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Short communication
SHORT
BBA
647
COMMUNICATIONS
53207
Fatty acid composition and biosynthesis in ferns The occurrence of arachidonic acid (cis-$3,Ir,I+eicosatetraenoic acid) in the plant kingdom, in doubt for some years due to its absence from the Angiosperms, and unsatisfactory methods used in early reports of its presence in lower plants, was positively established for several species of algae, bryophytes and pteridophytes by SCHLENK AND GELLERMAN'~~. This acid is well known in animals, where it appears to have an important physiological role, and its biosynthesis has been thoroughly studied. A dietary source of linoleic acid is essential to animals, as they are unable to desaturate oleate between the existing double bond and the methyl end of the chain (with the possible exception of some nematodes3). Starting from linoleic acid, the biosynthetic pathway in mammals has been shown4 to be via y-linolenic acid and homo-y-linolenic
18:zw (9.12)
acid :
y-18:3(6,9, 12)
y-20:3(8.11, '4)
cm:4 (5,8, 11, '4)
In rat liver6, a soil amoebae and Euglena graciW, occurs, in which chain elongation precedes desaturation: 18:2 (9,12)
20:2 (11,14)
a variant
of this pathway
y-20:3-----f 20:4 (5,8, 11, '4) (8,11, '4)
The purpose of the present work was to study the fatty acid composition of fern fronds with particular reference to polyunsaturated acids, and to attempt to elucidate the pathway of biosynthesis of arachidonate in this order of plants. In contrast to animals, all plants so far studied are able to produce the precursor linoleic acid by dehydrogenation of oleic acid 8. The production of oleic acid from stearic acid in plants has been harder to demonstrate, as higher plants appear to synthesise oleic acid by a system tightly coupled to the synthesis of long-chain saturated acids from acetateg. In the algae, it has been possible to demonstrate direct desaturation of exogenous stearate under suitable conditionslO. It was, therefore, of added interest to check whether direct desaturation of exogenous saturated fatty acids occurred in the ferns, a group at an intermediate level of complexity. I%-labelled fatty acids were obtained from the Radiochemical Centre, Amersham, England. Fronds of the male shield fern (Dryopteris filix-mas) were gathered locally. Plants of the lady fern (AthyriumJilix-foemina) and the harts-tongue fern (Scolopendrium vu&are) were purchased from Silverdale Nurseries, Carnforth, Lancashire, England, and were grown in a humidity tent in the greenhouse. Lipids were extracted by homogenising with chloroform-methanol (2: I, v/v) and fatty acid methyl esters were produced by transmethylation with methanolbenzene-cont. H&SO, (20: IO: I, v/v/v) under reflux. The fatty acid esters were identified by a combination of gas-liquid chromatography, argentation-thin-layer chromatography, hydrogenation and comparison with standards. Double bond position in 18 : 2,18 : 3 and 20 : 4 was determined by partial reduction with hydrazinell followed by isolation of the monoenes on argentation-thin-layer chromatography. Oxidation of the monoenes by the method of VON RUDLOFF'~ gave a mixture of mono- and dicarboxylic acids which could be identified by gas-liquid chromatography. Biochim.
Biophys.
Acta,
176 (1969) 647-650
SHORT COMMUNICATIONS
648
Incubations with radioactive substrates were initially in 0.2 M phosphate buffer (pH 6.0). Subsequently tap water was used as bathing medium, as this gave higher incorporation in these and other studiesIs. Gas chromatographic analyses were carried out on a Pye “104” with flameionisation detectors. Column coatings employed were ethylene glycol adipate and SE-30. Radiochemical gas-liquid chromatography was carried out in a system virtually identical to that described glycol adipate column. The fatty acid composition
by JAMES AND HITCHCOCK~~,utilizing
an ethylene
varies with age of the fern frond. Values given in
Table I are for mature fronds. The fatty acids identified in Dryopteris are shown in Table I, with C,, unsaturates comprising 48% and C,, unsaturates 8.8% of the total. The series of saturated acids from 20 : o to 28 : o had lower retention times than expected on ethylene glycol adipate columns. All the saturated acids, odd- and evennumbered alike, of more than 20 C atoms fell exactly on a straight line when logarithms of their retention times were plotted against equivalent chain length. SimiTABLE
1
FATTY ACID CoMPosITIoN OF FERN FRONDS t
(trace) = less than 0.5%.
Identity
Percentage Dryopteris
Equivalent A thyrium
Scolopendrium
t
12.00
t
14.00
0.6 t
14.50 15.00
I.5 26.9 0.8
15.55 16.00 16.40
I.4
16.55
12:o
0.8
t
Iq:O
2.5 t t I.5
0.6 0.8 t 2.1
22.0 t 0.9
19.5 1.0 2.1
0.6
1.0
0.7
17.00
4.9 t 1.6
9.9
4.9
17.80
I.2
0.6
17.45 18.00
4.6 10.4 t 32.9 t 2.8 t t 1.6
5.0 8.4 0.5 36.8
0.8 26.1
0.7
0.7
2.2 0.9 5.6
t I.4 12.2
I.5
0.7
t
I.5
‘4:
I Aat
15:o I 6 : o branched ? 16:o 16:1 r6:I d3t 16:~ 17:o 1 1613 17:1 18:o 18:1 18:2
18:3y 18:3 cc 18:4 2o:o 20:
I
20:2
20:3y 20:4 20: 4 iso20:5 21:o
3-9 0.7 2.6 t 0.9 t 2.2 t t 1.0
22:o
23:o 24:o 24:1 *5:0 26:o 28:o
B&him.
0.9
Biophys.
Acta,
176 (1969) 647-650
5.9 13.1
chain
length on ethylene glycol adipate
18.37 19.00 19.45 19.80 20.26 20.00 20.26 20.90 21.30 21.60 22.10 22.40 20.90 21.90 22.80 23.80 24.10 24.75 25.70 27.60
AgNO,-thinlayer chromatographic fraction Satd. Gtd. 43 t mono Satd. Satd. Satd. Mono d3t mono ( ::::e Triene Mono Satd. Mono Diene Triene Triene Tetra Satd.
Mono Diene Triene Tetra Tetra Penta Satd. Satd. Satd. Satd. Mono Satd. Satd. Satd.
649
SHORT COMMUNICATIONS
larly, all the saturated however,
had slightly
acids from C,, to C,, also fell on a straight different
slopes and crossed at
20: o.
line. The two lines,
A sample of the saturated
fraction was run on gas-liquid chromatographic mass spectrometer to determine whether the longer chain acids were branched. The fatty acids of equivalent chain length 20.0,21.9 and 23.8 were checked and found to be normal 20: o, 22 : o and 24: o. This phenomenon is still unexplained. The fatty acids of Athyrium and Scolopendrium are also listed in Table I, where both are seen to contain appreciable amounts of arachidonic acid. Incubations with labelled substrates were carried out on young opening fronds. [I-%]Palmitate and stearate were supplied to sliced fronds of Dryopteris. Neither acid was desaturated. Palmitate was elongated to 18 : o and 20 : o and stearate was elongated to 20: o. Thus, the acids were taken up and activated, but not dehydrogenated. Apparently the ferns resemble the higher plants rather than the algae in their handling of exogenous long-chain saturated acids. When [I-X]oleate was fed to Dryopteris, there was appreciable of radioactivity into linoleate, M- and y-linolenate, homo-y-linolenate
incorporation and arachi-
donate, in addition to some elongation to 20: I. [I-%]Linoleate was also converted to both 18:3 isomers, 20: 3 and 20:4. There was no appreciable label in 16: o or 18: o from either of these substrates, thus the conversions must have been direct, rather than by breakdown to acetate and resynthesis. These results, with labelling of ylinolenate, and no detectable labelling of 20: 2, suggest that synthesis of arachidonate in Dryopteris proceeds via the mammalian pathway. A point of interest is that while the ferns can be kept actively growing and producing new fronds throughout the winter in a greenhouse, they will not dehydrogenate exogenous oleate or linoleate to C,, unsaturated acids during the winter months. The new fronds are synthesising C,, polyunsaturates during this time, as confirmed by analysis, and will incorporate label from [2-%]acetate into homo-ylinolenate and arachidonate during the winter months. Oleate, however, is converted only to linoleate, and in low yield, while exogenous linoleate is not further desaturated. Use of different incubation media, including the sucrose-ficoll-dextran medium of HONDA et a1.l5 did not improve incorporation of long-chain acids. The most probable explanation of this seasonal effect is a change in cell permeability. Biochenaistry Division, U&lever Research Laboratory, Colworth House, Sharnbrook, Bedford (Great Britain)
W. G. HAIGH* R. SAFFORD A. T. JAMES
I J, L. GELLERMAN AND H. SCHLENK, Experientia, 20 (1964) 426. 2 H. SCHLENK AND J. L. GELLERMAN, J. Am. Oil Chemists’ Sot., 42 (1965) 504. 3 M. ROTHSTEIN AND P. GBTz, Arch. Biochem. Biophys., 126 (1968) 131. 4 J. F. MEAD AND D. HOWTON, J. Biol. Chem., zzg (1957) 575. 5 W. STOFFEL, Z. Physiol. Chem., 335 (1963) 71. 6 E. D. KORN, J. Biol. Chem., 239 (1964) 396. 7 D. MULANICKA, J. ERWIN AND K. BLOCH. J. Biol. Chem., 239 (Ig64j 2778. 8 R. V. HARRIS AND A. T. JAMES, Biochim. Biophys. Acta, 106 (1965) 456. g R. V. HARRIS, A. T. JAMES AND P. HARRIS, in T. W. GOODWIN, Biochemistry of Chloroplasts, Vol. 2, Academic Press, New York, 1967. IO R. V. HARRIS, P. HARRIS AND A. T. JAMES, Biochim. Biophys. Acta, 106 (1965) 465. II 0. S. PRIVETT AND E. C. NICKELL, Lipids, I (1966) 98. * Present address:
Division
of Biology,
National
Research
Council, Ottawa,
Biochim.
Biophys.
Acta,
Canada.
176 (1969) 647-650
650 12 13 x4 15
SHORT CO~~~U~~CATIO~S
E. VON RUDLOFF, Can. j. Chem., 34 (1956) 1413. W. G. HAIGH, L. J. MORRIS ANIJ A. T. JAMES, Lipids, 3 (1908) 307. A. T. JAMES AND C. HITCHCOCK, Kerntechnik, 7 (1965) 5. S. I. HONDA, T. HONGLADAROM AND G. G. LATIES, J.ExptE.Botan., 17(rg66) 460.
Received November rzth, 1968 Biockim. Biaphys. Acta, 176 (1969) 647-650
m3A 53210 Intermediary metabolism of phospholipids. The biosynthesis of phosphatidylglycerophosphate and phosphatidylglycerol in heart mitochondria Phosphatidylg~ycerophosphate is now known to be the precursor for the biosynthesis of phosphatidy~glycerol in liver r, brain2 and in ~sc~e~~c~~~co@; it is also known that phosphatidylglycerol is the precursor for the biosynthesis of cardiolipin in bacteria4. The biosynthesis of cardiolipin in animal tissue, however, is not as yet understood, although the occurrence, distribution and importance of this polyglycerophosphatide had been studied extensively in the past&. A recent report6 on the quantitative distribution of cardiolipin in the mitochondria of various tissues, has indicated that cardiolipin represents approx. 200; of the total phospholipids }:I esent in heart mitochondria. As a result of our efforts to establish the pathway(s) for the biosynthesis of cardiolipin in animal tissue, we wish now to report the biosynthesis in heart mitochondria of phosphatid~7lglycerophosphate and phosphatidylglycerol, two precursors very likely involved in the formation of cardiolipin. The capability of an extract obtained from rat heart to catalyze the conversion of L-a-glycerophosphate to lipid in the presence of CDP-diglyceride was briefly reported’, but the nature of biosynthesized lipid(s) was not examined in detail. A freshly prepared rat heart mitochondrial fraction’ was incubated with L-a-[z-SHjglycerophosphate (prepared as described in ref. 3), in the presence of TABLE
I
SYNTHESIS OF LABELLED
LIPIIE FROW ~-~-~Z-~H]GLYCEROPHOSP~~A~E AND
CDP-D-DIGLYCERIDE
The complete system contained 50 pmoles of Tris buffer (pH 7.4), z pmoles of MgCl,, 0.15 pmole of L-e-[z-SH]glycerophosphate (specific activity, 1.44. IO? disint./min per pmole), 0.50 #$,rnoleof CUP-o-diglyceride, 4 mg of Triton x-100 and 0.10 ml of rat heart mitochondria’ (0.93 mg of protein), in a final volume of 0.50 ml, The reaction mixture was incubated for IZO min at 37’. The reported radioactivity in the lipid phases represents an average of duplicates. ____“.._ Lipids formedlmg protein Reaction mixture -~ Disint. /min nmoles -. I. Complete system 57036 3.79 6r 856 4.12 2. Add IO mmoles of glycerol 0.16 3. Omit CDP-o-diglyceride 2451 0.15 4. Zero time control 2 303 Biochim. Biophys.
Acta, 176 (x969) 650-653
BBA
647
COMMUNICATIONS
53207
Fatty acid composition and biosynthesis in ferns The occurrence of arachidonic acid (cis-$3,Ir,I+eicosatetraenoic acid) in the plant kingdom, in doubt for some years due to its absence from the Angiosperms, and unsatisfactory methods used in early reports of its presence in lower plants, was positively established for several species of algae, bryophytes and pteridophytes by SCHLENK AND GELLERMAN'~~. This acid is well known in animals, where it appears to have an important physiological role, and its biosynthesis has been thoroughly studied. A dietary source of linoleic acid is essential to animals, as they are unable to desaturate oleate between the existing double bond and the methyl end of the chain (with the possible exception of some nematodes3). Starting from linoleic acid, the biosynthetic pathway in mammals has been shown4 to be via y-linolenic acid and homo-y-linolenic
18:zw (9.12)
acid :
y-18:3(6,9, 12)
y-20:3(8.11, '4)
cm:4 (5,8, 11, '4)
In rat liver6, a soil amoebae and Euglena graciW, occurs, in which chain elongation precedes desaturation: 18:2 (9,12)
20:2 (11,14)
a variant
of this pathway
y-20:3-----f 20:4 (5,8, 11, '4) (8,11, '4)
The purpose of the present work was to study the fatty acid composition of fern fronds with particular reference to polyunsaturated acids, and to attempt to elucidate the pathway of biosynthesis of arachidonate in this order of plants. In contrast to animals, all plants so far studied are able to produce the precursor linoleic acid by dehydrogenation of oleic acid 8. The production of oleic acid from stearic acid in plants has been harder to demonstrate, as higher plants appear to synthesise oleic acid by a system tightly coupled to the synthesis of long-chain saturated acids from acetateg. In the algae, it has been possible to demonstrate direct desaturation of exogenous stearate under suitable conditionslO. It was, therefore, of added interest to check whether direct desaturation of exogenous saturated fatty acids occurred in the ferns, a group at an intermediate level of complexity. I%-labelled fatty acids were obtained from the Radiochemical Centre, Amersham, England. Fronds of the male shield fern (Dryopteris filix-mas) were gathered locally. Plants of the lady fern (AthyriumJilix-foemina) and the harts-tongue fern (Scolopendrium vu&are) were purchased from Silverdale Nurseries, Carnforth, Lancashire, England, and were grown in a humidity tent in the greenhouse. Lipids were extracted by homogenising with chloroform-methanol (2: I, v/v) and fatty acid methyl esters were produced by transmethylation with methanolbenzene-cont. H&SO, (20: IO: I, v/v/v) under reflux. The fatty acid esters were identified by a combination of gas-liquid chromatography, argentation-thin-layer chromatography, hydrogenation and comparison with standards. Double bond position in 18 : 2,18 : 3 and 20 : 4 was determined by partial reduction with hydrazinell followed by isolation of the monoenes on argentation-thin-layer chromatography. Oxidation of the monoenes by the method of VON RUDLOFF'~ gave a mixture of mono- and dicarboxylic acids which could be identified by gas-liquid chromatography. Biochim.
Biophys.
Acta,
176 (1969) 647-650
SHORT COMMUNICATIONS
648
Incubations with radioactive substrates were initially in 0.2 M phosphate buffer (pH 6.0). Subsequently tap water was used as bathing medium, as this gave higher incorporation in these and other studiesIs. Gas chromatographic analyses were carried out on a Pye “104” with flameionisation detectors. Column coatings employed were ethylene glycol adipate and SE-30. Radiochemical gas-liquid chromatography was carried out in a system virtually identical to that described glycol adipate column. The fatty acid composition
by JAMES AND HITCHCOCK~~,utilizing
an ethylene
varies with age of the fern frond. Values given in
Table I are for mature fronds. The fatty acids identified in Dryopteris are shown in Table I, with C,, unsaturates comprising 48% and C,, unsaturates 8.8% of the total. The series of saturated acids from 20 : o to 28 : o had lower retention times than expected on ethylene glycol adipate columns. All the saturated acids, odd- and evennumbered alike, of more than 20 C atoms fell exactly on a straight line when logarithms of their retention times were plotted against equivalent chain length. SimiTABLE
1
FATTY ACID CoMPosITIoN OF FERN FRONDS t
(trace) = less than 0.5%.
Identity
Percentage Dryopteris
Equivalent A thyrium
Scolopendrium
t
12.00
t
14.00
0.6 t
14.50 15.00
I.5 26.9 0.8
15.55 16.00 16.40
I.4
16.55
12:o
0.8
t
Iq:O
2.5 t t I.5
0.6 0.8 t 2.1
22.0 t 0.9
19.5 1.0 2.1
0.6
1.0
0.7
17.00
4.9 t 1.6
9.9
4.9
17.80
I.2
0.6
17.45 18.00
4.6 10.4 t 32.9 t 2.8 t t 1.6
5.0 8.4 0.5 36.8
0.8 26.1
0.7
0.7
2.2 0.9 5.6
t I.4 12.2
I.5
0.7
t
I.5
‘4:
I Aat
15:o I 6 : o branched ? 16:o 16:1 r6:I d3t 16:~ 17:o 1 1613 17:1 18:o 18:1 18:2
18:3y 18:3 cc 18:4 2o:o 20:
I
20:2
20:3y 20:4 20: 4 iso20:5 21:o
3-9 0.7 2.6 t 0.9 t 2.2 t t 1.0
22:o
23:o 24:o 24:1 *5:0 26:o 28:o
B&him.
0.9
Biophys.
Acta,
176 (1969) 647-650
5.9 13.1
chain
length on ethylene glycol adipate
18.37 19.00 19.45 19.80 20.26 20.00 20.26 20.90 21.30 21.60 22.10 22.40 20.90 21.90 22.80 23.80 24.10 24.75 25.70 27.60
AgNO,-thinlayer chromatographic fraction Satd. Gtd. 43 t mono Satd. Satd. Satd. Mono d3t mono ( ::::e Triene Mono Satd. Mono Diene Triene Triene Tetra Satd.
Mono Diene Triene Tetra Tetra Penta Satd. Satd. Satd. Satd. Mono Satd. Satd. Satd.
649
SHORT COMMUNICATIONS
larly, all the saturated however,
had slightly
acids from C,, to C,, also fell on a straight different
slopes and crossed at
20: o.
line. The two lines,
A sample of the saturated
fraction was run on gas-liquid chromatographic mass spectrometer to determine whether the longer chain acids were branched. The fatty acids of equivalent chain length 20.0,21.9 and 23.8 were checked and found to be normal 20: o, 22 : o and 24: o. This phenomenon is still unexplained. The fatty acids of Athyrium and Scolopendrium are also listed in Table I, where both are seen to contain appreciable amounts of arachidonic acid. Incubations with labelled substrates were carried out on young opening fronds. [I-%]Palmitate and stearate were supplied to sliced fronds of Dryopteris. Neither acid was desaturated. Palmitate was elongated to 18 : o and 20 : o and stearate was elongated to 20: o. Thus, the acids were taken up and activated, but not dehydrogenated. Apparently the ferns resemble the higher plants rather than the algae in their handling of exogenous long-chain saturated acids. When [I-X]oleate was fed to Dryopteris, there was appreciable of radioactivity into linoleate, M- and y-linolenate, homo-y-linolenate
incorporation and arachi-
donate, in addition to some elongation to 20: I. [I-%]Linoleate was also converted to both 18:3 isomers, 20: 3 and 20:4. There was no appreciable label in 16: o or 18: o from either of these substrates, thus the conversions must have been direct, rather than by breakdown to acetate and resynthesis. These results, with labelling of ylinolenate, and no detectable labelling of 20: 2, suggest that synthesis of arachidonate in Dryopteris proceeds via the mammalian pathway. A point of interest is that while the ferns can be kept actively growing and producing new fronds throughout the winter in a greenhouse, they will not dehydrogenate exogenous oleate or linoleate to C,, unsaturated acids during the winter months. The new fronds are synthesising C,, polyunsaturates during this time, as confirmed by analysis, and will incorporate label from [2-%]acetate into homo-ylinolenate and arachidonate during the winter months. Oleate, however, is converted only to linoleate, and in low yield, while exogenous linoleate is not further desaturated. Use of different incubation media, including the sucrose-ficoll-dextran medium of HONDA et a1.l5 did not improve incorporation of long-chain acids. The most probable explanation of this seasonal effect is a change in cell permeability. Biochenaistry Division, U&lever Research Laboratory, Colworth House, Sharnbrook, Bedford (Great Britain)
W. G. HAIGH* R. SAFFORD A. T. JAMES
I J, L. GELLERMAN AND H. SCHLENK, Experientia, 20 (1964) 426. 2 H. SCHLENK AND J. L. GELLERMAN, J. Am. Oil Chemists’ Sot., 42 (1965) 504. 3 M. ROTHSTEIN AND P. GBTz, Arch. Biochem. Biophys., 126 (1968) 131. 4 J. F. MEAD AND D. HOWTON, J. Biol. Chem., zzg (1957) 575. 5 W. STOFFEL, Z. Physiol. Chem., 335 (1963) 71. 6 E. D. KORN, J. Biol. Chem., 239 (1964) 396. 7 D. MULANICKA, J. ERWIN AND K. BLOCH. J. Biol. Chem., 239 (Ig64j 2778. 8 R. V. HARRIS AND A. T. JAMES, Biochim. Biophys. Acta, 106 (1965) 456. g R. V. HARRIS, A. T. JAMES AND P. HARRIS, in T. W. GOODWIN, Biochemistry of Chloroplasts, Vol. 2, Academic Press, New York, 1967. IO R. V. HARRIS, P. HARRIS AND A. T. JAMES, Biochim. Biophys. Acta, 106 (1965) 465. II 0. S. PRIVETT AND E. C. NICKELL, Lipids, I (1966) 98. * Present address:
Division
of Biology,
National
Research
Council, Ottawa,
Biochim.
Biophys.
Acta,
Canada.
176 (1969) 647-650
650 12 13 x4 15
SHORT CO~~~U~~CATIO~S
E. VON RUDLOFF, Can. j. Chem., 34 (1956) 1413. W. G. HAIGH, L. J. MORRIS ANIJ A. T. JAMES, Lipids, 3 (1908) 307. A. T. JAMES AND C. HITCHCOCK, Kerntechnik, 7 (1965) 5. S. I. HONDA, T. HONGLADAROM AND G. G. LATIES, J.ExptE.Botan., 17(rg66) 460.
Received November rzth, 1968 Biockim. Biaphys. Acta, 176 (1969) 647-650
m3A 53210 Intermediary metabolism of phospholipids. The biosynthesis of phosphatidylglycerophosphate and phosphatidylglycerol in heart mitochondria Phosphatidylg~ycerophosphate is now known to be the precursor for the biosynthesis of phosphatidy~glycerol in liver r, brain2 and in ~sc~e~~c~~~co@; it is also known that phosphatidylglycerol is the precursor for the biosynthesis of cardiolipin in bacteria4. The biosynthesis of cardiolipin in animal tissue, however, is not as yet understood, although the occurrence, distribution and importance of this polyglycerophosphatide had been studied extensively in the past&. A recent report6 on the quantitative distribution of cardiolipin in the mitochondria of various tissues, has indicated that cardiolipin represents approx. 200; of the total phospholipids }:I esent in heart mitochondria. As a result of our efforts to establish the pathway(s) for the biosynthesis of cardiolipin in animal tissue, we wish now to report the biosynthesis in heart mitochondria of phosphatid~7lglycerophosphate and phosphatidylglycerol, two precursors very likely involved in the formation of cardiolipin. The capability of an extract obtained from rat heart to catalyze the conversion of L-a-glycerophosphate to lipid in the presence of CDP-diglyceride was briefly reported’, but the nature of biosynthesized lipid(s) was not examined in detail. A freshly prepared rat heart mitochondrial fraction’ was incubated with L-a-[z-SHjglycerophosphate (prepared as described in ref. 3), in the presence of TABLE
I
SYNTHESIS OF LABELLED
LIPIIE FROW ~-~-~Z-~H]GLYCEROPHOSP~~A~E AND
CDP-D-DIGLYCERIDE
The complete system contained 50 pmoles of Tris buffer (pH 7.4), z pmoles of MgCl,, 0.15 pmole of L-e-[z-SH]glycerophosphate (specific activity, 1.44. IO? disint./min per pmole), 0.50 #$,rnoleof CUP-o-diglyceride, 4 mg of Triton x-100 and 0.10 ml of rat heart mitochondria’ (0.93 mg of protein), in a final volume of 0.50 ml, The reaction mixture was incubated for IZO min at 37’. The reported radioactivity in the lipid phases represents an average of duplicates. ____“.._ Lipids formedlmg protein Reaction mixture -~ Disint. /min nmoles -. I. Complete system 57036 3.79 6r 856 4.12 2. Add IO mmoles of glycerol 0.16 3. Omit CDP-o-diglyceride 2451 0.15 4. Zero time control 2 303 Biochim. Biophys.
Acta, 176 (x969) 650-653