MEMBRANES AS ACCEPT 3RS FOR PALMITOYL teA IN FATTY ACID BIOSYNTHESIS M. SUMPER and H.

(1)

Volume 30. n u m b e r 1 FEBS LETTERS l'ebrt,ary 1973

M E M B R A N E S A S ACCEPT 3 R S F O R PALMITOYL t e A IN F A T T Y ACID B I O S Y N T H E S I S

M. SUMPER and H. T R A U B L E Max-Planek.Institut fi~r Bioph ysikalische Chemic,,

3400 Gi~tringen- Nikolau~berg. Germany Received ~. December 1972

Revised version received 20 December 1972

I. introduction

The biosynthesis o f phospholipids involves an in- teresting interplay between ble and membrane- bound enzymes. T h e f'mal assembly o f p h o s p h o l i p i d molecules is catalyzed by m e m b r a n e - b o u n d enzymes lli ;however the ~recursor substrates, palmitoyl ¢ o A and stearoyl C o A ~ . are synthesized (in yeast) by the

~01uble m u l t i e n z y m e complex fatty acid s y n t h e t a ~

|FAS). As is well k n o w n palmitoyl CoA associates easily and unspecifically with a variety of s o l u b l e en- t) n,es and causes their denaturation [2, 3 l . In line

~ t h this, the present study ~tows that newly synthe-

~i:ed pahnitoyl CoA cannot freely dissuciate l'tom Ihe F&S. Therefore a free diffusion o f individual palmi- ttyl CoA molecules ts highly improbable.

Thus rite question arises h o w is patmitoyl CoA tlansferred f r o m the FAS to the subsequent irletn- brahe-bound cnzytnes. The rate and m e c h a n i s m o1"

this transfer may '~¢ inaportant for the regulati~m o f tire lipid biosynthesis. The simplest mechanisnl might be the transfer through direct collis!ons b e t w e e n the FAS and the nlcmbrane-bound enzymes. A slightly illore sophisticated one ntisht involve lipid binding molecules, like bovine m r u m albumin (BSA), operat- ing us carriers o f pallnltoyl CoA, In both cases the transfer would be accomplished through the three- ttimensional diffusion of macromolecule.~

"'l"he term " p a l m i t o y l C o A " will be u m d bel,.~v, to denote bt~th contpotinds.

Alternatively, it is conceivable treat a c o m b i n e d mechanism operates in which palnlitoyl CoA is intro- duced intc~ target membranes and diffuses in the plane of the m e m b r a n e (cf. Adam arid Delbruek 14] ).This model presupposes that i) lipid membranes can serve as accepters (and as n reservoir) tot palmitoyl CoA and ii) that the pahnitoyl CoA molecules can rapidly diffuse within the plane of the membratle. T h e present paper provides experimental evidence in f.'tvour of this mechanism.

2. Materials and methods

The assay and purificatiotl of the fi~'tty acid svnthe.

tase frtml yeast fi~llowed procedures described in 15].

Accwyl C'oA, malonyi CoA and pahuitoyl CoA were prepared acct~rding Io t6 '-q.

liSA, CoASH and NADPH were commercial prod- nets frtma Behringwerke, Marburg mid Boehringer, Mannheim, respectively. [14C]Acetic anhydride was obtained from the Radiochemical Centre, Amersham, and I -onilino-napththalene-8-sul fonate (ANS ~ ) fiom Pierce Chemicals.

P,: coti K I062 pla-,ma a lembranes were kindly sup- plied by Dr. P. Overath, Ki~in. They were prepared according to Kabaek's me thod [ 10].

Dimyristoyllecithin was a product from Koch Light. The lipids were ultr.'tsomcalty dispers~'d at 30 ° for 3 - 5 mitt to yield optically clear solutions,

The kinetics o f pahnitoy[ CoA synthesis was measured by following NAi)Pil consumption u s i n g a

?¢"~'ttt !!otta~Jd l~sblL~hinl¢ C'ontpany ,,ttn,~rt'rdam 2~)

(2)

Volume 30, ntamber 1 FEBS L E T I E R S February 1973

i2

+ii

" ~ o s

g~ 0.7!

t

1

. . . . . . .

0 2 L 6 8

MINUTES

Fig. 1. E f f e c t o f lipid a c c e p t o r s t t b ; t a n c e s o n t h e k i n e t i c s o f f a t t y =cid s y f . t h e s i s (as m e a s u r e d b y fire+ N A D P H c o n s u m p -

t i o n ) . The incubation mixture (2 ml) contained: 200 umoles

potassium phosphate pl-I 7.5; 0.4 ~tmole NADPH; 0.2 ~moles acetyl CoA and 20 t.tg fatty arid synthetas¢ (2000 ruff/my).

.Ttte reaction Was initiated by the addition o f O-2 umoie m:~.lonyl C~;A. Curve (1): [ncu!yation mL,~ture without acceptor substances; (is): After a prolonged period of ~lILinhibi - tmn dimyristoyllecithin bilab'ers were added (3.8 × 10-:; M lipid). Curves (2)-(6): increasing concentrations of dimyris- myllecithin disl~.rsions: (2) 3.1 X I0 -6 M; (3) 6.2 X 10 -6 1~1;

(4) 12.5 x ~0- M;(5) 5 x I0 -s M;(6) l.l X 10 --4 M. Curve (7): ira the ;~resencc of t mg bovine serum albumin (about 7.5 × t 0 -~ :M). Curve (8): In the presence of E. coli plasma membranes in a concentration corresponding to 2.5 × lO -s ~i lipid.

Cary 14. A :total o f 14 N A D P H m o l e c u l e s is c o n s u m e d for ! p a l m i t o y l C o A . T h e f l u o r e s c e n c e d a t a were o b - tained with a Fica 55, an i n s t r u m e n t a l l o w i n g the re- c o r d i n g o f fully c o r r e c t e d differential spectra.

3. Results

10.

8

-~ £,.

E

o "

<~

FA.~

$ A

F ~

5 I0 15 20 FRACTION

Fil g. 2. Assay for the association o f the products of fatty acid s3-nthesis (Iong<ha/n | 14C|acyl CoA) witlt the fatty acid synthetase (FAS} and bovine .serum albumia (USA) by sucros~

density gradient centrifugation. The incubation mixture (2 rot} contained: 200 tamoles potassium phosphate pH 7.5:

0.+1 amole NADPH; 0.018 gmole [ "*Clacetyl CoA (10 ta?i//amole} and tOO tag fatty acid synthetase (2000 mU/mF).

"lille reaction wa'+ initiated by the addition o f 0.2 ~mole trmlonyl CoA- Incubation: 5 rain at 25 °. (A) Incubatitm in llt,,' presence o f 2 mg BSA. (B) Without BSA (producl-ir~Li- t/on occurs soon). Aliquots o f the incubation mixtures wer~

hyered on a linear s u c t o ~ density gradient ( 5 - 2 0 % ) in : 0. ]. M potassium phosphate pH 6.5. After centrifugation ft~i 5.,'i hr at 40 000 rpm and 10 ° (SW 40, Beckman L 2q55) the fractions were analysed for long chain [ taClacyi CoA in th. ~ following way: 0.2 mg BSA and 2 ml 5% tricbloroacetie acid

~ere added to each fraction; the precipitates were colIectt'd on millipore f'dters (HAWP 0.45 jam) washed 6 times with 5%

trichloroacetic acid and assayed for radioactivity after drying Palmitoyl C o A a n d s t e a r o y l f o A , t h e p r o d u c t s o f

the FAS, are effective i n h i b i t e r s o f the e n z y m a t i c activity o f F A S | 11 ] . T h i s is d e m o n s t r a t e d by curves 1 and 7 in fig. 1. T h e s e curvea s h o w the kinetics o f the fatty acid synthesis in the p r e s e n c e (curve 7) a n d in the absence (curve !} o f BSA w h i c h is a p o w e r f u l ae- c e p t o r o f f a t t y acid derivatives. In the a b s e n c e o f BSA tile e n z y m a t i c activity is b l o c k e d a l m o s t c o m - pletely after a s h o r t p e r i o d o f synthes~s (rest a c t i v i t y a b o u t 5%); this initial p e r i o d o f s y n t h e s i s will be de- noted in the f o l l o w i n g as the " a c t i v e p e r i o d " . In the

p r e s e n c e o f B S A the e f f e c t o f p r o d u c t - i n h i b i t i o n is n o t o b s e r v e d , i t c a n be s h o w n t h a t the paJmitoyl C o A m o l e c u l e s are readily t r a n s f e r r e d f r o m t h e FAS

to BSA a n d t h a t t h e y r e m a i n q u a n t i t a t i v e l y attached t o the F A S in the a b s e n c e o f B S A . Using

[ 1 4 C l a c e r y l C o A as s u b s t r a t e t h e s y n t h e s i s was carried o u t ( A ) in the p r e s e n c e a n d ( B ) in t h e absence of BSA a n d the a s s o c i a t i o n a n d spatial d i s t r i b u t i o n o . [ 14CJpalmitoyl C o A was a n a l y z e d b y Sucrose dc~.;i~' g r a d i e n t c e n t r i f u g a t i o n . T h e results o f this analysi.~

are p l o t t e d in fig. 2 . In case ( A ) the | t 4 C l p a l m i t o y l

(3)

Volta le 30, number I FEBS LETTERS Fe:Jruary t973

1

F-

IAJ ~O----4--

t.) 1tt

o 113

ut F- _J

0 1 2 3 4

.e-,-

P A L M I T O Y L COA CONCENll%'~TIf..W~

Fig- 3. Decrease it, A N S - f l u o r e s c e n c e i n t e n s i t y ( 3 4 0 n m , 500 n m ) u p o n a d d i t i o n o f p a l m i t o y l C o A t o d ! m y r i s t o y l - lecithin dis v e r s i o n s , 25 ~. A N S - c o n e . : 1.25 x tO - ¢ M ; O.l M potassium p h o s p h a t e p i t 7.5. C u r v e ( ! ) : ! . 2 5 X IO - a M lecithin; C a r v e ( 2 ) : 0 . 6 2 x IO - 4 M l e c i t h i n : C u r v e (3): I n t e r - zcti0u o f A N $ - w i t h p a l m i t o y l C o A .

CoA sediments q u a n t i t a t i v e l y with BSA w h e r e a s in case (B) it remains associated with the FAS. Palmi- toyl CoA was n o t f o u n d in d e t e c t a b l e a m o u n t s in free solution. This suggests t h a t direct collisions between the F A S a n d BSA are necessary to transfer the paknitoyl C o A m o l e c u l e s f r o m the F A S t o BSA.

From curve I o f fig. ! o n e e s t i m a t e s t h a t a b o u t 125-- 150 p a l m i t o y l C o A m o l e c u l e s are syilthesized per FAS c o m p l e x (M.W. = 2.3 X 106) in tile " a c t i v e period". Titus the n e t p r o d u c t i o n o f p a i m i t o y l C o A iu a ~Vetl i n c u b a t i o n m i x t u r e d u r i n g the " a c t i v e period'" is e x p e c t e d to increase linearly with the en- zyme c o n c e n t r a t i o n . This is fully s u p p o r t e d h ¥ the

relevant e x p e r i m e n t s ( n o t s h o w n ) .

Curves 2 to 6 in fig. I d e m o n s t r a t e t h a t natural membranes a n d lipid bilayers are also p o w e r f u l accep- tots o f long-chain acyl C o A c o m p o u n d s f r o m the FAS.

T'nese curves show the k i n e t i c s o f the f a t t y acid sytt- thesi~ in the presetice o f increasing a m o u n t s o f di- my! istoyllecithin bilayers. Curve 8 s h o w s the result o f ml;'togous e x p e r i m e n t s in which E. colt plasma mem- b,a.es served as a c c e p t o r s . T h e net a m o u n t o f pahni- toyi CoA t h a t can be s y n t h e s i z e d in t h e " a c t i v e period" increases linearly with the lipid c o n c e n t r a t i o n , indicating that a given lipid m a t r i x h a s a well-defined capacity f o r the a d s o r p t i o n o f p a l m i t o y l C o A . F r o m

1.2.

1.0

~: a6

C.3 -5.

0.4

. l~a/- f,oA t

0 2 4 6 B

MINUTES

Fig. 4. Comparison o* the kinezics of palmitoyi CoA synthesis (1) and of ~he incorporation of palmitoy! CoA into di- myristoyilecithin bilayers (2,1. Curve (2) shows the decrease in ANS- fluorescence intensity (410 mr*, 520 -am) in the course of the reaction (of. fig. 3). Identical incubation mixtures were used for both experiments containing 0.62 × 10 -4 M lipid and 5 × 10 -s M ANS- in addition to the substrales used for experiment I in fig_ I. The fluorescence decrease was measured against an identical inc[tbation mixture containing no ANS- (differential speetrumL

fig. I orte estimates that as m u c h as 1 7 - 2 0 p a l n l i t o y l CoA molecules can be i n c o r p o r a t e d p e r l OO lecithin molecules.

Fig. I shows that tile activity o f the p r o d u c t - i n h i b - ited e n z y m e can be restored by tim a d d i t i o n o f phos- phollpid vesizIes (curve la in fig. I).

T w o e x p e r i m e n t s were p e r f o r m e d to c h e c k w h e t h e r p a h n i t o y l CoA is actually i n c o r p o r a l e d into the lipid layers. A substitution titration was carried o u t using the fluorescence p r o b e ANS- as a s u b s t i t u e n t a n d in- dicator. S e c o n d , the e f f e c t o f palmitoyl CoA o n the t h e m m l phase transition o f d i n l y r i s t o y l l e c i t h i n was studied using optical m e a s u r e m e n t s [ 13, i 4 ! .

,~) A d s o r p t i o n o f ANS- to lipid m e m b r a n e s results in a strong e n h a n c e m e n t o f the fluorescence inten,';iLv (cf. review [ 12] ). When p a l m i t o y t C o A is a d d e d to a lipid dispersion c o n t a i n i n g A N S - . the f l u o r e s c e n c e intensity decreases, indicating the release o f A N S - f r o m the m e m b r a n e surface. One obvious rc~son f o r this effect is fire e l e c t r o s t a t i c repulsion o f the A N S - b y the i n c o r p o r a t e d palmitoyl CoA which carries 3--.4 negative charges per molecule. As s h o w n in fig. 3 the fluorescence intensity reaches a plateau for p a l m i t o y l _- CoA c o n c e n t r a t i o n s larger than .~ vahm c , c h a r a c t e r - istic for the lipid c o n c e n t r a t i o n . F r o l u file values o f

31

(4)

Volume 30, fiumber I

c determined by the curves i n fig. 3 we estimate t h a t a m~-.xlmum n u m b e r of" 2 5 p a l m i t o y l C o A m o l e c u l e s can be incorporated into the lipid layers per 100 lipid molecules. The fldoreseence decrease accompanying " ' th e incorporation o f palmitoyl CoA can.be utilized to measure ind~,~endently the rates o f synthesis (NADPH.consumption) and incorporation o f palmi-

toyl CoA. As d e m o n s t ~ t e d in fig. 4 these two pro- cesses are.synchronize&

• ii) A d d i t i o n o f p a l m i t o y l C o A to dispersions o f dimyristoyllecithin P.JUSes a significant broadening and a gradual disappearance of the lipid phase transition ~,t 24 °. This effect is characteristio o f the incor- porah 3n o f foreign molecules into a pure lipid matrix

[ 1 5 ! . .,

4. Discumsio~

The products o f th e fatty acid synthetase, palmitoyl CoA and stearoyI CoA, remain atta,,~ted .to the multienzyme complex and inhibit the enzymatic activity unless lipid accepting molecules, l ~ e BSP, or lipid membranes are'present in the soluffon. Blla'/ers o f dimydstoyllec[thin can incorporate as much as 20 palmitoy] CoA molecules per I O0 lipid molecules- The acceptor c z ~ a c i t y o f a given membrane is most probably determined by fixed m e m b r a n e charges and.

by the negative charge, of.the incorporated palmitoyl CoA.

"On the basis o f o u r findings we propose the follow- inl~ mechanism for the transfer o f palmitoyi CoA from the FAS to subsequent membrane-bound enzymes.

In a first step the palmitoyl CoA is carried to a place somewhere on the membrane by its synthesizingen-, zyme or, perhaps, by another lipid-carrier molecule.

(At present there is no evidence that the FAS is associated with membrane surfaces [18] ). Lateral diffu- s-ion within the plane of the membrane would be the mechanism by which the palmitoyl CoA is transferred to the subsequent membrane-bound enzymes. The following findings lend sup~ort to this hypothesis: i) In

a recent study-Admn and Delbruck. [4] _Ira.re shown

that a transfer mechanism, involving lateral diffusion in addition to free diffusion pern~its a much faster transfer o f a molecule from the cell cytoplasma to a small target on the

eell

membrane than.free diffusion alone. A detailed di~ussion o f this point is given in

F E B S £ ~ S ' " . + _~ - - F e ~ 19.73-

+

Io

Ill I

~e5 ,d-~ ou=Fusm~ • ~" I I I

• i l l l

" . ,:Ill

!°7' ' " " I

10

su, v~E o~mrv ~ umoe+s p , [ ~ " = ]

Flg. 5. C o n t o u r Rn_~_ enclodn 8 the regime in which the combined 3d--2d diffusion mechanism is more favourable than free cliff.ion alone (calculated according to eq..(4), Append~L q denotes the ratio 1"(-3~)/r0) o f the reep~ctlve transfer fimes;f t ts

the

target area. Simg~ contour lines are

obtained

if R is considered as constant.and ~creaslng values of D(3)/D(3 ) are.drawn on the o r d i n l t e scale.

the APpendix. (ii) The occurence o f rapid lateral diF- fusion ot" lipld soluble molecules iq~lipid brayers and' natural membranes has been est/tblished in recent ESR studies [16, 17l. T h e coefficients oFlatfral diffusion for androstane and fatiy acids are'as high as ( ] --3) X 10 -8 cruZ/see, corresponding to a ndt dis- placement o f these molecules b y a b o u t IO O00 A in one second. T h e ~ f o r e palmitoyl C o A m o l e c h l e s are also expected to be highly mobile within the plane of a lipid m~frix, in additi0n'to a higher t ~ s f e r rate the c o m b i n e d mechanism w o u l d also avoid compltca-.

tions due to the u n ~ e c i f i c association o f palmitoy]

CoA with' soluble protefiriS iwithin the cell (detergent properties o f p a ~ n i t o y l C o A ) . , "

+

. A ~ r d i n g t o ~ mock..[ a na+tural m e m b r a n e con- raining p .aL'nitoy! CoA ~ be visualized ~ a resev'oir o f substrates for the enzymes invotved in'tl/e furthe[

trm~r, erases, desatt/rases, etc.). ,The.rate o f fatty acid

biosynth .esi.'$

w o u l d ~ e n ' b e

gdcemdd~bythe acceptor

: capacity and the degree o[.sai...ufa~ u . o n o f t h e mem=

- b~ane ~ -s~. Oir

- in;close.~!o~y~i the'. !~~.estigated model systems. This interplay ." +n~ay Ue'- _imp o _rtmit for th~ regulation ofthe Hl~ICl bio~ynth~.

^{- i} ^" ^" ^"

32

(5)

VaJu/dz30,in~aber t Aeknowledsemmt,

We thank Prof. F- Lynen for helpful discussions

~ d s u p p o ~ , The interest o f Prof. M. Eigen in this work L~ gratefully acknowledged. We are indebted to D~..G. Adam for expert criticism and t o Dr. P.

Overath fo r providing E.-coli membranes.

Appendix: The a d ~ u ~ a g e o f laterat thffuslon in the transfer o f patmitoyl CoA f r o m tim f a t t y acid synthe-

to membrane bound o ~ y m e e

• We consider a spherical diffusion cell (radius R ) containing randomly diffusing FAS molecules which are ideally reflected b y the surrounding membrane. Thus,

macroscopic concentration gradients do not

exist

and

the average time ~'(3) required b y an FAS motecule to hit the membrane is governed by the self.diffusion o f the FAS. (The index (3) is used for three.d/mensional

diffusion).

T~3 ) is given by

J

- -

c e2/oo) (l)

where D(3 ) denotes the coefficient of free (three-di- mensional)

diffttsioct (/)(3) ( H 2 0 , 25 °) ~ 2 X

10 -7

c m 2 /

~ec for the F A S ) . L o w e r a n d upper limits for c are 1120 and 3/2. These values are obtained b y avera~nS ovei- the sho/test and longest times o f travel from all places in the cell to the enclosing membrane. In the following a mean value o f t - = 3 / 4 will be used.

We are interested in the average time ( r ) required for the transfer o f palmitoyl CoA from the FAS to target molecules on the membrane for the following two cases: i) the transfer is accomplis~,©d t h r o u ~ di- zect collisions between the freely d.itTusing FAS molecules and the

membrane

b o u n d targets (~O)); ii) the palmitoyt C o A L s / n c o r p o r a t e d randomly into the

membrane

(via

collisions

between

the

F A S and

the

membrane) and reaches the target molecules through lateral diffusion within the plane o f the membrane (T(3/2)). it w111 be kssumed: ( I ) that the area o f all tar- .gets i# negligibly small compared to the rest o f the membrane ~ (2) that the targets represent

"'sinks"

o f inf'mite capac~'ty and (3) that following the release o f peimitoyJ CoA f r o m the FAS new palmitoyl CoA m 0 l ~ are synthesized in a time which is short corollated

to

the transfer times 0-)- The average time

FEllS LETTERS

Febru azy

1973

1"(3 )

required by a freely diffusing FAS molecule to hit a target on the membrane is

"(3)

⁼

o)/pt

#

(2)

where Pr

denotes

the derLsity o f target

molecules

per

¢~n 2 o f the membrane surface a n d f t is the surf~ace -area o f one target molecule.

For the combined mechanism (free and lateral diffusion) the average time o f transfer is

where 1"(2 )

denotes

the average

time

of lateral diffu- sion

from an arbitrary place on the membrane to a target molecule. Since the target molecules represent sinks for the palmitoy! CoA, concentration gradients will be established within the plane o f the membrane.

For art exact treatment o f the diffusion.problem with- i n the membrane one has to stact from Fick's second

law o f diffusion and assumptions must be made about the distribution and density Of sinks and sources.

However, an approximate value for T(2 ) can be esti- mated easily if we consider the lateral diffusion of rite palmitoyl CoA as a random walk problem (self-diffusion) involving a series o f successive j u m p s o f length

?, within the two-dimensional lattice 'of lipid molecules.

One derives (eL [ 16] )

! ?,

= - ( 3 )

r(2)

2(rd+rr)ipt 4 ( r d + r t ) o t D ( 2 )

where i

denotes

the integrated diffusional path in ! sec (l=D(2)/2X) and (r d ÷ r t) is the sum o f the radiL o f the diffusing molecule (palrnitoyl CoA) and the target molecule. Using the values D(3 ) = 2 X l 0 -7 cm2/sec, /)(2 ) = 2 X 10 -8 cm2/sec. X = 8 A ( c f . [ 1 6 ] ] and as- suming (r d + rt) ~ 50 A we obtain for the ,-.'~H~z q o f the transfer times:

"r(3/2)-fp (l

X ^I

D(3)).~1t(~,t÷ 2 ~ ) q -

r(3) t t ~ R2

- - ' 3Pt(rd+rt) ~(2)

(4) For a given ratio o f tile diffusion c o e m c i e n t s

(Dt_3_)/Dt~l._. ~' !0 in our case) the ratio o f the transfer times is determined by the area (ft) and density (Pt) o f the targel moleculc~ and by the radius, R , o f the

diffusion

space.

33

(6)

Vblum~ 30; h~i.t~:r l

. , - _ • . - . . :

The

c6mbi~e'd

~ s f e r i ~ c h a n i s m bec~d~,s tnoge tar~.ts and in cTeasing ~i,~. :df t:he .diffudion,space. ! n .'

• fig. 5 c o n t o u r [~es are .drawn~.' an R v e ~ s ~o~-dia-~ : g r a m enclosing the ~ g i m ~ in which the c o m b i n e d . ~ mechanism is more fav..omble (q .-~ !).' F ° r a givenm'ea.

of th~ target molecules ffr) ~.e c 6 m b h ~ d ~_medm~igm..

is mo~.favorable " ifi).the, radius o f the diffusion '. -

'space ~ ~ , - ~ t~ana ~ a c ~ ~ u e ~ ' ~ d if u)-the

density'of tmget molecules is lower than a critical Val-.

Def'ming the " m o r e favorable'" regime by-q-_~ O. t w e

obtain

R cr ~ 200 A, p~r = 101 t / c r n 2 f o r

ft = 104 A2; an d R cr -~ 80 A, P~" = ! t)12/cm 2 for:- It = 103 A2._ • T ~ shows that in ~ e ca~e e o n d d e r e d

(Di3)/D(2)

"~ i 0) art extremely wlde range o f pare- - meters Oft; Pt,

R)

exists for which the combined" ""

mechanism (free and lateral diffu-~on) is m o i e favour-

able ~ --ee diffu.~on hlone. -

R e f e r e n c e s -

[ ! i W.C." MeMurray and W.L. M a ~ , Ann. Rev, Bloehem. 41 (1972) 129.

FESS ~ S ' t ~ t ~ ~97~:~

- . 7 ~ : ~ , ' : ' ~. . . " ' " ~ " _ : : . . . . -._. _

[3fw.~ .~,~: /~ ~.,,-.,~w.w.~,,. ~ . -

- [ 5 i F . - r s n m . " i n - _ m , t ~ , ~ ' , , d . ~ . . M . - . ~ o ~ , ~ , ~ , ~

. .. ( A ~ , d . ' . . t ~ . ~ w : C ~ k , ~ 9 ) 1.4, p . l ~ . : , . : .

[61.EJ.. ~ a.d

n . . ~ . . , ~ , ~

J. ~ ,"hem. ~

^7s

(tgs3)

2 5 2 0 . --. - . , : . . ; . -..

1:71 t ~ . ~ ~ d F_Lyn~, modt~.-Z.-3as (n~6~)

s 4 0 .

[81 .T.-W'm~m~ Natu~wlss.:38 (1951) 384:--, :~ ,.

. [ 9 1 T - W h ~ a m d ~ A a ~ v . ~ , C ~ a m ~ 6 - ~ . ( 1 9 ~ : 3 ) [ 8 6 . . ;

NeW" YO~k, 1971) 22,'p. 99..~

1 i21 G.K. ilxdda, "C~urr:-'rcHp[ci'Bioener 8. 4 (1"971) 81.--

!]31 H.-.Tt~I~. ; N a ~ 58.(1971) 2T?.-' ' .~ : [ 14] H. Trilmble, in: Biomemb~n~ 3, ~ F. gmuzer and

J.F.G. ~ Wienm.Pre~, 1972) p. 127.. "

[15] B.D.'Lad'orool~.~t.:M lO~]~tma.and D- Chapm',~, e i o -

~hem. Bio~ys. A c t a [ S O ( 1 9 5 8 ) 3 3 3 . + ~,, "'

! i 6 ] ~ t . . T r ~ t ~ a ~ E : , s , c ~ ; ~ , ~ , J. ~ o ~ . ~ 9 4

( 1 9 7 2 ) 4 4 9 9 . " " ,~ - .

[17] P. Devaux andFLM. McCmmdl, 1. Am. Chem. Soc. 94

- ( i 9 / 2 ) 4475: '~ -

[18] W. Ph'aon. Doclm-~ ~ ' M u n i ~ b 1970~

34