15.1. 1975
Specialia
o v e r c o m i n g b a c t e r i a l p e r m e a b i l i t y b a r r i e r s in clinical practice.
10 I am indebted to Dr. R. J. OLDS for his kind advice and to Dr H. B. F. DIXON for his generous gift of samples of pure TRIEN. This work was supported by the H. E. Durham Fund, King's College, Cambridge. 11 Present address: The Middlesex Hospital Medical School, London WIP 7PN, England.
85
Rdsumd. I n vivo aussi bien q u ' i n vitro, la t r i 6 t h y l 6 n e t 6 t r a m i u e ( T R I E N ) , a g e n t qui f o r m e des ch61ats avec les cations bivalents, renforce signifieativement l'activit6 a n t i b a c t 6 r i e n n e de l ' a n t i b i o t i q u e carb6nicilline envers la r~sistante Pseudomonas aeruginosa. C-. S M I T H 10, iX
St Catharine's College, University o/ Cambridge, Cambridge (Great Britain), 7 August 7974.
P h l o r i z i n B i n d i n g to Bilayer Vesicles of P h o s p h o l i p i d s and P h o s p h o l i p i d - C h o l e s t e r o l P h l o r i z i n i n h i b i t s s u g a r t r a n s p o r t in kidney, intestine, a n d e r y t h r o c y t e s ~,2. I n e r y t h r o e y t e s , it also i n h i b i t s a n i o n e x c h a n g e a n d increases a n i o n c o n d u c t a n c e 3,4. However,. t h e m e c h a n i s m of its i n t e r a c t i o n w i t h cell m e m b r a n e s is unclear. I n renal cell m e m b r a n e s , phlorizin was f o u n d t o b i n d n o t o n l y to a specific, h i g h - a f f i n i t y p r o t e i n r e c e p t o r ~, b u t also t o l o w - a f f i n i t y sites of unk n o w n n a t u r e ~, ~, p o s s i b l y i n c l u d i n g lipid. I n t h i s c o n t e x t , it s e e m e d useful t o s t u d y t h e b i n d i n g of p h l o r i z i n to s o n i c a t e d b i l a y e r vesicles of p h o s p h o l i p i d s a n d p h o s p h o lipid-cholesterol.
0.8 0.6 oo
% o
I
,
,
250
.
,
270
.
290
.
.
.
.
.
~ , - ~ ~
310 330 Wavelength (nm)
350
370
Fig. 1. Phlorizin absorbance spectra at pH 4.5 and 9.5 and the effect of PC vesicles. Pi~lorizin, 25 ~M; phospholipid, 2 mM. At pH 4.5: a) keto-phlorizin; aM, 1.62 • 104 M -1 cm-1 (285 nm); b) phlorizin and PC. At pH 9.5: e) enol-phlorizin; aM, 2.73• M 1 em-I (325 rim); d) pblorizin and PC. To amplify changes in spectra, lipid concentrations were increased to 4 times that used in the binding studies.
Methods. C h r o m a t o g r a p h i c a l l y p u r e egg yolk p h o s p h a t i d y l c h o l i n e (PC) a n d o x - b r a i n p h o s p h a t i d y l s e r i n e (PS) were o b t a i n e d f r o m Lipid P r o d u c t s (Sou t h Nutfield, U . K . ) . Cholesterol (99%) (Merck) was r e c r y s t a l l i z e d f r o m m e t h a nol. P h l o r i z i n (Roth) was p u r i f i e d to r e m o v e all traces of p h l o r e t i n 4. PC a n d P S d i s p e r s i o n s in 50 m M KC1 a n d 30 m M Glycylglycine buffer a t p H 7.2 were s o n i c a t e d a t 5~ u n d e r N 2 to m i n i m u m optical d e n s i t y T, s. Ph!orizin b i n d i n g to lipid vesicles was m e a s u r e d s p e c t r o p h o t o m e t r i c a l l y in d o u b l e - c h a m b e r m i x i n g c u v e t t e s (Hellma) a t 25~ The 2 c o m p a r t m e n t s of t h e reference c u v e t t e were filled w i t h e q u a l v o l u m e s (1 ml) of lipid vesicles a n d buffer. The s a m p l e c u v e t t e c o n t a i n e d vesicles in one c h a m b e r a n d phlorizin (20-75 /~M) in t h e other. The a b s o r b a n c e s of k e t o - p h l o r i z i n a t 285 n m a n d of enolphlorizin a t 325 n m were m e a s u r e d before m i x i n g a n d w i t h i n 2 m i d a f t e r w a r d s . On t h e a s s u m p t i o n t h a t t h e enolic f o r m of phlorizin is n o t b o u n d to t h e lipid vesicles, t h e c o n c e n t r a t i o n s of free a n d b o u n d k e t o - p h l o r i z i n were c a l c u l a t e d f r o m t h e s e a b s o r b a n c e s 9. Results and discussion. The a b s o r p t i o n s p e c t r a of t h e keto a n d enol t a u t o m e r s 1~of p h l o r i z i n are s h o w n in F i g u r e 1 (Curves a a n d c). Also s h o w n is t h e effect of PC vesicles on t h e spectra. The s p e c t r u m of p h l o r i z i n plus lipid a t p H 9.5 is identical t o t h a t of t h e enolic species alone. T h e s p e c t r u m of phlorizin plus lipid a t p H 4.5 differs slightly f r o m t h a t of t h e ketonic species alone. These o b s e r v a t i o n s suggest t h a t an association of t h e k e t o n i c species w i t h t h e p h o s p h o l i p i d occurs, a c c o m p a n i e d b y a c h a n g e in t h e m o l a r a b s o r b a n c y i n d e x (aM). The effect of P S on aM was q u a l i t a t i v e l y similar, b u t smaller. I n t h e calculation of free a n d b o u n d phlorizin c o n c e n t r a t i o n s , c o r r e c t i o n s were m a d e for this small c h a n g e in a~. T h e a d d i t i o n of liposomes t o phlorizin a t p H 7.2, close to t h e pK~ of phlorizin, lowers t h e a b s o r b a n c e a t 325 n m a n d increases t h a t a t 285 n m (Figure 2). T o g e t h e r w i t h
06
8
0.4
/'~ ~ / ~ ' o/" /'-
~ 0.2 9
0
/./
'
'
'
270
~
--\
-o-o-,o / "--0..._ N \ c ~,o \ \
~,~-
250
,~-,,./
O
~
,
,
,
290 310 Wavelength (nm)
,
330
,
,
350
-
~ 370
Fig. 2. Effect of PC and PS vesicles on the phlorizin absorbance spectrum at pH 7.2. Phlorizin, 25 FM; phospholipid, 2 mM. a) phlorizin; b) phlorizin and PS; e) phlorizin and PC.
1 A. KOTVK,J. KOLiNSKA,K. VERbs and J. SZAM~ER,Bioehem. Z. 342, 129 (1965). 2 p. G. LEFEvRE, Pharmac. Rev. 13, 39 (1961). 3 K. F. SCtINELL, S. GERHARDT, S. LEPKE and H. PASSOW,Biochim. biophys. Aeta 378, 474 (1973). 4 j. H. KAPLANand H. PASSOW,J. Membrane Biol., in press. 5 L. THOMAS, Bioehim. biophys. Acta 297,454 (1973). s H. GLOSSMANNand D. 3/[. NEVILLE JR., Biochim. biophys. Acta 323, 408 (1973). 7 E. G. FINER, A. G. FLOOK and H. HAUSER, Biochim. biophys. Acta 260, 49 (1972). s H. HAUSER,D. OLI~ANIand M. C. PHILLIPS,Biochemistry 12, 4507 (1973). 9 H. H. JAFF~ and M. ORCHIN, Theory and Applications o/ Ultraviolet Spectroscopy (J. Wiley, New York 1962), p. 557. 10 A. LAMBRECFITS,C.r. Acad. Sci., Paris 796, 295 (1933).
86
Specialia
EXPERIENTIA 31/1
Number and affinity of phlorizin binding sites of lipid bilayer vesicles
Preparations
K (I/mole) • 104
Pure Phospholipids (5) Phospholipids pIus Cholesterol (1:1) (5)
n (~zmoles/mmole PL)
PC
PS
PC
PS
2.1 • 0.2 1.5 ~_ 0.2
1.0 -[- 0.2 1.6 • 0.3
98 • 8 86 ~ 11
112 ~ 14 67 =t- 13
The association constant (K) and the number of binding sites (n) were calculated from the least squares regression lines of the Seatchard plot. Values are means ~ S.E. In parentheses number of measurements. PL represents total phospholipid. Lipid phosphorus was determined according to BARTLETT15.
t h e d a t a in F i g u r e 1, t h e i n v e r s e v a r i a t i o n of enolic a n d k e t o n i c p e a k s s t r o n g l y suggests t h a t t h e u n c h a r g e d , k e t o n i c f o r m of p h l o r i z i n b i n d s r a p i d l y to t h e p h o s p h o lipid bilayers. F i g u r e 3 shows t h e r e l a t i o n s h i p b e t w e e n b o u n d p h l o r i z i n a n d t h e c o n c e n t r a t i o n of t h e free k e t o n i c species. A S c a t c h a r d p l o t of t h e d a t a is s h o w n in t h e inset. As s h o w n in t h e Table, t h e a s s o c i a t i o n c o n s t a n t s of p u r e P C a n d P S b i l a y e r s differ s i g n i f i c a n t l y (p < 0.005), while t h e c o r r e s p o n d i n g n u m b e r of b i n d i n g sites do n o t (p > 0.4). Since a t p H 7.2 t h e vesicles of PC are isoelectric a n d t h o s e of P S n e g a t i v e l y c h a r g e d n, t h e s m a l l difference in affinities suggests t h a t t h e c h a r g e of t h e p h o s p h o l i p i d h e a d g r o u p s p l a y s a m i n o r role in t h e b i n d i n g process. Control experiments indicated that phlorizin penetrat i o n of t h e vesicles d u r i n g t h e b i n d i n g m e a s u r e m e n t s is negligible. T h u s , if i t is a s s u m e d t h a t all t h e p h o s p h o l i p i d is p r e s e n t in t h e f o r m of single b i l a y e r vesicles of 250 A d i a m e t e r s,~2, a b o u t 7 0 % of t b e t o t a l p h o s p h o l i p i d w o u l d b e o n t h e o u t e r vesicle surface ~, ~a, a n d h e n c e b e a v a i l a b l e for b i n d i n g . A c c o r d i n g l y , P C a n d P S b i l a y e r vesicles b i n d m a x i m a l l y a b o u t 150 # m o l e s / m m o l e p h o s p h o l i p i d or 1 p h l o r i z i n p e r 7 p h o s p h o l i p i d molecules.
Cholesterol (1:1 m o l a r ratio) lowers t h e p h l o r i z i n a f f i n i t y of P C b i l a y e r s a n d raises t h a t of P S t o r e s u l t a n t v a l u e s w h i c h are n o t s i g n i f i c a n t l y d i f f e r e n t f r o m e a c h o t h e r (p > 0.7). F o r t h e r a n g e of p h l o r i z i n c o n c e n t r a t i o n s studied, Scatchard analysis again indicated a homogeneous p o p u l a t i o n of b i n d i n g sites. T h e p r e s e n c e of cholesterol, does n o t a l t e r t h e b i n d i n g c a p a c i t y p e r p h o s p h o l i p i d molecule of P C b i l a y e r s s i g n i f i c a n t l y (p > 0.4) ; h o w e v e r , it t e n d s t o r e d u c e t h a t of P S b i l a y e r s b y a b o u t 4 0 % (p < 0.05). I f one a s s u m e s a size of a b o u t 250-300 ~_ for t h e c h o l e s t e r o l c o n t a i n i n g vesicles~% one c a n c a l c u l a t e t h a t c h o l e s t e r o l - P S vesicles b i n d m a x i m a l l y o n l y 96 /~moles k e t o p h l o r i z i n / m m o l e P S or 1 p h l o r i z i n p e r 10 P S molecules.
Zusammen/assung. S p e k t r o p h o t o m e t r i s c h e M e s s u n g e n a n P h o s p h o l i p i d - D o p p e l s c h i c h t e n zeigten, dass die K e t o - F o r m y o n P h l o r i z i n b e v o r z u g t g e b u n d e n wird. Phosphatidylcholin und Phosphatidylserin besitzen beide eine B i n d u n g s k a p a z i t g t y o n e t w a 1 Molekfil P h l o r i z i n p r o 7 Molekiile P h o s p h o l i p i d . I h r e A f f i n i t g t e n ftir P h l o r i z i n sind 2.1 • 10 4 bzw. 1.0 • 10 ~ 1/mole. Die A n w e s e n h e i t y o n C h o l e s t e r i n v e r / i n d e r t die B i n d u n g s p a r a m e t e r . G. ~ H R E N S P E C K 16
40
.2
.~ 3o
//
o_ E
PC/o
of /
Max-Planck- Institut /i~r Biophysik, Kennedyallee 70, D - 6 Franh/urt am Main 70 (German Federal Republic, B R D ) , 13 August 7974.
./
2o
.............o~.~...~C
~'~.~.
10
ps ...........
,.,/./'PS
"5 E
1; 10 ' 2'0 ' 3'0 Free keto*phlorizin {pM}
'
2;
bound
~'0
20'"
4;
Fig. 3. Concentration dependence of phlorizin binding. Graph depicts data of a representative experiment. A Seatchard plot of the data is shown in the inset with ordinate: bound phlorizin ([zmole/mmole)/free keto-phlorizin ([LM); abscissa: [zmoles phloriziI1 bound/mmole total phospholipid. Phlorizin, 20-75 ~zM; phospholipid, 0.5 raM.
11 A. D. BANGHAM, in Progress in Biophysics and Molecular Biology (Eds J. A. V. BUTLER and D. NOBLE; Pergamon Press, Oxford 1968), vol. 18, p. 29. lZ D . ATKINSON, H. HAUSER, G. P. SHIPLEY and J. M. STUBBS, Bioehim. biophys. Acta 339, 10 (1974). la S. M. JOHNSOX, A. D. BANGHA~I,M. W. HILL and E. D. KOR~, Bioehim. biophys. Acta 233, 820 (1971). 14 D. PAPAHADJOPOULOS,S. NIR and S. OHKI, Biochim. biophys. Acta 266, 561 (1971). 15 G. BARTLETT, J. biol. Chem. 234, 466 (1959). i6 Present Address: Dept. of Physiology and Biophysics, Mount Sinai School of Medicine, 100th St. & 5th Ave., New York, N.Y. 10029, USA.
Specialia
o v e r c o m i n g b a c t e r i a l p e r m e a b i l i t y b a r r i e r s in clinical practice.
10 I am indebted to Dr. R. J. OLDS for his kind advice and to Dr H. B. F. DIXON for his generous gift of samples of pure TRIEN. This work was supported by the H. E. Durham Fund, King's College, Cambridge. 11 Present address: The Middlesex Hospital Medical School, London WIP 7PN, England.
85
Rdsumd. I n vivo aussi bien q u ' i n vitro, la t r i 6 t h y l 6 n e t 6 t r a m i u e ( T R I E N ) , a g e n t qui f o r m e des ch61ats avec les cations bivalents, renforce signifieativement l'activit6 a n t i b a c t 6 r i e n n e de l ' a n t i b i o t i q u e carb6nicilline envers la r~sistante Pseudomonas aeruginosa. C-. S M I T H 10, iX
St Catharine's College, University o/ Cambridge, Cambridge (Great Britain), 7 August 7974.
P h l o r i z i n B i n d i n g to Bilayer Vesicles of P h o s p h o l i p i d s and P h o s p h o l i p i d - C h o l e s t e r o l P h l o r i z i n i n h i b i t s s u g a r t r a n s p o r t in kidney, intestine, a n d e r y t h r o c y t e s ~,2. I n e r y t h r o e y t e s , it also i n h i b i t s a n i o n e x c h a n g e a n d increases a n i o n c o n d u c t a n c e 3,4. However,. t h e m e c h a n i s m of its i n t e r a c t i o n w i t h cell m e m b r a n e s is unclear. I n renal cell m e m b r a n e s , phlorizin was f o u n d t o b i n d n o t o n l y to a specific, h i g h - a f f i n i t y p r o t e i n r e c e p t o r ~, b u t also t o l o w - a f f i n i t y sites of unk n o w n n a t u r e ~, ~, p o s s i b l y i n c l u d i n g lipid. I n t h i s c o n t e x t , it s e e m e d useful t o s t u d y t h e b i n d i n g of p h l o r i z i n to s o n i c a t e d b i l a y e r vesicles of p h o s p h o l i p i d s a n d p h o s p h o lipid-cholesterol.
0.8 0.6 oo
% o
I
,
,
250
.
,
270
.
290
.
.
.
.
.
~ , - ~ ~
310 330 Wavelength (nm)
350
370
Fig. 1. Phlorizin absorbance spectra at pH 4.5 and 9.5 and the effect of PC vesicles. Pi~lorizin, 25 ~M; phospholipid, 2 mM. At pH 4.5: a) keto-phlorizin; aM, 1.62 • 104 M -1 cm-1 (285 nm); b) phlorizin and PC. At pH 9.5: e) enol-phlorizin; aM, 2.73• M 1 em-I (325 rim); d) pblorizin and PC. To amplify changes in spectra, lipid concentrations were increased to 4 times that used in the binding studies.
Methods. C h r o m a t o g r a p h i c a l l y p u r e egg yolk p h o s p h a t i d y l c h o l i n e (PC) a n d o x - b r a i n p h o s p h a t i d y l s e r i n e (PS) were o b t a i n e d f r o m Lipid P r o d u c t s (Sou t h Nutfield, U . K . ) . Cholesterol (99%) (Merck) was r e c r y s t a l l i z e d f r o m m e t h a nol. P h l o r i z i n (Roth) was p u r i f i e d to r e m o v e all traces of p h l o r e t i n 4. PC a n d P S d i s p e r s i o n s in 50 m M KC1 a n d 30 m M Glycylglycine buffer a t p H 7.2 were s o n i c a t e d a t 5~ u n d e r N 2 to m i n i m u m optical d e n s i t y T, s. Ph!orizin b i n d i n g to lipid vesicles was m e a s u r e d s p e c t r o p h o t o m e t r i c a l l y in d o u b l e - c h a m b e r m i x i n g c u v e t t e s (Hellma) a t 25~ The 2 c o m p a r t m e n t s of t h e reference c u v e t t e were filled w i t h e q u a l v o l u m e s (1 ml) of lipid vesicles a n d buffer. The s a m p l e c u v e t t e c o n t a i n e d vesicles in one c h a m b e r a n d phlorizin (20-75 /~M) in t h e other. The a b s o r b a n c e s of k e t o - p h l o r i z i n a t 285 n m a n d of enolphlorizin a t 325 n m were m e a s u r e d before m i x i n g a n d w i t h i n 2 m i d a f t e r w a r d s . On t h e a s s u m p t i o n t h a t t h e enolic f o r m of phlorizin is n o t b o u n d to t h e lipid vesicles, t h e c o n c e n t r a t i o n s of free a n d b o u n d k e t o - p h l o r i z i n were c a l c u l a t e d f r o m t h e s e a b s o r b a n c e s 9. Results and discussion. The a b s o r p t i o n s p e c t r a of t h e keto a n d enol t a u t o m e r s 1~of p h l o r i z i n are s h o w n in F i g u r e 1 (Curves a a n d c). Also s h o w n is t h e effect of PC vesicles on t h e spectra. The s p e c t r u m of p h l o r i z i n plus lipid a t p H 9.5 is identical t o t h a t of t h e enolic species alone. T h e s p e c t r u m of phlorizin plus lipid a t p H 4.5 differs slightly f r o m t h a t of t h e ketonic species alone. These o b s e r v a t i o n s suggest t h a t an association of t h e k e t o n i c species w i t h t h e p h o s p h o l i p i d occurs, a c c o m p a n i e d b y a c h a n g e in t h e m o l a r a b s o r b a n c y i n d e x (aM). The effect of P S on aM was q u a l i t a t i v e l y similar, b u t smaller. I n t h e calculation of free a n d b o u n d phlorizin c o n c e n t r a t i o n s , c o r r e c t i o n s were m a d e for this small c h a n g e in a~. T h e a d d i t i o n of liposomes t o phlorizin a t p H 7.2, close to t h e pK~ of phlorizin, lowers t h e a b s o r b a n c e a t 325 n m a n d increases t h a t a t 285 n m (Figure 2). T o g e t h e r w i t h
06
8
0.4
/'~ ~ / ~ ' o/" /'-
~ 0.2 9
0
/./
'
'
'
270
~
--\
-o-o-,o / "--0..._ N \ c ~,o \ \
~,~-
250
,~-,,./
O
~
,
,
,
290 310 Wavelength (nm)
,
330
,
,
350
-
~ 370
Fig. 2. Effect of PC and PS vesicles on the phlorizin absorbance spectrum at pH 7.2. Phlorizin, 25 FM; phospholipid, 2 mM. a) phlorizin; b) phlorizin and PS; e) phlorizin and PC.
1 A. KOTVK,J. KOLiNSKA,K. VERbs and J. SZAM~ER,Bioehem. Z. 342, 129 (1965). 2 p. G. LEFEvRE, Pharmac. Rev. 13, 39 (1961). 3 K. F. SCtINELL, S. GERHARDT, S. LEPKE and H. PASSOW,Biochim. biophys. Aeta 378, 474 (1973). 4 j. H. KAPLANand H. PASSOW,J. Membrane Biol., in press. 5 L. THOMAS, Bioehim. biophys. Acta 297,454 (1973). s H. GLOSSMANNand D. 3/[. NEVILLE JR., Biochim. biophys. Acta 323, 408 (1973). 7 E. G. FINER, A. G. FLOOK and H. HAUSER, Biochim. biophys. Acta 260, 49 (1972). s H. HAUSER,D. OLI~ANIand M. C. PHILLIPS,Biochemistry 12, 4507 (1973). 9 H. H. JAFF~ and M. ORCHIN, Theory and Applications o/ Ultraviolet Spectroscopy (J. Wiley, New York 1962), p. 557. 10 A. LAMBRECFITS,C.r. Acad. Sci., Paris 796, 295 (1933).
86
Specialia
EXPERIENTIA 31/1
Number and affinity of phlorizin binding sites of lipid bilayer vesicles
Preparations
K (I/mole) • 104
Pure Phospholipids (5) Phospholipids pIus Cholesterol (1:1) (5)
n (~zmoles/mmole PL)
PC
PS
PC
PS
2.1 • 0.2 1.5 ~_ 0.2
1.0 -[- 0.2 1.6 • 0.3
98 • 8 86 ~ 11
112 ~ 14 67 =t- 13
The association constant (K) and the number of binding sites (n) were calculated from the least squares regression lines of the Seatchard plot. Values are means ~ S.E. In parentheses number of measurements. PL represents total phospholipid. Lipid phosphorus was determined according to BARTLETT15.
t h e d a t a in F i g u r e 1, t h e i n v e r s e v a r i a t i o n of enolic a n d k e t o n i c p e a k s s t r o n g l y suggests t h a t t h e u n c h a r g e d , k e t o n i c f o r m of p h l o r i z i n b i n d s r a p i d l y to t h e p h o s p h o lipid bilayers. F i g u r e 3 shows t h e r e l a t i o n s h i p b e t w e e n b o u n d p h l o r i z i n a n d t h e c o n c e n t r a t i o n of t h e free k e t o n i c species. A S c a t c h a r d p l o t of t h e d a t a is s h o w n in t h e inset. As s h o w n in t h e Table, t h e a s s o c i a t i o n c o n s t a n t s of p u r e P C a n d P S b i l a y e r s differ s i g n i f i c a n t l y (p < 0.005), while t h e c o r r e s p o n d i n g n u m b e r of b i n d i n g sites do n o t (p > 0.4). Since a t p H 7.2 t h e vesicles of PC are isoelectric a n d t h o s e of P S n e g a t i v e l y c h a r g e d n, t h e s m a l l difference in affinities suggests t h a t t h e c h a r g e of t h e p h o s p h o l i p i d h e a d g r o u p s p l a y s a m i n o r role in t h e b i n d i n g process. Control experiments indicated that phlorizin penetrat i o n of t h e vesicles d u r i n g t h e b i n d i n g m e a s u r e m e n t s is negligible. T h u s , if i t is a s s u m e d t h a t all t h e p h o s p h o l i p i d is p r e s e n t in t h e f o r m of single b i l a y e r vesicles of 250 A d i a m e t e r s,~2, a b o u t 7 0 % of t b e t o t a l p h o s p h o l i p i d w o u l d b e o n t h e o u t e r vesicle surface ~, ~a, a n d h e n c e b e a v a i l a b l e for b i n d i n g . A c c o r d i n g l y , P C a n d P S b i l a y e r vesicles b i n d m a x i m a l l y a b o u t 150 # m o l e s / m m o l e p h o s p h o l i p i d or 1 p h l o r i z i n p e r 7 p h o s p h o l i p i d molecules.
Cholesterol (1:1 m o l a r ratio) lowers t h e p h l o r i z i n a f f i n i t y of P C b i l a y e r s a n d raises t h a t of P S t o r e s u l t a n t v a l u e s w h i c h are n o t s i g n i f i c a n t l y d i f f e r e n t f r o m e a c h o t h e r (p > 0.7). F o r t h e r a n g e of p h l o r i z i n c o n c e n t r a t i o n s studied, Scatchard analysis again indicated a homogeneous p o p u l a t i o n of b i n d i n g sites. T h e p r e s e n c e of cholesterol, does n o t a l t e r t h e b i n d i n g c a p a c i t y p e r p h o s p h o l i p i d molecule of P C b i l a y e r s s i g n i f i c a n t l y (p > 0.4) ; h o w e v e r , it t e n d s t o r e d u c e t h a t of P S b i l a y e r s b y a b o u t 4 0 % (p < 0.05). I f one a s s u m e s a size of a b o u t 250-300 ~_ for t h e c h o l e s t e r o l c o n t a i n i n g vesicles~% one c a n c a l c u l a t e t h a t c h o l e s t e r o l - P S vesicles b i n d m a x i m a l l y o n l y 96 /~moles k e t o p h l o r i z i n / m m o l e P S or 1 p h l o r i z i n p e r 10 P S molecules.
Zusammen/assung. S p e k t r o p h o t o m e t r i s c h e M e s s u n g e n a n P h o s p h o l i p i d - D o p p e l s c h i c h t e n zeigten, dass die K e t o - F o r m y o n P h l o r i z i n b e v o r z u g t g e b u n d e n wird. Phosphatidylcholin und Phosphatidylserin besitzen beide eine B i n d u n g s k a p a z i t g t y o n e t w a 1 Molekfil P h l o r i z i n p r o 7 Molekiile P h o s p h o l i p i d . I h r e A f f i n i t g t e n ftir P h l o r i z i n sind 2.1 • 10 4 bzw. 1.0 • 10 ~ 1/mole. Die A n w e s e n h e i t y o n C h o l e s t e r i n v e r / i n d e r t die B i n d u n g s p a r a m e t e r . G. ~ H R E N S P E C K 16
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Fig. 3. Concentration dependence of phlorizin binding. Graph depicts data of a representative experiment. A Seatchard plot of the data is shown in the inset with ordinate: bound phlorizin ([zmole/mmole)/free keto-phlorizin ([LM); abscissa: [zmoles phloriziI1 bound/mmole total phospholipid. Phlorizin, 20-75 ~zM; phospholipid, 0.5 raM.
11 A. D. BANGHAM, in Progress in Biophysics and Molecular Biology (Eds J. A. V. BUTLER and D. NOBLE; Pergamon Press, Oxford 1968), vol. 18, p. 29. lZ D . ATKINSON, H. HAUSER, G. P. SHIPLEY and J. M. STUBBS, Bioehim. biophys. Acta 339, 10 (1974). la S. M. JOHNSOX, A. D. BANGHA~I,M. W. HILL and E. D. KOR~, Bioehim. biophys. Acta 233, 820 (1971). 14 D. PAPAHADJOPOULOS,S. NIR and S. OHKI, Biochim. biophys. Acta 266, 561 (1971). 15 G. BARTLETT, J. biol. Chem. 234, 466 (1959). i6 Present Address: Dept. of Physiology and Biophysics, Mount Sinai School of Medicine, 100th St. & 5th Ave., New York, N.Y. 10029, USA.
