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Plasma membrane lipid composition of Dictyostelium Discoideum during early development in aqueous suspension Withers, Howard Keith 1979

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PLASMA MEMBRANE L I P I D COMPOSITION OF DICTYQSTELIUM DISCQIDEUM DURING EARLY DEVELOPMENT IN AQUEOUS SUSPENSION  by HOWARD KEITH WITHERS B.Sc,  U n i v e r s i t y o f Birmingham, E n g l a n d , U.K., 1974.  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES Department of M i c r o b i o l o g y  We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA March 1979 ©  Howard K e i t h W i t h e r s , 1979  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r  an advanced d e g r e e a t the U n i v e r s i t y of B r i t i s h C o l u m b i a , I a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree that permission  f o r extensive copying of t h i s t h e s i s  f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s .  It i s understood that copying or p u b l i c a t i o n  o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my written  permission.  Department The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  DE-6  BP 75-51  1E  i.  ABSTRACT  C e l l - c e l l contact must be made and maintained  for normal  development and eventual d i f f e r e n t i a t i o n of J). discoideum to occur. Certain plasma membrane components are known to a l t e r i n a c t i v i t y or abundance during the organism's developmental cycle although no quantitative measurement of plasma membrane neutral l i p i d and phospholipid content has been reported to date. Optimal conditions for the extraction, separation and assay of l i p i d components were derived and tested by q u a n t i f i c a t i o n of the neutral l i p i d and phospholipid components of i n t a c t c e l l s of s t r a i n Ax-2.  Development was i n i t i a t e d i n JJ. discoideum populations  suspended i n aqueous buffer and plasma membrane f r a c t i o n s were p u r i f i e d from both exponentially growing and aggregation-phase c e l l s by a modified procedure which minimized phospholipid degradation during the plasma membrane i s o l a t i o n . phospholipid compositions  Neutral l i p i d and  of the plasma membrane fractions PM1  and PM2 from exponentially growing c e l l s and from those i n early aggregation phase were determined. Exponential phase c e l l s ' plasma membranes contained large proportions of phosphatidylethanolamine, lysophosphatidylethanolamine.  phosphatidylcholine and  Lysophosphatidylcholine was absent.  A s i g n i f i c a n t quantity of phosphatidylinositol was detected and c a r d i o l i p i n , phosphatidylglycerol, phosphatidic acid and l y s o phosphatidic acid were each present i n small amounts. of phosphatidylethanolamine proven.  The presence  plasmalogen was suspected but not  No a c y l g l y c e r o l components were detected, the major  neutral l i p i d  f r a c t i o n b e i n g t h a t of f r e e s t e r o l w h i c h l a r g e l y  comprised s t i g m a s t - 2 2 - e n - 3 3 - o l ; extremely  small quantities.  of plasma membranes was and  s t e r o l e s t e r was  present  in  An u n i d e n t i f i e d n e u t r a l l i p i d  d e t e c t e d by i t s c h a r a c t e r i s t i c  component  absorption  f l u o r e s c e n c e upon i r r a d i a t i o n a t u l t r a v i o l e t w a v e l e n g t h s .  A f t e r s i x t e e n hours a g g r e g a t i o n of the plasma membranes was  content  g r e a t l y reduced, a s i g n i f i c a n t p r o p o r t i o n  of the p h o s p h a t i d y l e t h a n o l a m i n e lysophosphatidylethanolamine, a c i d and  the p h o s p h a t i d y l c h o l i n e  appeared t o have been c o n v e r t e d  to  and p h o s p h a t i d y l g l y c e r o l , p h o s p h a t i d i c  l y s o p h o s p h a t i d i c a c i d were a l l i n g r e a t e r abundance than  i n growing c e l l s ' membranes.  The  f r e e s t e r o l component remained  r e l a t i v e l y c o n s t a n t but s t e r o l e s t e r had (7 t o 1 0 - f b l d ) and p h o s p h o l i p i d s was  increased dramatically  the f a t t y a c i d c o m p o s i t i o n  o f the plasma membrane  more s a t u r a t e d , p r i m a r i l y because of the accumu-  l a t i o n of p a l m i t a t e and  s t e a r a t e and a r e d u c t i o n of the  d i e n o i c f a t t y a c i d components.  octadeca-  iii.  TABLE OF CONTENTS Page  ABSTRACT  i  TABLE OF CONTENTS  i  LIST OF TABLES LIST OF FIGURES .  .  .  ACKNOWLEDGEMENTS  i  i  i  v  v v i  INTRODUCTION  1  MATERIALS AND METHODS  5  (a)  Materials  5  (b)  Organism and Membrane P r e p a r a t i o n  5  (c)  L i p i d E x t r a c t i o n and I s o l a t i o n  6  (d)  Assays  RESULTS  14  (a)  Comparison o f L i p i d E x t r a c t i o n P r o c e d u r e s  (b)  I s o l a t i o n and Q u a l i t a t i v e A n a l y s i s o f L i p i d Classes  (c)  (e)  14  20  Q u a n t i t a t i v e L i p i d Determinations of I n t a c t Cell Extracts  (d)  10  23  E v a l u a t i o n of P h o s p h o l i p i d Degradation During Plasma Membrane P r e p a r a t i o n  29  Plasma Membrane L i p i d C o m p o s i t i o n  37  DISCUSSION  45  BIBLIOGRAPHY  51  iv.  LIST OF TABLES Table  Page  1  Lipid-phosphorus extracted from exponentially growing c e l l s of JJ. discoideum Ax-2.  15  2  Fatty acid analysis of hydrolyzed t o t a l l i p i d from Folch ejt a l (41) and Bligh & Dyer (38) extractions of exponentially growing c e l l s of I), discoideum Ax-2.  16  3.  S t e r o l analysis of t o t a l l i p i d from Folch et al (41) and Bligh & Dyer (38) extractions of exponentially growing c e l l s of I), discoideum Ax-2.  y]  4  Fatty acid analysis of various f i n a l extractions (Fractions III) following i n i t i a l procedures of Folch et a l (41).  19  5  Lipid-phosphorus analysis of i n t a c t c e l l s of exponentially growing I), discoideum Ax-2 following phospholipid separation by thin-layer chromatography.  24  6  A c y l g l y c e r o l analysis of i n t a c t c e l l s of exponentially growing B. discoideum Ax-2.  26  7  Sterol analysis of the free s t e r o l s and s t e r o l esters extracted from i n t a c t c e l l s of exponentially growing ID. discoideum Ax-2.  28  8  Proportion of i n d i v i d u a l phospholipid components of I), discoideum Ax-2 crude membranes resuspended i n 8.6% sucrose-Tris-PMSF buffer, pH 7.4, incubated at either 4°C or 22°C as shown.  35  9  Plasma membrane lipid-phosphorus analysis at three stages of development of I), discoideum Ax-2 following phospholipid separation by thin-layer chromatography.  38  10  Plasma membrane free s t e r o l and s t e r o l ester s t e r o l analysis at two stages of development of I), discoideum Ax-2.  41  11  I), discoideum Ax-2 plasma membrane phospholipid f a t t y acid composition at two stages of the organism.' s development.  44  V.  LIST OF FIGURES Figure  Page  S e p a r a t i o n o f n e u t r a l l i p i d s by m o n o d i r e c t i o n a l , b i p h a s i c t h i n - l a y e r chromatography on s i l i c a g e l H plates.  21  Two-dimensional, b i p h a s i c t h i n - l a y e r chromatography of a m i x t u r e of s t a n d a r d p h o s p h o l i p i d s on s i l i c a g e lH plates with additives.  22  The e f f e c t o f temperature on t h e r a t e s of l o s s of l i p i d - p h o s p h o r u s from B. d i s c o i d e u m Ax-2 homogenates over an extended p e r i o d .  31  The e f f e c t o f temperature on t h e e a r l y l o s s o f l i p i d - p h o s p h o r u s from I), d i s c o i d e u m A x - 2 homogenates.  32  The e f f e c t of t h e s o l u b l e f r a c t i o n o f c e l l homogenates o f _D. d i s c o i d e u m Ax-2 on t h e l i p i d phosphorus c o n t e n t o f crude membrane p r e p a r a t i o n s i n c u b a t e d a t 22 C.  33  L i p i d - p h o s p h o r u s c o n t e n t of B. d i s c o i d e u m Ax-2 crude membranes d u r i n g extended i n c u b a t i o n a t 4 C.  35  ACKNOWLEDGEMENT S  I w i s h t o thank Dr. Thomas B u c k l e y ,  o f t h e Department o f B i o c h e m i s t r y  and M i c r o b i o l o g y , U n i v e r s i t y o f V i c t o r i a , B.C., f o r h i s c r i t i c a l  advice  on l i p i d e x t r a c t i o n and t h i n - l a y e r c h r o m a t o g r a p h i c t e c h n i q u e s , and Mrs.  Kathy LaRoy f o r h e r a s s i s t a n c e w i t h c e r t a i n s t a g e s o f t h e l a b o r a t o  work.  D r s . G e r a l d and C l a i r e Weeks and Dr. N e i l G i l k e s c o n t r i b u t e d  w i t h h e l p f u l d i s c u s s i o n s throughout t h e p r o j e c t .  Dr. G i l k e s p r o v i d e d  b l u e l o h i d e x and f i l i p i n was s u p p l i e d by Dr. J . Masson o f t h e Upjohn Company, T o r o n t o , O n t a r i o . redesigned and  I am g r a t e f u l t o Mr. James R i c h t e r who  and c o n s t r u c t e d equipment f o r t h i n - l a y e r sample a p p l i c a t i o n  t o Ms. Rosemary Morgan f o r a r r a n g i n g and t y p i n g t h e m a n u s c r i p t  w i t h such p r e c i s i o n .  T h i s t h e s i s c o u l d n o t have been w r i t t e n w i t h o u t t h e encouragement and support  o f c l o s e f r i e n d s , n o r w i t h o u t c o n s i d e r a b l e e f f o r t from Gerry  who has been v e r y , v e r y p a t i e n t .  1.  INTRODUCTION  D i c t y o s t e l i u m d i s c o i d e u m i s a c e l l u l a r s l i m e mould d i s t r i b u t e d i n t h e s o i l s and r o t t i n g v e g e t a t i o n o f temperate f o r e s t s t h r o u g h o u t the w o r l d .  This simple  e u c a r y o t i c organism i s amoeboid i n t h e  v e g e t a t i v e phase o f i t s l i f e c y c l e d u r i n g w h i c h i t i n g e s t s b a c t e r i a , but on s t a r v a t i o n p o p u l a t i o n s development. and  undergo a p r o c e s s o f synchronous  T h i s comprises a g g r e g a t i o n ,  f r u i t i n g body c o n s t r u c t i o n .  grex f o r m a t i o n  and m i g r a t i o n ,  W i t h i n the grex c e l l s d i f f e r e n t i a t e  i n t o d i s t i n c t a n t e r i o r and p o s t e r i o r t y p e s , and u l t i m a t e l y i n t o s t a l k and s p o r e c e l l s r e s p e c t i v e l y w i t h t h e f o r m a t i o n body.  of the f r u i t i n g  The s t a l k c e l l s a r e n o t v i a b l e b u t t h e s p o r e s g e r m i n a t e i n t o  s m a l l myxamoebae a f t e r d i s p e r s a l ( 1 , 2 ) . I n 1889 M a c B r i d e s t a t e d o f s l i m e moulds: r e t i r e s them from o r d i n a r y ken; t h e i r microscopic  " T h e i r minuteness  b u t such i s t h e extreme beauty o f  s t r u c t u r e , such t h e e x c e e d i n g i n t e r e s t o f t h e i r  l i f e h i s t o r y , t h a t f o r many y e a r s e n t h u s i a s t i c s t u d e n t s have found the group one o f p e c u l i a r f a s c i n a t i o n , i n some r e s p e c t s a t l e a s t , the most i n t e r e s t i n g and remarkable t h a t f a l l s beneath our l e n s e s " (3).  This d e s c r i p t i o n a p p l i e s t o the A c r a s i a l e s ( c e l l u l a r  slime  moulds) as w e l l as t o t h e Myxomycetes ( t r u e s l i m e moulds).  The  two have f r e q u e n t l y been c o n f u s e d a l t h o u g h major d i s t i n c t i o n s were n o t e d more t h a n a c e n t u r y  ago ( 3 , 4 ) .  T h e i r d i f f e r e n c e s and s i m i l a r i t i e s  have been r e v i e w e d r e c e n t l y ( 5 ) . I), d i s c o i d e u m has s e v e r a l advantages f o r t h e l a b o r a t o r y  study  of b i o l o g i c a l development, and i t s c e l l u l a r d i f f e r e n t i a t i o n mechanisms may be o f r e l e v a n c e  t o s t u d i e s o f development i n m u l t i c e l l u l a r  2.  organisms ( 1 , 2 ) ; e m b r y o l o g i c a l development, t i s s u e r e g e n e r a t i o n and c a n c e r o u s p r o l i f e r a t i o n b e i n g examples of the l a t t e r .  Certain  s t r a i n s of ID. d i s c o i d e u m  grow q u i t e r a p i d l y i n a x e n i c media a l l o w i n g  biochemical manipulation  d u r i n g growth and f a c i l i t a t i n g the  and c l o n i n g of mutants.  The o r g a n i s m has been the s u b j e c t of  isolation  d i v e r s e i n v e s t i g a t i o n s w h i c h have p r o v i d e d a comprehensive background of i n t e r r e l a t e d i n f o r m a t i o n ( 1 ) .  I n c o n t r a s t t o the m a j o r i t y of  d e v e l o p m e n t a l systems, t h a t of I), d i s c o i d e u m  i s not  complicated  by the e f f e c t s of growth and d i v i s i o n , b o t h of w h i c h cease b e f o r e a g g r e g a t i o n b e g i n s ; the d e v e l o p m e n t a l phenomena a r e thus r e s t r i c t e d to morphogenesis and d i f f e r e n t i a t i o n .  S l i m e mould development has  been d i s c u s s e d i n g e n e r a l terms by Bonner (6) and p a r t i c u l a r emphasis on ID. d i s c o i d e u m  elsewhere w i t h  (1,2,5,7).  C e l l - c e l l contacts e s t a b l i s h e d during aggregation coideum a r e m a i n t a i n e d  throughout development and a r e r e q u i r e d a t  a l l stages f o r the p r o c e s s  t o advance.  The p r e c i s e mechanisms of  these c e l l u l a r i n t e r a c t i o n s and t h e i r m o l e c u l a r determined, although  of ID. d i s -  b a s i s have not been  s e v e r a l plasma membrane p r o t e i n s , g l y c o p r o t e i n s  and a g l y c o l i p i d a r e known t o undergo q u a n t i t a t i v e a l t e r a t i o n s or a c t i v i t y changes d u r i n g d i f f e r e n t i a t i o n (8-17), and  univalent  a n t i b o d i e s to a g g r e g a t i o n - s p e c i f i c surface antigens b l o c k aggregation  and a r r e s t f u r t h e r development  Developmentally  (8,18).  r e g u l a t e d m o d i f i c a t i o n of plasma membrane  l i p i d components may processes.  cell  be a s s o c i a t e d w i t h c e l l - c e l l  interaction  S p e c i f i c q u a l i t a t i v e or q u a n t i t a t i v e l i p i d changes  might be n e c e s s a r y  t o a l l o w p r o t e i n s or g l y c o p r o t e i n s t o b i n d to  the c e l l s u r f a c e or t o be i n c o r p o r a t e d w i t h i n the membrane.  The  3.  a c t i v i t i e s or s p e c i f i c i t i e s of c e r t a i n developmentally regulated cell of  s u r f a c e components might be i n f l u e n c e d in s i t u by a l t e r a t i o n s  l i p i d s i n d i r e c t a s s o c i a t i o n w i t h these components o r t h r o u g h  more g e n e r a l membrane f l u i d i t y i n l i p i d composition.  e f f e c t s brought about by changes  I n d e v e l o p m e n t a l b i o l o g y t h e r e has been no  d i r e c t o b s e r v a t i o n o f such mechanisms, a l t h o u g h i n c e r t a i n c u l t u r e d a n i m a l c e l l s the p h y s i c a l s t a t e o f t h e plasma membrane a l t e r s d u r i n g c e l l a d h e s i o n (mouse L929 c e l l s ) ( 1 9 ) , c e l l f u s i o n ( a v i a n myoblasts)  (20) and c e l l u l a r d i f f e r e n t i a t i o n (neuroblastoma) ( 2 1 ) .  Moreover, s t u d i e s o u t s i d e t h e d e v e l o p m e n t a l f i e l d 'micro-domains'  suggest t h a t l i p i d  e x i s t i n n a t u r a l membranes and t h a t s p e c i f i c  lipid-  p r o t e i n i n t e r a c t i o n s as w e l l as membrane l i p i d phase t r a n s i t i o n s a r e a b l e t o a f f e c t c e r t a i n membrane enzyme a c t i v i t i e s and a c t i v e transport processes  (22-27).  There i s c i r c u m s t a n t i a l e v i d e n c e t h a t l i p i d c o m p o s i t i o n may be a c r i t i c a l  f a c t o r o f I), d i s c o i d e u m development.  The a n t i b i o t i c  c e r u l e n i n which i n h i b i t s f a t t y a c i d b i o s y n t h e s i s prevents aggregation and t h e e n s u i n g d i f f e r e n t i a t i o n o f t h e organism  ( 2 8 ) , and t h e f a t t y  a c i d c o m p o s i t i o n o f t h e a x e n i c s t r a i n o f I), d i s c o i d e u m can be m o d i f i e d d u r i n g growth such t h a t i t s subsequent impaired (29). lipid  development i s  Furthermore b o t h t h e p h o s p h o l i p i d (30) and n e u t r a l  (31) c o m p o s i t i o n s o f t h e i n t a c t organism have been shown t o  a l t e r d u r i n g development s u g g e s t i n g t h a t t h e r e i s e i t h e r a w i d e spread adjustment cells'  o f t h e s e components o c c u r r i n g throughout t h e  v a r i o u s membranes o r t h a t t h e r e a r e s i g n i f i c a n t  a l t e r a t i o n s w i t h i n p a r t i c u l a r membranes. involved i n c e l l - c e l l  lipid  Although i t i s d i r e c t l y  i n t e r a c t i o n , t h e 13. d i s c o i d e u m plasma membrane  4.  had not had i t s l i p i d composition investigated u n t i l this study was undertaken to determine whether s i g n i f i c a n t l i p i d changes occur during early stages of the organism's development.  5.  MATERIALS AND METHODS  a.  Materials  Standard  l i p i d s were of t h e h i g h e s t p u r i t y a v a i l a b l e c o m m e r c i a l l y  and were p u r c h a s e d from Supelco  I n c . and A p p l i e d S c i e n c e I n c . .  S o l v e n t s were r e d i s t i l l e d i n g l a s s and s t o r e d d r y over seives.  I n order to prevent  molecular  s o l v e n t o x i d a t i o n and f r e e r a d i c a l  f o r m a t i o n 0.005% (w/v) 2 , 6 - d i - t e r t - b u t y l - 4 - m e t h y l p h e n o l  (BHT) t w i c e  r e c r y s t a l l i z e d from c a r b o n t e t r a c h l o r i d e was added t o a l l e t h e r s : t h i s a d d i t i v e a l s o p r o t e c t s d i s s o l v e d l i p i d s from a u t o x i d a t i o n ( 3 2 ) . A l l chromatography, s t o r a g e and assay g l a s s w a r e was acid-washed i n 5M HC1.  Thick g l a s s chromatoplates  were a l s o washed i n 2M KOH  and t h o r o u g h l y r i n s e d w i t h d i s t i l l e d , d e i o n i z e d water b e f o r e  being  spread w i t h t h e a p p r o p r i a t e t h i n l a y e r .  b.  Organism and Membrane P r e p a r a t i o n  The a x e n i c J). d i s c o i d e u m medium HL-5 a t 22°C (33,34).  s t r a i n Ax-2 was grown i n the l i q u i d C e l l s were h a r v e s t e d d u r i n g e x p o n e n t i a l  growth a t a c o n c e n t r a t i o n of a p p r o x i m a t e l y c e n t r i f u g a t i o n f o r 6 min a t 700 xg and 4°C.  5 x 10^ c e l l s m l ^ by Three i c e - c o l d d i s t i l l e d  water washes and subsequent c e n t r i f u g a t i o n s were used t o remove a l l t r a c e s of HL-5 medium b e f o r e immediate l i p i d e x t r a c t i o n o f t h e intact cells. C e l l s d e s t i n e d f o r membrane f r a c t i o n a t i o n were h a r v e s t e d and washed t w i c e w i t h i c e - c o l d 8.6% (w/v) sucrose-5mM t r i s ( h y d r o x y m e t h y l )  6.  aminomethane h y d r o c h l o r i d e ( s u c r o s e - T r i s ) , pH c e n t r i f u g a t i o n s of 700 prepared  under s t a n d a r d  c e l l p e l l e t s i n 17mM ml ^ and  xg as above.  suspensions  6.0,  7.4,  cells  a t 22°C f o r exponential  resuspended i n i c e -  phenylmethylsulphonylfluoride  at 10  8  c e l l s ml"  In o r d e r to minimize l i p i d d e g r a d a t i o n membrane p u r i f i c a t i o n procedure (35,37) was 105,400 xg p e l l e t was  rpm  Harvested  and aggregation-phase c e l l s were immediately  of the washed  a t 7 x 10  a t 150  e i t h e r 8 or 16 hours b e f o r e r e h a r v e s t i n g .  cold sucrose-Tris saturated with  min  Aggregation-phase c e l l s were  phosphate b u f f e r , pH  pH  using 6  c o n d i t i o n s (36) by r e s u s p e n s i o n  r o t a t i n g these c e l l  (sucrose-Tris-PMSF),  7.4,  not washed but was  1  (35).  the standard modified  plasma  i n t h a t the  e i t h e r used d i r e c t l y  as  'crude membrane' m a t e r i a l f o r p r e l i m i n a r y experiments or resuspended i n 20%  (w/v)  sucrose-Tris-PMSF,  onto the d i s c o n t i n u o u s  pH  7.4,  and  immediately  sucrose d e n s i t y g r a d i e n t s .  layered  These g r a d i e n t s  were c e n t r i f u g e d f o r 16 hours at 75,800 xg, c e n t r i f u g a t i o n b e i n g i n i t i a t e d w i t h i n 45 minutes of the s t a r t of c e l l d i s t i n c t bands of membranous m a t e r i a l (PM1 as d e s c r i b e d p r e v i o u s l y (37) and immediately.  The  fracture.  and PM2)  Two  (35) were r e c o v e r e d  a l i q u o t s were assayed  or e x t r a c t e d  c r i t e r i a used to a s s e s s the p u r i t y of the plasma  membrane p r e p a r a t i o n s have been d i s c u s s e d by G i l k e s and Weeks (37).  c.  L i p i d E x t r a c t i o n and  Isolation  In p r e l i m i n a r y experiments l i p i d  e x t r a c t i o n was  carried  out  e i t h e r by the method of B l i g h and Dyer (38) a c c o r d i n g to Kates 40)  or by t h a t of F o l c h e t a l (40,41), the l a t t e r b e i n g  efficacious  (see R e s u l t s ) .  The  (39,  the more  aqueous wash s o l u t i o n f o r the  7.  F o l c h p r o c e d u r e comprised 4.5 i n t e r f a c e was  mM  C a C l ^ and  the r e s u l t i n g phase  r i n s e d a t l e a s t s i x times w i t h the F o l c h 'pure s o l v e n t s  upper phase' of c h l o r o f o r m - m e t h a n o l - 3 . 6 mM  aq. C a C ^  A second e x t r a c t i o n of each F o l c h r e s i d u e was  (3:48:47 by v o l ) .  performed  overnight  a t 4°C w i t h 15 times the o r i g i n a l sample volume of e i t h e r c h l o r o f o r m methanol (7:1 v/v)  s a t u r a t e d w i t h 5%  methanol-glacial a c e t i c acid-water m e t h a n o l - c o n c e n t r a t e d HC1 was  thoroughly  was  added t o i t ( 4 2 ) .  (w/v)  aq. NH^,  or  chloroform-  (8:4:2:1 by v o l . ) , or  (50:50:0.3 by v o l . ) .  d r i e d under n i t r o g e n and  The  second e x t r a c t  the washed F o l c h e x t r a c t  E x t r a c t s were c o n c e n t r a t e d  methanol (2:1 v/v) under n i t r o g e n and  chloroform-  i n chloroform-  stored, i f necessary,  i n Teflon  s e a l e d v i a l s a t -70°C. S e p a r a t i o n of e x t r a c t s i n t o a p h o s p h o l i p i d f r a c t i o n and n e u t r a l l i p i d c l a s s e s was  achieved  individual  by b i p h a s i c t h i n - l a y e r chromato-  graphy i n one d i m e n s i o n on a 500 ym l a y e r of s i l i c a g e l H (Merck & B e f o r e use the c h r o m a t o p l a t e s  were s u b j e c t e d t o an a s c e n d i n g  c h l o r o f o r m - m e t h a n o l (2:1 v/v)  f o r a t l e a s t 12 h o u r s .  d e p o s i t e d a t the o r i g i n of a c t i v a t e d c h r o m a t o p l a t e s o f dry n i t r o g e n and  the p l a t e s were i m m e d i a t e l y  Co.).  wash w i t h  Samples were (43) under a s t r e a m  developed i n a n i t r o g e n  atmosphere u s i n g the b i p h a s i c system of s o l v e n t s d e s c r i b e d by S k i p s k i and B a r c l a y ( 4 3 ) .  C h r o m a t o p l a t e s were s t o r e d i n a d e s s i c a t o r under  a s p i r a t i o n f o r 20 min a f t e r each development t o remove a l l t r a c e s of solvent. to  Routine n e u t r a l l i p i d  i d e n t i f i c a t i o n was  made by  the m o b i l i t y of s t a n d a r d l i p i d s d e v e l o p e d on each p l a t e .  6 G a t 0.05% sprayed  (w/v)  i n aqueous e t h a n o l (95% v/v) was  onto the s t a n d a r d s  reference Rhodamine  selectively  and the f l u o r e s c e n c e of the r e s u l t i n g  complexes viewed under u l t r a v i o l e t  radiation.  lipid  8.  Techniques f o r the e x t r a c t i o n of l i p i d s from the c h r o m a t o p l a t e s were d e r i v e d from methods proposed by S k i p s k i and B a r c l a y  (43).  Areas of s i l i c a g e l c o n t a i n i n g i n d i v i d u a l l i p i d f r a c t i o n s were t r a n s f e r r e d r a p i d l y and q u a n t i t a t i v e l y t o 15 ml c o n i c a l , g r o u n d - g l a s s stoppered  c e n t r i f u g e tubes and  of s o l v e n t f o r 10 min vortexing.  e x t r a c t e d w i t h t h r e e 5 ml a l i q u o t s  each a t room temperature w i t h  occasional  T r i a c y l g l y c e r o l s , 1 , 2 - d i a c y l g l y c e r o l s and  g l y c e r o l s were e x t r a c t e d w i t h d i e t h y l e t h e r whereas methanol (4:1 v/v) was  1,3-diacylchloroform-  used t o e x t r a c t m o n o a c y l g l y c e r o l s ,  e s t e r s , s t e r o l s and hydrocarbons from the s i l i c a g e l . neutral l i p i d  f r a c t i o n s were c o n c e n t r a t e d  i f necessary,  a t -70°C i n T e f l o n - s e a l e d v i a l s .  e x t r a c t e d a t 37°C from a band 1.5  sterol  The  extracted  under n i t r o g e n and  stored,  Phospholipids  were  cm i n w i d t h a t the c h r o m a t o p l a t e  o r i g i n under c o n d i t i o n s s i m i l a r t o those f o r n e u t r a l l i p i d e x t r a c t i o n but u s i n g 5 ml a l i q u o t s of the f o l l o w i n g s o l v e n t m i x t u r e s two  t r e a t m e n t s of c h l o r o f o r m - m e t h a n o l - g l a c i a l  i n sequence:  acetic acid-distilled,  d e i o n i z e d w a t e r (25:15:4:2 by v o l . ) , f o l l o w e d by methanol, and  then  by m e t h a n o l - g l a c i a l a c e t i c a c i d - d i s t i l l e d , d e i o n i z e d water (94:1:5 by v o l . ) .  The  n i t r o g e n and  e x t r a c t e d p h o s p h o l i p i d s were c o n c e n t r a t e d  s t o r e d under the same c o n d i t i o n s as the n e u t r a l l i p i d s .  P h o s p h o l i p i d s were s e p a r a t e d t h i n - l a y e r chromatography.  by b i p h a s i c , t w o - d i m e n s i o n a l  A t e c h n i q u e was  a l l f u l l y acylated phospholipids present  under  and  i n I), d i s c o i d e u m , a l t h o u g h  r e s o l v e d (30,44).  A 97.5  developed t h a t f r a c t i o n a t e d  l y s o p h o s p h o l i p i d s known t o  be  p l a s m a l o g e n forms were not  ml volume of 0.5  mM  aqueous magnesium  a c e t a t e was ,added r a p i d l y t o 45 g Camag s i l i c a g e l H and  shaken  v i g o r o u s l y f o r 90 seconds b e f o r e b e i n g s p r e a d onto c h r o m a t o p l a t e s  9.  i n a 300 ym wet  layer.  P h o s p h o l i p i d s c o n t a i n i n g v i c i n a l hydroxy1  groups c o u l d be s e l e c t i v e l y r e t a r d e d d u r i n g development by i n c o r p o r a t i n g b o r i c a c i d i n t o the aqueous s o l u t i o n a t 0.4  M (45).  d r y i n g s l o w l y the t h i n - l a y e r s were washed by a s c e n d i n g  After  development  w i t h acetone f o r a t l e a s t 12 h o u r s , d r i e d a g a i n and a c t i v a t e d o v e r n i g h t a t (115 ± 5)°C  u n t i l immediately  p r i o r t o use.  After  c o o l i n g , each c h r o m a t o p l a t e r e c e i v e d one p h o s p h o l i p i d sample o f to  300 ymol l i p i d phosphorus (optimum c.175  origin.  up  ymol.) a t i t s p o i n t  C o o l i n g , s p o t t i n g and development of the t h i n - l a y e r were  c a r r i e d out e n t i r e l y i n a d r y n i t r o g e n atmosphere u s i n g equipment s i m i l a r t o t h a t d e s c r i b e d by S k i p s k i and B a r c l a y ( 4 3 ) . chromatography t a n k s were l i n e d w i t h Whatman 3MM w i t h the s o l v e n t m i x t u r e ,  Thin-layer  paper and w e t t e d  the vapours of w h i c h were a l l o w e d  e q u i l i b r a t e w i t h the e n c l o s e d atmosphere f o r an hour b e f o r e Each c h r o m a t o p l a t e was form-methanol - 28% h e i g h t of 18 cm.  to use.  d e v e l o p e d i n the f i r s t d i m e n s i o n w i t h c h l o r o -  (w/v)  aqueous ammonia (65:25:8 by v o l . ) t o a  R e s i d u a l s o l v e n t was  immediately  e v a p o r a t e d by  a p o w e r f u l stream of c o l d a i r f o l l o w e d by a s p i r a t i o n i n a d e s s i c a t o r for  t h i r t y minutes.  The p l a t e was  t h e n d e v e l o p e d f o r 18 cm i n the  second d i m e n s i o n w i t h c h l o r o f o r m - a c e t o n e - m e t h a n o l - g l a c i a l a c i d - d i s t i l l e d , d e i o n i z e d water (35:35:7:10:3 by v o l . ) . s o l v e n t was  acetic Residual  removed as b e f o r e and i n d i v i d u a l p h o s p h o l i p i d s were  r o u t i n e l y l o c a t e d by b r i e f immersion of the c h r o m a t o p l a t e i n t o an i o d i n e - s a t u r a t e d atmosphere. The c h a r a c t e r i s t i c p o s i t i o n s of i n d i v i d u a l p h o s p h o l i p i d s the t h i n - l a y e r chromatogram were i d e n t i f i e d by a c o m b i n a t i o n  on of  methods a f t e r s e p a r a t i n g s t a n d a r d p h o s p h o l i p i d s w i t h a c y l c h a i n s  of  known s a t u r a t i o n .  These methods i n c l u d e d the f o l l o w i n g : r e l a t i v e  speed and r e v e r s i b i l i t y o f brown complex f o r m a t i o n d u r i n g immersion in  i o d i n e vapour, both f a c t o r s b e i n g dependent on the degree o f  s a t u r a t i o n of the l i p i d s '  fatty acid  substituents; detection of  phosphate e s t e r s w i t h t h e molybdenum-blue reagent Lester  (46); n i n h y d r i n reagent  phosphatidylethanolamine, tives  (43); D r a g e n d o r f f  t o d e t e c t the f r e e amino groups o f  p h o s p h a t i d y l s e r i n e and t h e i r l y s o d e r i v a reagent  to detect phosphatidylcholine,  l y s o p h o s p h a t i d y l c h o l i n e and sphingomyelin of  of D i t t m e r and  (47); t h e i n c o r p o r a t i o n  b o r i c a c i d i n t o the t h i n - l a y e r , as d e s c r i b e d above, i n order to  s e l e c t i v e l y r e t a r d p h o s p h a t i d y l g l y c e r o l m i g r a t i o n (45). I n d i v i d u a l p h o s p h o l i p i d s were e i t h e r e x t r a c t e d from the s i l i c a p r e v i o u s l y d e s c r i b e d or assayed  i n the presence  q u a n t i t a t i v e t r a n s f e r t o a Pyrex assay  d.  g e l by t h e method  of the g e l a f t e r  tube.  Assays  P r o t e i n was r o u t i n e l y assayed (48).  The ' b i u r e t technique'  by the method o f Lowry et_ al  (49) was used i n measurements o f  p r o t e o l y t i c a c t i v i t y d u r i n g membrane p u r i f i c a t i o n , volume b e i n g reduced  t o 1.0 ml.  the t o t a l  T o t a l p h o s p h o l i p i d was  reagent  estimated  by a s s a y i n g the e x t r a c t e d l i p i d - p h o s p h o r u s by Ames' method (50), a l t h o u g h t h i s assay c o u l d not be used f o r the d e t e r m i n a t i o n of i n d i v i d u a l p h o s p h o l i p i d s r e c o v e r e d d i r e c t l y from t h i n — l a y e r chromatop l a t e s because s i l i c a g e l i n t e r f e r e d w i t h b o t h the d i g e s t i o n step and  the c o l o r i m e t r i c r e a c t i o n .  assay  The B a r t l e t t  lipid-phosphorus  (51) was m o d i f i e d t o i n c r e a s e i t s s e n s i t i v i t y w h i l e  remaining  11.  u n a f f e c t e d by i o d i n e p r e t r e a t m e n t o f l i p i d s o r t h e p r e s e n c e o f s i l i c a gel.  These p r o p e r t i e s were determined by a s s a y i n g t h e  phosphorus c o n t e n t o f i d e n t i f i e d p h o s p h o l i p i d a l i q u o t s a f t e r removal o f t h e l i p i d from an i o d i n e - t r e a t e d c h r o m a t o p l a t e . m o d i f i e d B a r t l e t t p r o c e d u r e was as f o l l o w s :  The  0.50 ml 70% (w/v)  aqueous p e r c h l o r i c a c i d was added t o t h e sample i n a s m a l l , a c i d washed tube and heated f o r 2.5 hours a t 150°C ( 5 2 ) . tube had c o o l e d , 0.60 ml d i s t i l l e d , (w/v) aqueous ammonium molybdate reagent (51) were added.  A f t e r the  d e i o n i z e d w a t e r , 0.20 ml 5%  and 0.02 ml f r e s h Fiske-SubbaRow  Tubes were s e a l e d w i t h P a r a f i l m , t h e  c o n t e n t s mixed and heated f o r 30 m i n a t 70°C, a l l o w e d t o c o o l and c e n t r i f u g e d i n s w i n g i n g b u c k e t s f o r a t l e a s t 5 min a t 1250 x g . Each s u p e r n a t a n t was c a r e f u l l y removed w i t h a P a s t e u r p i p e t t e and i t s a b s o r p t i o n measured a t 807 nm v e r s u s a r e a g e n t b l a n k .  This  a b s o r p t i o n maximum d i f f e r s from t h e 830 nm maximum o f t h e phosphomolybdate  complex i n t h e s t a n d a r d B a r t l e t t assay ( 5 1 ) .  It is  e s p e c i a l l y i m p o r t a n t t h a t a l l g l a s s and q u a r t z a p p a r a t u s be a c i d washed f o r each of t h e s e phosphorus a s s a y s . N e u t r a l l i p i d s o f t h e plasma membranes were measured by g a s l i q u i d chromatography. of  A f t e r t h i n l a y e r chromatographic s e p a r a t i o n  the i n d i v i d u a l n e u t r a l l i p i d f r a c t i o n s i n t e r n a l standards of  e i c o s a n o i c a c i d ( a r a c h i d i c a c i d ) and c h o l e s t - 5 e n - 3 3 o l _  (cholesterol)  were added t o t h o s e f r a c t i o n s c o n t a i n i n g f a t t y a c i d and s t e r o l moieties respectively.  The i n d i v i d u a l n e u t r a l l i p i d  fractions  were s a p o n i f i e d i n 1.0 ml a l i q u o t s w i t h an e q u a l volume of 15% (w/v) K0H i n methanol f o r 1 hour a t 70°C: t h e methanol was t h e n e v a p o r a t e d under n i t r o g e n and 1.0 m l d i s t i l l e d  water was added.  Non-saponifiable  l i p i d s were e x t r a c t e d d i r e c t l y w i t h f o u r  equal  volumes o f n-pentane and s a p o n i f i a b l e l i p i d s were s i m i l a r l y e x t r a c t e d f o l l o w i n g a c i d i f i c a t i o n o f t h e h y d r o l y s a t e w i t h 0.25 m l 12 M H^SO^, The n o n - s a p o n i f i a b l e f r a c t i o n s were d r i e d s e p a r a t e l y under n i t r o g e n and a c e t y l a t e d w i t h 0.5 m l a c e t i c a n h y d r i d e i n T e f l o n - s e a l e d , screw-capped tubes.  f o r 30 min a t 135°C  Samples were t h e n d r i e d  under n i t r o g e n and r e d i s s o l v e d i n m i n i m a l q u a n t i t i e s o f hexanes i n p r e p a r a t i o n f o r i s o t h e r m a l g a s - l i q u i d chromatography.  The non-  s a p o n i f i a b l e f r a c t i o n s o f t h e s t e r o l e s t e r samples were rechromatographed by t h i n - l a y e r chromatography i n o r d e r t o s e p a r a t e a t i n g hydrocarbons from t h e d e r i v e d s t e r o l s .  contamin-  These s t e r o l s were  e l u t e d , a c e t y l a t e d and assayed by g a s - l i q u i d chromatography as d e s c r i b e d above.  S t e r o l e s t e r s were a l s o q u a n t i f i e d by i s o t h e r m a l  g a s - l i q u i d chromatography o f t h e i r f a t t y a c i d m e t h y l e s t e r d e r i v a t i v e s as were a c y l g l y c e r o l s , f r e e f a t t y a c i d s and c e r t a i n p h o s p h o l i p i d fractions.  These d e r i v a t i v e s were o b t a i n e d by d r y i n g t h e i n d i v i d u a l  s a p o n i f i a b l e f r a c t i o n s under n i t r o g e n , a d d i n g 1.0 ml b o r o n  tri-  f l u o r i d e i n methanol and b o i l i n g f o r 2 min i n T e f l o n - s e a l e d , screwcapped tubes:  1.0 ml o f d i s t i l l e d water was t h e n added and t h e  m e t h y l e s t e r s were e x t r a c t e d w i t h t h r e e e q u a l volumes o f n-pentane. The p o o l e d pentane e x t r a c t s o f each sample were d r i e d down under n i t r o g e n , resuspended i n a m i n i m a l volume o f hexanes and a n a l y z e d by g a s - l i q u i d chromatography.  A c e t y l a t e d s t e r o l s were  separated  u s i n g 3% (w/w) m e t h y l s i l i c o n e (SE-30) phase on 100/120 mesh Gas Chrom Q ( A p p l i e d S c i e n c e I n c . ) a t 245°C i n 6 f t , 2 mm i . d . s i a l y s e d g l a s s tubes w i t h h e l i u m as c a r r i e r gas. F a t t y a c i d m e t h y l e s t e r s were s e p a r a t e d  on 10% (w/w) d i e t h y l e n e g l y c o l s u c c i n a t e t r e a t e d  w i t h orthophosphoric a c i d  (DEGS-PS) on 80/100 mesh S u p e l c o p o r t  (Supelco I n c . ) a t 140°C i n 6 f t , 2 ma. i . d . m e t a l columns u s i n g h e l i u m as c a r r i e r gas. Intact c e l l s provided s u f f i c i e n t neutral l i p i d  f o r free  g l y c e r o l and a c y l g l y c e r o l t o be measured by t h e method o f E g g s t e i n (53) as m o d i f i e d by Schmidt et_ a l ( 5 4 ) , u s i n g a 0.001% (w/v) aqueous p i c r i c a c i d r e f e r e n c e s o l u t i o n . f u l l hydrolysis  I n order to accomplish  of the i n d i v i d u a l , separated n e u t r a l l i p i d  samples,  t h e KOH s a p o n i f i c a t i o n s t e p was lengthened t o 12 hours from t h e 20 minutes recommended i n t h e s t a n d a r d , c l i n i c a l Assay components were purchased  procedure.  from t h e Boehringer-Mannheim  Corporation. L i p i d measurements were s t a n d a r d i z e d by r e f e r e n c e t o t h e p r o t e i n c o n t e n t o f t h e whole c e l l , crude membrane o r plasma membrane p r e p a r a t i o n from w h i c h t h e l i p i d s were d e r i v e d .  14.  RESULTS  a.  Comparison of L i p i d E x t r a c t i o n Procedures  The t e c h n i q u e s of F o l c h e_t a l (41) and of B l i g h and Dyer a c c o r d i n g to Kates (39,40) were compared (Table 1 ) .  (38)  Incomplete  p h o s p h o l i p i d e x t r a c t i o n from t i s s u e s by the B l i g h and Dyer method, e s p e c i a l l y of a c i d i c p h o s p h o l i p i d s , has been r e p o r t e d by Palmer The amounts of p h o s p h o l i p i d (Table 1 ) , f a t t y a c i d (Table 2)  (18,55).  and  s t e r o l (Table 3) e x t r a c t e d from p_. d i s c o i d e u m c e l l s showed t h a t the method o f F o l c h et al was for  t h i s study.  s u p e r i o r t o t h a t of B l i g h and Dyer  I n v i e w of the r e s u l t s of the o r i g i n a l a n a l y s i s  of l i p i d e x t r a c t i o n by F o l c h e t a l ( 4 1 ) , l i t t l e of the phosphorus measured i n ' F o l c h f r a c t i o n I I ' (Table 1) i s l i k e l y t o have been d e r i v e d from l i p i d phosphorus.  S i m i l a r l y , most o f the phosphorus  i n the B l i g h and Dyer aqueous methanol phase was p r o b a b l y n o n - l i p i d phosphorus.  C o n f i r m a t i o n of these c o n c l u s i o n s was p r o v i d e d by  the f a t t y a c i d a n a l y s e s (Table 2 ) . i n each procedure o n l y 1.5% was 2.8%  l o s t t o ' F o l c h f r a c t i o n I I ' and  t o the ' B l i g h and Dyer f r a c t i o n I I ' and i t i s l i k e l y t h a t  i n b o t h cases t h i s was  l a r g e l y from g l y c o l i p i d s o u r c e s .  i n d i c a t e s t h a t the procedure for  Of the t o t a l f a t t y a c i d r e c o v e r e d  of F o l c h et a l was  a l s o more e f f i c i e n t  the e x t r a c t i o n o f s t e r o l s from I), d i s c o i d e u m Ax-2.  Long and Coe e s t i m a t e d t h a t s t i g m a s t - 2 2 - e n - 3 3 - o l  Table 3  Although  (stigmastenol)  comprised more than 99% of f r e e and e s t e r i f i e d s t e r o l i n such (31) more r e c e n t a n a l y s e s have measured o n l y 88% of t o t a l  cells  sterol  i n the plasma membrane of v e g e t a t i v e c e l l s as s t i g m a s t e n o l ( 3 7 ) .  Lipid-phosphorus extracted  T a b l e 1:  Extraction Procedure  Fraction No.  from e x p o n e n t i a l l y  growing c e l l s o f I), d i s c o i d e u m Ax-2  Description of F r a c t i o n  (1)  (2)  Phosphorus i n Fraction  Phosphorus i n Fraction  - x  F o l c h et. a l  I II III  IV  I  B l i g h & Dyer  II  III  I n i t i a l 19.0 v o l . C-M  3  (2:1 v/v)  extract  Aqueous C a C ^ phase and combined aqueous washings. 3  Second e x t r a c t o f 15.0 v o l . C-M (7:1 v/v) s a t u r a t e d w i t h 5% aq. NH^. Residue cl 1 s t and 2nd 6.25 v o l . aqueous C-M phase e x t r a c t s combined. Aqueous methanol phase a f t e r second extraction. Residue  lower C-M  3  -±  (nmol m g t o t a l protein)  (nmol m g t o t a l protein)  118.1  135.3 ± 20  ± 0  12.4 ± 0  9.3 ± 3.0  4.0 ± 1.5  4.5 ± 2.5  n o t determined  n o t determined  88.9 ± 1.5  9.5 ± 0.9  not determined  1.  E x t r a c t i o n of d u p l i c a t e samples by the t e c h n i q u e s o f F o l c h j^lt a l (41) ( i n c o r p o r a t i n g a second, ammoniacal e x t r a c t i o n ) and o f B l i g h & Dyer (38). R e s u l t s a r e t h emean o f two e x t r a c t i o n s .  2.  E x t r a c t i o n o f m u l t i p l e samples by t h e method o f F o l c h e t a l . R e s u l t s a r e t h e mean of independent e x t r a c t i o n s ± s t a n d a r d d e v i a t i o n . 1  C-M i n d i c a t e s c h l o r o f o r m - m e t h a n o l ' .  seven  T a b l e 2:  F a t t y a c i d a n a l y s i s o f h y d r o l y z e d t o t a l l i p i d from F o l c h et: a l (41) and B l i g h & Dyer (38) e x t r a c t i o n s o f e x p o n e n t i a l l y growing c e l l s o f I), d i s c o i d e u m Ax-2.  F o l c h et a l E x t r a c t i o n F r a c t i o n s Fatty Acid  I  II  III (Ug  fatty  trace  0.3 + 0.1  0.5 ± 0.1  0.7 ± 0.1  5.2 + 0.1  0.5 + 0.3  1.5 + 0.1  trace  trace  trace  1.3 + 0.7  trace  0.3 + 0.1  1.6 + 0.1  trace  trace  trace  1.3 + 0.7  trace  trace  1.7 + 0.1  0  0  0  2.0 + 0.3  0  0  30.1  +  0.7  0. 4 ± 0.1  0.7 ± 0.1  0.7 ± 0.1  26.5  +  0.8  0.8 + 0.3  1.5 + 0.1  49.5  +  1.1  0. 5 ± 0.3  0.7 ± 0.1  0.3 ± 0.1  43.6  +  1.1  0.8 + 0.4  0.5 + 0.1  0.9 + 0.4  trace  0  trace  0  0  1.5 + 0.7  trace  0.3 ± 0.2  0.3 ± 0.2  0.5 + 0.3  2.3 + 0.6  0.3 + 0.1  trace  trace  16:0  5.2 + 0.7  0. 4 ± 0.2  2.1 + 0.1 9 )  & 17:0  a  18:0 A9  18:1< > _ 1 8 : l A5  ( A l l ) a  9  18:2< ' > _ 1 8 : 2 < (A9,12) 1 8 : 2  0thers  b  A 5  >  1 1 ) a  protein)  1.5 + 0.3  Palmitaldehyde  >  III  trace  trace  ( A 5  •A " I total a c i d mg  II  0  trace  16:2  I  trace  0.5 + 0.2  A9  IV  2.9 + 0.4  14:0  16:1< >  B l i g h & Dyer E x t r a c t i o n F r a c t i o n s  . trace  trace 3.2 + 0.5  Total fatty acid per f r a c t i o n (lig mg~l p r o t e i n )  93.7  +  4  1. 5 ± 1  2.4 ± 1  2.3 ± 1  87.5  +  5  2.7 + 2  6.4 + 1  % T o t a l Recovered  93.8  +  4  1. 5 ± 1  2.4 ± 1  2.3 ± 1  90.6  +  5  2.8 + 2  6.6 + 1  L i p i d e x t r a c t i o n f r a c t i o n s were as d e s c r i b e d i n T a b l e 1. R e s u l t s a r e p r e s e n t e d as t h e mean o f t h r e e d e t e r m i n a t i o n s ± s t a n d a r d d e v i a t i o n ; ' t r a c e ' i n d i c a t e s <0.1 Ug f a t t y a c i d mg-'- t o t a l p r o t e i n . -  a  These f a t t y a c i d s were n o t s e p a r a t e d under t h e c o n d i t i o n s used b u t were shown t o be p r e s e n t by D a v i d o f f & Korn ( 5 6 ) .  b  S e v e r a l u n i d e n t i f i e d minor components.  T a b l e 3:  S t e r o l a n a l y s i s of t o t a l l i p i d from F o l c h e t a l (41) and B l i g h & Dyer (38) e x t r a c t i o n s of e x p o n e n t i a l l y growing c e l l s of IL d i s c o i d e u m Ax-2.  Folch ^ t a l Extraction Fractions Retention  a  I  II  III  B l i g h & Dyer E x t r a c t i o n F r a c t i o n s IV  I  II  (yg c h o l e s t e r o l e q u i v a l e n t  1.17  1.6  +  0.1  1.30  1.8  +  0.1  0.2  ±  0.1  1.43  17.1  +  3.5  0.2  ±  0.0  1.59  0.5  +  0.1  trace  0.4  +  0.1  0  T o t a l s t e r o l per f r a c t i o n (yg mg~l p r o t e i n )  21.4  +  3.9  0.4  ±  0.1  0.3  ±  % T o t a l Recovered  96.8  +  17.6  1.8  ±  0.5  1.4  ±  Others  b  trace  0.1  +  0.0  trace  -1 mg  +  protein)  0  trace  III  0 trace  trace  .2.0  ± 0.4  trace  13.6  ± 2.1  trace  trace  0.2  ± 0.2  trace  trace  0.5  ± 0.1  0.4  ±  0.3  0.1  trace  16.3  ± 2.8  2.0  ±  1.6  0.5  trace  89.1  ± 15.3  0.2  ±  0.1  1.6  ±  1.3  0  10.9  ±8.7  L i p i d e x t r a c t i o n f r a c t i o n s were as d e s c r i b e d i n T a b l e 1. R e s u l t s a r e p r e s e n t e d as the mean of t h r e e d e t e r m i n a t i o n s ± s t a n d a r d d e v i a t i o n ; ' t r a c e ' i n d i c a t e s <0.1 yg c h o l e s t e r o l e q u i v a l e n t mg~l p r o t e i n . A b s o l u t e v a l u e s v a r y more t h a n '% t o t a l r e c o v e r e d ' f o r the l a t t e r a r e measured w i t h i n each e x p e r i m e n t . R e t e n t i o n f i g u r e s a r e the r a t i o of ( s t e r o l a c e t a t e : c h o l e s t e r y l a c e t a t e ) g a s - l i q u i d chromatography c o n d i t i o n s used. Several  . u n i d e n t i f i e d minor components.  r e t e n t i o n under  the  The  present  study c o n f i r m e d  s i g n i f i c a n t proportions stigmastenol  t h a t o t h e r s t e r o l s were p r e s e n t  (Table 3) and  in  showed t h a t b o t h  ( s t a n d a r d r e t e n t i o n 1.43)  and minor s t e r o l s remained  a f t e r e x t r a c t i o n of the c e l l s by the B l i g h and Dyer  technique.  Having determined t h a t f o r v e g e t a t i v e c e l l s of I), d i s c o i d e u m the F o l c h l i p i d e x t r a c t i o n p r o c e d u r e was  s u p e r i o r t o t h a t of  B l i g h and Dyer, the e f f i c i e n c y of the ' F o l c h f i n a l e x t r a c t i o n ' ( F o l c h f r a c t i o n I I I ) was  analyzed.  n o t a b l y the p o l y p h o s p h o i n o s i t i d e s ,  Certain residual l i p i d s , may  not have been q u a n t i t a t i v e l y  e x t r a c t e d by the ammoniacal s o l v e n t s (55-57) used i n the e a r l y experiments and a c i d c o n d i t i o n s have been found n e c e s s a r y t o remove such l i p i d s from t i s s u e s (30,56,58). f a t t y a c i d analyses  T a b l e 4 shows the  of v a r i o u s f i n a l e x t r a c t i o n s of the  r e s i d u e ' i n w h i c h e x t r a c t i o n t i m e and volumes were  standardized  t o p e r m i t d i r e c t comparison of e x t r a c t i o n e f f i c i e n c y . c h l o r o f o r m - m e t h a n o l - c o n c e n t r a t e d HC1  'Folch  The  (50:50:0.3 by v o l . ) s o l v e n t  m i x t u r e has been used r o u t i n e l y f o r e x t r a c t i n g p h o s p h a t i d y l i n o s i t o l and r e l a t e d compounds from v a r i o u s t i s s u e s (T. p e r s o n a l communication).  Buckley,  B o t h a c i d c o n d i t i o n s t e s t e d were  s u p e r i o r to the a l k a l i n e s o l v e n t m i x t u r e  and  c o n d i t i o n s were the most e f f i c i e n t .  r e a s o n s f o r these  The  the s t r o n g l y a c i d i c  d i f f e r e n c e s were not a p p a r e n t . As a r e s u l t of t h e s e s t u d i e s , I), d i s c o i d e u m l i p i d s were e x t r a c t e d i n a l l subsequent e x p e r i m e n t s by the p r o c e d u r e of F o l c h et al_ ( 4 1 ) , f o l l o w e d by an e x t r a c t i o n w i t h m e t h a n o l - c o n c e n t r a t e d HC1  (50:50:0.3 by  vol.).  chloroform-  Fatty a c i d a n a l y s i s of various f i n a l e x t r a c t i o n s ( F r a c t i o n s I I I ) f o l l o w i n g i n i t i a l procedures of F o l c h e t a l (41).  T a b l e 4:  Fraction I  Fraction III A  Fatty Acid  C  B  (yg f a t t y a c i d )  14:0  0.58  0.01  0.03  0.08  Palmitaldehyde  0.85  0.02  0.02  0.03  16:0  3.62  0.06  0.34  0.29  1.76  0.01  0.02  0.04  0.80  0.04  0.03  0.03  2.59  0.06  0.11  0.10  23.75  0.09  0.20  0.57  34.76  0.12  0.16  0.18  0.45  0.00  0.04  0.05  1.26  0.02  0.11  0.47  70.42  0.43  1.06  1.84  A9  16:1< > . (A5,9) 1 6  2  &  1  7  :  Q  a  18:0 18:l  ( A 9 ) &  ( A 5  18:l  9 )  ( A 1 1 ) a  18:2 ' & 18:2 (A9,12)  ( A 5  '  1 8 : 2  Others  0  Total Fatty Acid  1 1 ) a  A l l e x t r a c t i o n s were o f e q u i v a l e n t samples from one p r e p a r a t i o n of e x p o n e n t i a l l y growing I), d i s c o i d e u m d e s c r i b e d i n T a b l e 1.  Ax-2.  F r a c t i o n I was as  F r a c t i o n I I I was an o v e r n i g h t e x t r a c t i o n  a t 4°C c o m p r i s i n g : A, 15 v o l ( c h l o r o f o r m - m e t h a n o l  (7:1 v/v)  s a t u r a t e d w i t h 5% (w/v) aq. NH3); B, 15 v o l ( c h l o r o f o r m - m e t h a n o l - g l a c i a l a c e t i c acid-water  (8:4:2:1 by v o l ) ) ;  C, 15 v o l ( c h l o r o f o r m - m e t h a n o l - c o n c e n t r a t e d  HC1  (50:50:0.3 by v o l ) ) . V a l u e s a r e t h e mean o f t h r e e d e t e r m i n a t i o n s . These f a t t y a c i d s were n o t s e p a r a t e d under t h e c o n d i t i o n s used but were shown t o be p r e s e n t by D a v i d o f f and Korn (60). a  b  S e v e r a l u n i d e n t i f i e d minor components.  20.  b.  I s o l a t i o n and Q u a l i t a t i v e A n a l y s i s of L i p i d  Classes  S e p a r a t i o n o f p h o s p h o l i p i d from i n d i v i d u a l n e u t r a l l i p i d c l a s s e s by t h i n - l a y e r chromatography i s i l l u s t r a t e d i n F i g u r e 1.  Figure 2 ( i )  shows r o u t i n e t h i n — l a y e r c h r o m a t o g r a p h i c s e p a r a t i o n of i n d i v i d u a l p h o s p h o l i p i d c l a s s e s and F i g u r e 2 ( i i ) an a l t e r n a t i v e p h o s p h o l i p i d s e p a r a t i o n i n the presence of b o r i c a c i d w h i c h r e t a r d s the m i g r a t i o n of p h o s p h a t i d y l g l y c e r o l and a s s i s t e d i d e n t i f i c a t i o n of the c h a r a c t e r i s t i c p o s i t i o n s of p a r t i c u l a r p h o s p h o l i p i d s a f t e r t h e i r development on b o t h  chromatoplates.  E x p o n e n t i a l l y growing i n t a c t c e l l s of p_. d i s c o i d e u m  Ax-2  were  found t o c o n t a i n a l l major p h o s p h o l i p i d groups w i t h the e x c e p t i o n o f sphingomyelin.  W h i l e W i l h e l m s et^ al have shown g l y c o s p h i n g o l i p i d s  to be a c t i v e as s u r f a c e a n t i g e n s of JJ. d i s c o i d e u m  ( 3 6 ) , most g l y c o -  l i p i d s would have been removed from the l i p i d e x t r a c t by the aqueous wash i n c o r p o r a t e d i n the e x t r a c t i o n method of F o l c h et al i n t h i s i n v e s t i g a t i o n . Lysophosphatidylethanolamine, c h o l i n e and  l y s o p h o s p h a t i d i c a c i d were d e t e c t e d  (41) used  lysophosphatidyl-  i n the i n t a c t  e x t r a c t s , i n agreement w i t h p r e v i o u s s t u d i e s ( 3 0 ) .  cell  Phosphatidyl-  g l y c e r o l and p h o s p h a t i d y l s e r i n e were shown t o be components of I), d i s c o i d e u m  grown under s t a n d a r d  t r i a c y l g l y c e r o l s , ubiquinones,  axenic c o n d i t i o n s .  f r e e s t e r o l s and  Mono-, d i - and  s t e r o l e s t e r s were  a l l p r e s e n t , s t i g m a s t e n o l b e i n g a major component of b o t h the f r e e s t e r o l and s t e r o l e s t e r f r a c t i o n s . Q u a l i t a t i v e l y s i m i l a r p h o s p h o l i p i d r e s u l t s were o b t a i n e d plasma membrane samples as from i n t a c t c e l l e x t r a c t s .  from  The q u a n t i t i e s  of f r e e s t e r o l and s t e r o l e s t e r i n the plasma membranes were measured  21.  _b  Solvent 11 <-  o -a. Solvent 1<-  o. o*^  0  0'  0  0  0  0  0  0~ —»—i—i—i—i—i—•—f»j ©  Origin-4 2  3  4  5  Individual  Fig.  1;  6  7  8  9  1 0 II 12  A B  C  neutral lipids  Separation of neutral l i p i d s by monodirectional, biphasic thin-layer chromatography on s i l i c a g e l H plates.  Lipids 1 and 3 appear dark under short wavelength u l t r a v i o l e t i r r a d i a t i o n . L i p i d s 1, 4 and 8 y i e l d blue fluorescence under long wavelength u l t r a v i o l e t i r r a d i a t i o n . c Standard l i p i d s : . 1. phospholipid 2. monoacylglycerol 3. ubiquinone 4. unidentified l i p i d i c free s t e r o l 5. 6. 1,2-diacylglycerol A. B. C.  compound  7. 8. 9. 10. 11. 12.  1,3-diacylglycerol free f a t t y acid triacylglycerol f a t t y acid ester s t e r y l ester hydrocarbon  standard neutral l i p i d mixture D. discoideum Ax-2 whole c e l l l i p i d extract D. discoideum Ax-2 plasma membrane (PM1) l i p i d extract  Solvent  I  comprised: isopropyl e t h e r - g l a c i a l acetic acid (96:4 v/v).  Solvent I I comprised: n-hexane-diethyl e t h e r - g l a c i a l a c e t i c acid (90:10:1 by v o l . ) . Each l i p i d was i d e n t i f i e d as described under Materials and Methods. This compound was i s o l a t e d from JJ. discoideum membrane as stated under Results.  preparations  22.  (i)  (ii)  V  f  ?  r  r eg)  CD® CD  ® CD® CD <z> ® CD  +  +  . Solvent ll  Fig. 2:  (i)  Solvent  b  n  b  Two-dimensional, biphasic thin-layer chromatography of a mixture of standard phospholipids on s i l i c a gel H plates with additives.  magnesium a c e t a t e - s i l i c a gel H thin layer of 300 ym thickness ,  ( i i ) magnesium acetate-boric a c i d - s i l i c a gel H thin layer of 300 thickness .  ym  c  Standard l i p i d s : 1. 2. 3. 4. 5. 6.  lysophosphatidylethanolamine phosphatidylethanolamine lysophosphatidylcholine phosphatidylcholine phosphatidic acid lysophosphatidic acid  Solvent I  7. 8. 9. 10. 11. i.  phosphatidylinositol phosphatidylserine phosphatidylglycerol sphingomyelin cardiolipin chromatoplate impurities  comprised: chloroform-methanol-28% (w/v) ammonia (65:25:8 by v o l . ) .  acqueous  Solvent II comprised:  chloroform-acetone-methanol-glacial acetic a c i d - d i s t i l l e d , deionised water (35:35:7:10:3 by v o l . ) .  Each phospholipid was and Methods.  i d e n t i f i e d as described under Materials  but only trace amounts of a c y l g l y c e r o l were present and quinone derivatives were not detected. Short wavelength u l t r a v i o l e t absorption revealed the presence of a currently unidentified neutral l i p i d compound on chromatoplates following thin layer chromatography of crude and plasma membrane l i p i d extracts (Fig. 1). I n i t i a l l y this was thought to be one of the  organism's less common s t e r o l components migrating with a  d i f f e r e n t mobility than the standard cholesterol, f o r stigmastenol has a marginally lower chromatographic system.  than cholesterol i n t h i s thin layer However, the compound's u l t r a v i o l e t  absorption spectrum features r a d i c a l differences from that.of stigmastenol and gas-liquid chromatographic analysis following hydrolytic procedures showed that the unidentified neutral l i p i d contained a negligable amount of either s t e r o l or f a t t y acid. The compound emitted blue fluorescence upon i r r a d i a t i o n with long wavelength u l t r a v i o l e t energy.  Fluorescence was also observed  under both long and short wavelength u l t r a v i o l e t radiation after treatment of the unknown l i p i d with rhodamine 6G, from which i t was dissociated by solution i n chloroform-methanol (4:1 v/v).  c.  Quantitative L i p i d Determinations of Intact C e l l Extracts  To enable comparison of intact c e l l l i p i d data with that i n the  l i t e r a t u r e (1,30,31,59-62) protein and dry weight measurements  were taken from exponentially growing c e l l s and a r a t i o of 0.59 (protein mass:mass of dry c e l l s ) was obtained. Table 5 displays the i n d i v i d u a l and t o t a l phospholipid content  T a b l e 5:  L i p i d - p h o s p h o r u s a n a l y s i s of i n t a c t c e l l s of e x p o n e n t i a l l y growing I), d i s c o i d e u m Ax-2 f o l l o w i n g p h o s p h o l i p i d s e p a r a t i o n by t h i n - l a y e r chromatography. Current Analysis  Phospholipid  Ellingson Analysis  (nmol mg (molar (molar t o t a l p r o t e i n ) percentage) percentage) Ly sopho spha t i d y l e thanolamine  11.4  +  7.7  8.9  +  6.0  10. 6  b  Phosphatidylethanolamine  47.1  +  8.0  36.9  +  6.3  47. 2  b  3.1  +  0.5  2.4  +  0.4  3.4  41.3  +  7.3  32.4  +  5.7  16.8  2.3  +  0.5  1.7  +  0.4  3.4  b  3.5  +  2.4  2.8  +  1.9  1.4  b  Phosphatidylinositol  6.8  +  1.1  5.3  +  0.9  8.7  Phosphatidylserine  4.2  +  3.3  3.3  +  2.6  -  Phosphatidylglycerol  2.2  +  0.7  1.7  +  0.6  0  Lysophosphatidylcholine Phosphatidylcholine Phosphatidic  acid  Lysophosphatidic  acid  Sphingomyelin Cardiolipin  0 3.6  +  1.7  2.8  +  Lysocardiolipin  0  0  3.  3.5  +  +  129.0  ±  Others  Total Phospholipid  -  0  2.5  2.7  1.5  2.6 1.3  1.9  8.3  16.9  Data from the c u r r e n t a n a l y s i s a r e the mean of f i v e d e t e r m i n a t i o n s standard  deviation.  Minor phospholipid a c i d (30). b  ±  components, p o s s i b l y i n c l u d i n g l y s o b i s p h o s p h a t i d i c  Undetermined amounts of p l a s m a l o g e n forms a l s o p r e s e n t  (30).  of e x p o n e n t i a l l y growing i n t a c t c e l l s o f I), d i s c o i d e u m  Ax-2.  In  comparison w i t h E l l i n g s o n ' s d a t a f o r Ax-2 ( 3 0 ) , a l s o shown i n T a b l e 5, the c u r r e n t a n a l y s i s y i e l d e d major d i f f e r e n c e s . t i d y l c h o l i n e was r e c o v e r e d phospholipids  i n much g r e a t e r p r o p o r t i o n t o o t h e r  (32.4 moles %) and p h o s p h a t i d y l e t h a n o l a m i n e  s i g n i f i c a n t l y lower e x t e n t p r e v i o u s study  P r i m a r i l y phospha-  to a  (36.9 moles %) than they were i n t h e  (16.8 moles % and 47.2 moles % r e s p e c t i v e l y ) ,  although  they were t h e two most abundant p h o s p h o l i p i d s i n b o t h a n a l y s e s . However, t h e e a r l i e r r e p o r t p r o v i d e d p e r c e n t a g e v a l u e s o n l y ( 3 0 ) , p r e v e n t i n g p r e c i s e measurement these two s t u d i e s .  of q u a n t i t a t i v e d i f f e r e n c e s between  E l l i n g s o n d e t e c t e d no p h o s p h a t i d y l g l y c e r o l  i n Ax-2 and a t t r i b u t e d t h e t r a c e found i n c e l l s o f s t r a i n NC-4 to b a c t e r i a l s o u r c e s whereas i n t h e p r e s e n t  study  phosphatidyl-  g l y c e r o l comprised 1.7 moles % o f t h e t o t a l p h o s p h o l i p i d : t h e reasons for  these d i s c r e p a n c i e s a r e not apparent.  E l l i n g s o n d i d not record  any d a t a f o r p h o s p h a t i d y l s e r i n e (30) a l t h o u g h i n many s i m i l a r t h i n - l a y e r chromatography systems t h i s compound i s p o o r l y r e s o l v e d from p h o s p h a t i d y l i n o s i t o l .  The p r e v i o u s l y p u b l i s h e d  phosphatidyl-  i n o s i t o l f i g u r e i s s i m i l a r t o the t o t a l of p h o s p h a t i d y l s e r i n e p l u s p h o s p h a t i d y l i n o s i t o l detected i n t h i s a n a l y s i s , suggesting E l l i n g s o n d i d n o t s e p a r a t e t h e s e two components. lysophosphatidylethanolamine, tidic  that  The l e v e l s o f  l y s o p h o s p h a t i d y l c h o l i n e , lysophospha-  a c i d , p h o s p h a t i d i c a c i d and c a r d i o l i p i n were s i m i l a r t o t h e  previous a n a l y s i s (30). A c y l g l y c e r o l a s s a y s o f l i p i d e x t r a c t s from i n t a c t c e l l s were u n d e r t a k e n b e f o r e and a f t e r t h e i r s e p a r a t i o n i n t h e n e u t r a l l i p i d t h i n - l a y e r chromatography system.  T a b l e 6 shows t h a t t h e t o t a l  T a b l e 6:  A c y l g l y c e r o l a n a l y s i s of i n t a c t c e l l s of e x p o n e n t i a l l y growing JJ_. d i s c o i d e u m Ax-2.  Neutral Lipid  I n t a c t C e l l Content (nmol mg total) ( protein )  (molar p e r c e n t a g e )  Monoacylglycerol  5.9  32  1.2- d i a c y l g l y c e r o l  4.2  23  1.3- d i a c y l g l y c e r o l  1.1  6  Triacylglycerol  7.4  40  3.  Total Acylglycerol Recovered  18.2 ±  0.4  T o t a l a c y l g l y c e r o l was measured as an independent parameter.  27.  q u a n t i t y of a c y l g l y c e r o l i n v e g e t a t i v e p_. d i s c o i d e u m Ax-2 r e l a t i v e to p h o s p h o l i p i d lipid  (cf. Table 1).  i n most e u c a r y o t i c c e l l s , was  was  T r i a c y l g l y c e r o l , a storage  the most abundant but s i g n i f i c a n t  q u a n t i t i e s of the o t h e r a c y l g l y c e r o l s were a l s o p r e s e n t . Coe  q u a n t i f i e d t r i a c y l g l y c e r o l i n c e l l s of s t r a i n NC-4  e x p o n e n t i a l l y on b a c t e r i a and (31)  ( c . 7 nmol mg  t h e i r f i g u r e of 3.2  The  and  growing g ^ dry w e i g h t nmol mg  c e l l s grown under a x e n i c c o n d i t i o n s .  i n t a c t c e l l f r e e s t e r o l a n a l y s i s shown i n T a b l e 7 c o n f i r m e d  t h a t the major f r e e s t e r o l was 85.5  mg  Long  ^ p r o t e i n ) agrees c l o s e l y w i t h the 7.4  p r o t e i n determined here i n Ax-2  small  moles % of the t o t a l .  stigmastenol  Two  (31,37), w h i c h comprised  o t h e r components of the f r e e s t e r o l  f r a c t i o n were found i n s i g n i f i c a n t amounts, t h e i r a c e t a t e s  having  g a s - l i q u i d chromatography r e t e n t i o n v a l u e s r e l a t i v e t o c h o l e s t e r y l acetate  of 1.30  (7.5 moles % t o t a l f r e e s t e r o l ) and  % t o t a l free sterol). distinguished.  (4.2 moles  Three o t h e r minor c o n s t i t u e n t s c o u l d  Long and  Coe,  p r e s e n t a t a p p r o x i m a t e l y 40 nmol mg (31) compared t o the t o t a l of 56.8  as s t i g m a s t e n o l . p r o t e i n (9 mg nmol mg  This  99% was  g ^ dry weight)  ^ p r o t e i n reported  in  Ax-2.  S t e r o l e s t e r a n a l y s i s of l i p i d  e x t r a c t s from i n t a c t c e l l s  q u a n t i t a t i v e g a s - l i q u i d chromatography y i e l d e d a f i g u r e of nmol mg  be  however, measured i n e x c e s s of  of the f r e e s t e r o l from s t r a i n NC-4  t h i s s t u d y of  1.59  ^ p r o t e i n , 2.3  1.28  moles % of the f r e e s t e r o l t o t a l ( T a b l e 7 ) .  P r e v i o u s d e t e r m i n a t i o n s of s t e r o l e s t e r c o n t e n t of i n t a c t c e l l s s t r a i n NC-4 2 mg  g  y i e l d e d v a l u e s from 1 t o 3 nmol mg  d r y w e i g h t ) (31).  that stigmastenol  was  by  In contrast  ^ p r o t e i n (up  t o Long and  of  to  Coe's f i n d i n g  the s o l e s t e r o l p r e s e n t i n the s t e r o l e s t e r  T a b l e 7:  Retention  S t e r o l a n a l y s i s o f t h e f r e e s t e r o l s and s t e r o l e s t e r s e x t r a c t e d from i n t a c t c e l l s o f e x p o n e n t i a l l y growing D. d i s c o i d e u m Ax-2.  3  Free S t e r o l of I n t a c t C e l l s , as S t i g m a s t e n o l  nmol mg protein  D  S t e r o l Ester S t e r o l of I n t a c t C e l l s , as S t i g m a s t e n o l ^ , -1 nmol mg protein  molar percentage  31.2 ± 1.0  1.17  trace  1.30  4.3 ± 0.8  .7.5 ± 1 . 4  0  48.5 ± 1.0  85.5 ± 1.8  0.71 ± 0.01  55.5 ± 0.8  2.4 ± 1.0  4.2 ± 1.7  0.17 ± 0.02  13.5 ± 1.7  1.43  c  1.59 Others  trace  Total Sterol Recovered  56.8 ± 2.8  trace  molar percentage  . 0.40 ± 0..01  0  trace  0  1.28 ± 0.04  Free s t e r o l v a l u e s a r e t h e mean o f f i v e d e t e r m i n a t i o n s deviation;  0  -  ' t r a c e ' i n d i c a t e s <0.4 nmol mg"'" p r o t e i n .  ± standard Sterol ester  v a l u e s a r e t h e mean of two d e t e r m i n a t i o n s .  R e t e n t i o n f i g u r e s a r e the r a t i o o f ( s t e r o l a c e t a t e : c h o l e s t e r y l a c e t a t e ) r e t e n t i o n under t h e q u a n t i t a t i v e g a s - l i q u i d chromatography c o n d i t i o n used.  The r e l a t i v e amounts o f each s t e r o l were determined by r e f e r e n c e to an i n t e r n a l c h o l e s t e r o l s t a n d a r d and were c o n v e r t e d t o m o l a r i t i e s u s i n g t h e m o l e c u l a r w e i g h t o f s t i g m a s t e n o l , the most abundant s t e r o l o f D.  c  Stigmastenyl acetate.  discoideum.  f r a c t i o n o f NC-4 (31) t h e p r e s e n t s t u d y shows t h a t i t c o n s t i t u t e s o n l y 55.5 % o f t h e s t e r o l m o i e t i e s o f Ax-2 s t e r o l e s t e r s ( T a b l e 7 ) . The second most abundant s t e r o l o f t h e Ax-2 s t e r o l e s t e r f r a c t i o n was a minor component o f t h e f r e e s t e r o l f r a c t i o n and had a r e t e n t i o n v a l u e o f 1.17.  The second most abundant f r e e s t e r o l  (retention  v a l u e 1.30) was absent from t h e s t e r o l e s t e r f r a c t i o n . s t e r o l p r o f i l e s o f these two l i p i d c l a s s e s were v e r y  d.  Thus t h e different.  E v a l u a t i o n o f P h o s p h o l i p i d D e g r a d a t i o n D u r i n g Plasma Membrane Preparation.  Membrane l i p i d measurements have been r e c o r d e d r e l a t i v e t o the p r o t e i n c o n t e n t o f t h e i r r e s p e c t i v e membrane f r a c t i o n s .  Crude  membrane p r e p a r a t i o n s underwent m i n i m a l p r o t e i n l o s s i n t h e t w e l v e hours f o l l o w i n g h o m o g e n i z a t i o n ,  indicating  that p r o t e i n degradation  was i n s i g n i f i c a n t under t h e c o n d i t i o n s o f p r e p a r a t i o n . F e r b e r et al d i s c o v e r e d h i g h a c t i v i t i e s o f p h o s p h o l i p a s e A and l y s o p h o s p h o l i p a s e i n I), d i s c o i d e u m v l 2 / M l grown i n a s s o c i a t i o n w i t h b a c t e r i a ( 6 3 ) . These two a c t i v i t i e s would l e a d t o t h e f o r m a t i o n from p h o s p h o g l y c e r i d e s o f n o n - a c y l a t e d ( s n ) g l y c e r o l - 3 - p h o s p h a t i d y l d e r i v a t i v e s w h i c h would be f u l l y s o l u b l e i n t h e aqueous phase o f t h e s t a n d a r d e x t r a c t i o n washing procedure subsequent l i p i d - p h o s p h o r u s a n a l y s i s .  (41) and e x c l u d e d  from  No e v i d e n c e o f p h o s p h o l i p a s e  C a c t i v i t y has been found i n c e l l homogenates ( 6 3 ) .  Any l i p a s e  a c t i v i t y was o f g r e a t s i g n i f i c a n c e t o t h i s d e t e r m i n a t i o n o f plasma membrane l i p i d c o m p o s i t i o n f o r t h e membrane p r e p a r a t i o n procedure r e q u i r e d a minimum o f 17% h o u r s .  F i g u r e 3 demonstrates t h a t p h o s p h o l i p i d d e g r a d a t i o n was observed i n c e l l homogenates a t 22°C b u t t h a t t h i s was reduced a t 4°C. When l i p i d - p h o s p h o r u s i s expressed remaining  as t h e l o g a r i t h m o f t h e p e r c e n t a g e  a t l e a s t two d i s t i n c t r a t e s o f l o s s a r e r e v e a l e d  (Fig. 3).  T h i s s u g g e s t s t h a t i f e n z y m a t i c d e g r a d a t i o n was r e s p o n s i b l e  there  was e i t h e r more t h a n one p o o l o f p h o s p h o l i p i d as s u b s t r a t e o r t h a t l i p a s e s were i s o l a t e d w i t h i n p a r t i c u l a r s u b f r a c t i o n s . and  the long-term  Figure 4 confirms  degradation  Both the short  r a t e s increased a t the higher  temperature  t h a t t h e e a r l y p h o s p h o l i p i d l o s s f o l l o w i n g homo-  g e n i z a t i o n was c o n s i d e r a b l y g r e a t e r a t 22°C t h a n a t 4°C. C o n s e q u e n t l y the e n t i r e plasma membrane p r e p a r a t i o n p r o c e d u r e was c a r r i e d out a t 4°C o r on i c e and each s t e p performed as r a p i d l y as p o s s i b l e .  The  PMSF used t o i n h i b i t s e r i n e - p r o t e a s e a c t i v i t y d u r i n g plasma membrane p r e p a r a t i o n had no apparent e f f e c t on t h e r a t e o f l o s s o f l i p i d phosphorus from homogenates.  A l s o i n e f f e c t i v e was t h e p o l y e n e  a n t i b i o t i c f i l i p i n w h i c h F e r b e r et al had found t o i n h i b i t t h e phospholipase  A and l y s o p h o s p h o l i p a s e  a c t i v i t i e s o f I), d i s c o i d e u m  vl2/Ml (63). Crude membranes were p r e p a r e d  r a p i d l y a t 4°C and then  immediately  i n c u b a t e d a t 22°C w i t h e i t h e r 8.6% s u c r o s e - T r i s - P M S F b u f f e r , pH 7.4, or w i t h t h e 105,400 xg s u p e r n a t a n t Methods ( b ) ) .  f r a c t i o n (see M a t e r i a l s and  F i g u r e 5 shows t h a t t h e r a t e o f l i p i d - p h o s p h o r u s  l o s s from t h e c r u d e membranes was i n c r e a s e d a p p r o x i m a t e l y by t h e p r e s e n c e o f t h e s u p e r n a t a n t  suggesting  four-fold  t h a t much o f t h e l i p a s e  a c t i v i t y observed i n t h e e a r l i e r e x p e r i m e n t s was n o t membrane bound. Crude membranes were a l s o prepared  and i n c u b a t e d  of e x c e s s o f t h e f o l l o w i n g p u t a t i v e 13. d i s c o i d e u m  i n the presence phospholipase  31.  Fig. 3:  The e f f e c t of temperature on the rates of loss of l i p i d phosphorus from I), discoideum Ax-2 homogenates over an extended period.  Intact c e l l s were removed from the homogenate by centrifugation at 700 xg and incubation was c a r r i e d out at 4°C ( O ) or at 22°C (• ) i n 8.6% sucrose-Tris-PMSF buffer, pH 7.4, as described under Materials and Methods.  F i g . 4:  The e f f e c t of temperature on the early loss of l i p i d phosphorus from I), discoideum Ax-2 homogenates.  Intact c e l l s were removed from the homogenate by centrifugation at 700 xg and incubation was carried out at 4°C i n 8.6% sucroseTris-PMSF buffer, pH 7.4, as described under Materials and Methods (O). After 65 minutes half of the preparation was transferred from 4°C to 22°C ( • ).  33.  6  0  7  0  8  Time  F i g . 5:  0  post  9  100m  0  homogenization  (minutes)  The e f f e c t of the soluble f r a c t i o n of c e l l homogenates of p_. discoideum Ax-2 on the lipid-phosphorus content of crude membrane preparations incubated at 22 C.  The crude membrane samples were prepared at 4°C as described under Materials and Methods. (1)  Crude membranes resuspended i n 8.6% sucrose-Tris-PMSF buffer, pH 7.4, and incubated at 22°C ( • ) . ^ _ Rate of lipid-phosphorus l o s s =0.6 nmol mg protein min x  3  (2)  Crude membranes resuspended i n the supernatant from the 105,400 xg preparatory centrifugation step (homogenate soluble fraction) and incubated at 22°C ( • ) . _ _^ Rate of lipid-phosphorus l o s s = 2.3 nmol mg protein min x  a  Data was subjected to linear regression analysis i n order to obtain the average rate of change of lipid-phosphorus over the sampling period.  inhibitors:  4 - c h l o r o m e r c u r i b e n z o i c a c i d , sodium  4-hydroxymercuri-  b e n z o a t e , d i g i t o n i n i n b o t h aqueous s u s p e n s i o n and m e t h a n o l i c solution, ethylenediaminetetraacetic acid e f f e c t i v e a t r e d u c i n g l i p i d phosphorus  ( 6 3 ) . However, none were  l o s s from crude membranes.  F i g u r e 6 shows t h a t t h i s l o s s was m i n i m a l over a 20 hour s a m p l i n g p e r i o d when t h e i n c u b a t i o n t e m p e r a t u r e was m a i n t a i n e d a t 4°C. Plasma membranes were r o u t i n e l y i s o l a t e d w i t h i n t h i s t i m e . In o r d e r t o d e t e r m i n e whether p a r t i c u l a r p h o s p h o l i p i d s were m o d i f i e d crude membranes were i n c u b a t e d i n 8.6% s u c r o s e - T r i s - P M S F b u f f e r , pH 7.4, f o r a t i m e p e r i o d e q u i v a l e n t t o t h a t of a plasma membrane p r e p a r a t i o n ( T a b l e 8 ) . to  P h o s p h a t i d y l c h o l i n e was observed  d e c r e a s e v e r y d r a m a t i c a l l y from 95.0 t o 30.5 nmol mg ^ p r o t e i n  i n 20 hours a t 4°C and even f u r t h e r a t 22°C. L y s o p h o s p h a t i d y l c h o l i n e remained a t a low l e v e l throughout and p h o s p h a t i d i c a c i d and lysophosphatidic a c i d d i d not increase g r e a t l y suggesting that a p h o s p h o l i p a s e C o r a c o m b i n a t i o n of p h o s p h o l i p a s e s were f u n c t i o n i n g s p e c i f i c a l l y . Phosphatidylethanolamine a l s o decreased  significantly,  v i r t u a l l y a l l o f t h e l o s s o c c u r r i n g i n t h e f i r s t 10 hours o f t h e incubation.  L y s o p h o s p h a t i d y l e t h a n o l a m i n e i n c r e a s e d t o a degree  t h a t was a l m o s t e q u i v a l e n t t o t h e l o s s o f p h o s p h a t i d y l e t h a n o l a m i n e i n d i c a t i n g t h a t the d i s a p p e a r a n c e o f l i p i d i c c h o l i n e from t h e i n c u b a t i o n may have been due t o a s e l e c t i v e a c t i v i t y . minor components p h o s p h a t i d y l i n o s i t o l and c a r d i o l i p i n  Of t h e quantities  f l u c t u a t e d t o some e x t e n t , t h e amount of p h o s p h a t i d y l s e r i n e s m a l l and p h o s p h a t i d y l g l y c e r o l was e n t i r e l y degraded. of  remained  The c a t e g o r y  ' o t h e r s ' c o n t a i n s an i n c r e a s i n g amount of m a t e r i a l w i t h t i m e .  I t i n c l u d e s u n i d e n t i f i e d new s p o t s w h i c h appeared on the chromato-  in en  C 0  2 o _  1  5 0 0  P  Ui  E  "o E c w  (/> 3  a o  •5-  1—*  T  4 0 0  3ooj  2 0 0  100  i 8  10  12  Time post homogenization F i g . 6:  14  16  18  2 0  (hours)  L i p i d - p h o s p h o r u s c o n t e n t o f I), d i s c o i d e u m Ax-2 crude membranes d u r i n g extended i n c u b a t i o n a t 4 C.  Data was s u b j e c t e d t o l i n e a r r e g r e s s i o n a n a l y s i s u s i n g t h e l e a s t squares p r o c e d u r e . l i p i d - p h o s p h o r u s = -2.2 n mol mg~l p r o t e i n h o u r ~ l .  Rate o f change of  Table 8:  P r o p o r t i o n o f i n d i v i d u a l p h o s p h o l i p i d components o f JJ. d i s c o i d e u m Ax-2.crude membranes resuspended i n 8.6% sucrose-Tris-PMSF b u f f e r , pH 7.4, i n c u b a t e d a t e i t h e r 4°C o r 22°C as shown.  Phospholipid  Zero Time  10 h r at: 4°C  (nmol mg  Lysophosphatidylethanolamine  128.5 + 36.0  Phosphatidylethanolamine  120.5 + 23.5  143.5 + 23.0  20 h r a t 4°C  20 h r a t 22°C  protein)  151.0 + 14.0  72.0 +  9.0  69.0 +  4.0  164.5 + 31.0 71.0 + 18.5  Lysophosphatidylcholine  11.5 +  5.0  8.0 +  3.5  5.0 +  3.5  Phosphatidylcholine  95.0 +  4.5  44.0 +  6.0  30.5 +  8.0  21.5 + 11.0  8.0 +  8.0  13.0 +  8.5  20.5 + 12.5  25.5 + 18.0  22.0 +  2.5  43.0 +  2.0  19.0 + 18.5  19.5 + 13.0  Phosphatidic acid Lysophosphatidic  acid  Phosphatidylinositol  41.5 + 14.5  38.5 + 23.0  54.0 + 3.0 +  9.0 ) * 3.0 J  1.0 +  48.5 + 15.0  Phosphatidylserine  6.5 +  5.0  2.5 +  Phosphatidylglycerol  •5.5 +  4.5  0  0  0  0  0  0  Sphingomyelin Cardiolipin Others  0 16.5 +  6.5  17.5 +  6.5  34.5 + 18.5  15.5 + 10.5  9.0 +  8.0  55.5 + 34.5  30.0 + 14.0  23.0 + 14.0  3  Total Phospholipid  2.5  1.0  464.5  437.5  416.5  b  389.0  V a l u e s o f l i p i d - p h o s p h o r u s r e c o v e r e d a r e p r e s e n t e d as t h e mean o f d u a l l i p i d e x t r a c t i o n s . The p h o s p h o l i p i d s were s e p a r a t e d by t h i n - l a y e r chromatography b e f o r e b e i n g Si  U n i d e n t i f i e d minor components, k  These two p h o s p h o l i p i d s d i d n o t s e p a r a t e f u l l y i n t h i s a n a l y s i s .  assayed.  grams as t h e i n c u b a t i o n proceeded and p r o b a b l y lated degradation  products.  comprised phosphory-  I n c u b a t i o n a t 22°C g e n e r a l l y enhanced  the changes o b s e r v e d a t 4°C. Thus t h e plasma membrane p h o s p h o l i p i d r e s u l t s a r e p r o b a b l y accurate f o r phosphatidic a c i d , lysophosphatidic a c i d , phosphatidyls e r i n e , p h o s p h a t i d y l i n o s i t o l and c a r d i o l i p i n whereas p h o s p h a t i d y l choline, l y s o p h o s p h a t i d y l c h o l i n e , phosphatidylethanolamine p h o s p h a t i d y l g l y c e r o l a r e p o s s i b l y underestimated e t h a n o l a m i n e may be  e.  and  and l y s o p h o s p h a t i d y l -  overestimated.  Plasma Membrane L i p i d C o m p o s i t i o n  The  i n d i v i d u a l p h o s p h o l i p i d s , t h e s t e r o l s and t h e p h o s p h o l i p i d  f a t t y a c i d compositions  o f t h e two plasma membrane f r a c t i o n s PM1  and PM2 (35) a t e a r l y s t a g e s o f t h e organism's development a r e shown i n Tables  9, 10 and 11.  I t has been suggested t h a t PM1 may be a  p u r e r plasma membrane p r e p a r a t i o n t h a n PM2 o r t h a t t h e two may represent d i f f e r e n t areas of s u r f a c e s p e c i a l i z a t i o n (37). The  t o t a l p h o s p h o l i p i d o f b o t h PM1 and PM2 decreased  i n t h e f i r s t e i g h t hours o f development (Table 9 ) . 29% i n PM1 (from 690.3 t o 491.5 nmol mg" (from 583.3 t o 362.4 nmol mg  1  protein).  1  substantially  The l o s s was  p r o t e i n ) and 38% i n PM2 T h i s r e d u c t i o n i n phospho-  l i p i d : p r o t e i n r a t i o might r e f l e c t the s y n t h e s i s o r i n c o r p o r a t i o n o f membrane p r o t e i n o r t h e d e g r a d a t i o n  o r removal o f membrane l i p i d .  I t i s n o t p o s s i b l e t o d i s t i n g u i s h between t h e s e p o s s i b i l i t i e s from the d a t a i n T a b l e 9 a l t h o u g h p r o t e i n c o n t e n t p e r c e l l has been shown to d e c r e a s e over t h i s p e r i o d ( 1 ) .  Both plasma membrane f r a c t i o n s  T a b l e 9:  Plasma membrane l i p i d - p h o s p h o r u s a n a l y s i s a t t h r e e s t a g e s o f development o f D_. d i s c o i d e u m Ax-2 f o l l o w i n g p h o s p h o l i p i d s e p a r a t i o n by t h i n - l a y e r chromatography.  PMl Phospholipid  0 hrs ( %  a  '  PM2  8 hrs  16 h r s  l i p i d phosphorus r e c o v e r e d )  0 hrs ( %  a  8 hrs  16 h r s  l i p i d phosphorus r e c o v e r e d )  Lysophosphatidylethanolamine  38.5 ± 1.5  17.9  42. 8 ± 1.0  27.9 ± 4.4  36. 7  37.4 + 0.3  Phosphatidylethanolamine  19.4 ± 1.0  17.9  13. 8 ± 3.5  29.7 ± 11.2  17. 7  12.3 + 3.1  0  0  0  trace  5. 9  1.8 + 0.3  31.1 ± 2.0  43.4  5. 3 ± 1.1  25.6 ± 6.0  25. 0  4.1 + 2.0  0  0  trace  trace  0  16.0 + 6.0  0  1.9  8. 9 ± 5.7  4.6 ± 4.4  0  4.5 + 0.2  6.5 ± 2.1  8. 8  7.8 + 0.7  Lysophosphatidylcholine Phosphatidylcholine Phosphatidic  acid  Lysophosphatidic  acid  Phosphatidylinositol Phosphatidylserine Phosphatidylglycerol Sphingomyelin Cardiolipin Others  b  Total Phospholipid (nmol mg~l p r o t e i n )  J  8.6 ± 0 . 5  14.2  trace  C  4 ± 3.7 trace  0  0  0  8. 7 ± 2.6  trace  0  0  0  0  0  0  0  trace  trace  0  trace  5. 9  0  7. 9 ± 1 . 7  3.4 ± 1.8  1.2 ± 0.2 690.3 ± 27.2  3.8 491.5  487. 4 ± 66.5  583.3 ± 41.9  0 362. 4  0 14.8 + 0.9  1.2 + 1.2 360.5 + 12.5  '0 h r s ' i n d i c a t e s t h a t c e l l s were growing e x p o n e n t i a l l y and were e i t h e r h a r v e s t e d f o r plasma membrane a n a l y s i s o r washed f r e e o f n u t r i e n t s and p l a c e d i n b u f f e r t o i n i t i a t e development t o p e r m i t a n a l y s i s a t l a t e r time p o i n t s . 0-, 16- and 8 h r d e t e r m i n a t i o n s a r e t h e mean o f f o u r , two and one s e t o f plasma membrane l i p i d e x t r a c t i o n s r e s p e c t i v e l y . E r r o r s a r e r e c o r d e d as s t a n d a r d d e v i a t i o n s where a p p r o p r i a t e ' t r a c e ' i n d i c a t e s <1% t o t a l p h o s p h o l i p i d . a  b  : PM1 and PM2 a r e t h e two plasma membrane p r e p a r a t i o n s M a t e r i a l s and Methods. : U n i d e n t i f i e d minor components.  obtained  by t h e t e c h n i q u e d e s c r i b e d  under  maintained  t h e i r e i g h t hour p h o s p h o l i p i d : p r o t e i n t o t a l a f t e r s i x t e e n  hours of development, y e t numerous membrane-associated p r o t e i n changes a r e known t o occur d u r i n g e a r l y development  (1,9-12).  Compared w i t h the whole c e l l e x p o n e n t i a l l y grown Ax-2  PM1  discoideum  f r a c t i o n s ( c f . T a b l e 5, column 2; T a b l e 9, column 1) con-  t a i n e d a f a r g r e a t e r p e r c e n t a g e of  lysophosphatidylethanolamine  (38.5 moles %) and a s u b s t a n t i a l l y lower p r o p o r t i o n of p h o s p h a t i d y l ethanolamine (19.4 moles %) .  Lysophosphatidylcholine,  a c i d and l y s o p h o s p h a t i d i c a c i d were absent from PM1 was  cardiolipin  equivalent proportions, approximately Lysophosphatidic  a c i d and  were p r e s e n t  in  t r a c e s of l y s o p h o s p h a t i d y l c h o l i n e  and  Phosphatidylcholine  i n s i m i l a r p r o p o r t i o n ( c . 30 moles %, r e l a t i v e t o t o t a l  phospholipid)  i n the i n t a c t c e l l s , PM1  The p h o s p h a t i d y l c h o l i n e i n PM1  and PM2  was  fractions alike.  found t o d e c r e a s e d r a m a t i c a l l y  r e l a t i v e t o p r o t e i n between the e i g h t h and s i x t e e n t h hours of development (Table 9, columns 1 t o 3 ) .  p r o g r e s s i v e l y from z e r o time. c o n t e n t of PM1  The  a f t e r 16 hours development a p p r o x i m a t e l y contained  occurred  lysophosphatidylethanolamine  fluctuated considerably.  p h o s p h o l i p i d i n PM1  Ax-2  Phosphatidylethanolamine  a l s o decreased by a f a c t o r o f two but t h i s r e d u c t i o n  In vegetative c e l l s 57 moles % of the  and  total  e t h a n o l a m i n e , a l t h o u g h the p r o p o r t i o n  dropped t o 36 moles % a t the 8 hour s t a g e . was  PM2  29 moles % each (Table 9, column  p h o s p h a t i d i c a c i d were a l s o d e t e c t e d i n PM2. present  The  i n t h a t b e f o r e development began phospha-  t i d y l e t h a n o l a m i n e and l y s o p h o s p h a t i d y l e t h a n o l a m i n e  was  and  reduced i n b o t h plasma membrane f r a c t i o n s (0.9 moles % ) .  f r a c t i o n d i f f e r e d from PM1  4).  phosphatidic  not d e t e c t e d a t any t i m e i n PM1,  Lysophosphatidylcholine  y e t p h o s p h a t i d i c a c i d and  lysophosphatidic acid increased as development progressed.  In  view of the substantial loss of phosphatidylcholine i n the l a t t e r half of the observed period these r e s u l t s may indicate that one or more phospholipases  acted p r e f e r e n t i a l l y on phosphatidylcholine  i n the plasma membrane during development.  Previous data had  shown that crude membranes were capable of degrading  phosphatidyl-  choline i n the presence of lysophosphatidylethanolamine an accumulation  of lysophosphatidylcholine (Table 8).  without Phosphatidyl-  glycerol and c a r d i o l i p i n were present i n small quantities i n the PMI f r a c t i o n of exponentially growing c e l l s , although after 16 hours phosphatidylglycerol had increased s u b s t a n t i a l l y to 8.7 moles % of t o t a l phospholipid.  The proportion of u n i d e n t i f i e d compounds  also increased as development progressed  (Table 9, 'others').  Phosphatidylinositol and phosphatidylserine remained r e l a t i v e l y constant i n PMI during the observed period. Plasma membrane f r a c t i o n PM2 provided similar phospholipid r e s u l t s to PMI during early development, the major differences being a greater reduction of phosphatidylethanolamine a smaller amount of lysophosphatidylethanolamine  with time,  i n i t i a l l y and  more of the minor components although phosphatidylserine was absent throughout and there was less unidentified phospholipid (Table 9). Table 10 shows the s t e r o l compositions  of the free s t e r o l and  s t e r o l ester fractions of PMI and PM2 from both exponentially growing c e l l s and those which had undergone 16 hours development. The t o t a l quantity of free s t e r o l i n PMI dropped s l i g h t l y during  T a b l e 10:  Plasma membrane f r e e s t e r o l and s t e r o l e s t e r s t e r o l a n a l y s i s a t two s t a g e s o f development o f D. d i s c o i d e u m Ax-2.  Plasma Membrane F r a c t i o n PM2  Plasma Membrane F r a c t i o n PMI  Retention  0 hours sterol ester  free sterol (nmol mg  1  0 hours  16 hours  protein)  0  free sterol  sterol ester  (nmol mg  protein)  0.85  11.4 ±  6.4  0  1.9 ±  0.7  0  1.17  16.8 ±  4.1  1.5 ± 0. 1  2.8 ±  0.2  4.6 + 0.8  1.30  25.1 ±  0.5  0  22.8 ±  6.1  0  3.7 ± 0. 3  200.3 ±  7.7  37.1 ± 0.6  5.6 ±  0.0  2.8 + 1.2  1.43  d  170.1 ± 32.2  1.59  17.8 ±  8.4  1.0 ± 0. 2  1.83  12.9 ±  6.5  0  Total Sterol  253.8 ± 19.3  6.2 ± 0.9  234.5 ± 14.3  (nmol mg 8.5 ±  44.4 ± 8.2  1  protein)  2.8  18.6 ± 11.6 19.6 +  2.6  189.4 ± 23.5 21.7 ±  0  1.4 +. 0.4  sterol ester  free sterol C  16 hours  8.5  0  free sterol  sterol ester  (nmol mg  • \c protexn)  0  18.1 ±  8.1  0  0.9 ± 0.5  17.4 ±  7.2  6.6 ± 2.1  0  12.2 ±  2.1  0  1.7 ± 0.4 0.6 ± 0.2  275.9 ± 12.5 5.3 ±  0.0  0  0  0  257.8 ± 10.6  3.2 ± 1.8  328.5 ± 67.3  '0 hour' and '16 hour' d a t a were determined from t h r e e and two s e t s o f plasma membrane l i p i d s  22.3 ± 1.5 0.7 ± 0.7 0 29.6 ± 2.5  respectively.  V a l u e s a r e e x p r e s s e d as t h e mean ± s t a n d a r d d e v i a t i o n . PMI and PM2 a r e t h e two plasma membrane p r e p a r a t i o n s Methods. k  Retention f i g u r e s are the r a t i o of ( s t e r o l acetate: chromatography c o n d i t i o n s used.  °  Molar equivalents content.  were c a l c u l a t e d as d e s c r i b e d  d Stigmastenyl  acetate.  o b t a i n e d by t h e t e c h n i q u e d e s c r i b e d  under M a t e r i a l s and  c h o l e s t e r y l a c e t a t e ) , r e t e n t i o n under the g a s - l i q u i d  i n Table 7 ^ \  These v a l u e s were r e l a t e d t o membrane p r o t e i n  t h i s p e r i o d , whereas t h a t of PM2 stigmastenol  The  proportion  of  i n the f r e e s t e r o l f r a c t i o n of v e g e t a t i v e c e l l s ' plasma  membranes was and  increased.  l e s s t h a n t h a t of i n t a c t c e l l e x t r a c t s ( c f . T a b l e s 7  10), being  67 moles % and  74 moles % i n PMI  and PM2  respectively.  However, a f t e r 16 hours development the f r e e s t e r o l c o m p o s i t i o n of b o t h membrane f r a c t i o n s resembled t h a t of e x p o n e n t i a l l y growing whole c e l l s , each c o n t a i n i n g 84-86 moles % s t i g m a s t e n o l . a component w i t h a s t a n d a r d f r a c t i o n PMI  r e t e n t i o n of 1.83  not observed i n  D u r i n g e x p o n e n t i a l growth of s t r a i n Ax-2 v i r t u a l l y t w i c e as l a r g e i n PMI  y e t , as i n the i n t a c t c e l l , was  o n l y 1-3  was  present i n  a t zero t i m e , y e t became a v e r y minor component a f t e r  16 hours development: i t was  e s t e r was  w h i c h was  There  PM2. the q u a n t i t y of  as i n PM2  sterol  (Table 10)  and  the plasma membrane s t e r o l e s t e r c o n t e n t  moles % of the t o t a l s t e r o l .  The  s t e r o l compositions  of the plasma membrane s t e r o l e s t e r f r a c t i o n s were a l s o s i m i l a r to t h a t of whole c e l l s t e r o l e s t e r s ( c f . T a b l e s 7 and s t e r o l e s t e r mg  1  p r o t e i n r e c o v e r e d i n PMI  10).  The  the c e l l .  that  the  t o membranous s t r u c t u r e s w i t h i n  A f t e r 16 hours development the plasma membranes' s t e r o l  e s t e r c o n t e n t had and  nmol  a t zero time c o n s t i t u t e s  a f i v e - f o l d enrichment from the homogenate, s u g g e s t i n g s t e r o l ester i s l a r g e l y confined  6.2  t h a t of PM2  i n c r e a s e d d r a m a t i c a l l y ; t h a t of PMI  nine-fold.  Only t h r e e s t e r o l m o i e t i e s were  i n the e s t e r f r a c t i o n : i n i t i a l l y s t i g m a s t e n o l of the s t e r o l e s t e r s t e r o l i n PMI f i g u r e s r i s i n g t o 84 and hours development.  The  of the f r e e s t e r o l and  seven-fold  and  detected  comprised 60 moles %  54 moles % i n PM2,  these  75 moles % r e s p e c t i v e l y a f t e r s i x t e e n d i f f e r e n c e s between the s t e r o l c o m p o s i t i o n s  s t e r o l e s t e r f r a c t i o n s of whole c e l l s ,  and  plasma membranes a t b o t h t h e z e r o and a t t h e 16 hour time p o i n t s , suggest t h a t a s e l e c t i v e e s t e r i f i c a t i o n mechanism may be o p e r a t i n g ( T a b l e s 7 and 1 0 ) . The f a t t y a c i d c o m p o s i t i o n s o f t h e t o t a l p h o s p h o l i p i d  isolated  from t h e two plasma membrane f r a c t i o n s o f JJ. d i s c o i d e u m a t z e r o and s i x t e e n hours development  a r e d i s p l a y e d i n T a b l e 11.  I n expo-  n e n t i a l l y growing c e l l s t h e p h o s p h o l i p i d a c y l c h a i n s o f b o t h PM1 and PM2 were a p p r o x i m a t e l y 14% f u l l y s a t u r a t e d and 86% u n s a t u r a t e d , whereas a f t e r s i x t e e n hours development  t h e s a t u r a t e d components  had r i s e n t o a p p r o x i m a t e l y 29% l e a v i n g 71% u n s a t u r a t e d . development  Before  began almost 80% o f b o t h membrane f r a c t i o n s ' a c y l c h a i n s  . . . (A9 o r A l l ) . (A5,9 o r A 5 , l l ) . . comprised 18:1 o r 18:2 , these two b e i n g represented i n s i m i l a r q u a n t i t i e s . the  A f t e r s i x t e e n hours  development  t o t a l o f these f a t t y a c i d s had dropped t o 53% i n PM1 and 64%  i n PM2.  Moreover, most o f t h i s l o s s was from t h e 18:2 p o o l .  Consequently one might expect t h e membrane f l u i d i t y t o d e c r e a s e s u b s t a n t i a l l y over t h e same p e r i o d .  Table 11:  I), discoideum Ax-2 plasma membrane phospholipid fatty acid composition at two stages of the organism's development.  Fatty Acid  Plasma Membrane Fraction PM1 0 hours  16 hours  Plasma Membrane Fraction PM2 0 hours  (%. recovered)  .'(.% recovered)  14:0  0.9  1.7 + 0.8  0.9  1.2 + 0.0  Palmitaldehyde  0.4  3.1 + 1.9  0.6  1.0 + 0.4  16:0  9.4  14.3 + 1.2  10.9  12.9 + 2.9  3.0  3.5 + 0.8  2.2  1.6 + 0.6  1.7  4.2 + 0.7  1.8  2.1 + 0.4  3.9  13.5 + 2.0  2.5  12.3 + 3.5  40.9  35.2 + 2.1  37.4  45.0 + 1.2  37.5  17.9 + 2.5  39.9  19.0 + 0.9  1.2  2.1 + 2.1  1.4  2.1 + 0.5  1.1  4.5 + 1.4  2.4  2.8 + 1.2  16:  ( A 9 )  16:2  ( A 5  '  9 ) &  17:0  b  18:0 18:l  ( A 9 )  A5  & 18:l 9  ( A l l ) b  18:2^ ' > 1 8 : 2 (A9,12) &  18:2  Others  0  ( A 5  »  1 1  >  b  (% recovered)  16 hours (% recovered)  PM1 and PM2 are the two plasma membrane preparations obtained by the technique described under Materials and Methods. These fatty acids were not separated under the conditions used but were shown to be present by Davidoff and Korn (61). Several unidentified minor components.  DISCUSSION  In  t h i s a n a l y s i s D. d i s c o i d e u m Ax-2 l i p i d q u a n t i t a t i o n d a t a  has been e x p r e s s e d i n b o t h molar p e r c e n t a g e s and i n r e l a t i o n t o the p r o t e i n c o n t e n t o f t h e i n t a c t c e l l s o r the membrane p r e p a r a t i o n from w h i c h t h e l i p i d s were e x t r a c t e d .  Although decreases i n both  p r o t e i n and d r y w e i g h t per c e l l a r e among many parameters known to a l t e r d u r i n g t h e organism's development (1,59,64), t h e c r i t e r i a of  r e l i a b i l i t y and r e p r o d u c i b i l i t y (31) f a v o u r e d t h e use o f t h e  p r o t e i n standard f o r the c u r r e n t i n v e s t i g a t i o n . The s u p e r i o r i t y o f t h e l i p i d e x t r a c t i o n method used i n t h i s s t u d y , m o d i f i e d from t h a t o f F o l c h e± _al ( 4 1 ) , i s apparent  from  the d a t a i n T a b l e s 1, 2, 3 and 4, a l t h o u g h a d i s a d v a n t a g e o f a c i d e x t r a c t i o n c o n d i t i o n s i s the p o s s i b i l i t y of plasmalogen (65).  hydrolysis  Throughout t h i s i n v e s t i g a t i o n t h e a c i d e x t r a c t i o n f o l l o w i n g  each F o l c h t r e a t m e n t was k e p t s e p a r a t e from t h e i n i t i a l c h l o r o f o r m methanol e x t r a c t u n t i l t h e HC1 had been removed i n a stream o f nitrogen. Nevertheless i t i s possible that s u f f i c i e n t acid to degrade plasmalogens  remained  t o t h e i r l y s o d e r i v a t i v e s when t h e two  e x t r a c t s were p o o l e d and t h a t p r e v i o u s r e p o r t s o f a h i g h l y s o p h o s p h a t i d y l e t h a n o l a m i n e c o n t e n t o f i n t a c t ID. d i s c o i d e u m c e l l s  (30,61)  may a l s o be due t o h y d r o l y s i s o f t h e p h o s p h a t i d y l e t h a n o l a m i n e plasmalogen. E l l i n g s o n d e t e c t e d plasmalogens  i n certain phospholipid  i s o l a t e s ( T a b l e 5) b u t d i d n o t r e p o r t t h e q u a n t i t i e s p r e s e n t ( 3 0 ) . Thus t h e h i g h e r l y s o p h o s p h a t i d y l e t h a n o l a m i n e c o n t e n t o f l i p i d e x t r a c t s o f crude membranes and plasma membranes compared w i t h those o f i n t a c t c e l l s ( T a b l e s 5, 8 and 9) may i n d i c a t e t h a t t h e  phosphatidylethanolamine plasmalogen i s enriched i n these membranes at the early developmental stages tested. to contain plasmalogens  Membranes might be expected  rather than lysophospholipids because of  the powerful detergent effect of the l a t t e r . for  There i s no evidence  the presence of phosphatidylcholine plasmalogen i n J). discoideum  (30). The majority of plasma membrane p u r i f i c a t i o n methods, including those for obtaining plasma membranes of D. discoideum (35,37), are of extended duration and consequently allow the p o s s i b i l i t y of considerable phospholipid degradation.  Extremely active phospholipases  have been detected i n the organism by Ferber ej: j t l (63) and i n the present study substantial phospholipid degradation occurred during incubation of c e l l - f r e e homogenates ixi v i t r o  (Figures 3 and 4 ) .  There was f a r less phospholipid degradation during incubation of crude membrane preparations In v i t r o (Figure 5) indicating that under these conditions most of the phospholipase a c t i v i t y was not membrane bound.  The higher lysophosphatidylethanolamine content  of both crude and plasma membranes compared with that of intact c e l l s (Tables 5, 8 and 9) may  indicate that a certain amount of  l y s o l i p i d formed after c e l l breakage and this proposal i s reinforced by the apparent conversion of phosphatidylethanolamine to lysophosphatidylethanolamine which was revealed upon prolonged incubation of crude membranes (Table 8 ) . However, the l y s o l i p i d may have been produced from plasmalogen during the extraction procedure, a p o s s i b i l i t y discussed above.  Upon incubation of the crude membranes  there was also a substantial loss of phospholipid containing choline i n contrast to the r e l a t i v e l y small loss of l i p i d i c  ethanolamine,  w h i c h suggests t h a t a mechanism may e x i s t i n t h e s e membranes f o r the s p e c i f i c removal o r d e g r a d a t i o n of c h o l i n e - c o n t a i n i n g phospholipids. P h o s p h o l i p i d d e g r a d a t i o n d u r i n g plasma membrane p u r i f i c a t i o n was m i n i m i z e d by s h o r t e n i n g t h e p r e v i o u s l y p u b l i s h e d procedure (37) wherever p o s s i b l e .  The method o f s u c r o s e g r a d i e n t c e n t r i f u g a t i o n  used t o p u r i f y plasma membranes from crude membranes may e l i m i n a t e t h e r e s i d u a l l i p i d d e g r a d a t i o n by p h y s i c a l s e p a r a t i o n o f t h e plasma membrane f r a c t i o n s from t h e enzymes r e s p o n s i b l e .  However, any  l i p a s e a c t i v i t y s p e c i f i c a l l y a s s o c i a t e d w i t h t h e plasma membrane would be s i m u l t a n e o u s l y e n r i c h e d , w h i c h might l e a d t o an exaggera t i o n of l i p i d  changes brought about by d e v e l o p m e n t a l l y  plasma membrane enzyme a c t i v i t i e s .  controlled  There a r e p r e c e d e n t s f o r phospho-  l i p a s e a c t i v i t i e s i n plasma membranes: p h o s p h a t i d i c a c i d  phosphorylase  i s p r e s e n t i n c h i c k embryonic muscle c e l l plasma membranes (66) and p h o s p h o l i p a s e A, l y s o p h o s p h o l i p a s e , a c y l - C o A palmitoyl-CoA  h y d r o l a s e and  s y n t h e t a s e a c t i v i t i e s have been measured i n plasma  membranes o f Acanthamoeba c a s t e l l a n i i ( 6 7 ) .  N e v e r t h e l e s s plasma  membrane p h o s p h o l i p a s e s do n o t appear t o i n t e r f e r e w i t h t h e a n a l y s i s of  E>. d i s c o i d e u m plasma membrane l i p i d c o m p o s i t i o n f o r p r o l o n g e d  i n c u b a t i o n o f t h i s organism's plasma membranes ^Ln v i t r o causes no decrease i n t h e amount o f membrane bound p h o s p h o l i p i d n o r a l t e r a t i o n of  t h e membranes' p h o s p h o l i p i d c o m p o s i t i o n (G. Weeks, u n p u b l i s h e d  observations). A number of D. d i s c o i d e u m plasma membrane l i p i d  changes o c c u r r e d  d u r i n g e a r l y development o f t h e c e l l s i n b u f f e r e d shake s u s p e n s i o n . A f t e r 16 hours a c o n s i d e r a b l e r e d u c t i o n o f t h e membranes' phospha-  t i d y l c h o l i n e c o n t e n t had  taken p l a c e ( T a b l e 9 ) .  In a d d i t i o n there  were s e v e r a l s m a l l e r a l t e r a t i o n s of o t h e r p h o s p h o l i p i d s . These changes comprised a d e c r e a s e i n the p r o p o r t i o n of w h i c h was  phosphatidylethanolamine,  accompanied by an i n c r e a s e i n the p r o p o r t i o n s of l y s o p h o s p h a -  t i d y l e t h a n o l a m i n e and p h o s p h a t i d y l g l y c e r o l and by the a c c u m u l a t i o n  of  l y s o p h o s p h a t i d i c a c i d and p h o s p h a t i d i c a c i d i n plasma membrane f r a c t i o n s PM1  and PM2  r e s p e c t i v e l y . A p a r t from the i n c r e a s e o f p h o s p h a t i d y l -  g l y c e r o l these r e s u l t s r e f l e c t an o v e r a l l d e g r a d a t i o n  of p h o s p h o l i p i d  species. The most d r a m a t i c m o d i f i c a t i o n of plasma membrane l i p i d e a r l y development was c o n t e n t w h i c h was  the e i g h t - f o l d i n c r e a s e i n the s t e r o l e s t e r  accompanied by a s l i g h t r e d u c t i o n i n the amount  of f r e e s t e r o l ( T a b l e There was  during  10).  a l s o a s u b s t a n t i a l a l t e r a t i o n o f the plasma membrane  phospholipid f a t t y a c i d composition  a f t e r 16 hours development  (Table  11). The p r o p o r t i o n s o f p a l m i t a t e and s t e a r a t e i n c r e a s e d markedly a t A5 9 the expense of the o c t a d e c a d i e n o i c A5,ll^  f a t t y a c i d s (18:2  '  and  Thus the s a t u r a t i o n of the p h o s p h o l i p i d a c y l c h a i n s  s u b s t a n t i a l l y over t h i s p e r i o d . s p e c i f i c degradation  increased  Whether these changes a r e caused by  or by s p e c i f i c s y n t h e s i s of p a r t i c u l a r  a c i d s i s unknown, a l t h o u g h  18:2  fatty  the t o t a l p h o s p h o l i p i d of b o t h PM1  and  PM2  plasma membrane f r a c t i o n s d e c r e a s e d s u b s t a n t i a l l y r e l a t i v e t o p r o t e i n d u r i n g the f i r s t e i g h t hours of development ( T a b l e 9 ) . E l e c t r o n s p i n resonance and f l u o r e s c e n c e d e p o l a r i z a t i o n a n a l y s e s of p_. d i s c o i d e u m  plasma membranes prepared  from c e l l s t h a t  had developed f o r 16 hours under the c o n d i t i o n s used i n the study r e v e a l e d a pronounced d e c r e a s e i n membrane f l u i d i t y F.G.  and I . T a t i s c h e f f , u n p u b l i s h e d  present  (Herring,  observations) which i s i n  k e e p i n g w i t h the changes i n l i p i d c o m p o s i t i o n r e p o r t e d above.  I n the  s u s p e n s i o n b u f f e r a g g r e g a t i o n was i n i t i a t e d w i t h i n 8 hours o f t r a n s f e r and a t t h i s time t h e c e l l s e x h i b i t no a l t e r a t i o n o f plasma membrane f l u i d i t y ( H e r r i n g , F.G. and I . T a t i s c h e f f , u n p u b l i s h e d o b s e r v a t i o n s ) a l t h o u g h changes i n membrane p h o s p h o l i p i d were apparent  (Table 9 ) .  Thus t h e e s t a b l i s h m e n t o f c e l l - c e l l c o n t a c t s does n o t appear t o be c o r r e l a t e d w i t h o v e r a l l changes i n plasma membrane f l u i d i t y even though a l t e r a t i o n s o f p h o s p h o l i p i d c o m p o s i t i o n a r e a s s o c i a t e d w i t h t h i s s t a g e o f development under these c o n d i t i o n s .  I t i s possible  t h a t t h e f l u i d i t y changes o c c u r r i n g between 8 and 16 hours a r e unconnected w i t h normal development s i n c e plasma membranes p r e p a r e d from c e l l s d e v e l o p i n g on a s o l i d s u r f a c e f o r t h e same time p e r i o d show no a l t e r a t i o n o f f l u i d i t y a t any s t a g e .  The 16 hour  formed i n s u s p e n s i o n were o f normal s i z e and proceeded  aggregates  t o develope  and d i f f e r e n t i a t e n o r m a l l y when t r a n s f e r r e d t o a s o l i d s u r f a c e d e s p i t e t h e i r d e c r e a s e d membrane f l u i d i t y .  Thus t h e a l t e r e d membrane  f l u i d i t y imposed under t h e s t a n d a r d i s e d b u t a r t i f i c i a l c o n d i t i o n s of  s u s p e n s i o n c u l t u r e (36) do n o t appear t o i m p a i r normal  cell-cell  i n t e r a c t i o n a l t h o u g h a t p r e s e n t i t i s unknown whether t h e membrane f l u i d i t y r e v e r t s t o 'normal' when t h e d i f f e r e n t i a t i o n o f t h e s e aggregates i s p e r m i t t e d on a s o l i d s u r f a c e . The r e s u l t s p r e s e n t e d i n t h i s study i n d i c a t e t h a t s u b s t a n t i a l l i p i d changes o c c u r i n t h e plasma membrane o f I), d i s c o i d e u m d u r i n g e a r l y development i n shake s u s p e n s i o n . large increase i n s t e r o l ester.  Most i n t r i g u i n g i s t h e  A p o s s i b l e f u n c t i o n f o r such a  m o l e c u l e i n development i s n o t e a s i l y v i s u a l i z e d a t p r e s e n t a l t h o u g h s t a l k d i f f e r e n t i a t i o n has been i n d u c e d i n c e l l monolayers o f t h e  organism lipid  (68), and t h e i n d u c i n g component appears t o be a n e u t r a l  ( S t a n f i e l d , E. and G Weeks, u n p u b l i s h e d o b s e r v a t i o n s ) .  S u f f i c i e n t q u a n t i t i e s of t h i s m a t e r i a l f o r the d e t e r m i n a t i o n of i t s p r e c i s e molecular nature a r e c u r r e n t l y u n a v a i l a b l e . The plasma membrane l i p i d measurements o u t l i n e d i n t h i s s t u d y p r o v i d e a background development.  f o r f u r t h e r a n a l y s i s throughout t h e organism's  I f t h e p h o s p h o l i p i d changes, i n c l u d i n g those o f t h e  f a t t y a c i d m o i e t i e s , w h i c h have been observed i n t h e p r e s e n t s t u d y a r e o f d e v e l o p m e n t a l importance t h e n they s h o u l d a l s o be observed d u r i n g development o f I), d i s c o i d e u m on a s o l i d s u r f a c e .  I t may be  e s p e c i a l l y p e r t i n e n t t o determine the s t e r o l e s t e r content of the plasma membrane d u r i n g development under these c o n d i t i o n s .  51.  BIBLIOGRAPHY  1.  Loomis, W.F. system'.  2.  (1975).  'Dictyostelium discoideum, a developmental  Academic Press Inc., New  Ashworth, J.M.  (1973).  York.  Studies on c e l l d i f f e r e n t i a t i o n i n the  c e l l u l a r slime mould I), discoideum.  Biochem. Soc. 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