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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 LIPID COMPOSITION OF DICTYQSTELIUM DISCQIDEUM DURING EARLY DEVELOPMENT IN AQUEOUS SUSPENSION by HOWARD KEITH WITHERS B . S c , U n i v e r s i t y of Birmingham, England, U.K., 1974. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of M i c r o b i o l o g y We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA March 1979 © Howard K e i t h Withers, 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 degree a t the U n i v e r s i t y o f 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 a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes 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 . I t i s u n d e r s t o o d t h a t c o p y i n g o r 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 w r i t t e n p e r m i s s i o n . 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 1 E i . ABSTRACT C e l l - c e l l contact must be made and maintained for normal development and eventual differentiation of J). discoideum to occur. Certain plasma membrane components are known to alter in activity 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 quantification of the neutral l i p i d and phospholipid components of intact cells of strain Ax-2. Development was initiated in JJ. discoideum populations suspended i n aqueous buffer and plasma membrane fractions were purified from both exponentially growing and aggregation-phase cells by a modified procedure which minimized phospholipid de-gradation during the plasma membrane isolation. Neutral l i p i d and phospholipid compositions of the plasma membrane fractions PM1 and PM2 from exponentially growing cells and from those in early aggregation phase were determined. Exponential phase cel l s ' plasma membranes contained large proportions of phosphatidylethanolamine, phosphatidylcholine and lysophosphatidylethanolamine. Lysophosphatidylcholine was absent. A significant quantity of phosphatidylinositol was detected and cardiolipin, phosphatidylglycerol, phosphatidic acid and lyso-phosphatidic acid were each present in small amounts. The presence of phosphatidylethanolamine plasmalogen was suspected but not proven. No acylglycerol components were detected, the major n e u t r a l l i p i d f r a c t i o n being that of f r e e s t e r o l which l a r g e l y comprised stigmast-22-en-33-ol; s t e r o l e s t e r was present i n extremely s m a l l q u a n t i t i e s . 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 component of plasma membranes was detected by i t s c h a r a c t e r i s t i c a b s o r p t i o n and fluorescence upon i r r a d i a t i o n at u l t r a v i o l e t wavelengths. A f t e r s i x t e e n hours aggregation the p h o s p h a t i d y l c h o l i n e content of the plasma membranes was 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 phosphatidylethanolamine appeared to have been converted to lysophosphatidylethanolamine, and p h o s p h a t i d y l g l y c e r o l , phosphatidic 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 a l l i n greater 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 constant but s t e r o l e s t e r had increased d r a m a t i c a l l y (7 to 10-fbld) and the f a t t y a c i d composition of the plasma membrane phospholipids was more sa 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 octadeca-d i e n o i c f a t t y a c i d components. i i i . TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i i i LIST OF TABLES i v LIST OF FIGURES . . . v ACKNOWLEDGEMENTS v i INTRODUCTION 1 MATERIALS AND METHODS 5 (a) M a t e r i a l s 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 10 RESULTS 14 (a) Comparison of L i p i d E x t r a c t i o n Procedures 14 (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 20 (c) 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 C e l l E x t r a c t s 23 (d) E v a l u a t i o n of Ph 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 (e) Plasma Membrane L i p i d Composition 37 DISCUSSION 45 BIBLIOGRAPHY 51 iv. LIST OF TABLES Table Page 1 Lipid-phosphorus extracted from exponentially 15 growing cells of JJ. discoideum Ax-2. 2 Fatty acid analysis of hydrolyzed total l i p i d 16 from Folch ejt a l (41) and Bligh & Dyer (38) extractions of exponentially growing cells of I), discoideum Ax-2. 3. Sterol analysis of total l i p i d from Folch et al y] (41) and Bligh & Dyer (38) extractions of exponentially growing cells of I), discoideum Ax-2. 4 Fatty acid analysis of various f i n a l extractions 19 (Fractions III) following i n i t i a l procedures of Folch et a l (41). 5 Lipid-phosphorus analysis of intact cells of 24 exponentially growing I), discoideum Ax-2 following phospholipid separation by thin-layer chromatography. 6 Acylglycerol analysis of intact cells of 26 exponentially growing B. discoideum Ax-2. 7 Sterol analysis of the free sterols and sterol 28 esters extracted from intact cells of exponentially growing ID. discoideum Ax-2. 8 Proportion of individual phospholipid components 35 of I), discoideum Ax-2 crude membranes resuspended in 8.6% sucrose-Tris-PMSF buffer, pH 7.4, incubated at either 4°C or 22°C as shown. 9 Plasma membrane lipid-phosphorus analysis at 38 three stages of development of I), discoideum Ax-2 following phospholipid separation by thin-layer chromatography. 10 Plasma membrane free sterol and sterol ester 41 sterol analysis at two stages of development of I), discoideum Ax-2. 11 I), discoideum Ax-2 plasma membrane phospholipid 44 fatty acid composition at two stages of the organism.' s development. V. LIST OF FIGURES Fig u r e Page Separation of n e u t r a l l i p i d s by monodirectional, 21 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 p l a t e s . Two-dimensional, b i p h a s i c t h i n - l a y e r chromato- 22 graphy of a mixture of standard phospholipids on s i l i c a g e l H p l a t e s w i t h a d d i t i v e s . The e f f e c t of temperature on the r a t e s of l o s s 31 of lipid-phosphorus from B. discoideum Ax-2 homogenates over an extended p e r i o d . The e f f e c t of temperature on the e a r l y l o s s of 32 lipid-phosphorus from I), discoideum Ax-2 homogenates. The e f f e c t of the s o l u b l e f r a c t i o n of c e l l 33 homogenates of _D. discoideum Ax-2 on the l i p i d -phosphorus content of crude membrane preparations incubated at 22 C. Lipid-phosphorus content of B. discoideum Ax-2 35 crude membranes during extended i n c u b a t i o n a t 4 C. ACKNOWLEDGEMENT S I wish to thank Dr. Thomas Buckley, of the Department of Biochemistry and M i c r o b i o l o g y , U n i v e r s i t y of 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 chromatographic techniques, and Mrs. Kathy LaRoy f o r her a s s i s t a n c e w i t h c e r t a i n stages of the labor a t o work. Drs. Gerald 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 the p r o j e c t . Dr. G i l k e s provided blue l o h i d e x and f i l i p i n was su p p l i e d by Dr. J . Masson of the Upjohn Company, Toronto, Ontario. I am g r a t e f u l to Mr. James R i c h t e r who redesigned and constructed 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 and to Ms. Rosemary Morgan f o r arranging and t y p i n g the manuscript w i t h such p r e c i s i o n . This t h e s i s could not have been w r i t t e n without the encouragement and support of c l o s e f r i e n d s , nor without considerable e f f o r t from Gerry who has been very, very p a t i e n t . 1. INTRODUCTION D i c t y o s t e l i u m discoideum i s a c e l l u l a r slime mould d i s t r i b u t e d i n the s o i l s and r o t t i n g v e g e t a t i o n of temperate f o r e s t s throughout the world. This simple e u c a r y o t i c organism i s amoeboid i n the ve g e t a t i v e phase of i t s l i f e c y c l e during which 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 populations undergo a process of synchronous development. This comprises aggregation, grex formation and m i g r a t i o n , and f r u i t i n g body c o n s t r u c 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 types, and u l t i m a t e l y i n t o s t a l k and spore c e l l s r e s p e c t i v e l y w i t h the formation of the f r u i t i n g body. The s t a l k c e l l s are not v i a b l e but the spores germinate i n t o small myxamoebae a f t e r d i s p e r s a l (1,2). In 1889 MacBride s t a t e d of slime moulds: "Their minuteness r e t i r e s them from or d i n a r y ken; but such i s the extreme beauty of t h e i r microscopic s t r u c t u r e , such the exceeding i n t e r e s t of t h e i r l i f e h i s t o r y , that f o r many years e n t h u s i a s t i c students have found the group one of p e c u l i a r f a s c i n a t i o n , i n some respects at l e a s t , the most i n t e r e s t i n g and remarkable that f a l l s beneath our lenses" (3). This d e s c r i p t i o n a p p l i e s to 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 to the Myxomycetes (true slime moulds). The two have f r e q u e n t l y been confused although major d i s t i n c t i o n s were noted more than a century ago (3,4). Their 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 reviewed r e c e n t l y (5). I), discoideum has s e v e r a l advantages f o r the 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 of relevance to s t u d i e s of development i n m u l t i c e l l u l a r 2. organisms (1,2); embryological development, t i s s u e regeneration and cancerous p r o l i f e r a t i o n being examples of the l a t t e r . C e r t a i n s t r a i n s of ID. discoideum grow q u i t e r a p i d l y i n axenic media a l l o w i n g biochemical manipulation during growth and f a c i l i t a t i n g the i s o l a t i o n and c l o n i n g of mutants. The organism has been the subject of d i v e r s e i n v e s t i g a t i o n s which have provided 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). In c o n t r a s t to the m a j o r i t y of developmental systems, that of I), discoideum i s not complicated by the e f f e c t s of growth and d i v i s i o n , both of which cease before aggregation begins; the developmental phenomena are 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 . Slime mould development has been discussed i n general terms by Bonner (6) and elsewhere w i t h p a r t i c u l a r emphasis on ID. discoideum (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 of ID. d i s - coideum are maintained throughout development and are r e q u i r e d at a l l stages f o r the process to 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 molecular b a s i s have not been determined, although 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 are known to 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 during d i f f e r e n t i a t i o n (8-17), and u n i v a l e n t 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 c e l l aggregation and a r r e s t f u r t h e r development (8,18). Developmentally regulated m o d i f i c a t i o n of plasma membrane l i p i d components may be a s s o c i a t e d w i t h c e l l - c e l l i n t e r a c t i o n processes. 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 necessary to 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 to bind to the c e l l surface or to be incorporated 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 c e l l surface 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 of 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 or through more general membrane f l u i d i t y e f f e c t s brought about by changes i n l i p i d composition. In developmental b i o l o g y there has been no d i r e c t observation of such mechanisms, although i n c e r t a i n c u l t u r e d animal c e l l s the p h y s i c a l s t a t e of the plasma membrane a l t e r s during c e l l adhesion (mouse L929 c e l l s ) (19), c e l l f u s i o n (avian 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) (21). Moreover, s t u d i e s outside the developmental f i e l d suggest that l i p i d 'micro-domains' e x i s t i n n a t u r a l membranes and that s p e c i f i c l i p i d -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 are able to 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 t r a n s p o r t processes (22-27). There i s c i r c u m s t a n t i a l evidence that l i p i d composition may be a c r i t i c a l f a c t o r of I), discoideum 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 the ensuing d i f f e r e n t i a t i o n of the organism (28), and the f a t t y a c i d composition of the axenic s t r a i n of I), discoideum can be modified during growth such that i t s subsequent development i s impaired (29). Furthermore both the p h o s p h o l i p i d (30) and n e u t r a l l i p i d (31) compositions of the i n t a c t organism have been shown to a l t e r during development suggesting that there i s e i t h e r a wide-spread adjustment of these components o c c u r r i n g throughout the c e l l s ' v a r i o u s membranes or that there are s i g n i f i c a n t l i p i d 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. Although i t i s d i r e c t l y i n v o l v e d i n c e l l - c e l l i n t e r a c t i o n , the 13. discoideum 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 significant l i p i d changes occur during early stages of the organism's development. 5. MATERIALS AND METHODS a. M a t e r i a l s Standard l i p i d s were of the highest p u r i t y a v a i l a b l e commercially and were purchased from Supelco Inc. and Ap p l i e d Science Inc.. Solvents were r e d i s t i l l e d i n g l a s s and stored dry over molecular s e i v e s . I n order to prevent solvent o x i d a t i o n and f r e e r a d i c a l formation 0.005% (w/v) 2,6-di-tert-butyl-4-methylphenol (BHT) twice r e c r y s t a l l i z e d from carbon t e t r a c h l o r i d e was added to a l l ethers: 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 (32). A l l chromatography, storage and assay glassware 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 thoroughly r i n s e d w i t h d i s t i l l e d , deionized water before being spread w i t h the appropriate 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 axenic J). discoideum s t r a i n Ax-2 was grown i n the l i q u i d medium HL-5 at 22°C (33,34). C e l l s were harvested during exponential growth at a co n c e n t r a t i o n of approximately 5 x 10^ c e l l s ml ^ by c e n t r i f u g a t i o n f o r 6 min at 700 xg and 4°C. 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 to remove a l l t r a c e s of HL-5 medium before immediate l i p i d e x t r a c t i o n of the i n t a c t c e l l s . C e l l s destined f o r membrane f r a c t i o n a t i o n were harvested and washed twice w i t h i c e - c o l d 8.6% (w/v) sucrose-5mM tris(hydroxymethyl) 6. aminomethane hydrochloride (sucrose-Tris), pH 7.4, using 6 min centrifugations of 700 xg as above. Aggregation-phase c e l l s were prepared under standard conditions (36) by resuspension of the washed c e l l p e l l e t s i n 17mM phosphate buffer, pH 6.0, at 7 x 10 c e l l s ml ^ and r o t a t i n g these c e l l suspensions at 150 rpm at 22°C for either 8 or 16 hours before reharvesting. Harvested exponential and aggregation-phase c e l l s were immediately resuspended i n i c e -cold sucrose-Tris saturated with phenylmethylsulphonylfluoride (sucrose-Tris-PMSF), pH 7.4, at 10 8 c e l l s ml" 1 (35). In order to minimize l i p i d degradation the standard plasma membrane p u r i f i c a t i o n procedure (35,37) was modified i n that the 105,400 xg p e l l e t was not washed but was e i t h e r used d i r e c t l y as 'crude membrane' material for preliminary experiments or resuspended i n 20% (w/v) sucrose-Tris-PMSF, pH 7.4, and immediately layered onto the discontinuous sucrose density gradients. These gradients were centrifuged f o r 16 hours at 75,800 xg, c e n t r i f u g a t i o n being i n i t i a t e d within 45 minutes of the s t a r t of c e l l f racture. Two d i s t i n c t bands of membranous material (PM1 and PM2) (35) were recovered as described previously (37) and aliquots were assayed or extracted immediately. The c r i t e r i a used to assess the p u r i t y of the plasma membrane preparations have been discussed by Gilkes and Weeks (37). c. L i p i d E x traction and I s o l a t i o n In preliminary experiments l i p i d e xtraction was c a r r i e d out ei t h e r by the method of B l i g h and Dyer (38) according to Kates (39, 40) or by that of Folch et a l (40,41), the l a t t e r being the more e f f i c a c i o u s (see Results). The aqueous wash s o l u t i o n f or the 7. F o l c h procedure comprised 4.5 mM CaCl^ and the r e s u l t i n g phase i n t e r f a c e was r i n s e d at 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 chloroform-methanol-3.6 mM aq. CaC^ (3:48:47 by v o l ) . 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 performed overnight at 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 chloroform-methanol (7:1 v/v) saturated w i t h 5% (w/v) aq. NH^, or chloroform-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 . ) , or chloroform-methanol-concentrated HC1 (50:50:0.3 by v o l . ) . The second e x t r a c t was thoroughly d r i e d under n i t r o g e n and the washed F o l c h e x t r a c t was added to i t (42). E x t r a c t s were concentrated i n chloroform-methanol (2:1 v/v) under n i t r o g e n and s t o r e d , i f necessary, i n T e f l o n sealed v i a l s at -70°C. Separation of e x t r a c t s i n t o a p h o spholipid f r a c t i o n and 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 was achieved by b i p h a s i c t h i n - l a y e r chromato-graphy i n one dimension on a 500 ym l a y e r of s i l i c a g e l H (Merck & Co.). Before use the chromatoplates were subjected to an ascending wash w i t h chloroform-methanol (2:1 v/v) f o r at l e a s t 12 hours. Samples were deposited at the o r i g i n of a c t i v a t e d chromatoplates (43) under a stream of dry n i t r o g e n and the p l a t e s were immediately developed i n a n i t r o g e n atmosphere using the b i p h a s i c system of solvents described by S k i p s k i and B a r c l a y (43). Chromatoplates were stored 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 to remove a l l t r a c e s of solv e n t . 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 reference to the m o b i l i t y of standard l i p i d s developed on each p l a t e . Rhodamine 6 G at 0.05% (w/v) i n aqueous ethanol (95% v/v) was s e l e c t i v e l y sprayed onto the standards and the fluorescence of the r e s u l t i n g l i p i d complexes viewed under u l t r a v i o l e t r a d i a t i o n . 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 chromatoplates were derived 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 to 15 ml c o n i c a l , ground-glass stoppered c e n t r i f u g e tubes and e x t r a c t e d w i t h three 5 ml a l i q u o t s of solvent f o r 10 min each at room temperature w i t h o c c a s i o n a l v o r t e x i n g . 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 1 , 3 - d i a c y l -g l y c e r o l s were extracted w i t h d i e t h y l ether whereas chloroform-methanol (4:1 v/v) was used to e x t r a c t monoacylglycerols, s t e r o l 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 . The e x t r a c t e d n e u t r a l l i p i d f r a c t i o n s were concentrated under n i t r o g e n and s t o r e d , i f necessary, at -70°C i n Te f l o n - s e a l e d v i a l s . Phospholipids were extr a c t e d at 37°C from a band 1.5 cm i n width at the chromatoplate o r i g i n under c o n d i t i o n s s i m i l a r to 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 using 5 ml a l i q u o t s of the f o l l o w i n g solvent mixtures i n sequence: two treatments of chloroform-methanol-glacial 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 (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 e x t r a c t e d phospholipids were concentrated under n i t r o g e n and stored 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 hospholipids were separated by b i p h a s i c , two-dimensional t h i n - l a y e r chromatography. A technique was developed that f r a c t i o n a t e d a l l f u l l y a c y l a t e d phospholipids and l y s o p h o s p h o l i p i d s known to be present i n I), discoideum, although plasmalogen forms were not res o l v e d (30,44). A 97.5 ml volume of 0.5 mM aqueous magnesium acetate was ,added r a p i d l y to 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 before being spread onto chromatoplates 9. i n a 300 ym wet l a y e r . Phospholipids c o n t a i n i n g v i c i n a l hydroxy1 groups could be s e l e c t i v e l y retarded during 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 at 0.4 M (45). A f t e r 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 ascending development w i t h acetone f o r at l e a s t 12 hours, d r i e d again and a c t i v a t e d overnight at (115 ± 5)°C u n t i l immediately p r i o r to use. A f t e r c o o l i n g , each chromatoplate r e c e i v e d one p h o s p h o l i p i d sample of up to 300 ymol l i p i d phosphorus (optimum c.175 ymol.) at i t s p o i n t o r i g i n . 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 dry n i t r o g e n atmosphere usi n g equipment s i m i l a r to that described by S k i p s k i and B a r c l a y (43). T h i n - l a y e r chromatography tanks were l i n e d w i t h Whatman 3MM paper and wetted w i t h the solvent mixture, the vapours of which were allowed to e q u i l i b r a t e w i t h the enclosed atmosphere f o r an hour before use. Each chromatoplate was developed i n the f i r s t dimension w i t h c h l o r o -form-methanol - 28% (w/v) aqueous ammonia (65:25:8 by v o l . ) to a height of 18 cm. R e s i d u a l solvent was immediately evaporated by a powerful stream of c o l d a i r followed by a s p i r a t i o n i n a d e s s i c a t o r f o r t h i r t y minutes. The p l a t e was then developed f o r 18 cm i n the second dimension w i t h chloroform-acetone-methanol-glacial 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 (35:35:7:10:3 by v o l . ) . R e s i d u a l solvent was removed as before and i n d i v i d u a l phospholipids 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 chromatoplate 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 phospholipids on the t h i n - l a y e r chromatogram were i d e n t i f i e d by a combination of methods a f t e r s e p a r a t i n g standard phospholipids w i t h a c y l chains of known saturation. These methods included the following: r e l a t i v e speed and r e v e r s i b i l i t y of brown complex formation during immersion i n iodine vapour, both factors being dependent on the degree of saturation of the l i p i d s ' f a t t y a c i d substituents; detection of phosphate esters with the molybdenum-blue reagent of Dittmer and Lester (46); ninhydrin reagent to detect the free amino groups of phosphatidylethanolamine, phosphatidylserine and t h e i r lyso deriva-t i v e s (43); Dragendorff reagent to detect phosphatidylcholine, lysophosphatidylcholine and sphingomyelin (47); the incorporation of b o r i c aci d into the th i n - l a y e r , as described above, i n order to s e l e c t i v e l y retard phosphatidylglycerol migration (45). Ind i v i d u a l phospholipids were eit h e r extracted from the s i l i c a g e l by the method previously described or assayed i n the presence of the gel a f t e r quantitative tr a n s f e r to a Pyrex assay tube. d. Assays Pro t e i n was r o u t i n e l y assayed by the method of Lowry et_ al (48). The 'biuret technique' (49) was used i n measurements of p r o t e o l y t i c a c t i v i t y during membrane p u r i f i c a t i o n , the t o t a l reagent volume being reduced to 1.0 ml. T o t a l phospholipid was estimated by assaying the extracted lipid-phosphorus by Ames' method (50), although t h i s assay could not be used f o r the determination of i n d i v i d u a l phospholipids recovered d i r e c t l y from t h i n — l a y e r chromato-plates because s i l i c a g e l i n t e r f e r e d with both the dig e s t i o n step and the c o l o r i m e t r i c reaction. The B a r t l e t t lipid-phosphorus assay (51) was modified to increase i t s s e n s i t i v i t y while remaining 11. unaffected by i o d i n e pretreatment of l i p i d s or the presence of s i l i c a g e l . These p r o p e r t i e s were determined by assaying the phosphorus content of 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 of the l i p i d from an i o d i n e - t r e a t e d chromatoplate. The modified B a r t l e t t procedure was as f o l l o w s : 0.50 ml 70% (w/v) aqueous p e r c h l o r i c a c i d was added to the sample i n a s m a l l , a c i d -washed tube and heated f o r 2.5 hours at 150°C (52). A f t e r the tube had cooled, 0.60 ml d i s t i l l e d , d eionized water, 0.20 ml 5% (w/v) aqueous ammonium molybdate and 0.02 ml f r e s h Fiske-SubbaRow reagent (51) were added. Tubes were sealed w i t h P a r a f i l m , the contents mixed and heated f o r 30 min at 70°C, allowed to c o o l and c e n t r i f u g e d i n swinging buckets f o r at l e a s t 5 min at 1250 xg. Each supernatant was c a r e f u l l y removed w i t h a Pasteur p i p e t t e and i t s a b s o r p t i o n measured at 807 nm versus a reagent blank. This abs o r p t i o n maximum d i f f e r s from the 830 nm maximum of the phospho-molybdate complex i n the standard B a r t l e t t assay (51). I t i s e s p e c i a l l y important that a l l g l a s s and quartz apparatus be a c i d -washed f o r each of these phosphorus assays. N e u t r a l l i p i d s of the plasma membranes were measured by gas-l i q u i d chromatography. 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 of 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 eicosanoic 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 ( c h o l e s t e r o l ) were added to those 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 r e s p e c t i v e l y . 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 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 equal volume of 15% (w/v) K0H i n methanol f o r 1 hour at 70°C: the methanol was then evaporated under n i t r o g e n and 1.0 ml 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 four equal volumes of 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 of the hydrolysate w i t h 0.25 ml 12 M H^SO^, The non-saponifiable 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 ml a c e t i c anhydride f o r 30 min at 135°C i n T e f l o n - s e a l e d , screw-capped tubes. Samples were then 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 minimal q u a n t i t i e s of 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 of the s t e r o l e s t e r samples were rechromato-graphed by t h i n - l a y e r chromatography i n order to separate contamin-a t i n g hydrocarbons from the derived s t e r o l s . 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 described 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 isoth e r m a l g a s - l i q u i d chromatography of t h e i r f a t t y a c i d methyl 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 f r a c t i o n s . These d e r i v a t i v e s were obtained by d r y i n g the 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 , adding 1.0 ml boron t r i -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 Te f l o n - s e a l e d , screw-capped tubes: 1.0 ml of d i s t i l l e d water was then added and the methyl e s t e r s were ext r a c t e d w i t h three equal volumes of n-pentane. The pooled pentane e x t r a c t s of each sample were d r i e d down under n i t r o g e n , resuspended i n a minimal volume of hexanes and analyzed 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 using 3% (w/w) methyl s i l i c o n e (SE-30) phase on 100/120 mesh Gas Chrom Q (Applied Science Inc.) at 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 helium as c a r r i e r gas. F a t t y a c i d methyl e s t e r s were separated 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 Supelcoport (Supelco Inc.) a t 140°C i n 6 f t , 2 ma. i . d . metal columns using helium as c a r r i e r gas. I n t a c t c e l l s provided s u f f i c i e n t n e u t r a l l i p i d f o r f r e e g l y c e r o l and a c y l g l y c e r o l to be measured by the method of Eggstein (53) as modified by Schmidt et_ a l (54), using a 0.001% (w/v) aqueous p i c r i c a c i d reference s o l u t i o n . In order to accomplish f u l l h y d r o l y s i s 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, the KOH s a p o n i f i c a t i o n step was lengthened to 12 hours from the 20 minutes recommended i n the standard, c l i n i c a l procedure. Assay components were purchased from the Boehringer-Mannheim Corporation. L i p i d measurements were standardized by reference to the p r o t e i n content of the whole c e l l , crude membrane or plasma membrane p r e p a r a t i o n from which the l i p i d s were der 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 techniques of F o l c h e_t a l (41) and of B l i g h and Dyer (38) according to Kates (39,40) were compared (Table 1). 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 reported by Palmer (18,55). The amounts of phospholipid (Table 1 ) , f a t t y a c i d (Table 2) and s t e r o l (Table 3) e x t r a c t e d from p_. discoideum c e l l s showed that the method of F o l c h et al was s u p e r i o r to that of B l i g h and Dyer f o r t h i s study. In view 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 et a l (41), l i t t l e of the phosphorus measured i n 'Folch f r a c t i o n I I ' (Table 1) i s l i k e l y to have been derived from l i p i d phosphorus. S i m i l a r l y , most of the phosphorus i n the B l i g h and Dyer aqueous methanol phase was probably n o n - l i p i d phosphorus. Confirmation of these conclusions was provided by the f a t t y a c i d analyses (Table 2). Of the t o t a l f a t t y a c i d recovered i n each procedure only 1.5% was l o s t to 'Folch f r a c t i o n I I ' and 2.8% to 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 that i n both cases t h i s was l a r g e l y from g l y c o l i p i d sources. Table 3 i n d i c a t e s that the procedure of F o l c h et a l was a l s o more e f f i c i e n t f o r the e x t r a c t i o n of s t e r o l s from I), discoideum Ax-2. Although Long and Coe estimated that stigmast-22-en-33-ol (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 c e l l s (31) more recent analyses have measured only 88% of t o t a l s t e r o l i n the plasma membrane of v e g e t a t i v e c e l l s as stigmastenol (37). Table 1: Lipid-phosphorus e x t r a c t e d from e x p o n e n t i a l l y growing c e l l s of I), discoideum Ax-2 (1) (2) E x t r a c t i o n F r a c t i o n D e s c r i p t i o n of F r a c t i o n Phosphorus i n Phosphorus i n Procedure No. F r a c t i o n F r a c t i o n (nmol mg - x (nmol mg - ± t o t a l p r o t e i n ) t o t a l p r o t e i n ) F o l c h et. a l I I n i t i a l 19.0 v o l . C-M3 (2:1 v/v) e x t r a c t 118.1 ± 0 135.3 ± 20 I I Aqueous C a C ^ phase and combined aqueous 12.4 ± 0 9.3 ± 3.0 washings. I I I Second e x t r a c t of 15.0 v o l . C-M3 (7:1 4.0 ± 1.5 4.5 ± 2.5 v/v) saturated w i t h 5% aq. NH^. IV Residue not determined not determined B l i g h & Dyer I cl 1st and 2nd 6.25 v o l . aqueous C-M lower 88.9 ± 1.5 phase e x t r a c t s combined. I I Aqueous methanol phase a f t e r second C-M3 9.5 ± 0.9 e x t r a c t i o n . I I I Residue 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 techniques of 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 of B l i g h & Dyer (38). Results are the mean of two e x t r a c t i o n s . 2. E x t r a c t i o n of m u l t i p l e samples by the method of Folch et a l . R e s u l t s are the mean of seven independent e x t r a c t i o n s ± standard d e v i a t i o n . C-M i n d i c a t e s 1chloroform-methanol'. Table 2: F a t t y a c i d a n a l y s i s of hydrolyzed 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 of e x p o n e n t i a l l y growing c e l l s of I), discoideum Ax-2. Fo 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 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 F a t t y A c i d I I I I I I IV I I I I I I (Ug f a t t y •A " I a c i d mg t o t a l p r o t e i n ) 14:0 0.5 + 0.2 trace t r a c e . t r a c e 2.9 + 0.4 tr a c e 0 Palmitaldehyde 0.3 + 0.1 tr a c e t r a c e t r a c e 1.5 + 0.3 tr a c e 0.3 + 0.1 16:0 5.2 + 0.7 0. 4 ± 0. 2 0.5 ± 0.1 0.7 ± 0.1 5.2 + 0.1 0.5 + 0.3 1.5 + 0.1 16:1<A9> 2.1 + 0.1 tr a c e t r a c e t r a c e 1.3 + 0.7 t r a c e 0.3 + 0.1 1 6 : 2 ( A 5 > 9 ) & 17:0 a 1.6 + 0.1 t r a c e t r a c e t r a c e 1.3 + 0.7 t r a c e t r a c e 18:0 1.7 + 0.1 0 0 0 2.0 + 0.3 0 0 18:1<A9> _ 1 8 : l ( A l l ) a 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 18:2< A 5' 9> _ 1 8 : 2 < A 5 > 1 1 ) a 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 1 8 : 2 ( A 9 , 1 2 ) 0.9 + 0.4 t r a c e 0 t r a c e t r a c e 0 0 0 t h e r s b 1.5 + 0.7 tra c e 0.3 ± 0.2 0.3 ± 0.2 3.2 + 0.5 0.5 + 0.3 2.3 + 0.6 T o t a l f a t t y a c i d per f r a c t i o n 93.7 + 4 1. 5 ± 1 2.4 ± 1 2.3 ± 1 87.5 + 5 2.7 + 2 6.4 + 1 (lig mg~l p r o t e i n ) % 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 described i n Table 1. Re s u l t s are presented as the mean of three determinations ± standard 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 not separated under the c o n d i t i o n s used but were shown to be present by Davi d o f f & Korn (56). b Several u n i d e n t i f i e d minor components. Table 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 et 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 discoideum Ax-2. F o l c h ^ t a l E x t r a c t i o n F r a c t i o n s 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 Retention a I I I I I I IV I I I + I I I (yg c h o l e s t e r o l equivalent -1 mg p r o t e i n ) 1.17 1.6 + 0.1 t r a c e 0.1 + 0.0 t r a c e 0 0 1.30 1.8 + 0.1 0.2 ± 0.1 t r a c e t r a c e .2.0 ± 0.4 t r a c e 1.43 17.1 + 3.5 0.2 ± 0.0 0.2 ± 0.1 t r a c e 13.6 ± 2.1 1.6 ± 1.3 1.59 0.5 + 0.1 t r a c e t r a c e t r a c e 0.2 ± 0.2 0 Others b 0.4 + 0.1 0 t r a c e t r a c e 0.5 ± 0.1 0.4 ± 0.3 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 ± 0.1 t r a c e 16.3 ± 2.8 2.0 ± 1.6 % T o t a l Recovered 96.8 + 17.6 1.8 ± 0.5 1.4 ± 0.5 t r a c e 89.1 ± 15.3 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 Table 1. R e s u l t s are presented as the mean of three determinations ± standard 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 equivalent mg~l p r o t e i n . Absolute values vary more than '% t o t a l recovered' f o r the l a t t e r are measured w i t h i n each experiment. Retention f i g u r e s are 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 acetate) r e t e n t i o n under the 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. The present study confirmed that other s t e r o l s were present i n s i g n i f i c a n t p r o p o r t i o n s (Table 3) and showed that both stigmastenol (standard 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), discoideum the F o l c h l i p i d e x t r a c t i o n procedure was s u p e r i o r to that of B l i g h and Dyer, the e f f i c i e n c y of the 'Folch f i n a l e x t r a c t i o n ' (Folch f r a c t i o n I I I ) was analyzed. C e r t a i n r e s i d u a l l i p i d s , notably the p o l y p h o s p h o i n o s i t i d e 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 solv 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 necessary to remove such l i p i d s from t i s s u e s (30,56,58). Table 4 shows the f a t t y a c i d analyses 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 'Folch residue' i n which e x t r a c t i o n time and volumes were standardized to permit 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 . The chloroform-methanol-concentrated HC1 (50:50:0.3 by v o l . ) solvent mixture 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 phosphatidyl-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. Buckley, personal communication). Both a c i d c o n d i t i o n s t e s t e d were superi o r to the a l k a l i n e solvent mixture and the s t r o n g l y a c i d i c c o n d i t i o n s were the most e f f i c i e n t . The reasons f o r these d i f f e r e n c e s were not apparent. As a r e s u l t of these s t u d i e s , I), discoideum l i p i d s were ex t r a c t e d i n a l l subsequent experiments by the procedure of F o l c h et al_ (41), followed by an e x t r a c t i o n w i t h chloroform-methanol-concentrated HC1 (50:50:0.3 by v o l . ) . Table 4: F a t t y a c i d a n a l y s i s of v a r i o u s 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 et a l (41). F r a c t i o n F r a c t i o n I I I I F a t t y A c i d A B C (yg f a t t y a cid) 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 16:1< A 9> 1.76 0.01 0.02 0.04 1 6 . 2 ( A 5 , 9 ) & 1 7 : Q a 0.80 0.04 0.03 0.03 18:0 2.59 0.06 0.11 0.10 1 8 : l ( A 9 ) & 1 8 : l ( A 1 1 ) a 23.75 0.09 0.20 0.57 1 8 : 2 ( A 5 ' 9 ) & 1 8 : 2 ( A 5 ' 1 1 ) a 34.76 0.12 0.16 0.18 1 8 : 2 ( A 9 , 1 2 ) 0.45 0.00 0.04 0.05 Others 0 1.26 0.02 0.11 0.47 T o t a l F a t t y A c i d 70.42 0.43 1.06 1.84 A l l e x t r a c t i o n s were of equivalent samples from one pr 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), discoideum Ax-2. F r a c t i o n I was as described i n Table 1. F r a c t i o n I I I was an overnight e x t r a c t i o n at 4°C comprising: A, 15 v o l (chloroform-methanol (7:1 v/v) saturated w i t h 5% (w/v) aq. NH3); B, 15 v o l (chloroform-methanol-glacial a c e t i c acid-water (8:4:2:1 by v o l ) ) ; C, 15 v o l (chloroform-methanol-concentrated HC1 (50:50:0.3 by v o l ) ) . Values are the mean of three determinations. a These f a t t y a c i d s were not separated under the c o n d i t i o n s used but were shown to be present by Davidoff and Korn (60). b Several 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 Separation of 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 Figure 1. Figure 2 ( i ) shows r o u t i n e t h i n — l a y e r chromatographic s e p a r a t i o n of i n d i v i d u a l p hospholipid 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 phospholipid separation i n the presence of b o r i c a c i d which 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 phospholipids a f t e r t h e i r development on both 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_. discoideum Ax-2 were found to c o n t a i n a l l major ph o s p h o l i p i d groups w i t h the exception of sphingomyelin. While Wilhelms 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 surface antigens of JJ. discoideum (36), 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 incorporated i n the e x t r a c t i o n method of F o l c h et al (41) used i n t h i s i n v e s t i g a t i o n . Lysophosphatidylethanolamine, l y s o p h o s p h a t i d y l -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 detected i n the i n t a c t c e l l e x t r a c t s , i n agreement w i t h previous s t u d i e s (30). P h o s p h a t i d y l -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 to be components of I), discoideum grown under standard axenic c o n d i t i o n s . Mono-, d i - and t r i a c y l g l y c e r o l s , ubiquinones, f r e e s t e r o l s and s t e r o l e s t e r s were a l l present, stigmastenol being a major component of both 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 obtained from plasma membrane samples as from i n t a c t c e l l e x t r a c t s . 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 <--a. Solvent 1<-Origin-4 o o. 0 o *^ 0 0' 0 0 0 0 0 ~ 2 3 4 5 6 — » — i — i — i — i — i — • — f » j © 7 8 9 10 II 12 A B C Individual neutral lipids Fig. 1; Separation of neutral lipi d s by monodirectional, biphasic thin-layer chromatography on s i l i c a gel H plates. Lipids 1 and 3 appear dark under short wavelength ultraviolet irradiation. Lipids 1, 4 and 8 yield blue fluorescence under long wavelength ultraviolet irradiation. c Standard l i p i d s : . 1. phospholipid 7. 1,3-diacylglycerol 2. monoacylglycerol 8. free fatty acid 3. ubiquinone 9. triacylglycerol 4. unidentified l i p i d i c compound 10. fatty acid ester 5 . free sterol 11. steryl ester 6. 1,2-diacylglycerol 12. hydrocarbon A. standard neutral l i p i d mixture B. D. discoideum Ax-2 whole c e l l l i p i d extract C. D. discoideum Ax-2 plasma membrane (PM1) l i p i d extract Solvent I comprised: isopropyl ether-glacial acetic acid (96:4 v/v). Solvent II comprised: n-hexane-diethyl ether-glacial acetic acid (90:10:1 by vol.). Each l i p i d was identified as described under Materials and Methods. This compound was isolated from JJ. discoideum membrane preparations as stated under Results. 22. (i) (ii) V f C D ® eg) CD + r ? r ® CD® CD <z> ® CD + . So lvent l l b Solvent n b Fig. 2: Two-dimensional, biphasic thin-layer chromatography of a mixture of standard phospholipids on s i l i c a gel H plates with additives. (i) magnesium acetate-silica 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 ym thickness c. Standard lipid s : 1. lysophosphatidylethanolamine 7. phosphatidylinositol 2. phosphatidylethanolamine 8. phosphatidylserine 3. lysophosphatidylcholine 9. phosphatidylglycerol 4. phosphatidylcholine 10. sphingomyelin 5. phosphatidic acid 11. cardiolipin 6. lysophosphatidic acid i . chromatoplate impurities Solvent I comprised: chloroform-methanol-28% (w/v) acqueous ammonia (65:25:8 by vol.). Solvent II comprised: chloroform-acetone-methanol-glacial acetic acid- d i s t i l l e d , deionised water (35:35:7:10:3 by vol.). Each phospholipid was identified as described under Materials and Methods. but only trace amounts of acylglycerol were present and quinone derivatives were not detected. Short wavelength ultraviolet 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 sterol components migrating with a different mobility than the standard cholesterol, for stigmastenol has a marginally lower than cholesterol in this thin layer chromatographic system. However, the compound's ultraviolet absorption spectrum features radical 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 sterol or fatty acid. The compound emitted blue fluorescence upon irradiation with long wavelength ultraviolet energy. Fluorescence was also observed under both long and short wavelength ultraviolet radiation after treatment of the unknown l i p i d with rhodamine 6G, from which i t was dissociated by solution in chloroform-methanol (4:1 v/v). c. Quantitative Lipid Determinations of Intact Cell Extracts To enable comparison of intact c e l l l i p i d data with that in the literature (1,30,31,59-62) protein and dry weight measurements were taken from exponentially growing cells and a ratio of 0.59 (protein mass:mass of dry cells) was obtained. Table 5 displays the individual and total phospholipid content Table 5: Lipid-phosphorus 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), discoideum 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 E l l i n g s o n P h o s p h o l i p i d A n a l y s i s A n a l y s i s (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 Lysophosphatidylcholine 3.1 + 0.5 2.4 + 0.4 3.4 P h o s p h a t i d y l c h o l i n e 41.3 + 7.3 32.4 + 5.7 16.8 Phosphatidic a c i d 2.3 + 0.5 1.7 + 0.4 3.4 b Lysophosphatidic a c i d 3.5 + 2.4 2.8 + 1.9 1.4 b P h o s p h a t i d y l i n o s i t o l 6.8 + 1.1 5.3 + 0.9 8.7 P h o s p h a t i d y l s e r i n e 4.2 + 3.3 3.3 + 2.6 -P h o s p h a t i d y l g l y c e r o l 2.2 + 0.7 1.7 + 0.6 0 Sphingomyelin 0 0 -C a r d i o l i p i n 3.6 + 1.7 2.8 + 1.5 2.6 L y s o c a r d i o l i p i n 0 0 1.3 3. Others 3.5 + 2.5 2.7 + 1.9 8.3 T o t a l P h o s p h o l i p i d 129.0 ± 16.9 Data from the current a n a l y s i s are the mean of f i v e determinations ± standard d e v i a t i o n . Minor ph o s p h o l i p i d 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 a c i d (30). b Undetermined amounts of plasmalogen forms a l s o present (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 of I), discoideum Ax-2. In comparison w i t h E l l i n g s o n ' s data f o r Ax-2 (30), a l s o shown i n Table 5, the current 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 . P r i m a r i l y phospha-t i d y l c h o l i n e was recovered i n much greater p r o p o r t i o n to other phospholipids (32.4 moles %) and phosphatidylethanolamine to a s i g n i f i c a n t l y lower extent (36.9 moles %) than they were i n the previous study (16.8 moles % and 47.2 moles % r e s p e c t i v e l y ) , although they were the two most abundant phospholipids i n both analyses. However, the e a r l i e r r e p o r t provided percentage values only (30), preventing p r e c i s e measurement 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 these two s t u d i e s . E l l i n g s o n detected 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 the t r a c e found i n c e l l s of s t r a i n NC-4 to b a c t e r i a l sources whereas i n the present study p h o s p h a t i d y l -g l y c e r o l comprised 1.7 moles % of the t o t a l p h o s p h o l i p i d : the reasons f o r these d i s c r e p a n c i e s are not apparent. E l l i n g s o n d i d not record any data f o r p h o s p h a t i d y l s e r i n e (30) although 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 poor l y resolved 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 published p h o s p h a t i d y l -i n o s i t o l f i g u r e i s s i m i l a r to 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 that E l l i n g s o n d i d not separate these two components. The l e v e l s of lysophosphatidylethanolamine, l y s o p h o s p h a t i d y l c h o l i n e , lysophospha-t i d i c a c i d , phosphatidic a c i d and c a r d i o l i p i n were s i m i l a r to the previous a n a l y s i s (30). A c y l g l y c e r o l assays 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 were undertaken before and a f t e r t h e i r s e p a r a t i o n i n the n e u t r a l l i p i d t h i n - l a y e r chromatography system. Table 6 shows that the t o t a l Table 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_. discoideum Ax-2. N e u t r a l L i p i d I n t a c t C e l l Content (nmol mg t o t a l ) (molar percentage) ( p r o t e i n ) 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 T r i a c y l g l y c e r o l 7.4 40 3. T o t a l A c y l g l y c e r o l 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_. discoideum Ax-2 was s m a l l r e l a t i v e to p h o s p h o l i p i d ( c f . Table 1). T r i a c y l g l y c e r o l , a storage l i p i d i n most e u c a r y o t i c c e l l s , was 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 other a c y l g l y c e r o l s were a l s o present. Long and 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 growing e x p o n e n t i a l l y on b a c t e r i a and t h e i r f i g u r e of 3.2 mg g ^ dry weight (31) (c. 7 nmol mg ^ p r o t e i n ) agrees c l o s e l y w i t h the 7.4 nmol mg p r o t e i n determined here i n Ax-2 c e l l s grown under axenic c o n d i t i o n s . The 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 Table 7 confirmed that the major f r e e s t e r o l was stigmastenol (31,37), which comprised 85.5 moles % of the t o t a l . Two other 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 acetates having g a s - l i q u i d chromatography r e t e n t i o n values r e l a t i v e to 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 1.59 (4.2 moles % t o t a l f r e e s t e r o l ) . Three other minor c o n s t i t u e n t s could be d i s t i n g u i s h e d . Long and Coe, however, measured i n excess of 99% of the f r e e s t e r o l from s t r a i n NC-4 as stigmastenol. This was present at approximately 40 nmol mg p r o t e i n (9 mg g ^ dry weight) (31) compared to the t o t a l of 56.8 nmol mg ^ p r o t e i n reported i n t h i s study of 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 by 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 1.28 nmol mg ^ p r o t e i n , 2.3 moles % of the f r e e s t e r o l t o t a l (Table 7). Previous determinations of s t e r o l e s t e r content of i n t a c t c e l l s of s t r a i n NC-4 y i e l d e d values from 1 to 3 nmol mg ^ p r o t e i n (up to 2 mg g dry weight) (31). In c o n t r a s t to Long and Coe's f i n d i n g that stigmastenol was the s o l e s t e r o l present i n the s t e r o l e s t e r Table 7: S t e r o l a n a l y s i s of the 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 of e x p o n e n t i a l l y growing D. discoideum Ax-2. R e t e n t i o n 3 Free S t e r o l of I n t a c t S t e r o l E s t e r 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 D C e l l s , as Stigmastenol^ nmol mg molar , -1 nmol mg molar p r o t e i n percentage p r o t e i n percentage 1.17 tr a c e t r a c e . 0.40 ± 0..01 31.2 ± 1.0 1.30 4.3 ± 0.8 .7.5 ±1.4 0 0 1.43 c 48.5 ± 1.0 85.5 ± 1.8 0.71 ± 0.01 55.5 ± 0.8 1.59 2.4 ± 1.0 4.2 ± 1.7 0.17 ± 0.02 13.5 ± 1.7 Others t r a c e t r a c e 0 0 T o t a l S t e r o l Recovered 56.8 ± 2.8 1.28 ± 0.04 Free s t e r o l values are the mean of f i v e determinations ± standard d e v i a t i o n ; 'trace' i n d i c a t e s <0.4 nmol mg-"'" p r o t e i n . S t e r o l e s t e r values are the mean of two determinations. Retention f i g u r e s are 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 acetate) r e t e n t i o n under the q u a n t i t a t i v e g a s - l i q u i d chromato-graphy c o n d i t i o n used. The r e l a t i v e amounts of each s t e r o l were determined by reference to an i n t e r n a l c h o l e s t e r o l standard and were converted to m o l a r i t i e s using the molecular weight of stigmastenol, the most abundant s t e r o l of D. discoideum. c Stigmastenyl acetate. f r a c t i o n of NC-4 (31) the present study shows that i t c o n s t i t u t e s only 55.5 % of the s t e r o l m o i e t i e s of Ax-2 s t e r o l e s t e r s (Table 7). The second most abundant s t e r o l of the 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 of the 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 value of 1.17. The second most abundant f r e e s t e r o l ( r e t e n t i o n value 1.30) was absent from the s t e r o l e s t e r f r a c t i o n . Thus the s t e r o l p r o f i l e s of these two l i p i d c l a s s e s were very d i f f e r e n t . d. E v a l u a t i o n of Ph o s p h o l i p i d Degradation During Plasma Membrane  Pre p a r a t i o n . Membrane l i p i d measurements have been recorded r e l a t i v e to the p r o t e i n content of 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 preparations underwent minimal p r o t e i n l o s s i n the twelve hours f o l l o w i n g homogenization, i n d i c a t i n g that p r o t e i n degradation was i n s i g n i f i c a n t under the c o n d i t i o n s of pr e p a r a t i o n . Ferber et al discovered high a c t i v i t i e s of phospholipase A and lysophospholipase i n I), discoideum 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 (63). These two a c t i v i t i e s would lead to the formation from phosphoglycerides of non-acylated (sn) 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 which would be f u l l y s o l u b l e i n the aqueous phase of the standard e x t r a c t i o n washing procedure (41) and excluded from subsequent lipid-phosphorus a n a l y s i s . No evidence of phospholipase C a c t i v i t y has been found i n c e l l homogenates (63). Any l i p a s e a c t i v i t y was of great s i g n i f i c a n c e to t h i s determination of plasma membrane l i p i d composition f o r the membrane pr e p a r a t i o n procedure r e q u i r e d a minimum of 17% hours. F i g u r e 3 demonstrates that p h o s p h o l i p i d degradation was observed i n c e l l homogenates at 22°C but that t h i s was reduced a t 4°C. When lipid-phosphorus i s expressed as the lo g a r i t h m of the percentage remaining at l e a s t two d i s t i n c t r a t e s of l o s s are revealed ( F i g . 3). This suggests that i f enzymatic degradation was r e s p o n s i b l e there was e i t h e r more than one pool of pho s p h o l i p i d as sub s t r a t e or th 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 . Both the short and the long-term degradation r a t e s increased at the higher temperature Figure 4 confirms that the 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 consider a b l y greater at 22°C than at 4°C. Consequently the e n t i r e plasma membrane p r e p a r a t i o n procedure was c a r r i e d out at 4°C or on i c e and each step performed as r a p i d l y as p o s s i b l e . The PMSF used to i n h i b i t serine-protease a c t i v i t y during plasma membrane pr e p a r a t i o n had no apparent e f f e c t on the r a t e of l o s s of l i p i d -phosphorus from homogenates. Al s o i n e f f e c t i v e was the polyene a n t i b i o t i c f i l i p i n which Ferber et al had found to i n h i b i t the phospholipase A and lysophospholipase a c t i v i t i e s of I), discoideum v l 2 / M l (63). Crude membranes were prepared r a p i d l y a t 4°C and then immediately incubated at 22°C w i t h e i t h e r 8.6% sucrose-Tris-PMSF b u f f e r , pH 7.4, or w i t h the 105,400 xg supernatant f r a c t i o n (see M a t e r i a l s and Methods ( b ) ) . F i g u r e 5 shows that the r a t e of lipid-phosphorus l o s s from the crude membranes was increased approximately f o u r - f o l d by the presence of the supernatant suggesting that much of the l i p a s e a c t i v i t y observed i n the e a r l i e r experiments was not membrane bound. Crude membranes were a l s o prepared and incubated i n the presence of excess of the f o l l o w i n g p u t a t i v e 13. discoideum phospholipase 31. Fig. 3: The effect 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 cells were removed from the homogenate by centrifugation at 700 xg and incubation was carried out at 4°C (O) or at 22°C (• ) in 8.6% sucrose-Tris-PMSF buffer, pH 7.4, as described under Materials and Methods. Fig. 4: The effect of temperature on the early loss of l i p i d -phosphorus from I), discoideum Ax-2 homogenates. Intact cells were removed from the homogenate by centrifugation at 700 xg and incubation was carried out at 4°C in 8.6% sucrose-Tris-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 0 9 0 100m T i m e post h o m o g e n i z a t i o n (minutes) Fig. 5: The effect of the soluble fraction 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 in 8.6% sucrose-Tris-PMSF buffer, pH 7.4, and incubated at 22°C (•). ^ _ x Rate of lipid-phosphorus l o s s 3 =0.6 nmol mg protein min (2) Crude membranes resuspended in the supernatant from the 105,400 xg preparatory centrifugation step (homogenate soluble fraction) and incubated at 22°C (•). _ x _^ Rate of lipid-phosphorus l o s s a = 2.3 nmol mg protein min Data was subjected to linear regression analysis in order to obtain the average rate of change of lipid-phosphorus over the sampling period. i n h i b i t o r s : 4-chloromercuribenzoic a c i d , sodium 4-hydroxymercuri-benzoate, d i g i t o n i n i n both aqueous suspension and methanolic s o l u t i o n , e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d (63). However, none were e f f e c t i v e at reducing l i p i d phosphorus l o s s from crude membranes. Figure 6 shows that t h i s l o s s was minimal over a 20 hour sampling pe r i o d when the i n c u b a t i o n temperature was maintained at 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 time. In order to determine whether p a r t i c u l a r phospholipids were modified crude membranes were incubated i n 8.6% sucrose-Tris-PMSF b u f f e r , pH 7.4, f o r a time p e r i o d equivalent to that of a plasma membrane p r e p a r a t i o n (Table 8). P h o s p h a t i d y l c h o l i n e was observed to decrease very d r a m a t i c a l l y from 95.0 to 30.5 nmol mg ^ p r o t e i n i n 20 hours at 4°C and even f u r t h e r at 22°C. Lysophosphatidylcholine remained at a low l e v e l throughout and phosphatidic a c i d and lysoph o s p h a t i d i c a c i d d i d not increase g r e a t l y suggesting that a phospholipase C or a combination of phospholipases 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 s i g n i f i c a n t l y , v i r t u a l l y a l l of the l o s s o c c u r r i n g i n the f i r s t 10 hours of the in c u b a t i o n . Lysophosphatidylethanolamine increased to a degree that was almost equivalent to the l o s s of phosphatidylethanolamine i n d i c a t i n g that the disappearance of l i p i d i c c h o l i n e from the in c u b a t i o n may have been due to a s e l e c t i v e a c t i v i t y . Of the 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 q u a n t i t i e s f l u c t u a t e d to some extent, the amount of p h o s p h a t i d y l s e r i n e remained 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. The category of 'others' contains 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 time. I t i n c l u d e s u n i d e n t i f i e d new spots which appeared on the chromato-in en C 0 o _ 2 5 0 0 1 Ui E 4 0 0 "o E c w 3ooj (/> 3 P •5- 1—* T a o 2 0 0 1 0 0 i 8 1 0 1 2 1 4 1 6 1 8 2 0 Time post homogenization (hours) F i g . 6: Lipid-phosphorus content of I), discoideum Ax-2 crude membranes during extended i n c u b a t i o n a t 4 C. Data was subjected to l i n e a r r e g r e s s i o n a n a l y s i s using the l e a s t squares procedure. Rate of change of lipid-phosphorus = -2.2 n mol mg~l p r o t e i n h o u r ~ l . Table 8: P r o p o r t i o n of i n d i v i d u a l p h o s p holipid components of JJ. discoideum Ax-2.crude membranes resuspended i n 8.6% sucrose-Tris-PMSF b u f f e r , pH 7.4, incubated at e i t h e r 4°C or 22°C as shown. Ph o s p h o l i p i d Zero Time 10 hr at : 4°C 20 hr at 4°C 20 hr at 22°C (nmol mg pr o t e i n ) Lysophosphatidylethanolamine 128.5 + 36.0 143.5 + 23.0 151.0 + 14.0 164.5 + 31.0 Phosphatidylethanolamine 120.5 + 23.5 72.0 + 9.0 69.0 + 4.0 71.0 + 18.5 Lysophosphatidylcholine 11.5 + 5.0 8.0 + 3.5 5.0 + 3.5 1.0 + 1.0 P h o s p h a t i d y l c h o l i n e 95.0 + 4.5 44.0 + 6.0 30.5 + 8.0 21.5 + 11.0 Phosphatidic a c i d 8.0 + 8.0 13.0 + 8.5 20.5 + 12.5 25.5 + 18.0 Lysophosphatidic a c i d 22.0 + 2.5 43.0 + 2.0 19.0 + 18.5 19.5 + 13.0 P h o s p h a t i d y l i n o s i t o l 41.5 + 14.5 38.5 + 23.0 54.0 + 9.0 ) 3.0 * 48.5 + 15.0b P h o s p h a t i d y l s e r i n e 6.5 + 5.0 2.5 + 2.5 + 3.0 J P h o s p h a t i d y l g l y c e r o l •5.5 + 4.5 0 0 0 Sphingomyelin 0 0 0 0 C a r d i o l i p i n 16.5 + 6.5 17.5 + 6.5 34.5 + 18.5 15.5 + 10.5 O t h e r s 3 9.0 + 8.0 55.5 + 34.5 30.0 + 14.0 23.0 + 14.0 T o t a l Phospholipid 464.5 437.5 416.5 389.0 Values of lipid-phosphorus recovered are presented as the mean of dual l i p i d e x t r a c t i o n s . The phospholipids were separated by t h i n - l a y e r chromatography before being assayed. Si U n i d e n t i f i e d minor components, k These two phospholipids d i d not separate f u l l y i n t h i s a n a l y s i s . grams as the i n c u b a t i o n proceeded and probably comprised phosphory-l a t e d degradation products. Incubation at 22°C g e n e r a l l y enhanced the changes observed at 4°C. Thus the plasma membrane pho s p h o l i p i d r e s u l t s are probably accurate f o r phosphatidic a c i d , l y s o p h o s p h a t i d i c a c i d , p h o s p h a t i d y l -s 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 ph o s p h a t i d y l -c h o l i n e , l y s o p h o s p h a t i d y l c h o l i n e , phosphatidylethanolamine and p h o s p h a t i d y l g l y c e r o l are p o s s i b l y underestimated and l y s o p h o s p h a t i d y l -ethanolamine may be overestimated. e. Plasma Membrane L i p i d Composition The i n d i v i d u a l p h o s p h o l i p i d s , the s t e r o l s and the phospholipid f a t t y a c i d compositions of the two plasma membrane f r a c t i o n s PM1 and PM2 (35) at e a r l y stages of the organism's development are shown i n Tables 9, 10 and 11. I t has been suggested that PM1 may be a purer plasma membrane p r e p a r a t i o n than PM2 or that the two may represent d i f f e r e n t areas of surface 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 of both PM1 and PM2 decreased s u b s t a n t i a l l y i n the f i r s t e ight hours of development (Table 9). The l o s s was 29% i n PM1 (from 690.3 to 491.5 nmol mg"1 p r o t e i n ) and 38% i n PM2 (from 583.3 to 362.4 nmol mg 1 p r o t e i n ) . This 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 sy n t h e s i s or i n c o r p o r a t i o n of membrane p r o t e i n or the degradation or removal of membrane l i p i d . I t i s not p o s s i b l e to d i s t i n g u i s h between these p o s s i b i l i t i e s from the data i n Table 9 although p r o t e i n content per c e l l has been shown to decrease over t h i s p eriod (1). Both plasma membrane f r a c t i o n s Table 9: Plasma membrane lipid-phosphorus a n a l y s i s at three stages of development of D_. discoideum Ax-2 f o l l o w i n g phospholipid separation by t h i n - l a y e r chromatography. P M l a ' PM2 a P h o s p h o l i p i d 0 hrs 8 hrs 16 hrs 0 hrs 8 hrs 16 hrs ( % l i p i d phosphorus recovered ) ( % l i p i d phosphorus recovered) 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 Lysophosphatidylcholine 0 0 0 t r a c e 5. 9 1.8 + 0.3 Phosph a t i d y l c h o l i n e 31.1 ± 2.0 43.4 5. 3 ± 1.1 25.6 ± 6.0 25. 0 4.1 + 2.0 Phosphatidic a c i d 0 0 tr a c e t r a c e 0 16.0 + 6.0 Lysophosphatidic a c i d 0 1.9 8. 9 ± 5.7 4.6 ± 4.4 0 4.5 + 0.2 P h o s p h a t i d y l i n o s i t o l P h o s p h a t i d y l s e r i n e J 8.6 ±0.5 14.2 C 4 ± 3.7 tr a c e 6.5 ± 2.1 0 8. 0 8 7.8 + 0 0.7 P h o s p h a t i d y l g l y c e r o l t r a c e 0 8. 7 ± 2.6 t r a c e 0 14.8 + 0.9 Sphingomyelin 0 0 0 0 0 0 C a r d i o l i p i n t r a c e t r a c e 0 t r a c e 5. 9 0 Oth e r s b 1.2 ± 0.2 3.8 7. 9 ±1.7 3.4 ± 1.8 0 1.2 + 1.2 T o t a l Phospholipid (nmol mg~l protein) 690.3 ± 27.2 491.5 487. 4 ± 66.5 583.3 ± 41.9 362. 4 360.5 + 12.5 '0 hrs' i n d i c a t e s that 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 harvested f o r plasma membrane a n a l y s i s or washed f r e e of n u t r i e n t s and placed i n b u f f e r to i n i t i a t e development to permit a n a l y s i s at l a t e r time p o i n t s . 0-, 16- and 8 hr determinations are the mean of f o u r , two and one set of 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 are recorded as standard d e v i a t i o n s where appropriate 'trace' i n d i c a t e s <1% t o t a l phospholipid. a : PM1 and PM2 are the two plasma membrane preparations obtained by the technique described under M a t e r i a l s and Methods. b : U n i d e n t i f i e d minor components. maintained t h e i r eight 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, yet numerous membrane-associated p r o t e i n changes are known to occur during 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 discoideum Ax-2 PM1 f r a c t i o n s ( c f . Table 5, column 2; Table 9, column 1) con-tained a f a r greater percentage 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, phosphatidic 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 and c a r d i o l i p i n was reduced i n both plasma membrane f r a c t i o n s (0.9 moles % ) . The PM2 f r a c t i o n d i f f e r e d from PM1 i n that before development began phospha-tidylethanolamine and lysophosphatidylethanolamine were present i n equivalent p r o p o r t i o n s , approximately 29 moles % each (Table 9, column 4). Lysophosphatidic a c i d and 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 phosphatidic a c i d were a l s o detected i n PM2. P h o s p h a t i d y l c h o l i n e was present 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 to t o t a l phospholipid) i n the i n t a c t c e l l s , PM1 and PM2 f r a c t i o n s a l i k e . The p h o s p h a t i d y l c h o l i n e i n PM1 was found to decrease d r a m a t i c a l l y r e l a t i v e to 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 Ax-2 development (Table 9, columns 1 to 3). Phosphatidylethanolamine a l s o decreased by a f a c t o r of two but t h i s r e d u c t i o n occurred p r o g r e s s i v e l y from zero time. The lysophosphatidylethanolamine content of PM1 f l u c t u a t e d c o n s i d e r a b l y . In v e g e t a t i v e c e l l s and a f t e r 16 hours development approximately 57 moles % of the t o t a l p h o s p h o l i p i d i n PM1 contained ethanolamine, although the p r o p o r t i o n dropped to 36 moles % at the 8 hour stage. Lysophosphatidylcholine was not detected at any time i n PM1, yet phosphatidic a c i d and lysophosphatidic acid increased as development progressed. In view of the substantial loss of phosphatidylcholine i n the latter half of the observed period these results may indicate that one or more phospholipases acted preferentially on phosphatidylcholine in the plasma membrane during development. Previous data had shown that crude membranes were capable of degrading phosphatidyl-choline in the presence of lysophosphatidylethanolamine without an accumulation of lysophosphatidylcholine (Table 8). Phosphatidyl-glycerol and cardiolipin were present in small quantities in the PMI fraction of exponentially growing ce l l s , although after 16 hours phosphatidylglycerol had increased substantially to 8.7 moles % of total phospholipid. The proportion of unidentified compounds also increased as development progressed (Table 9, 'others'). Phosphatidylinositol and phosphatidylserine remained relatively constant in PMI during the observed period. Plasma membrane fraction PM2 provided similar phospholipid results to PMI during early development, the major differences being a greater reduction of phosphatidylethanolamine with time, a smaller amount of lysophosphatidylethanolamine 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 sterol compositions of the free sterol and sterol ester fractions of PMI and PM2 from both exponentially growing cells and those which had undergone 16 hours development. The total quantity of free sterol in PMI dropped slightly during Table 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 at two stages of development of D. discoideum Ax-2. Retention Plasma Membrane F r a c t i o n PMI 0 hours 16 hours Plasma Membrane F r a c t i o n PM2 0 hours 16 hours f r e e s t e r o l f r e e s t e r o l f r e e s t e r o l f r e e s t e r o l s t e r o l ester s t e r o l ester s t e r o l e s t e r s t e r o l e s t e r (nmol mg 1 p r o t e i n ) 0 (nmol mg p r o t e i n ) C (nmol mg 1 p r o t e i n ) 0 (nmol mg • \ c protexn) 0.85 11.4 ± 6.4 0 1.9 ± 0.7 0 8.5 ± 2.8 0 18.1 ± 8.1 0 1.17 16.8 ± 4.1 1.5 ± 0. 1 2.8 ± 0.2 4.6 + 0.8 18.6 ± 11.6 0.9 ± 0.5 17.4 ± 7.2 6.6 ± 2.1 1.30 25.1 ± 0.5 0 22.8 ± 6.1 0 19.6 + 2.6 0 12.2 ± 2.1 0 1.43 d 170.1 ± 32.2 3.7 ± 0. 3 200.3 ± 7.7 37.1 ± 0.6 189.4 ± 23.5 1.7 ± 0.4 275.9 ± 12.5 22.3 ± 1.5 1.59 17.8 ± 8.4 1.0 ± 0. 2 5.6 ± 0.0 2.8 + 1.2 21.7 ± 8.5 0.6 ± 0.2 5.3 ± 0.0 0.7 ± 0.7 1.83 12.9 ± 6.5 0 1.4 +. 0.4 0 0 0 0 0 T o t a l S t e r o l 253.8 ± 19.3 6.2 ± 0.9 234.5 ± 14.3 44.4 ± 8.2 257.8 ± 10.6 3.2 ± 1.8 328.5 ± 67.3 29.6 ± 2.5 '0 hour' and '16 hour' data were determined from three and two sets of plasma membrane l i p i d s r e s p e c t i v e l y . Values are expressed as the mean ± standard deviation. PMI and PM2 are the two plasma membrane preparations obtained by the technique described under M a t e r i a l s and 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: 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 chromatography co n d i t i o n s used. ° Molar equi v a l e n t s were c a l c u l a t e d as described i n Table 7 ^ \ These values were r e l a t e d to membrane p r o t e i n content. d Stigmastenyl acetate. t h i s p e r i o d , whereas that of PM2 increased. The p r o p o r t i o n of stigmastenol 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 l e s s than that of i n t a c t c e l l e x t r a c t s ( c f . Tables 7 and 10), being 67 moles % and 74 moles % i n PMI and PM2 r e s p e c t i v e l y . However, a f t e r 16 hours development the f r e e s t e r o l composition of both membrane f r a c t i o n s resembled that 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 % stigmastenol. There was a component w i t h a standard r e t e n t i o n of 1.83 which was present i n f r a c t i o n PMI at zero time, yet became a very minor component a f t e r 16 hours development: i t was not observed i n PM2. During e x p o n e n t i a l growth of s t r a i n Ax-2 the q u a n t i t y of s t e r o l e s t e r was v i r t u a l l y twice as l a r g e i n PMI as i n PM2 (Table 10) and yet, as i n the i n t a c t c e l l , the plasma membrane s t e r o l e s t e r content was only 1-3 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 that of whole c e l l s t e r o l e s t e r s ( c f . Tables 7 and 10). The 6.2 nmol s t e r o l e s t e r mg 1 p r o t e i n recovered i n PMI at 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, suggesting that the s t e r o l e s t e r i s l a r g e l y confined to membranous s t r u c t u r e s w i t h i n the c e l l . A f t e r 16 hours development the plasma membranes' s t e r o l e s t e r content had increased d r a m a t i c a l l y ; that of PMI seven-fold and that of PM2 n i n e - f o l d . Only three s t e r o l m o i e t i e s were detected 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 stigmastenol comprised 60 moles % of the s t e r o l e s t e r s t e r o l i n PMI and 54 moles % i n PM2, these f i g u r e s r i s i n g to 84 and 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 hours development. The d i f f e r e n c e s between the s t e r o l compositions of 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 of whole c e l l s , and plasma membranes at both the zero and at the 16 hour time p o i n t s , suggest that 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 operating (Tables 7 and 10). The f a t t y a c i d compositions of the t o t a l p h o s p h o l i p i d i s o l a t e d from the two plasma membrane f r a c t i o n s of JJ. discoideum at zero and s i x t e e n hours development are di s p l a y e d i n Table 11. In expo-n e n t i a l l y growing c e l l s the phospholipid a c y l chains of both PM1 and PM2 were approximately 14% f u l l y saturated and 86% unsaturated, whereas a f t e r s i x t e e n hours development the saturated components had r i s e n to approximately 29% l e a v i n g 71% unsaturated. Before development began almost 80% of both membrane f r a c t i o n s ' a c y l chains . . . (A9 or A l l ) . (A5,9 or A 5 , l l ) . . comprised 18:1 or 18:2 , these two being represented i n s i m i l a r q u a n t i t i e s . A f t e r s i x t e e n hours development the t o t a l of these f a t t y a c i d s had dropped to 53% i n PM1 and 64% i n PM2. Moreover, most of t h i s l o s s was from the 18:2 pool. Consequently one might expect the membrane f l u i d i t y to decrease s u b s t a n t i a l l y over the same per 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 Plasma Membrane Fraction PM2 0 hours 16 hours 0 hours 16 hours (%. recovered) .'(.% recovered) (% 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 1 6 : ( A 9 ) 3.0 3.5 + 0.8 2.2 1.6 + 0.6 16:2 ( A 5' 9 ) & 17:0b 1.7 4.2 + 0.7 1.8 2.1 + 0.4 18:0 3.9 13.5 + 2.0 2.5 12.3 + 3.5 1 8 : l ( A 9 ) & 1 8 : l ( A l l ) b 40.9 35.2 + 2.1 37.4 45.0 + 1.2 18:2^ A 5' 9> & 1 8 : 2 ( A 5 » 1 1 > b 37.5 17.9 + 2.5 39.9 19.0 + 0.9 1 8 : 2(A9,12) 1.2 2.1 + 2.1 1.4 2.1 + 0.5 Others 0 1.1 4.5 + 1.4 2.4 2.8 + 1.2 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. discoideum Ax-2 l i p i d q u a n t i t a t i o n data has been expressed i n both molar percentages and i n r e l a t i o n to the p r o t e i n content of the i n t a c t c e l l s or the membrane p r e p a r a t i o n from which the 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 dry weight per c e l l are among many parameters known to a l t e r during the organism's development (1,59,64), the 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) favoured the use of the p r o t e i n standard f o r the current 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 of the l i p i d e x t r a c t i o n method used i n t h i s study, modified from that of F o l c h e± _al (41), i s apparent from the data i n Tables 1, 2, 3 and 4, although a disadvantage of 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 h y d r o l y s i s (65). Throughout t h i s i n v e s t i g a t i o n the 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 treatment was kept separate from the i n i t i a l chloroform-methanol e x t r a c t u n t i l the HC1 had been removed i n a stream of nit r o g e n . Nevertheless i t i s p o s s i b l e that s u f f i c i e n t a c i d remained to degrade plasmalogens to t h e i r l y s o d e r i v a t i v e s when the two e x t r a c t s were pooled and that previous r e p o r t s of a high lysophospha-tidylethanolamine content of i n t a c t ID. discoideum c e l l s (30,61) may a l s o be due to h y d r o l y s i s of the phosphatidylethanolamine plasmalogen. E l l i n g s o n detected plasmalogens i n c e r t a i n p h o s p h o l i p i d i s o l a t e s (Table 5) but d i d not r e p o r t the q u a n t i t i e s present (30). Thus the higher lysophosphatidylethanolamine content of l i p i d e x t r a c t s of crude membranes and plasma membranes compared w i t h those of i n t a c t c e l l s (Tables 5, 8 and 9) may i n d i c a t e that the phosphatidylethanolamine plasmalogen is enriched in these membranes at the early developmental stages tested. Membranes might be expected to contain plasmalogens rather than lysophospholipids because of the powerful detergent effect of the latter. There is no evidence for the presence of phosphatidylcholine plasmalogen in J). discoideum (30). The majority of plasma membrane purification methods, including those for obtaining plasma membranes of D. discoideum (35,37), are of extended duration and consequently allow the possibility of con-siderable phospholipid degradation. Extremely active phospholipases have been detected in the organism by Ferber ej: jtl (63) and in the present study substantial phospholipid degradation occurred during incubation of cell-free homogenates ixi vitro (Figures 3 and 4). There was far less phospholipid degradation during incubation of crude membrane preparations In vitro (Figure 5) indicating that under these conditions most of the phospholipase activity was not membrane bound. The higher lysophosphatidylethanolamine content of both crude and plasma membranes compared with that of intact cells (Tables 5, 8 and 9) may indicate that a certain amount of lysolipid formed after c e l l breakage and this proposal is reinforced by the apparent conversion of phosphatidylethanolamine to lyso-phosphatidylethanolamine which was revealed upon prolonged incubation of crude membranes (Table 8). However, the lysolipid may have been produced from plasmalogen during the extraction procedure, a possibility discussed above. Upon incubation of the crude membranes there was also a substantial loss of phospholipid containing choline in contrast to the relatively small loss of l i p i d i c ethanolamine, which suggests that a mechanism may e x i s t i n these membranes f o r the s p e c i f i c removal or degradation of c h o l i n e - c o n t a i n i n g phospho-l i p i d s . P h o s p h o l i p i d degradation during plasma membrane p u r i f i c a t i o n was minimized by shortening the p r e v i o u s l y published procedure (37) wherever p o s s i b l e . The method of sucrose gradient c e n t r i f u g a t i o n used to p u r i f y plasma membranes from crude membranes may e l i m i n a t e the r e s i d u a l l i p i d degradation by p h y s i c a l s e paration of the plasma membrane f r a c t i o n s from the 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 the plasma membrane would be simultaneously enriched, which might lead to an exagger-a t i o n of l i p i d changes brought about by developmentally c o n t r o l l e d plasma membrane enzyme a c t i v i t i e s . There are precedents f o r phospho-l i p a s e a c t i v i t i e s i n plasma membranes: phosphatidic a c i d phosphorylase i s present i n c h i c k embryonic muscle c e l l plasma membranes (66) and phospholipase A, lysophospholipase, acyl-CoA hydrolase and palmitoyl-CoA synthetase a c t i v i t i e s have been measured i n plasma membranes of Acanthamoeba c a s t e l l a n i i (67). Nevertheless plasma membrane phospholipases do not appear to i n t e r f e r e w i t h the a n a l y s i s of E>. discoideum plasma membrane l i p i d composition f o r prolonged i n c u b a t i o n of t h i s organism's plasma membranes ^ Ln v i t r o causes no decrease i n the amount of membrane bound p h o s p h o l i p i d nor a l t e r a t i o n of the membranes' pho s p h o l i p i d composition (G. Weeks, unpublished o b s e r v a t i o n s ) . A number of D. discoideum plasma membrane l i p i d changes occurred during e a r l y development of the c e l l s i n b u f f e r e d shake suspension. A f t e r 16 hours a cons i d e r a b l e r e d u c t i o n of the membranes' phospha-t i d y l c h o l i n e content had taken p l a c e (Table 9). In a d d i t i o n there were s e v e r a l smaller a l t e r a t i o n s of other phospholipids. These changes comprised a decrease i n the p r o p o r t i o n of phosphatidylethanolamine, which was accompanied by an increase i n the p r o p o r t i o n s of lysophospha-tidylethanolamine and p h o s p h a t i d y l g l y c e r o l and by the accumulation of l y s o p h o s p h a t i d i c a c i d and phosphatidic 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 . Apart from the increase of 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 degradation of p h o s p h o l i p i d species. The most dramatic m o d i f i c a t i o n of plasma membrane l i p i d during e a r l y development 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 content which was 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 (Table 10). There was 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 of the plasma membrane phos p h o l i p i d 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 of p a l m i t a t e and s t e a r a t e increased markedly at A5 9 the expense of the octadecadienoic f a t t y a c i d s (18:2 ' and 18:2 A 5 , l l ^ 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 chains increased s u b s t a n t i a l l y over t h i s p e r i o d . Whether these changes are caused by s p e c i f i c degradation 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 f a t t y a c i d s i s unknown, although the t o t a l p h o s p h o l i p i d of both PM1 and PM2 plasma membrane f r a c t i o n s decreased s u b s t a n t i a l l y r e l a t i v e to p r o t e i n during the f i r s t e ight hours of development (Table 9). E l e c t r o n s p i n resonance and fluorescence d e p o l a r i z a t i o n analyses of p_. discoideum 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 present study revealed a pronounced decrease i n membrane f l u i d i t y ( H erring, F.G. and I . T a t i s c h e f f , unpublished observations) which i s i n keeping w i t h the changes i n l i p i d composition reported above. In the suspension b u f f e r aggregation was i n i t i a t e d w i t h i n 8 hours of t r a n s f e r and at t h i s time the c e l l s e x h i b i t no a l t e r a t i o n of plasma membrane f l u i d i t y ( H e rring, F.G. and I. T a t i s c h e f f , unpublished observations) although changes i n membrane ph o s p h o l i p i d were apparent (Table 9). Thus the establishment of c e l l - c e l l contacts does not appear to 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 of p h o s p h o l i p i d composition are a s s o c i a t e d w i t h t h i s stage of development under these c o n d i t i o n s . I t i s p o s s i b l e that the f l u i d i t y changes o c c u r r i n g between 8 and 16 hours are un-connected w i t h normal development s i n c e plasma membranes prepared from c e l l s developing on a s o l i d surface f o r the same time p e r i o d show no a l t e r a t i o n of f l u i d i t y a t any stage. The 16 hour aggregates formed i n suspension were of normal s i z e and proceeded to develope and d i f f e r e n t i a t e normally when t r a n s f e r r e d to a s o l i d surface d e s p i t e t h e i r decreased membrane f l u i d i t y . Thus the a l t e r e d membrane f l u i d i t y imposed under the standardised but a r t i f i c i a l c o n d i t i o n s of suspension c u l t u r e (36) do not appear to impair normal c e l l - c e l l i n t e r a c t i o n although at present i t i s unknown whether the membrane f l u i d i t y r e v e r t s to 'normal' when the d i f f e r e n t i a t i o n of these aggregates i s permitted on a s o l i d s urface. The r e s u l t s presented i n t h i s study i n d i c a t e that s u b s t a n t i a l l i p i d changes occur i n the plasma membrane of I), discoideum during e a r l y development i n shake suspension. Most i n t r i g u i n g i s the lar g e increase i n s t e r o l e s t e r . A p o s s i b l e f u n c t i o n f o r such a molecule i n development i s not e a s i l y v i s u a l i z e d a t present although s t a l k d i f f e r e n t i a t i o n has been induced i n c e l l monolayers of the organism (68), and the inducing component appears to be a n e u t r a l l i p i d ( S t a n f i e l d , E. and G Weeks, unpublished 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 determination of i t s p r e c i s e molecular nature are 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 study provide a background f o r f u r t h e r a n a l y s i s throughout the organism's development. 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