- Email: [email protected]
Separation and quantitative determination of 32P-labelled lipids from brain particulates by thin-layer chromatography
NOTES
as starting material generally moved to the solvent front, while the unsaturated alcohols, being more polar, had lower RI;I values. Apart from providing useful TLC separation procedures, these results also demonstrate that addition of mercaptoacetic acid to nonoene fatty acids, esters and alcohols yields both isomers, with the exception of undecenoic acid which gives rise to a single product probably the II-carboxymethylthioundecanoic acid. Regional Research Laboralory, Hyderabad-g (Ivadia)
R. KANNAN M. R. SUBBARAM. I<. T. ACHAYA
AND D. SWXRN, J. Am. Cltem. Sot., 79 (1957) 362. AND L. A. GOLDBLATT, Jo Am. Oil CIwnisls’ Sec., 35 (Ig3S) 225. 3 L. R. ESHELMAN, E. Y. M;\Nzo, S. J. MARCUS, A.E. DECOTEAU AND E. G. HAMMOND, Anal.
1 N. W. KOENIG
2 S. I?. FORE, R. T. O'CONNOR Chem.,
32 (1960)
844.
M. R. SUBBARAM, M. W. ROOMI AND I<.T. ACIIAYA, J. Chvomalog., 5 R. SUBBARAO, M. W. ROOMI, M. R. SUBBARAM AND I<.T. ACHAYA, G M. W. ROOMI, M. R. SUBBARAM AND I<.T. ACHAYA, J. Chvomatog., 4
Received
April
Izth,
21 J.
(1966)
16 (1964)
particulates
IOG.
g (xgG2) 295.
1966 J. CIwomalog,,
Separation
324.
Chromalog.,
and quantitative by thin-layer
determination
24 (IgGG)
of 32P-labelled lipids from
433-435
brain
chromatography
The separation and isolation of phospholipids by thin-layer chromatography (TLC) on silica gel has been reported by several investigatorsl-6. Although a distinct separation of some phospholipids was achieved by these techniques, they yield separations with overlap among such ph.ospholipids as phosphatidic acid and neutral lipids or phosphatidyl inositol and phosphatidyl serine. During our work on the effect of neurohormones and metabolic inhibitors of phosphate metabolism in nerve-ending particulates of rat brain, it was found that phospholipids were highly labelled when incubated in an oxidative phosphorylation medium containing 32P-orthophosphateO. By incorporating certain features of previously described techniquesls4, nerve endings or mitochondrial 32P-labelled phospholipids, including the highly labelled phosphatidic acid and phosphatidyl inositol, were effectively separated on Silica Gel G by means of two-dimensional TLC, and the radioactive specific activities of the individual phospholipids were determined.
Li$id extracts. Total lipids were extracted from rat brain nerve-ending particles or purified mitochondria with chloroform-methanol (z : I) after incubation in an oxidative phosphorylation medium containing IOO-z-50 PC of a2P-orthophosphate for I h at 37 Oas described previously7. The extract was filtered, concentrated irt VUCZ~O J.
Chvovn~alog,, 24 (1966)
435-439
’
436
NOTES
and the lipid concentrate taken up in chloroform. Solutions of lipid in chloroform were adjusted so that I ml solution contamed 27 ,~rnoles of lipicl phosphorus. Phospholipid standards were products of Pierce Chemical Company, Rockford, Ill. Prc$aration of chro??zatogra#ic @ztes alacE solve&s. To 40 g of Silica Gel G (Brinkmann Instruments), 80 ml of deionized water were added, and the resultant slurry was mixed with constant stirring for 4 min. Five glass plates, 20 x 20 cm, were coated with this slurry to a depth of 0.25 mm using the Desaga adjustable applicator. The plates were air-dried and activated for I h at IIOO just before use. Two different solvent systems were used for the development of the twodimensional thin-layer chromatograms : Solvent A : chloroform-methanol-acetic acid-water (25 : 15 : 4 : 2)l. Solvent,B : butanol-pyridine-water (45 : 5 :zo)~. Sjmtting and dsvelo~mcnt. For one-dimensional thin-layer chromatograms, 40&o ,ul of lipid extract were applied to the TLC plates with micropipettes, and allowed to develop in equilibrated jars containing Solvent A. The solvent was allowed to rise within 2 cm of the top of the adsorbent, Average running time was 1.1 h. The plates were removed from the chromatography j ars, dried for 30 min and placed in Solvent B after clockwise rotation through go”. Average running time was 3 11. Detection of spots. Lipids were detected on dried chromatograms in the following manner: The plates were exposed to iodine vapors and the spots were immediately outlined with the point of aneedle. For the detection of phosphatidyl serine, phosphatidyl ethanolamine, andplasmalogens, the plates were sprayed with ninhydrin. Phosphatidic acid was identified, after elution from the thin-layer chromatogram, by deacylation according to the procedure of DAWSON*. The radioactive product and standard a-glycerophosphate were run in paper electrophoresis at pE1 10.8 as described previously’. Other phospholipids of nerve-ending particles or purified mitochondria were identified by chromatographing standard phospholipids and comparison with previously published data using the same solvent systeml. For the detection of radioactive lipids the chromatoplates were wrapped in Saferan film, and then exposecl to Kodak No-Screen X-ray film for 3 days. phospholipid spots Analysis of the 32P-ZabeZZed$hos$hoZi$Gds. The radioactive cletected by autoradiography, and the non-radioactive lipids detected by iodine vapor, were scraped off with a razor blade and the radioactivity was measured with a gasflow counter and scaler. After counting, the silica gel from each planchette was poured into test tubes and 0.25 ml of concentrated sulfuric acid was added to all tubes including blank silica gel, reagent blanks and inorganic phosphate standards. The tubes were placed in a heating block and digested according to the procedure of PARICERAND
PETERS~N~~.
Phospholipid phosphorus was determined by a modification of the methods of MINGLE and BARTLETT~~. To all. the tubes, 4.05 ml of water, 0.5 ml of 5 O/~ammonium molybdate solution and 0.2 ml of I-amino-2-naphthol-4-sulfonic acid reagent were added. The latter reagent was prepared according to the method of KINGLY. After heating in a boiling water bath for IO min, the tubes containing silica gel were centrifuged at 300 x g for 40 min. The color was read at wavelength of 620 rnp (see Fig, 2) on a Beckman Model B Spectrophotometer using glass cells with a I cm light path. J. Cltvomalo~.,
24
(IgGG)
435-439
NOTES
437
Resz6lls
amI
discz6ssion
The resolution of mixtures of “2l?-labclled phospholipids, extracted either from brain mitochondria or nerve-ending particles was found to be incomplete after running in one-dimensional TLC (Fig. I A). However these mixtures were separated into ‘16 components by two-dimensional TLC (Fig. I B), and furthermore a clear __...
,,,.,_
,,.._..I
“.
.,.
:
;
I ; I
Fig. I. An autoradiograph showing the separation of rat brain mitochondrial lipids by means one-clinwnsional TLC (A) and two-climensionnl TLC (B). Compouncls (clotted lines) 8, 12, 14 and were cletcctecl with iodine. Numbers refer to those given in Table I.
of 16
separation of phosphatidyl inositol and phosphaticlic acid was achieved (Fig. I B, Compounds II and 15 respectively). Since these separations were clear and highly reproducible, the present method could be empl.oyed in studies on the turnover of the individual phospholipids in brain particulates (Table I).
)‘!a
I
OS50
650
750 Wavelength
(mp)
850
of color given by phosphorus assay Fig. 2. Spectra -/- IOO mg Silica Gel G: (2) 0~2 ktmole phosphorus;
procedure (see text). (I) 0.2 ,xmole (3) xoo mg Silica Gel G. J.
Cl~ovrzdog.,
phosphorus
24 (1966)
435-439
OF RADIOACTIVE
CHROMATOGRAPHY
16
13 ‘4 15
I2
II
IO
z 9
a
3
2
I
76.2
-
-! 14.0 16.0 23.0 33.4 24-o 43-I
B
0
I.974 5.370 67 302 87 359 123 2,000 1,974 1,026 13s 548 IO 7.360
4.460~
distance of center of spot from starting-point ’ distance of solvent from starting-point
96.1
-
14.5
71-j
” 17.8 31.2 48.0 59-o 65.2 77-o
A
6.3 6.3 14-7 28.6 17.5 39 31.5 20.3 35 36 24.4 54-S 44
0
B
39 I4
E?o)
0
35: 43
454 140 (210)
(75) (1.9S5) (0)
110
3: 71 29.2 14 18.7
14
(43) (216)
AFTER SEPARATION
(20)
(400) (21470) (29) (1%) (31)
(9001d
PHOSPHOLIPIDS
0
0.03 172
-
4 .2 14.1 73.I 3-j
II.2
19.2
10.4 6.2
o-95
I49
319 -
(254-3) (o-33)
I-1
BY TWO-DIXIENSIONI\L
b
RF values of standard lipids. CMitochondrial lipids. d Xerve-ending lipids. e Was not determined. f There was no detectable phosphorus in this spot. The radioactivity could have been due to contamination from the 32P-orthophosphate.
a Rp values of mitochondrial lipids; RF =
34-5 16.4 17.0 34.1 27.8 39.2 50.0 50-5 61.2 63.2 75 7s go.5 97-j
g-2
0
A
AND NERVE-ENDING
Rp x IOO it& solventsb
OF IIITOCHONDRIAL
RF x ror,itz solve&a
SPECIFIC ACTIVITIES
Proteolipids + 32P f ganglioside 32P ? Unknown Lysolecithin Unknown Sphingomyelin Unknown Plasmalogen ? Lecithin Unknown Phosphatidyl inositol Phosphatidyl serine Unknown Phosphatidyl ethanolamine Phosphatidic acid Cerebroside
Spot No. Cosrapowad (Fig. IB)
THIN-LAYER
DETERIIINATION
TABLE I
NOTES
439
As can be seen from Table I, it has not been possible to identify all the lipid components on the thin-layer chromatogram. Compounds 3, 5 and 13 did not stain with iodine but contained a considerable amount of activity. Compounds 3 and 5 could be the deacylated derivative of phosphatidic acid (lysophosphatidic acid) and the phosphorylated derivative of phosphatidyl inositol (diphosphoinositide) respectively, In agreement with the work of other investigatorsl3sla phosphatidic acid and phosphatidyl inositol were found to be highly labelled in both nerve-ending particles and mitochondria (Table I). On the other hand structural lipids such as phosphatidyl serine and phosphatidyl ethanolamine had little activity. Unlike Silica Gel I-PO,washed Silica Gel G gave poor resolution of phospholipids. This resulted in higher readings of the Silica Gel G blanks (Fig. 2). However, the blank readings could not have been due to the phosphorus-molybdate complex as can be seen from the silica gel spectrum (Fig. 2). The modification which was made in the developing reagents resulted in a shift of the spectral peak from a maximum at 830 rnp12 to a maximum at 620 rnp (Fig. 2). The separation and determination of the radioactive specific activities of lipids reported in this communication could be applied to the whole brain as well as particulates from other tissues. Achaowledgements This work was supported by the State of Illinois Mental Wealth Fund and the U.S. Public EIealth Service Research Grant NB 06628-01. The authors wish to thank Mrs. MARILYN MAYFIELD for technical assistance and Mr. JOHN DULANEY for suggesting the Skipski system to us. ATA A. ABDEL-LATIP FLORA E. CHANG
Illigzois State Pediatric Imtitzcte, and D&artment of Psychiatry, U?ziversii!y of Ih?inois Cohh.Jgeof ~edicim, Chicago,
Ill. (U.S.A.)
I V. P. SICIPSKI.R. I?.PETERSON
AND M. BARCLAS, Biochem.
J.,
QO (19G4)
z W. D, SKIDMORE AND C. ENTENMAN, J. Lipid Res., 3 (~1962)471. 3 s. N. PAYNE,J. ChYomatO~., I.5(1964) 173. 4 L. G. ABOOD, 1. KOYAMA ANIJ V. THOMAS. Am. J. Pltysiol., 207 (1964) 5 C. W. REDMAN AND R. W. KIZNAN,JC&O~~~O~., 15 (1~64) 180. 6 A. A. ABDIX-LATIP, Nature, 211 (~QGG) 1530. 7 A, A. ABIXZL-LATIP AND L. G. ABOOD, J0 Nezwocl~em., 12 (196.5)157.
374.
1435.
A. SIMS AND J, A. G. LAROSE,J.A~. OiEClaemisls’ So&, 39 (1962) 232. Q R. M. C. DATVSON, Biocltem. J., 75 (1960) 45. IO I?. FARIUZR AND N. I?. PETERSON,J. LLipidRes.,6 (1965) 455. IL E. J. KING, Biochem.J., 2G (1932)292. 12 G. R. BARTLETT, J.Riol. CJccm.,234 (19.59)466. 13 J, GARBUS, W. I?.DELTJCA,M.E. LOOMANS AND F. M. STRONG,J. Viol. Chem., 14 J. DURELL AND M. A. SODD.J. Biol. Ckcm., 239 (1964) 747.; , 8 R. I?.
Received
238
(1963)59.
April r5th, 1966 J.
Clwomatog.,
24 (x96(5)
435-439
as starting material generally moved to the solvent front, while the unsaturated alcohols, being more polar, had lower RI;I values. Apart from providing useful TLC separation procedures, these results also demonstrate that addition of mercaptoacetic acid to nonoene fatty acids, esters and alcohols yields both isomers, with the exception of undecenoic acid which gives rise to a single product probably the II-carboxymethylthioundecanoic acid. Regional Research Laboralory, Hyderabad-g (Ivadia)
R. KANNAN M. R. SUBBARAM. I<. T. ACHAYA
AND D. SWXRN, J. Am. Cltem. Sot., 79 (1957) 362. AND L. A. GOLDBLATT, Jo Am. Oil CIwnisls’ Sec., 35 (Ig3S) 225. 3 L. R. ESHELMAN, E. Y. M;\Nzo, S. J. MARCUS, A.E. DECOTEAU AND E. G. HAMMOND, Anal.
1 N. W. KOENIG
2 S. I?. FORE, R. T. O'CONNOR Chem.,
32 (1960)
844.
M. R. SUBBARAM, M. W. ROOMI AND I<.T. ACIIAYA, J. Chvomalog., 5 R. SUBBARAO, M. W. ROOMI, M. R. SUBBARAM AND I<.T. ACHAYA, G M. W. ROOMI, M. R. SUBBARAM AND I<.T. ACHAYA, J. Chvomatog., 4
Received
April
Izth,
21 J.
(1966)
16 (1964)
particulates
IOG.
g (xgG2) 295.
1966 J. CIwomalog,,
Separation
324.
Chromalog.,
and quantitative by thin-layer
determination
24 (IgGG)
of 32P-labelled lipids from
433-435
brain
chromatography
The separation and isolation of phospholipids by thin-layer chromatography (TLC) on silica gel has been reported by several investigatorsl-6. Although a distinct separation of some phospholipids was achieved by these techniques, they yield separations with overlap among such ph.ospholipids as phosphatidic acid and neutral lipids or phosphatidyl inositol and phosphatidyl serine. During our work on the effect of neurohormones and metabolic inhibitors of phosphate metabolism in nerve-ending particulates of rat brain, it was found that phospholipids were highly labelled when incubated in an oxidative phosphorylation medium containing 32P-orthophosphateO. By incorporating certain features of previously described techniquesls4, nerve endings or mitochondrial 32P-labelled phospholipids, including the highly labelled phosphatidic acid and phosphatidyl inositol, were effectively separated on Silica Gel G by means of two-dimensional TLC, and the radioactive specific activities of the individual phospholipids were determined.
Li$id extracts. Total lipids were extracted from rat brain nerve-ending particles or purified mitochondria with chloroform-methanol (z : I) after incubation in an oxidative phosphorylation medium containing IOO-z-50 PC of a2P-orthophosphate for I h at 37 Oas described previously7. The extract was filtered, concentrated irt VUCZ~O J.
Chvovn~alog,, 24 (1966)
435-439
’
436
NOTES
and the lipid concentrate taken up in chloroform. Solutions of lipid in chloroform were adjusted so that I ml solution contamed 27 ,~rnoles of lipicl phosphorus. Phospholipid standards were products of Pierce Chemical Company, Rockford, Ill. Prc$aration of chro??zatogra#ic @ztes alacE solve&s. To 40 g of Silica Gel G (Brinkmann Instruments), 80 ml of deionized water were added, and the resultant slurry was mixed with constant stirring for 4 min. Five glass plates, 20 x 20 cm, were coated with this slurry to a depth of 0.25 mm using the Desaga adjustable applicator. The plates were air-dried and activated for I h at IIOO just before use. Two different solvent systems were used for the development of the twodimensional thin-layer chromatograms : Solvent A : chloroform-methanol-acetic acid-water (25 : 15 : 4 : 2)l. Solvent,B : butanol-pyridine-water (45 : 5 :zo)~. Sjmtting and dsvelo~mcnt. For one-dimensional thin-layer chromatograms, 40&o ,ul of lipid extract were applied to the TLC plates with micropipettes, and allowed to develop in equilibrated jars containing Solvent A. The solvent was allowed to rise within 2 cm of the top of the adsorbent, Average running time was 1.1 h. The plates were removed from the chromatography j ars, dried for 30 min and placed in Solvent B after clockwise rotation through go”. Average running time was 3 11. Detection of spots. Lipids were detected on dried chromatograms in the following manner: The plates were exposed to iodine vapors and the spots were immediately outlined with the point of aneedle. For the detection of phosphatidyl serine, phosphatidyl ethanolamine, andplasmalogens, the plates were sprayed with ninhydrin. Phosphatidic acid was identified, after elution from the thin-layer chromatogram, by deacylation according to the procedure of DAWSON*. The radioactive product and standard a-glycerophosphate were run in paper electrophoresis at pE1 10.8 as described previously’. Other phospholipids of nerve-ending particles or purified mitochondria were identified by chromatographing standard phospholipids and comparison with previously published data using the same solvent systeml. For the detection of radioactive lipids the chromatoplates were wrapped in Saferan film, and then exposecl to Kodak No-Screen X-ray film for 3 days. phospholipid spots Analysis of the 32P-ZabeZZed$hos$hoZi$Gds. The radioactive cletected by autoradiography, and the non-radioactive lipids detected by iodine vapor, were scraped off with a razor blade and the radioactivity was measured with a gasflow counter and scaler. After counting, the silica gel from each planchette was poured into test tubes and 0.25 ml of concentrated sulfuric acid was added to all tubes including blank silica gel, reagent blanks and inorganic phosphate standards. The tubes were placed in a heating block and digested according to the procedure of PARICERAND
PETERS~N~~.
Phospholipid phosphorus was determined by a modification of the methods of MINGLE and BARTLETT~~. To all. the tubes, 4.05 ml of water, 0.5 ml of 5 O/~ammonium molybdate solution and 0.2 ml of I-amino-2-naphthol-4-sulfonic acid reagent were added. The latter reagent was prepared according to the method of KINGLY. After heating in a boiling water bath for IO min, the tubes containing silica gel were centrifuged at 300 x g for 40 min. The color was read at wavelength of 620 rnp (see Fig, 2) on a Beckman Model B Spectrophotometer using glass cells with a I cm light path. J. Cltvomalo~.,
24
(IgGG)
435-439
NOTES
437
Resz6lls
amI
discz6ssion
The resolution of mixtures of “2l?-labclled phospholipids, extracted either from brain mitochondria or nerve-ending particles was found to be incomplete after running in one-dimensional TLC (Fig. I A). However these mixtures were separated into ‘16 components by two-dimensional TLC (Fig. I B), and furthermore a clear __...
,,,.,_
,,.._..I
“.
.,.
:
;
I ; I
Fig. I. An autoradiograph showing the separation of rat brain mitochondrial lipids by means one-clinwnsional TLC (A) and two-climensionnl TLC (B). Compouncls (clotted lines) 8, 12, 14 and were cletcctecl with iodine. Numbers refer to those given in Table I.
of 16
separation of phosphatidyl inositol and phosphaticlic acid was achieved (Fig. I B, Compounds II and 15 respectively). Since these separations were clear and highly reproducible, the present method could be empl.oyed in studies on the turnover of the individual phospholipids in brain particulates (Table I).
)‘!a
I
OS50
650
750 Wavelength
(mp)
850
of color given by phosphorus assay Fig. 2. Spectra -/- IOO mg Silica Gel G: (2) 0~2 ktmole phosphorus;
procedure (see text). (I) 0.2 ,xmole (3) xoo mg Silica Gel G. J.
Cl~ovrzdog.,
phosphorus
24 (1966)
435-439
OF RADIOACTIVE
CHROMATOGRAPHY
16
13 ‘4 15
I2
II
IO
z 9
a
3
2
I
76.2
-
-! 14.0 16.0 23.0 33.4 24-o 43-I
B
0
I.974 5.370 67 302 87 359 123 2,000 1,974 1,026 13s 548 IO 7.360
4.460~
distance of center of spot from starting-point ’ distance of solvent from starting-point
96.1
-
14.5
71-j
” 17.8 31.2 48.0 59-o 65.2 77-o
A
6.3 6.3 14-7 28.6 17.5 39 31.5 20.3 35 36 24.4 54-S 44
0
B
39 I4
E?o)
0
35: 43
454 140 (210)
(75) (1.9S5) (0)
110
3: 71 29.2 14 18.7
14
(43) (216)
AFTER SEPARATION
(20)
(400) (21470) (29) (1%) (31)
(9001d
PHOSPHOLIPIDS
0
0.03 172
-
4 .2 14.1 73.I 3-j
II.2
19.2
10.4 6.2
o-95
I49
319 -
(254-3) (o-33)
I-1
BY TWO-DIXIENSIONI\L
b
RF values of standard lipids. CMitochondrial lipids. d Xerve-ending lipids. e Was not determined. f There was no detectable phosphorus in this spot. The radioactivity could have been due to contamination from the 32P-orthophosphate.
a Rp values of mitochondrial lipids; RF =
34-5 16.4 17.0 34.1 27.8 39.2 50.0 50-5 61.2 63.2 75 7s go.5 97-j
g-2
0
A
AND NERVE-ENDING
Rp x IOO it& solventsb
OF IIITOCHONDRIAL
RF x ror,itz solve&a
SPECIFIC ACTIVITIES
Proteolipids + 32P f ganglioside 32P ? Unknown Lysolecithin Unknown Sphingomyelin Unknown Plasmalogen ? Lecithin Unknown Phosphatidyl inositol Phosphatidyl serine Unknown Phosphatidyl ethanolamine Phosphatidic acid Cerebroside
Spot No. Cosrapowad (Fig. IB)
THIN-LAYER
DETERIIINATION
TABLE I
NOTES
439
As can be seen from Table I, it has not been possible to identify all the lipid components on the thin-layer chromatogram. Compounds 3, 5 and 13 did not stain with iodine but contained a considerable amount of activity. Compounds 3 and 5 could be the deacylated derivative of phosphatidic acid (lysophosphatidic acid) and the phosphorylated derivative of phosphatidyl inositol (diphosphoinositide) respectively, In agreement with the work of other investigatorsl3sla phosphatidic acid and phosphatidyl inositol were found to be highly labelled in both nerve-ending particles and mitochondria (Table I). On the other hand structural lipids such as phosphatidyl serine and phosphatidyl ethanolamine had little activity. Unlike Silica Gel I-PO,washed Silica Gel G gave poor resolution of phospholipids. This resulted in higher readings of the Silica Gel G blanks (Fig. 2). However, the blank readings could not have been due to the phosphorus-molybdate complex as can be seen from the silica gel spectrum (Fig. 2). The modification which was made in the developing reagents resulted in a shift of the spectral peak from a maximum at 830 rnp12 to a maximum at 620 rnp (Fig. 2). The separation and determination of the radioactive specific activities of lipids reported in this communication could be applied to the whole brain as well as particulates from other tissues. Achaowledgements This work was supported by the State of Illinois Mental Wealth Fund and the U.S. Public EIealth Service Research Grant NB 06628-01. The authors wish to thank Mrs. MARILYN MAYFIELD for technical assistance and Mr. JOHN DULANEY for suggesting the Skipski system to us. ATA A. ABDEL-LATIP FLORA E. CHANG
Illigzois State Pediatric Imtitzcte, and D&artment of Psychiatry, U?ziversii!y of Ih?inois Cohh.Jgeof ~edicim, Chicago,
Ill. (U.S.A.)
I V. P. SICIPSKI.R. I?.PETERSON
AND M. BARCLAS, Biochem.
J.,
QO (19G4)
z W. D, SKIDMORE AND C. ENTENMAN, J. Lipid Res., 3 (~1962)471. 3 s. N. PAYNE,J. ChYomatO~., I.5(1964) 173. 4 L. G. ABOOD, 1. KOYAMA ANIJ V. THOMAS. Am. J. Pltysiol., 207 (1964) 5 C. W. REDMAN AND R. W. KIZNAN,JC&O~~~O~., 15 (1~64) 180. 6 A. A. ABDIX-LATIP, Nature, 211 (~QGG) 1530. 7 A, A. ABIXZL-LATIP AND L. G. ABOOD, J0 Nezwocl~em., 12 (196.5)157.
374.
1435.
A. SIMS AND J, A. G. LAROSE,J.A~. OiEClaemisls’ So&, 39 (1962) 232. Q R. M. C. DATVSON, Biocltem. J., 75 (1960) 45. IO I?. FARIUZR AND N. I?. PETERSON,J. LLipidRes.,6 (1965) 455. IL E. J. KING, Biochem.J., 2G (1932)292. 12 G. R. BARTLETT, J.Riol. CJccm.,234 (19.59)466. 13 J, GARBUS, W. I?.DELTJCA,M.E. LOOMANS AND F. M. STRONG,J. Viol. Chem., 14 J. DURELL AND M. A. SODD.J. Biol. Ckcm., 239 (1964) 747.; , 8 R. I?.
Received
238
(1963)59.
April r5th, 1966 J.
Clwomatog.,
24 (x96(5)
435-439