physiological pH values carries a full negative charge on thephosphate and a full positive charge on the quaternary ammonium. Itthus is zwitterionic, but electrically neutral. Phosphatidylethanolamine isconstructed similarly. It carries a positive charge on the amine (whichcan be deprotonated as described above) as well as the negative chargeon the phosphate.

Phosphatidylserine contains, in addition to the negatively charged phos-phate, a positively charged amino group and a negatively charged car-boxyl. Therefore this lipid exhibits an overall negative charge at neutralpH. The amino group ofPS can also be deprotonated at high pH, whereasits carboxyl can be protonated at low pH.

The group of negatively charged lipids include phosphatidylglycerol andphosphatidylinositol. These phospholipids carry a net negative charge,because the sugar carries no positive charge to balance the negative chargeof the phosphate. Diphosphatidylglycerol normally carries two negativecharges, because of its two phosphates.

Interestingly, phosphatidylinositol sometimes appears in a phosphory-lated form with extra phosphates esterified to hydroxyls on the inositol.It then is referred to as di- or triphosphatidylinositol (PIP and PIP2,respectively), depending on the number of phosphates. Consequentlyphosphorylated Pis carry additional negative charge. The phosphatemonoesters are often esterified to positions 4 and 5 of the inositol (althoughother combinations are possible), whereas the diester occurs at position1 of the inositol.

The preceding discussion explains why some phospholipids carry netcharges. Those charges are held at the surface by the organization ofthe membrane lipid bilayer. Phospholipids can therefore be important indetermining the surface charge of the membrane.

As suggested by the variety of phospholipids just described, the phos-pholipid composition of cellular membranes can be complex. Individual

H@HH ' \ /HH~C, ...C HH-C...NH'I \ _AH C-H <J.

H/ ~c-O~p. 0

H~ ~Ii.. °'"c-O~C,H ,H

H'C/C:t! /C'HH' '\,..,H <\C.OIi.. /"H H..c/H'~C,H W 'C,HH,C/ 'H H,C/ 'HW ,-,H W '-.II

H, );:tt H'~C'HH'~ ,H H' ~H..C/C'H H,~ 'HH' :c,H W '~HH..-/ 'H H, / HWC, ,H WC'.H

WC'H H, /C'HWC, .II

CH,C/ 'IIW'H

A1\~H

H-~\ 1=1C-H r;r

HI ::;c-O ~.OH I I

H 0. --Ii;:""C'H

H O~C,~C,H.IIH- /~H /~HW~ ,H 0,-H, /C'" H );'0WC, ,H H):,-.HH../C'H H /!;"H

C XH' 'C,H H'';C

.HH../ 'H H, 'H

H'~C,H WC~HH,C/ 'H H'r! HIf' '-,H W-'-H

H~/C'H H,C/C'HW 'C,H H' 'c'H

Ii' 'H H'C/ 'HIi' 'c-liH'~ 'HH' 'H

H2C-OH C

HO~/HH B OH~ O'c_C_~ .-O.p,-O-I 1-o-p.O \C

,H1'06 H H eO/'1 0 / H0, 'H H-/0 H, ;C-O-C,H.H

o /SH H'C' H'~ .II Q"<::H

,\~'c~O-~H ~;C'O'c~~ H..C/~H CeOH, / 'H / H W"'O H 'C'H W-'C.H II. IH'~H <\C=O O.C/ H,C/ 'H H,C/ 'H H'~~H~'H ~/ ',H W,," H'~H 11./ HW-".c-HH'~H H /C'H H ;C'H "' / 'H H'C~HH..(;"-'H H.. / 'H H''C,-,H H)::: ,H H'C,.H II. / HH' ~H WC~H Ii.. /!;"H H);"''c'H II. /C'H H'C'c.HH'< 'H Y~H H'~C'H H' 'C.H H'~C,H H,C/ 'HH' C,H ~r~H H,c" 'H H'CF'H H,C/ 'H H' 'c:H"'C/ 'H "'C/ 'H Ii' );'H If' ):,H H' 'C,H H,C/ 'HH' 'c:H W 'c.H H,c.' 'H H~ 'H "'C/ 'H H' 'C.HH,C/ H H,C/ 'H Ii' 'c'H H' 'c,H H' 'C,H H,C/ 'HH' 'c,H W-'c,H H'C/ 'H H~/ 'H H..\:

/ 'H H' 'C.HW'H H,/'H H"oH H"H H' .II H,/'H

H'~c:H H' /C'H II. / H H'C,.HH,C/ 'H H'C, 'H H'C'H H, /C'HH''H ~'H ~

Fig. 2.2. (A) Chemical structure oC phosphalidycholine (leCt)and phosphatidyethano-lamine (right). (B) Chemical structure oCdiphosphatidyglycerol (cardiolipin). (C) Chemicalstructure oCphosphatidylinositol.

22 .2. The Lipids of Cell Membranes

membranes often exhibit distinct phospholipid composition with respect toheadgroup classes. For example, the phospholipid composition of severalbiological membranes are compared in Table 2.1. Such distinctions inmembrane phospholipid composition may be important to membrane func-tion. Some examples wi1l appear in Chapter 7.

C. Fatty Acid Composition

The division of phospholipids into classes according to their head groupstructure is only one level of complexity for biological membrane lipidcomposition. Even greater complexity is observed in the hydrocarbonchain composition within each phospholipid class. In one common formof phospholipids the hydrocarbon chains are esterified fatty acids. Bothsaturated and unsaturated fatty acids are found esterified to the glycerol.The length of the commonly found fatty acids varies from as few as 12carbons to as many as 26 carbons. The number of double bonds per fattyacid commonly ranges from none to as many as six. It is interesting tonote that in the case offatty acids with multiple double bonds, the doublebonds are almost never conjugated. (In a rare case from a plant source,a multiply conjugated fatty acid has been isolated, called parinaric acid.Parinaric acid is fluorescent and is commonly used as a fluorescent probefor studies of lipids in membranes.)- Common names are given to many of the fatty acids and these commonnames are frequently encountered in the literature. Examples includemyristic acid, with 14 carbon atoms and no double bonds, and oleic acid,with 18 carbon atoms and one double bond. In addition, the fatty acidsare often referred to by a numbering scheme. In this scheme, the numberbefore the colon describes the length of the fatty acid in carbon atoms,and the number after the colon refers to the number of carbon-carbondouble bonds. Thus 16: 0 refers to palmitic acid, a 16-carbon atom satu-rated fatty acid, and 18: I refers to oleic acid, an 18-carbon atom unsatu-rated fatty acid containing one double bond. Fatty acids are numberedfrom the carboxyl terminal, with the carboxyl carbon labeled as number1. The position of the double bond is indicated by the ~ nomenclature.As an example, ~9 refers to a double bond between carbons 9 and 10of the hydrocarbon chain. Thus, 18: I, ~9 refers to oleic acid. Anothernomenclature to indicate the position of the double bond is the (n- ) formatof the IUPAC-IUB. Here the position of the first double bond from theterminal methyl is indicated. Thus 18: I (n-9) designates oleic acid. A listof some of the common fatty acids and their common names appears inTable 2.2.

I!!

.51

e.....

,"=...f.~'" e\II"..,):0=....< C>~ =

~C>...eC>u...

'is.

~

::a(J)

'"

f""'I~NV"I!:; ~

u'" !::~IO~~ "''''1.1'\0"'''' <o:f'"

e~"'0.=U

11")00000'"- - \OOf'f'\V\-v v

3 e i~~ -a s~c:: 0'" (I)~ ':::--e e E E

8

/

v£ .~ -s-stt5 ~ ~ .g.gg=,o 0 ~ - .'" ~ 0 Q)'- 0en ...::J0..:::: - ... ....--

J:o(,J'~II)g. oo~«ft- '8 ~ ~.5 ~ 's 's :s ~o ....~ '" > ~ t ~ ~ 5 S~ .5 ~ :§ ~ :s~ gog. g g=,~~~.5.g«i-g].~.~:I:=~"'(J)~~\IIp.j::a::a

I I

.... ....

'" 0"':

I::;c:I

12 I

'"

M'"

C"I:!"Q,

I I00 ,.., -MM-

E=.9

lie:: II M

....oo...o\O

e0<)...'15.

;.j11$f16g!:;\0 - '" f""'I-MNN

ilon... on I I I. Phospholipids 25

MO\-\O"'O;t"'io\..o

Cf'f'\V")-o\V"I\O\Ot---oo1-111"1 I: TABLE2.3

Fally Acids Found in Egg Phosphatldylcholineand HumanErythrocyte Phosphatidylethanolamine

0

IA. Fally Acid Composition of Phospholipids'

g v I

0 . 0 oS Fatty acid Mol% in egg PC Mol% in erythrocyte PEE E .g. 0 . "0 .!:!'aE ..

'§'C 'g 0f;"fi g 'g.-S-S 16:0 33 19o Cu

jJ5;j 6;j;j< 16:I 2 -18:0 15 1318: 1 32 2218:2 17.8 720:4 4.3 19

.. 0 . 22:4 - 5.g '5 § §'g. § 22:6 1.7 4.. v aE E

B. Fatty Acid Combination Found in Egg PC'.!! .. g-g]fCR, R, Mol%:2 >- -g u U . ti

.. entiH-;,:J;:9 ":11-:'< ::: ::: t: t: t: t: 16:0 18: 1 45

C 16:0 18:2 31;: ." 'g'u 18:0 18:2 12.. ?;>"' 18:0 18:1 10.... 'E ." :r: 18:0 20:4 8"..:Ii! ." .!! 0v r:

I

=0 e

I

.From P. R. Cullis and B. De Kruijff, Biochim Biophys."::>Acta 513 (1978):31-42.r: ;;; :£:r:en C

. FromN. A.Porter,R. A.Wolf,andJ. R.Nixon,Lipids" ::> :r:80..: ,:Q o:r:O 14 (1979): 20-24.OU..II-;:'E -;:.:r::r:0en :r:u8

u N:r:The distribution of fatty acids in membrane phospholipids is peculiar:r::r:i5:5u::>

00 Yi:r: II to the class of phospholipid and the membrane type. As an example,u2 00 u:r:ci5 :r: II Table 2.3 lists the fatty acids found in egg phosphatidylcholine and humanu:r:

erythrocyte phosphatidylethanolamine.3 The mixtures are complex. AI-:£:£:£u:£88u :£u though the importance of a particular blend of fatty acids to membrane.:r::r::r:u:r:

:r::r::r::r::r::r: uuu:r:u function is not fully understood, maintenance of a suitable membrane000000 II II IIu888888 :r::r::r:llu environment by the fatty acid composition of the membrane phospholipids

:!; tt.tLC{:t:does appear to be important. Some specificideas on this subject willbe:£:£:£:£:£:£:£:£:£:£discussed in Chapters 4 and 7. Furthermore, arachidonic acid (20 :4) is888888 888u8

i'i'i'i'i'i' i'i'i'i'i' important metabolicallyas a precursor to prostaglandins.uuuuuu uuuuuAlthough a distribution of fatty acids is apparent in the examples given,

these fatty acids are not randomlyattached to positions I' and 2' of theglycerol.Unsaturated fatty acids are esterifiedpredominantlyat position

II r2' and the saturated fatty acids occur mostly at position l' (Table 2.3).4

000000 :::::1 This distribution of fatty acids arises from the specificity of enzymes.. .. .. ,. .. ..MOI:t\OOOO involvedin the biosynthesisof the phospholipids.----('IN ----(",I

26 2. The Lipids of Cell Membranes

Two isomers are possible for the double bonds found in the fatty acids.The isomers are referred to as cis and trans. As can be seen in Fig. 2.3,the two isomers can produce quite different looking molecules, especiallyif the double bond is in the middle of the fatty acid. The cis isomerintroduces a kink or bend in the molecule. This is a permanent structuralfeature of the fatty acid. On the other hand, the trans double bond doesnot introduce a strong structural perturbation into the fatty acid. As wiIlbe seen later, fatty acids with a trans double bond behave similarly tosaturated fatty acids of the same length. The isomer most commonly foundin biological membranes is the cis isomer. As a result, fatty acids withdouble bonds tend to perturb the membrane structure more than theirsaturated cousins because the double bonds necessarily force a kink toform in the hydrocarbon chain. In contrast, saturated fatty acids arecapable of a straight-chain all-trans configuration about the carbon-carbonbonds. This is one of the ways in which the fatty acid content of themembrane can affect membrane structure and properties.

Although many positional isomers exist for fatty acids containingcarbon-carbon double bonds, there are a relatively few isomers that aremost common. When a hydrocarbon chain contains one double bond, itusually occurs, in mammalian cell membranes, between carbons 9 and10. If there are two double bonds, they usually occur (in the longer fattyacids) between positions 9 and 10 and positions 12 and 13. Thus linoleic

. acid (18: 2) is cis-octadeca-9,12-dienoic acid. Oleic acid (18: I) is cis-octadeca-9-enoic acid. When more than two double bonds are includedin a fatty acid, this pattern does not necessarily hold. For example, arachi-donic acid (20: 4) is cis-eicosa-5,8,I1,14-tetraenoic acid. The positions

~eAlltransStearicacid

~ iSdoublebondOleic acid

e

trans double bondElaidic acid

~eFig. 2.3. Comparison of the cis and trans carbon-carbon double bond in a fatty acid.

I. Phospholipids 27

A e~

8 e~Fig. 2.4. Examples of branched-chain fatty acids. (A) Isopalmitate, (B) cis-II ,12-meth-

yleneoctadecanoic acid.

of these double bonds are determined during their biosynthesis by the.desaturating and elongating enzyme systems.

Not all the phospholipids contain hydrocarbon chains with the abovestructure. Some branched-chain fatty acids are found such as isopalmitateand isomyristate. Another variation is the inclusion of a cyclopropanering in the fatty acid. An example is seen in cis-II, 12-methyleneoctadeca-noic acid, shown in Fig. 2.4. Fatty acids containing cyclopropane can befound in bacteria like Escherichia coli when in an arrested state of theircell cycle.

D. StructuraUy Related Phosphorus-Containing Lipids

A further variation in lipid structure is found in a close relative ofthe phospholipid, the plasmalogen. The structure is quite analogous tophospholipids, with the exception occurring at position 2' of the glycerol.The hydrocarbon chain is attached through an unsaturated ether linkage.An example with ethanolamine as a headgroup appears in Fig. 2.5. Plasmal-ogens can constitute a major class of polar lipids in some membranes,including those of heart and brain in some species. Ethanolamine andcholine headgroups are found on plasmalogens.

Platelet-activating factor (PAF) is a particularly potent biological lipid,which also is of the general structure l-alkyl-2-acyl-glycerolphospholipid.The acyl group in this case is only an acetate. The structure of PAF isfound in Fig. 2.5.

Phosphonolipids, common in protozoa such as Tetrahymena pyri-formis,5 exhibit a different headgroup structure. Here the bonding betweenthe phosphate and the ethanolamine, found in PE, is replaced with a directcarbon-phosphorus bond (see Fig. 2.6). The l-alkyl-2-acyl-glycerolphos-phonolipid is commonly found, in which the alkyl group is connected tothe glycerol by an ether linkage.

28 2. The Lipids of Cell Membranes

H,,Ii 0 AW,N,,H I

H'C,C'-O...p.O H @H11'1 I H...I \ IH

H O,~ H~c~N-C'HH'C-O-CtH H H'I \ _A

H,p. '~H H IC,H 0:--C'C.H cf 'H H H-C-O~P=O

H, , 'H , ~ IH'C'C,H H..c'C=O ~H,C' 'H tr 'C.Ii II..O"c-O..c,HHH".Ii H,"H C;- C:H, ,C'H H'C, ,H If' H cf HH'C\. ,H H ,C'H 11..'H, ,C'H H;C,.Ii H~~H'C, ,H H, ,C'H H,C' H

H'C,C'H H'C'c.li II' ',...HII' 'c.li H,' 'H H'<'''II

H, "H H'C'.Ii H' ,..IiwC, ,H H, ,C'H H, /"11

H'I/'H H'C, ,H we, .HW ',H H, ,C'H H-C'~HH, ,C'H H'C, ,H H' , ..H

H'C'H H'C,C'H H,C'C);II' ';C,H II' 'c..H

H, 'H H,''HWC'H H'C'H

Fig. 2.5. (A) An example of plasmalogen. (B). Platelet-activating factor.

B

E. Characterization

The structures of the common membrane phospholipids have been eluci-dated through several approaches to structural analysis. A discussion ofthis topic provides the opportunity to get acquainted with common meth-ods of analysis in lipid biochemistry. 6Because of the differences in head-group structure, the various classes of phospholipids run differently whenanalyzed by thin-layer chromatographY- on silicic acid. An example ofsuch an analysis is shown in Fig. 2.7.' This is a two-dimensional thin-layer analysis, which is acidic in one direction and basic in the other.Spots appear after visualization with 50% (v/v) sulfuric acid spray followedby heating. (Aminophospholipids, such as phosphatidylserine and phos-phatidylethanolamine, can be developed using a ninhydrin spray, beforethe acid charring.) As can be seen, phospholipids with different head-groups are separated, providing a means for analyzing the phospholipidcontent of a membrane.

In order to obtain the phospholipids for such an analysis, the membranepreparation is subjected to a chloroform-methanol-water (8: 4 : I (v/v»extraction. In this two-phase system the phospholipids and neutral, hy-

I. Phospholipids 29

H,,Ii 0H"No..Ii I

wc::yP.OH'H b

H,C-O-C?~,p ,,:.Ii

H-C,~ /C,HH,C' 'H <\.C'OH",H ~'H..'C::H tr,.Ii

H'C,.Ii H)(C:HH, 'C:H II' ~HH'C\.c:H H~ HH,C' H II' 'c.IiII' ,-,H H,J' 'HH, '{;'H WC'-.IiH'c'_.Ii H,j\;'HH, '{;'H WC'.IiWC'_,H H, ,C'H

H'rf{;'H WC'c,HH' 'C,H H,c' 'HII..''H W'.IiWC'H H, ,C'H

H'C):

.Ii

H,C 'HH' 'H

Fig. 2.6. A phosphonolipid.

t

I

FFA £zPE :e. ...;; g

DPG PG Ph, _Sph

PA- PS.; LPC-(2) C/A/M/HAc/Hz\}

Fig. 2.7. An example of thin-layer chromatography on silica gel plates. Dimension I isrun in chloroform/methanol/ammonia (65/25/5, v/v},.anddimension 2 is run in chloroform/acetone/methanol/acetic acid/water (3/41111/0.5,v/v). Reproduced .with permission fromG. Rouser, S. Fleisher, and A. Yamamoto, Lipids 5 (1970):494.

30 2. The Lipids of Cell Membranes

drophobic lipids are extracted into the lower chloroform-rich phase. Verypolar lipids, like some glycolipids, are not effectively extracted with thisprocedure and remain in the aqueous layer. Proteins, including membraneproteins, precipitate at the interface between the two phases.

As discussed above, a wide variety of fatty acids are esterified to thephospholipids. This is an important observation for the properties of cellmembranes. How is information obtained on the fatty acid content of thephospholipid? To analyze the fatty acid content of a particular phospho-lipid from a membrane preparation requires three steps. First the phospho-lipid must be extracted and purified. Then the fatty acids must be hy-drolyzed in KOH and methanol from the glycerol and converted to theirmethyl esters. This done, the fatty acid methyl esters are ready for separa-tion by gas chromatography. Such an analysis for phosphatidylcholine16: 0, 18: I is presented in Fig. 2.8. By using standards, the original fattyacids can be both identified and quantitated. Recently, lipid analysis hasprogressed further with the application of high-performance liquid chroma-tography (HPLC), which permits the separation of each headgroup classof phospholipids. Furthermore, a single headgroup class of the naturalphospholipids may be separated into subclasses, each with different fattyacid compositions.s

F. Phospholipases

Phospholipids are substrate for a class of enzymes called phospholi-pases, which can hydrolyze the phospholipidat several differentplaces.

Retention Time ~J!1g.1.8. Example ora gas chromatograph run with 16:0 fatty acid (secondpeak)derived

by hydrolysis from 16:0, 18: I PC.

I. Phospholipids 31

Consequently, these enzymes can be useful tools in phospholipid struc-tural analysis and in studies on biological membranes. Phospholipasesalso play roles in lipid metabolism. For example, the release of arachidonicacid for prostaglandin biosynthesis and the release of diacylglycerol forprotein kinase activation result from the action ofphospholipases. Further-more, chemists have used phospholipases to assist in the synthesis ofphospholipids.

Examine the activities of these enzymes more closely. PhospholipaseA2 hydrolyzes at position 2' of the glycerol. These enzymes produce aderivative of phospholipids found in small quantities in membranes. Forexample, the action of phospholipase A2converts phosphatidylcholine toIysophosphatidylcholine, a phospholipid with only one hydrocarbon chainin position I' of the glycerol (see Fig. 2.9). This creates a molecule withproperties considerably different from those of the original phospholipid.For example, Iysophosphatidylcholine runs differently on a thin-layer si-licic acid chromatography plate in some solvent systems than does theparent phosphatidylcholine. Furthermore, Iysophosphatidylcholine hasdetergent-like properties, because of its tendency to form micelles (see

H H H HH l '<-/H H l \ IHH"'~N-c"'H H"'~N",C-HH-C \ H-C \H/A C-H rP. H/A C-H rP.

HI ~-o~p.o HI 'c_o~p.oH-j ~ H'j IH H H

o -..c1'H c~u £C--\r 'C::: H'o-y

:rz~ S.o ~c~o ~~.oIt'~ X~ PhospholipaseA.~c::: ~52

X;G

.. It'~ + H;C, .H~ 'Ii H ;c;H

-It ~~ ~~ H~~ ;;c'~ ~; :::C',.,H~ H /"'H ~ /"Ii H' /"'HIt' c.H It''c(;; H~c:~ t(C'c.-H

It' 'Ii ~Ai :x-~ :X-li~/c;: ~..}"'Ii H,i::::It' 'H H''''H H''''H

J!1g. 1.9. Representation of the action of phospholipase A on phosphatidylcholine. pro-ducing Iysophosphatidylcholine and a fatty acid.

'4".-,~;::~.:,',jJ': .:, '\~~" > "

32 ;I..The Lipids of Cell Membranes

Fig. 2.10.

CC ,£ D,\ c.. 0...,N 'C P~o

C CJ( .c,co I

A, r==> 'C_O-CC~ 'c

I IC 0

'c 'c=oI I

C C\ \C CI

C\CI

Representation of the sites of cleavage for the common phospholipases.

Chapter 4). Receptor-stimulated phospholipase A2 leads to the mobiliza-tion of arachidonic acid for prostaglandin synthesis.

Two other classes of phospholipase are commonly encountered. Phos-pholipase C cleaves the phospholipid between the phosphate and the 3'position of the glycerol, removing, for example, from phosphatidylcholinethe phosphorylcholine group and diacylglycerol. In the case of phosphati-dylinositol bisphosphate, phospholipase C releases inositol trisphosphate,an important second messenger involved in intracellular calcium release.The other product of phospholipase C hydrolysis, diacylglycerol, canstimulate protein kinase C activity.

Phospholipase D cleaves the phospholipid between the phosphate andthe alcohol and leaves phosphatidic acid. Referring again to phosphatidyl-choline, phospholipase D removes the choline alcohol (see Fig. 2.10).This reaction can playa role in phospholipid metabolism.

To act on the phospholipid substrate residing in a biological membrane,the phospholipase must gain access to the cleavage site on the lipid. Thismay entail modest penetration of the membrane bilayer surface and/or alimited extraction of the phospholipid substrate out of the bilayer surface.Figure 2.11 shows a representation of an interaction between phospholi-pase A2 and substrate. Major molecular contact is observed between thephospholipid headgroup and the enzyme.

Figure 2.11 suggests there might be some modest specificity of certainphospholipases for particular phospholipid headgroup classes. This expec-tation is fulfilled for some phospholipases. Together the phospholipasesoffer the biochemist a variety of ways to degrade selectively phospholip-

II. Sphingolipids 33

Fig. 2.11. X-ray crystal structure of the interaction between phospholipase A, andsubstrate. Major molecular contact is observed between the phospholipid headgroup andthe enzyme. Figure courtesy of Edward A. Dennis. Brent Segelke. David Fremont. Nguyen-Huu Xuong. and Ian A. Wilson.

ids.9 This can be useful, for instance, when it is necessary to determinethe accessibility of phospholipids from the aqueous medium, or to confirmthe influence of particular phospholipids on certain membrane behavior.

II. SPHINGOLIPIDS

Sphingomyelin is a sphingolipid and has a structure that is related tothe structure of phospholipids. Sphingomyelin is found in some plasmamembranes, including the human erythrocyte membrane where it appearsin approximately equal frequency to phosphatidylcholine.

I~

The structure of sphingomyelin appears in Fig. 2.12. This lipid containsthe phosphorylcholine headgroup and thus is zwitterionic like phosphati-dylcholine. It also contains two hydrocarbon chains like phosphatidylcho-line. A closer look however reveals several differences from the diacyl-phosphatidylcholines. The backbone of sphingomyelin is sphingosine,whose structure is also shown in Fig. 2.12. The most common form ofsphingosine contains 18 carbon atoms in the hydrocarbon chain. Somevariation is possible in the length of the chain. The only double bond isnear the polar end of the molecule and thus the hydrocarbon chain behavesin a manner similar to that of saturated fatty acid. The double bond issaturated in some sphingomyelins, creating dihydrosphingosine. Anotherdifference from phospholipids can be seen in the free hydroxyl group onthe sphingosine.

In sphingomyelin, the amino group of the sphingosine is esterified tofatty acids. These fatty acids exhibit considerable variability, as do thefatty acids in phospholipids, although the pattern of the composition isdifferent. Multiple-unsaturated fatty acids are not commonly found in

CH-CH-OH

~ ~C'O HC?Hz ?HzCHz CHzCHz CHzCHz CHzCHz CHzCHz CHzCHz CHzCHz CHz?Hz ?HzCHz CHzCHz CHzCHz CHzCHz CHI?Hz~2CHzCHs

Chemical structure of sphingosine and sphingomyelin.

34

Sphingosine

011

CHzHzN-C-H

HO-C-HCHCH

CHzCHzCHzCHzCHzCHzCHzCHzCHzCHzCHz

~:

Fig.2.U.

2. The Lipids of Cell Membranes II. Sphingolipid. 35

TABLE 2.4Fatty Acid Composilion of Bovine

Brain Sphingomyelin'

. From C. F. Schmidt, Y. Baren-holz, and T. E. Thompson, Biochem-istry 16 (1977): 2649-2656.

Sphingomyelin

'(CHs)s?HzCHz6

<2b-P'o6CHz-

sphingomyelin. Furthermore, the chain length is, on the average, some-what longer than the fatty acids of egg phosphatidylcholine discussedearlier. The fatty acid composition of bovine brain sphingomyelin appearsin Table 2.4.10(To obtain the fatty acids from sphingomyelin for analysis,acid hydrolysis must be used. The amide bond is reasonably stable tobase hydrolysis.) Because of the cumulative effects of these structuraldifferences from phosphatidylcholine, sphingomyelin runs on thin-layerchromatography in a manner different from that of phosphatidylcholine,even though both contain choline in their headgroups.

Other derivatives of sphingosine appear as lipids in membranes. Cera-mides consist of a fatty acid esterified to sphingosine. Adding a sugar tothe alcohol of the ceramide yields a cerebroside. Glucose and galactoseare frequently found as the sugar in the cerebrosides. A further derivativeof the cerebrosides adds a sulfate group to the sugar. These lipids arethen called sulfatides and are commonly found in brain tissue.

Among the fatty acids of these sphingolipids are some with free hydroxylgroups a to the carboxyl. These a-hydroxy fatty acids exhibit longerretention times during gas chromatographic analysis than do their nonhy-droxylated counterparts. Biosynthetically, these hydroxyls are added afterthe synthesis of the fatty acids.

Gangliosides are found at low levels in membranes (relative to phospho-lipids) but constitute an important component of the lipids of membranes.Gangliosides are derivatives of the ceramides with multiple additions ofsugars, ending with N-acetylneuraminic acid (sialic acid), a sugar acid

.~..-

Fatty acid Mol%

16:0 318:0 3620:0 I22:0 423:0 424:0 1025:0 524: I 3125: I 4

36 .2. The Lipids of Cell Membranes IV. Sterols 37

carrying a negative charge. The roles of these gangliosides are not allknown. However, Tay-Sachs disease is known to be a lipid storage diseaseinvolving one of the gangliosides. The binding of cholera toxin to mem-branes is believed to involve a ganglioside in the receptor-mediated interac-tion. An example of a gangliosideis given in Fig. 2.13.

Monogalactosyldiglyceridegalactose-0

CHt-CH-CH" "Coo Coo~z 9Hz, ,, ,

The last classes of sphingolipids discussed can also be termed glycolip-ids, because their polar groups contain carbohydrate. Lipids with otherstructures are also found in this class. Sugars can be attached to a glycerolvia a glycosidic linkage. If fatty acids are esterified to the other twopositions of the glycerol, glycosyldiglycerides result. The general structureis given in Fig. 2.14. These lipids are more closely related to the phospho-lipid structure than the sphingolipids; a sugar replaces the phosphate andalcohol. As with the gangliosides, a number of different derivatives canbe obtained through alterations in the structure of the polar headgroups

III. GLYCOLIPIDS

N-acetylgalactasaminegalactosegalactose . .--

sialic acid

sialic acid

Fig. 2.14.amide.

Monogaloclosylceromide

galactose-0CHt - ~ - ~HOH

NH CHCoO HC

?H (?Ht)ot: CHI

Chemical structure of monogaJactosyldiglyceride and monogalactosylcer-

glucose----CH20H

~°'v-~H2-~H - ~HOH~ NHCHOH CO HC

CHt CH2CH2 CHtCH2 CH2?H2 ~H2CHt CH2CH2 CH2CH2 CH2CH2 CH2CHt CH2CH2 CH2CHt CH2CHt CHtCH2 CHICH2CHtCHtCHI

and in the fatty acids. Glycolipids of this sort are often found in bacterialmembranes. The lipid composition of Acholeplasma laidlawii is given inTable 2.5.11Those lipids are also found in the thylakoid membranes. Thelipid composition of the thylakoid membrane may be important to itsstructure because of interesting behavior of the individual membrane com-ponents.

IV. STEROLS

Cholesterol is the most common sterol of mammalianplasma mem-branes. The structure of cholesterol appears in Fig. 2.15. It consists of a

TABLE2.sLipid Composition of Acholeplasma laldlawii Membranes

Grown on 18: 1 trans"

Lipid Mol%

MonoglucosyldiglycerideDiglucosyldiglyceridePhosphatidylglycerolGlycerophosphorylmonoglycosyldiglycerideGlycerophosphoryldiglucosyldiglyceride

1848222

10

Fig. 2.13. Chemical structure of ganglioside..From A. Weislander, J. Ulmius, G. Lindblom, and K.

Fontell, Biochim. Biophys. Acta S12 (1978): 241-253.

38 -2. The Lipids of Cell Membranes

Cholesterol

ffHO

HOCII. CH.

Fig. Z.1S. Chemical structure of cholesterol, ergosterol, and lanosterol.

planar, fused-ring nucleus, a polar hydroxyl group, and a hydrocarbontail. Cholesterol is quite different in structure from the other membranelipids that have been discussed. However, as with the phospholipids,cholesterol is an amphipathic molecule, containing both a hydrophobicand a hydrophilic portion.

The physical properties of cholesterol have been well studied and aredescribed in Chapter 5. Many of these properties derive from the rigidstructure of cholesterol, conferred on the molecule by the fused ringsystem. Modifications to the chemical structure, like removal of the tailor addition of methyl groups on the ring system (thereby rendering itnonplanar), destroy the ability of the derivative to behave like cholesterol.This last observation led to the suggestion that enzymatic removal of theextra methyl on lanosterol, an intermediate in cholesterol biosynthesis,to produce cholesterol was obligatory for proper membrane assembly.

Mammalian cells cannot grow properly without including cholesterolin their membranes, suggesting that cholesterol plays a crucial role inmammalian cell biology. Cholesterol is not found in all cell membranes.In some membranes, close relatives of cholesterol, like ergosterol or stig-masterol, take its place. In Tetrahymena a sterol-like compound, tetrahy-menal, takes the place and plays the role of sterol.

V. Detergents 39

V. DETERGENTS

Detergents have far-ranging use in membrane studies. Because deter-gents are amphipa:thic molecules, like lipids, some of the same rules gov-erning lipid behavior also apply to the detergents. Detergents come in twoclasses: nonionic and ionic detergents.

A. Nonionic Detergents

Nonionic detergents have polar headgroups that carry no charge. Inrecent years, the alkylglycosides have attracted much attention in mem-brane studies as nonionic detergents. Octylglucoside (OG) is one of themost highly used of these detergents. Its structure appears in Fig. 2.16.Relatives, such as laurylmaltoside, have also been used. One reason fortheir popularity is that a single molecular species can be obtained. There-fore, one only needs to worry about the stability of the detergent and itspurity with respect to the alcohols from which it is formed.

Other nonionic detergents include the Triton and the Tween species.Both of these detergents suffer from the fact that they usually are obtainedas a mixture of molecular species. This makes defining the behavior ofthe detergent rather difficult. There is, however, a series of similar com-pounds that are available in purer form. An example of these is CI2Es.Structures of these detergents appear in Fig. 2.16.

B. Ionic Detergents

As their name indicates, ionic detergents carry a net charge as part oftheir polar moiety. Sodium dodecyl sulfate (SDS) may be the most familiarof the ionic detergents, since it is used extensively in polyacrylamide gel

Octyl GlucosideCH20H

,);-O,C;>-CH -CH -CH -CH -C'L-CH -CH -CH~ 2 2 2 2 ", 2 2 3OH OH

Triton X-tOOCnH2n+, -Q-0-<CH2CH20ImH

C'2Ee CH3 - O::H2~- 0- [CH2- CH2- O]e-H

Fig. Z.16. Chemical structures of nonionic detergents.

1t

40 2. The .Lipids of Cell Membranes

electrophoresi.s. However, SDS suffers (as far as membrane studies areconcerned) from a tendency to denature proteins irreversibly. Further-more, SDS is difficult to remove from a solution. Interestingly, however,the Na+,K+-ATPase of plasma membranes can be effectively purifiedwith the aid of low concentrations of SDS which help to remove proteinsthat are only loosely associated with the membranes. The structure ofSDS is given in Fig. 2.17.

Other ionic detergents more commonly used in membrane studies in-clude cholate, deoxycholate (DOC), 3-[(3-cholamidopropyl)dimethylam-monio]-I-propane sulfonate (CHAPS), and dodecyltrimethylammoniumbromide (DTAB). The structures of these detergents are shown in Fig.2.17. Phospholipid analogs that act as detergents have also been de-veloped.

C. Chaotropic Agents

Chaotropic agents that change the properties of the aqueous phase facedby the membrane lipids are also useful in disrupting membrane structures.Examples of such agents include lithium diiodosalicylate (LIS) and guani-

CHAPS £ H /'00. I~so-N"""-"-""""'N :5~ I

HO H OH

SOS CH.- (CH. ),,- OSO; N.@

OTAB CD eCH.-(CH.),,- N(CH.). Br

Cholate(Sodium)

HOH

Fig. 2.17. Chemical structures of ionic detergents.

References 41

dine hydrochloride. These chemicals sufficiently change the thermody-namics of partitioning of the hydrophobic membrane components betweenthe membrane phase and the aqueous phase so that some membranecomponents can be removed from the membrane. LIS, for example, isused to extract glycoproteins from the human erythrocyte membrane.Because of the large carbohydrate (i.e., polar) moiety of these glycopro-teins, they are more easily solubilized than are other intrinsic membraneproteins of the erythrocyte, such as band 3. Thus this procedure achievesa selective solubilization. Proteins like band 3 require a more stringenttreatment, such as that achieved by an ionic detergent.

VI. SUMMARY

A wide variety oflipids are found in biological membranes. The varietyin structures leads to a collection of thousands of unique lipid chemicalspecies in cell membranes. Phospholipids are among the most common.Phospholipids are classed according to their polar headgroup structure.In addition they have considerable variability in the structure of the hydro-carbon chains esterified to the glycerol of the phospholipid (or in somecases linked by nonester chemistry). The chemical structures ofthe lipidscontrol the behavior of these lipids. The phospholipids can be degradedby phospholipases. Sphingolipids, built on the sphingosine base, and gly-colipids are also important components of biological membranes. Choles-terol is an essential constituent of mammalian cell membranes. Giventhe structural variety, thousands of lipid species are found in biologicalmembranes. As research continues, the roles of these many lipid speciesare gradually being defined. Detergents are useful tools for the study ofmembranes, since they assist in the purification of membrane proteins.Detergents can be classed as ionic and nonionic.

REFERENCES

1. Fox, C. F., and A. D. Keith, Membrane Molecular Biology (Stamford. Connecticut:Sinauer, 1972).

2. Chapman, D., An Introduction to Lipids (New York: McGraw-Hili, 1968).3. Cullis, P. R., and B. de Kruijff, "The polymorphic phase behaviour of phosphatidyletha-

nolamines of natural and synthetic origin," Biochim. Biophys. Acta 513 (1978): 31-42.4. Vance, D., and J. Vance, The Biochemistry of Lipids (New York: Benjamin, 1985).5. Pieringer, J., and R. L. Conner, "Positional distribution of fatty acids in the glycerophos-

pholipids of Tetrahymena pyriformis." J. Lipid Res. 20 (1979): 363-370.6. Kates, M., Techniques of Lipidology (Am. Elsvier, Amsterdam: North-Holland, 1972).