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γ-Butyrobetaine derivative in phospholipids of Phormia
353
SHORT COMMUNICATIONS
de-acylated p h o s p h a t i d y l choline suggests t h a t lactobacillic acid is incorporated directly into the phospholipids a n d is n o t broken down a n d resynthesised from a 1-carbon pool. I would like to t h a n k Dr. M. G. MACFARLANEfor a gift of cardiolipin. I a m grateful to Dr E. KODICEK for advice a n d encouragement, a n d I acknowledge with t h a n k s tile assistance of Mr. D. R. ASHBY with the t h i n - l a y e r chromatography.
D u n n Nutritional Laboratory, University of Cambridge and Medical Research Council, Cambridge (Great Britain)
KAREEN J. I. THORNE
z A. It. GILBY, A. V. FEW AND K. McQUILLEN, Biochim. Biophys. Acta, 29 (1958) 21. 2 M. G. MACFARLANE,Biochem. J., 79 (1961) 4P. 3 M. G. MACFARLANE,Biochem. J., 80 (1961) 45P. 4 M. G. MACFARLANE,Biochem. J., 82 (1962) 4oP. 5 M. I~ATES, D . J . I~USHNER AND A. T. JAMES, Can. J. Biochem. Physiol., 4° (1962) 83. 6 U. M. T. HOUTSMULLER AND L. L. M. VAN DEENEN, Biochem. J., 88 (1963) 43 P. 7 C. WEIBULL, Aeta Chem. Scan&, i i (I957) 881. 8 M. D . YUDKIN, Biochem. J., 82 ( I 9 6 2 ) 4 o P . 9 M. G. MACFARLANE, Nature, 196 (1962) 136.
10 T. I~ANESHIROAND A. G. MARR, J. Lipid Res., 3 (1962) I84. 11 j . I£ANFER AND E. P . KENNEDY, J. Biol. Chem., 238 (1963) 2919. 12 H . ZALKIN, J. H . L A W AND H . GOLDFINE, J. Biol. Chem., 238 (1963) 1242. 13 S. N . SEHGAL, M. t{ATES AND N. E . GIBBONS, Can. J. Biochem. Physiol., 4 ° (1962) 69. 14 15 16 17
K . J . [. THORNE AND E . KODICEK, Biochim. Biophys. Acta, 5 9 (1962) 273I{. J . I. THORNE AND E . KODICEK, Biochim. Biophys. Acla, 59 (1962) 280. H . WAGNER, L. HORHAMMER AND P . WOLFF, Biochem. Z., 3 3 4 (1961) 175. j . A. CIFONELLI AND F . SMITH, Anal. Chem., 26 (1954) 1132.
IS IV[.IKAWA,Federation Proc., 21 (1962) 281. LA Du, B. B. LEVY, J. M. STEELE AND B. B. BRODIE, J. Biol. Chem., 205 (1953) 803. 2o R. S. BANDURSKIAND B. AXELROD, J. Biol. Chem., I93 (1951) 4o5 . 21 D. E. BOWYER, Vq. M. LEAT, A. N . HOWARD AND G. A. GRESHAM, Biochem. J., 89 (1963) 2 4 P . 22 D . E . BOWYER, \V. M. LEAT, A. •. HOWARD AND G. A. GRESHAM, Biochim. Biophys. Acta, 7 ° (1963) 423 • 23 t{. J. I. THORNE AND E. t(ODICEK, Biochim. Biophys. Acta, 59 (1962) 3o6.
19 H . D . APPLETON, B . N .
Received December 2nd, 1963 Revised m a n u s c r i p t received J a n u a r y 9th, 1964
Biochim. Biophys. Acta, 84 (1964) 350-353
sc 5 3 O l 3
~,-Butyrobetaine derivative in phospholipids of Phormia Previous studies in this l a b o r a t o r y I showed t h a t )J-butyrobetaine a n d some related c o m p o u n d s can s u b s t i t u t e for a choline r e q u i r e m e n t in a chemically defined diet for larvae of the blowfly, Phormia regina (Meig.). Later it was shown t h a t the choline in the phospholipids of larvae, fed carnitine in the place of choline, was replaced b y fl-methylcholine~,3. C a r n i t i n e - c o n t a i n i n g phospholipids were not detected, although recently the incorporation of labeled carnitine a n d its derivative, fl-methylcholine, into phospholipids of e m b r y o n i c tissues, was reported 4. Since 7 - b u t y r o b e t a i n e b e h a v e d
Biochim. Biophys. Acta, 84 (1964) 353-355
354
SHORT COMMUNICATIONS
in a m a n n e r similar to carnitine in n u t r i t i o n studies, it was of interest to determine if it evoked a similar change in the phospholipid p a t t e r n . This possibility was investigated b y feeding [Me-14CI~-butyrobetaine to larvae, grown on a modified diet as described earlier z, except t h a t choline or carnitine was replaced b y 12.5 m g ~ - b u t y r o b e t a i n e per flask. I n addition each flask was supplied with 5.3/~C [Me-14Cl)J-butyrobetaine, which was synthesized from )J-aminobutyric acid a n d [14Clmethyliodide. The phospholipids were fractionated on a silicic acidhyflo column as described earlier a. Each t u b e was assayed for phosphorus, radioa c t i v i t y a n d phospholipids. For the latter, microscope slides, without further cleaning, were dipped in a suspension of silica-gel G in chloroform 5 a n d a c t i v a t e d for 5 min at I I 0 °. I n contrast to PEIFER'Sa experience good separations of phospholipids were o b t a i n e d on these microslides. Pooled fractions were ehromatographed on 20 cm × 20 cm plates, using c h l o r o f o r m - m e t h a n o l - o . 8 % aq. NaC1-28 % NH4OH (65 : 35:8 : I, v/v) as the solvent system. The following RF values were o b t a i n e d : p h o s p h a t i d y l ethanola m i n e 0.54, lecithin 0.46, p h o s p h a t i d y l methylcholine 0.49, p h o s p h a t i d y l serine o.31, a n d inositol phosphatide o.15. I f there is a real difference in RF value between lecithin a n d p h o s p h a t i d y l methylcholine, it m a y be due to several other factors t h a n the TABLE I DISTRIBUTION
OF P H O S P H O R U S
AND RADIOACTIVITY
IN P H O S P H O L I P I D S
The larvae were reared under aseptic conditions on a chemically defined diet with [Me-14C]vbutyrobetaine as a choline substitute. The washed phospholipid extract from 34.5 g larvae (wet wt.) was chromatographed on a column of 12 g silicic acid and 6 g Hyflo, and 4o0 drops were collected per tube. Abbreviations: PE, phosphatidyl ethanolanine; PMC, phosphatidyl methylcholine; PS, phosphatidyl serine; PI, inositol phosphatide. Pooled tube fractions
5-13203° -
Total P (t,moles)
I2 19 29 5o
199.2 40.6 14.3 64.6
51- 75 76- 85 86-1oo
22.0 4.5 5.1
Predominant phospholipido in thin-layer chromalogram
PE** PMC PE, ]?MC, PS Lyso PE, PMC, PS, PI PMC PMC PMC
Total disintegrations] rain ( × IO~)*
192.o 53.1 237.4 147.5 29.4 33-5
* Corrections were made for tube volume, quenching and efficiency of different chloroformmethanol mixtures in the p-dioxane scintillator7. *" Although every fraction consisted of a mixture of more phospholipids, only the main identified phospholipids are listed (cf. ref. 8). s t r u c t u r e of the bases, e.g. load effect a n d f a t t y acid composition. The bases were detected with I~ vapors, n i n h y d l i n , a n d D r a g e n d o r f ' s solution; the phospholipids with 12 vapors a n d a dilute Zinzadse reagent 6 (I part Zinzadse - i part H20 ). The results are s u m m a r i z e d in Table I. The phospholipids of each pooled fraction were hydrolyzed in 6 N HC1 for 17 h a n d the bases c h r o m a t o g r a p h e d either on KCl-treated paper 2 or i n a c t i v a t e d silica-gel-G chromatoplates. A satisfactory separation on the chromatoplates was o b t a i n e d with the solvent p h e n o l - b u t a n o l - 2 8 % N H I O H (5:5:2, v/v), with the following RF values: fl-methylcholine 0.60, choline 0.45, ~ - b u t y r o b e t a i n e 0.36,
Biochim. Biophys. Acta, 84 (1964) 353-355
SHORT COMMUNICATIONS
355
e t h a n o l a m i n e o.28, carnitine o.17, a n d serine o.o3. The spots on all c h r o m a t o g r a m s were s c a n n e d w i t h a T r a c e r l a b gas-flow counter (tube TGC 14) to d e t e c t r a d i o a c t i v i t y . I n t h e p h o s p h o l i p i d c h r o m a t o g r a m s only the p h o s p h a t i d y l m e t h y l c h o l i n e was radioactive. I n a g r e e m e n t w i t h this was the a d d i t i o n a l finding t h a t t h e only r a d i o a c t i v e s p o t on p a p e r a n d t h i n - l a y e r c h r o m a t o g r a m s of t h e bases was fl-methylcholine. HC1. R a d i o a u t o g r a m s were m a d e of a c h r o m a t o g r a m of t h e h y d r o l y s a t e s o f all pooled fractions a n d also of t h e h y d r o l y s a t e of F r a c t i o n 51-76, s p o t t e d w i t h each of t h e references. Here again, only t h e spots w i t h an R F identical w i t h t h a t of fl-methylcholine were r a d i o a c t i v e . F r o m these results we m a y conclude t h a t during t h e p o s t e m b r y o n i c d e v e l o p m e n t of P. regina no carnitine is i n c o r p o r a t e d in the phospholipids. F u r t h e r m o r e , these e x p e r i m e n t s p r o v i d e definite p r o o f t h a t fl-methylcholine, i n c o r p o r a t e d in t h e phospholipids, is d e r i v e d from ~ - b u t y r o b e t a i n e fed to the larvae. The a u t h o r s are i n d e b t e d to Dr. R. W. NEWBURGH for his interest a n d suggestions t h r o u g h o u t t h e course of this s t u d y . The w o r k was s u p p o r t e d b y A r m y Chemical Center a n d U.S. Public H e a l t h Service, N a t i o n a l I n s t i t u t e s of H e a l t h ; a n d an 0 . E . C . D . P o s t g r a d u a t e T r a v e l G r a n t (N.W.H.) from t h e N e t h e r l a n d s O r g a n i z a t i o n for t h e A d v a n c e m e n t of P u r e R e s e a r c h (Z.W.0.). Science Research Institute, Oregon State University, Corvallis (Oreg.) U.S.A.
H. C. AGARWAL* N. W. H. Houx**
i E. t-IODGSON, V. H. CHELDELIN AND R. W. NEWBURGH, Arch. Biochem. Biophys., 87 (196o) 48. L. L. BIEBER, V. J. BROOKES, V. H. CHELDELIN AND R. W. NEWBURGH, Biochem. Biophys. Res. Commun., 6 (1961) 237. a L. L. BIEBER, V. H. CHELDELIN AND R. W. NEWBURGH, J. Biol. Chem., 238 (1963) 1262. 4 M. A. MEHLMANAND G. WOLF, Arch. Biochem. Biophys., lO2 (1963) 346. 5 j. j. PEIFER, Mihrochim. Acta, (1962) 529 . e G. J. M. HOOGHWlNKEL AND H. P. G. A. VAN NIEKERK, Konink!. Ned. Akad. Wetenschap. Proc., Ser. B, 63 (196o) 4757 G. A. BRAY, Anal. Biochem., i (196o) 279. s L. L. BIEBER, E. HODGSON, V. H. CHELDELIN, V. J. BROOKES AND R. W. NEWBURGH, J. Biol. Chem., 236 (1961) 2590. R e c e i v e d J a n u a r y 7th, 1964 * Present address: University of Delhi, Delhi (India). ** Present address: Laboratory of Comparative Physiology, State University, Utrecht (The Netherlands). Biochim. Biophys. Acta, 84 (1964) 353-355
SHORT COMMUNICATIONS
de-acylated p h o s p h a t i d y l choline suggests t h a t lactobacillic acid is incorporated directly into the phospholipids a n d is n o t broken down a n d resynthesised from a 1-carbon pool. I would like to t h a n k Dr. M. G. MACFARLANEfor a gift of cardiolipin. I a m grateful to Dr E. KODICEK for advice a n d encouragement, a n d I acknowledge with t h a n k s tile assistance of Mr. D. R. ASHBY with the t h i n - l a y e r chromatography.
D u n n Nutritional Laboratory, University of Cambridge and Medical Research Council, Cambridge (Great Britain)
KAREEN J. I. THORNE
z A. It. GILBY, A. V. FEW AND K. McQUILLEN, Biochim. Biophys. Acta, 29 (1958) 21. 2 M. G. MACFARLANE,Biochem. J., 79 (1961) 4P. 3 M. G. MACFARLANE,Biochem. J., 80 (1961) 45P. 4 M. G. MACFARLANE,Biochem. J., 82 (1962) 4oP. 5 M. I~ATES, D . J . I~USHNER AND A. T. JAMES, Can. J. Biochem. Physiol., 4° (1962) 83. 6 U. M. T. HOUTSMULLER AND L. L. M. VAN DEENEN, Biochem. J., 88 (1963) 43 P. 7 C. WEIBULL, Aeta Chem. Scan&, i i (I957) 881. 8 M. D . YUDKIN, Biochem. J., 82 ( I 9 6 2 ) 4 o P . 9 M. G. MACFARLANE, Nature, 196 (1962) 136.
10 T. I~ANESHIROAND A. G. MARR, J. Lipid Res., 3 (1962) I84. 11 j . I£ANFER AND E. P . KENNEDY, J. Biol. Chem., 238 (1963) 2919. 12 H . ZALKIN, J. H . L A W AND H . GOLDFINE, J. Biol. Chem., 238 (1963) 1242. 13 S. N . SEHGAL, M. t{ATES AND N. E . GIBBONS, Can. J. Biochem. Physiol., 4 ° (1962) 69. 14 15 16 17
K . J . [. THORNE AND E . KODICEK, Biochim. Biophys. Acta, 5 9 (1962) 273I{. J . I. THORNE AND E . KODICEK, Biochim. Biophys. Acla, 59 (1962) 280. H . WAGNER, L. HORHAMMER AND P . WOLFF, Biochem. Z., 3 3 4 (1961) 175. j . A. CIFONELLI AND F . SMITH, Anal. Chem., 26 (1954) 1132.
IS IV[.IKAWA,Federation Proc., 21 (1962) 281. LA Du, B. B. LEVY, J. M. STEELE AND B. B. BRODIE, J. Biol. Chem., 205 (1953) 803. 2o R. S. BANDURSKIAND B. AXELROD, J. Biol. Chem., I93 (1951) 4o5 . 21 D. E. BOWYER, Vq. M. LEAT, A. N . HOWARD AND G. A. GRESHAM, Biochem. J., 89 (1963) 2 4 P . 22 D . E . BOWYER, \V. M. LEAT, A. •. HOWARD AND G. A. GRESHAM, Biochim. Biophys. Acta, 7 ° (1963) 423 • 23 t{. J. I. THORNE AND E. t(ODICEK, Biochim. Biophys. Acta, 59 (1962) 3o6.
19 H . D . APPLETON, B . N .
Received December 2nd, 1963 Revised m a n u s c r i p t received J a n u a r y 9th, 1964
Biochim. Biophys. Acta, 84 (1964) 350-353
sc 5 3 O l 3
~,-Butyrobetaine derivative in phospholipids of Phormia Previous studies in this l a b o r a t o r y I showed t h a t )J-butyrobetaine a n d some related c o m p o u n d s can s u b s t i t u t e for a choline r e q u i r e m e n t in a chemically defined diet for larvae of the blowfly, Phormia regina (Meig.). Later it was shown t h a t the choline in the phospholipids of larvae, fed carnitine in the place of choline, was replaced b y fl-methylcholine~,3. C a r n i t i n e - c o n t a i n i n g phospholipids were not detected, although recently the incorporation of labeled carnitine a n d its derivative, fl-methylcholine, into phospholipids of e m b r y o n i c tissues, was reported 4. Since 7 - b u t y r o b e t a i n e b e h a v e d
Biochim. Biophys. Acta, 84 (1964) 353-355
354
SHORT COMMUNICATIONS
in a m a n n e r similar to carnitine in n u t r i t i o n studies, it was of interest to determine if it evoked a similar change in the phospholipid p a t t e r n . This possibility was investigated b y feeding [Me-14CI~-butyrobetaine to larvae, grown on a modified diet as described earlier z, except t h a t choline or carnitine was replaced b y 12.5 m g ~ - b u t y r o b e t a i n e per flask. I n addition each flask was supplied with 5.3/~C [Me-14Cl)J-butyrobetaine, which was synthesized from )J-aminobutyric acid a n d [14Clmethyliodide. The phospholipids were fractionated on a silicic acidhyflo column as described earlier a. Each t u b e was assayed for phosphorus, radioa c t i v i t y a n d phospholipids. For the latter, microscope slides, without further cleaning, were dipped in a suspension of silica-gel G in chloroform 5 a n d a c t i v a t e d for 5 min at I I 0 °. I n contrast to PEIFER'Sa experience good separations of phospholipids were o b t a i n e d on these microslides. Pooled fractions were ehromatographed on 20 cm × 20 cm plates, using c h l o r o f o r m - m e t h a n o l - o . 8 % aq. NaC1-28 % NH4OH (65 : 35:8 : I, v/v) as the solvent system. The following RF values were o b t a i n e d : p h o s p h a t i d y l ethanola m i n e 0.54, lecithin 0.46, p h o s p h a t i d y l methylcholine 0.49, p h o s p h a t i d y l serine o.31, a n d inositol phosphatide o.15. I f there is a real difference in RF value between lecithin a n d p h o s p h a t i d y l methylcholine, it m a y be due to several other factors t h a n the TABLE I DISTRIBUTION
OF P H O S P H O R U S
AND RADIOACTIVITY
IN P H O S P H O L I P I D S
The larvae were reared under aseptic conditions on a chemically defined diet with [Me-14C]vbutyrobetaine as a choline substitute. The washed phospholipid extract from 34.5 g larvae (wet wt.) was chromatographed on a column of 12 g silicic acid and 6 g Hyflo, and 4o0 drops were collected per tube. Abbreviations: PE, phosphatidyl ethanolanine; PMC, phosphatidyl methylcholine; PS, phosphatidyl serine; PI, inositol phosphatide. Pooled tube fractions
5-13203° -
Total P (t,moles)
I2 19 29 5o
199.2 40.6 14.3 64.6
51- 75 76- 85 86-1oo
22.0 4.5 5.1
Predominant phospholipido in thin-layer chromalogram
PE** PMC PE, ]?MC, PS Lyso PE, PMC, PS, PI PMC PMC PMC
Total disintegrations] rain ( × IO~)*
192.o 53.1 237.4 147.5 29.4 33-5
* Corrections were made for tube volume, quenching and efficiency of different chloroformmethanol mixtures in the p-dioxane scintillator7. *" Although every fraction consisted of a mixture of more phospholipids, only the main identified phospholipids are listed (cf. ref. 8). s t r u c t u r e of the bases, e.g. load effect a n d f a t t y acid composition. The bases were detected with I~ vapors, n i n h y d l i n , a n d D r a g e n d o r f ' s solution; the phospholipids with 12 vapors a n d a dilute Zinzadse reagent 6 (I part Zinzadse - i part H20 ). The results are s u m m a r i z e d in Table I. The phospholipids of each pooled fraction were hydrolyzed in 6 N HC1 for 17 h a n d the bases c h r o m a t o g r a p h e d either on KCl-treated paper 2 or i n a c t i v a t e d silica-gel-G chromatoplates. A satisfactory separation on the chromatoplates was o b t a i n e d with the solvent p h e n o l - b u t a n o l - 2 8 % N H I O H (5:5:2, v/v), with the following RF values: fl-methylcholine 0.60, choline 0.45, ~ - b u t y r o b e t a i n e 0.36,
Biochim. Biophys. Acta, 84 (1964) 353-355
SHORT COMMUNICATIONS
355
e t h a n o l a m i n e o.28, carnitine o.17, a n d serine o.o3. The spots on all c h r o m a t o g r a m s were s c a n n e d w i t h a T r a c e r l a b gas-flow counter (tube TGC 14) to d e t e c t r a d i o a c t i v i t y . I n t h e p h o s p h o l i p i d c h r o m a t o g r a m s only the p h o s p h a t i d y l m e t h y l c h o l i n e was radioactive. I n a g r e e m e n t w i t h this was the a d d i t i o n a l finding t h a t t h e only r a d i o a c t i v e s p o t on p a p e r a n d t h i n - l a y e r c h r o m a t o g r a m s of t h e bases was fl-methylcholine. HC1. R a d i o a u t o g r a m s were m a d e of a c h r o m a t o g r a m of t h e h y d r o l y s a t e s o f all pooled fractions a n d also of t h e h y d r o l y s a t e of F r a c t i o n 51-76, s p o t t e d w i t h each of t h e references. Here again, only t h e spots w i t h an R F identical w i t h t h a t of fl-methylcholine were r a d i o a c t i v e . F r o m these results we m a y conclude t h a t during t h e p o s t e m b r y o n i c d e v e l o p m e n t of P. regina no carnitine is i n c o r p o r a t e d in the phospholipids. F u r t h e r m o r e , these e x p e r i m e n t s p r o v i d e definite p r o o f t h a t fl-methylcholine, i n c o r p o r a t e d in t h e phospholipids, is d e r i v e d from ~ - b u t y r o b e t a i n e fed to the larvae. The a u t h o r s are i n d e b t e d to Dr. R. W. NEWBURGH for his interest a n d suggestions t h r o u g h o u t t h e course of this s t u d y . The w o r k was s u p p o r t e d b y A r m y Chemical Center a n d U.S. Public H e a l t h Service, N a t i o n a l I n s t i t u t e s of H e a l t h ; a n d an 0 . E . C . D . P o s t g r a d u a t e T r a v e l G r a n t (N.W.H.) from t h e N e t h e r l a n d s O r g a n i z a t i o n for t h e A d v a n c e m e n t of P u r e R e s e a r c h (Z.W.0.). Science Research Institute, Oregon State University, Corvallis (Oreg.) U.S.A.
H. C. AGARWAL* N. W. H. Houx**
i E. t-IODGSON, V. H. CHELDELIN AND R. W. NEWBURGH, Arch. Biochem. Biophys., 87 (196o) 48. L. L. BIEBER, V. J. BROOKES, V. H. CHELDELIN AND R. W. NEWBURGH, Biochem. Biophys. Res. Commun., 6 (1961) 237. a L. L. BIEBER, V. H. CHELDELIN AND R. W. NEWBURGH, J. Biol. Chem., 238 (1963) 1262. 4 M. A. MEHLMANAND G. WOLF, Arch. Biochem. Biophys., lO2 (1963) 346. 5 j. j. PEIFER, Mihrochim. Acta, (1962) 529 . e G. J. M. HOOGHWlNKEL AND H. P. G. A. VAN NIEKERK, Konink!. Ned. Akad. Wetenschap. Proc., Ser. B, 63 (196o) 4757 G. A. BRAY, Anal. Biochem., i (196o) 279. s L. L. BIEBER, E. HODGSON, V. H. CHELDELIN, V. J. BROOKES AND R. W. NEWBURGH, J. Biol. Chem., 236 (1961) 2590. R e c e i v e d J a n u a r y 7th, 1964 * Present address: University of Delhi, Delhi (India). ** Present address: Laboratory of Comparative Physiology, State University, Utrecht (The Netherlands). Biochim. Biophys. Acta, 84 (1964) 353-355