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Biosynthesis of lecithins and phosphatidyl ethanolamines from various radioactive 1,2-diglycerides in rat liver microsomes
BIOCHIMICA ET BIOPHYSICA ACTA
249
**A 55777
BIOSYNTHESIS FROM
VARIOUS
OF LECITHINS
AND
RADIOACTIVE
PHOSPHATIDYL
I,Z-DIGLYCERIDES
ETHANOLAMINES IN RAT
LIVER
MICROSOMES HIDE0
KX?;OH*
Department
(Japan)
of Biochenzistvy,
Sappovo
,Wedical
College, South
I. ITTest I 7, Sappovo.
06 o, Hokkaido
(Received i&y .+th, 1970) (Revised manuscript received August Ioth, 1970)
SUMMARY Various
species of r,n-diglycerides
labeled with [@HIglycerol
or with [I-%]-
glycerol were obtained from biosynthetically labeled rat liver lecithins. The modes of utilization of the radioactive I,z-diglyceride species in the biosynthesis of lecithins and phosphatidyl ethanolamines were compared in rat liver microsomes. When each molecular species of I,z-diglycerides was incubated separately, no obvious difference of utilization was found between the formations of the two phospholipids. However, relatively higher utilization of hexaenoic I,ZZ-diglyceride was found to occur in phosphatidyl ethanolamine formation in contrast to lecithin biosynthesis when incorporation of various mixtures of I,z-diglycerides was studied.
INTRODUCTION Since the finding of characteristic distribution of fatty acids in rat liver glycerophospholipids’y2, the mechanism of formation of specific phospholipids has been studied in many laboratories3-7. In the experiments in vivo, metabolic heterogeneity of molecular species of glycerophospholipids has been reported+rr. In the previous paper, it was suggested the substrate specificity of CDPcholine : I ,a-diglyceride choline phosphotransferase (EC 2.7.8.2.) and CDPethanolamine: r,a-diglyceride ethanolaminephosphotransferase (EC 2.7.8.1.) occur towards different species of r,2-diglycerides, in view of the high reactivities of di- or monoenoic lecithin and hexaenoic phosphatidyl ethanolamine in rat liver slices12. The same findings were also reported by ARVIDSON~~~~,although contradictory results were obtained by HILL et a1.4 in phosphatidyl ethanolamine formation. However, no selectivity of utilization of 1,2diglyceride species was observed in the formation of lecithins in vitro13. In the present experiment, rat liver lecithins biosynthetically labeled with [@HIglycerol or [1-r4C]* Present address : Institute of Animal Physiology, Babraham, Cambridge, ISngland. Biochim.
Biophys.
Acta,
218
(1970)
z4g-258
H. KANOH
250
glycerol were directly subfractionated by argentation thin-layer chromatography and were used as sources of the radioactive r,z-diglycerides. It became possible to obtain specific and highly unsaturated radioactive r,z-diglycerides, such as r-saturated, z-arachidonoyl or r-saturated, a-hexaenoyl, with known specific radioactivities minimizing the chance of isomerization or oxidation of fatty acids. To consider the possible differences of solubilities of r,a-diglyceride species, the incorporations of r,z-diglycerides into lecithins and phosphatidyl ethanolamines were compared, substrates being taken from the same emulsion, and remarkable differences of utilization of the substrates were observed between the formations of the two phospholipids. MATERIALS Wistar
female
rats
of rso-zoog
body
weight,
maintained
with
commercial
experimental diets, were used after overnight fasting. Lz-3H]glycerol, [I-Xlglycerol and [Me-%]choline were obtained from the Radiochemical Centre (Amersham) and were used without further purification. CDP [Me-%]choline (specific activity : 20500 counts per min/pmole) and cold CDPethanolamine were prepared from phosphoryl[Me-%]choline and phosphorylethanolamine, respectively, by KENNEDY’S methodr4. Phosphoryl[Me-14C]choline was prepared by the method of SCHNEIDER et a1.15 and phosphorylethanolamine was purchased from Sigma (U.S.A.). CDP[Me-14C]choline and CDPethanolamine were purified by column chromatography on Dowex-r formate by the method of KENNEDY~~, and their purity was confirmed by paper chromatography by the method of SCHNEIDER et a1.15. Paper chromatographically pure, nonlabeled CDPcholine was a generous gift of Takeda Chemical Industries (Japan). Phospholipase C (Clostridium welchii) was obtained from Sigma. Solvents of analytical grade were distilled and deaerated with N, gas before use. No antioxidants were used. METHODS Preparation of biosynthetically labeled rat liver lecithins and subfractionation according to degree of unsaturation Rat liver lecithins were purified by thin-layer chromatography on Kiesel gel G (E. Merk A.G.), developed with chloroform-methanol-water (70: 30: 5, v/v/v), after removal of acidic lipids through DEAE-cellulose column, as described in the previous pape?. Lecithins were directly subfractionated according to the degree of unsaturation by ARVIDSON’S methodic. The results of the subfractionation were essentially the same as reported previouslylz. Mono-, di-, tetra- and hexaenoic species were the 4 major components and other minor species could not be detected clearly. Considerable contamination was found between mono- and dienoic species separated by this procedure12, but no correction was made in presenting the data. Although more strict subfractionation of r,a-diglycerides released from glycerophospholipids was reported 17,18, direct subfractionation of intact lecithins was performed, because of its rapidity in minimizing transacylation and oxidation of fatty acids in the r,z-diglyceride unit of the phospholipid. Biochim.
Biophys.
Ada,
218 (1970)
z4g-258
BIOSYNTHESIS
OF GLYCEROPHOSPHOLIPIDS
ifavitro
2sr
Subfractionation of lecithins and phosphatidyl ethanolamines, purified from rat liver microsomes after incubation, was also performed to study the formations of the subfractions of the phospholipids. Fig. I shows changes of specific radioactivities of molecular species of rat liver
13
6
12
Time after administratlon
24 (h)
TimeCmln)
Fig. r. Changes of specific radioactivities of subfractions of rat liver lecithins after intraperitoneal administration of [GWjglycerol in long-term experiments. 5 female rats of r5og body weight were injected intraperitoneaily with 1.0 mC of rz-*Hlglyeerol after overnight fasting. Lecithins __were purified from iivers at the time indicated and further subfractionated-into 4 major species. C-U, total lecithins: O-O, monoenoic lecithin; O-O, dienoic lecithin; A-A,, tetraenoic lecithin; n-A ; hexaenoic lecithin. Fig. 2. Effect of incubation time on the formation of lecithins and nhosnhatidvl ethanolamines from radioactive r,z-diglyceride mixture. r,z-Diglyceride mixture eqkvafent to’r pmole of original rat liver lecithins labeled with [z-3HS]glycerol (specific radioactivity: 4.52. ro4 counts per min/ jcmole P) was incubated in the standard reaction mixture. See text for details. O-O, lecithins; O-O, phosphatidyl ethano~amines.
lecithins, in intraperitoneal administration of I mC [z?H)glycerol in a preliminary experiment. Dienoic species had the highest reactivity, and the teraenoic subfraction was found to have a directly opposite metabolic behavior. These findings are consistent with the results of the previous studyl2. To obtain satisfactory radioactivity in tetraenoic species, rats were sacrificed 6 h after administration of 2.5 mC of [2-3H]glycerol in standard experiments. About 0.7% of radioactivity administered was recovered in rat liver lecithins. Throughout these experiments, specific radioactivities of unfractionated lecithins were in the range of 3.5-5.5 .104 counts per min/pmole and the lowest specific radioactivity was constantly found in the tetraenoic subfraction. In some experiments, ~o,LI,Cof [r-l*C]glycerol were injected into rats, and labeled rat liver lecithins were obtained after I h, as described before. Lecithins were stored in chloroform under nitrogen at -20~ and were used within a week after purification. Preparation of radioactive I,2diglycerides 3-5 pmoles of total or subfractionated
lecithins with known specific radiowere hydrolyzed with Fhospholipase C by the method of HANAHAN AND VERCAMER". After a 3 h incubation with vigorous stirring at room temperature, J,Zdiglycerides were extracted with ether and washed twice with water. An ether layer was evaporated under nitrogen, dissolved in IO ml of chloroform-methanol (98: 2, v/v) activities
Eiochfm.
Biofihys.
Acta, 218 (197”)
249-258
H. KANOH
252
and applied on a silicic acid column (I I 10 cm) with successive elution with 50 ml of the same solvent. Sg-zoo~~ of the radioactivity was recovered in this eluate. Aliquots were loaded on a thin-layer plate with carrier djpalmitin and cold rat liver lecithins, as described before. Over 95% of the loaded dipalmitin which moves with the solvent front, the lecithin area. On gas-liquid chromatographic analysis, the same fatty acid composition as the original glycerides are the representatives of the original
radioactivity was found with carrier and no radioactivity was detected in I,z-diglycerides
thus
obtained
had
lecithins, indicating that the r,z-dilecithins. In some experiments, slight
loss of hexaenoic fatty acids was detected in r,a-diglycerides. Although stereochemical analysis of r,a-diglycerides was not performed, diglycerides were prepared at the day of experimentation and used immediately.
I,Z-
Ether solution of radioactive r,z-diglycerides was dried under a nitrogen Bow in small tubes made of cellulose nitrate. Appropriate amounts of 19; Tween-2o and solutions of chemicals other than enzymes were added, and the tubes were sonicated twice for 3 min each time a.t o” with a Kubota Sonifier (IO ks. IOO IV, Kubota Co., Japan). The sonicated volumes were made constant (2.0 ml). The emulsion obtained was slightly tur-bid and radioacti~Iities of the aliquots were measured to determine the amounts of I+diglycerides actually incubated. Preparation of rat liver ~microsomes Rats were sacrificed by decapitation after overnight fasting. Livers were homogenized in 9 volumes of 0.25 M sucrose-r m&L EDTA in a teflon pestle--glass homogenizer. The homogenate was centrifuged at ~oooxg for IO min to remove cell debris and nuclei. The supernatant was centrifuged at IOOOO~,a for 30 min and the pellets were discarded. The ~ooooxg supernatant was centrifuged for 60 min at ~ogoooxg by a Hitachi Ultracentrifuge Model 65-P (Hitachi Co., Japan). The pellet was resuspended in the same solution at the concentration of 50 mg protein/ml and was used as enzymes.
The following reaction conditions were used in the standard experiments unless otherwise indicated : I+‘or the biosynthesis of lecithins, the reaction mixture contained radioactive x,2-diglycerides (equivalent to 1.0 ,umole of the original lecithins), 1.0 ,umole of CDPcholine (free form), 20 pmoles of MgCI,, 50 pmoles of Tris-HCl (pH T.j), I mg of Tween-2o and IO mg protein microsomes in a final volume of 1.0 ml. Incubations were performed in glass test tubes (1.5 .IO cm) at 37O for 30 min. The formation of phosphatidyl ethanolamines from the r,a-diglycerides was studied in parallel to lecithin biosynthesis. Experimental details were the same as in lecithin biosynthesis except that 1.0 ymole of CDPethanolamine was used instead of CDPcholine. The reactions were stopped by the addition of 5 ml of chloroformmethanol (I : 2, v/v). After standing overnight at -4O, microsomal lipids were further extracted with 4 ml of the same solvent and finally with IO ml of chloroform. The extracts were washed with z ml of 0.9 9,4 saline. The chloroform layer was evaporated Biochim.
Bin~hys.
.4&z,
218
(1970) 249-258
BIOSYNTHESIS OF GLY~EROPHOSPHO~~FIDS i?Z 7&O to dryness under nitrogen quots was fractionated
and redissolved
on a thin-layer
253
in 1.0 ml of chloroform.
0.5 ml of the ali-
plate of Kiesel gel G as described
before.
Radioactivity of phospholipid-containing silica gel was measured to calculate the amounts of phospholipids synthesized. In some experiments, two incubations were combined and phospholipids were quantitatively recovered from the gel by ARVIDSON’S method8. Lecithins and phosphatidyl ethanolamines obtained were further subfractionated by argentation thin-layer chromatography, and the radioactivity of each subfraction was determined. Although the data are not presented, additions of IO @moles of reduced glutathione, IO ,umoles of ATP and I mg of bovine serum albumin (Fraction V) were not markedly effective in the formation of phospholipids in this experiment. The amounts of newly formed phospholipids were calculated from the specific radioactivities of r,z-diglycerides (or the original rat liver lecithins) incubated. Retermi~zation of Ya~~o~ct~~ity Radioactivity was measured in dioxane-water scintillatorzO, using a liquid scintillation counter model LS-500, Horiba, Japan. Quenching was monitored by the external standard method and counting efficiency was 20% for %I and 55% for 14C, being fairly constant through the experiments. In the experiments shown in Fig. I, radioactivity of purified lecithins was determined in the toluene scintillator, as described elsewhere12. In some experiments, phospholipid-containing silica gel was scraped carefully into counting vials after detection with iodine vapor, and the radioactivity was determined with a good recovery. In double label experiments, radioactivity was determined by the channels ratio method.
0th.m Protein was determined by the biuret method Z*. Fatty acids were analyzed by gas-liquid chromatography as described in the previous paper. Other methods were the same as described in the previous report12. RESULTS Studies of the conditions of incubations The influence of incubation time on the formation of phospholipids is shown in Fig. 2. x,2-Diglycerides were incorporated linearly into lecithins during the period studied, while maximum incorporation was reached in 30 min in the case of phosphatidy1 ethanolamine formation. In Fig. 3, the effects of enzyme concentrations were presented. The reason for the slight decrease observed in the formations of both phospholipids in higher enzyme concentration is unknown. The biosynthesis of lecithins and phosphatidyl ethanolamines were also shown to be different in the study of the effects of r,a-diglyceride concentrations, as given in Fig. 4. The effect of Tween-zo concentration in the reaction mixture was studied and the results are presented in Fig. 5. In these experiments, radioactive r,2-diglycerides were obtained from unfractionated rat liver lecithins. The amounts of r,2-diglycerides and newly formed phospholipids were calculated from the specific radioactivity of the original lecithins. As seen in Figs. 2-5, considerable variability of substrate effectivity of the r,2-diglyceride emulsion was observed in different experiments. It B&whim.
Biophys.
A&,
218 (1970)
249-258
H. KANOH
254
;i
s!
E
e 400 E 6 3 3 E 2oc % a 0c IOC
f
a
Mlcrosomal
protein
hg)
/,.’
05 10 25 20 1,2-Dlglycerlde concentmtlon(wnOie equwalent to orlglnal rat liver leclthlns)
Fig. 3. Effect of enzyme concentration on the formations of lecithins and phosphatidyl ethanolamines. Concentrations of microsomal protein were changed in the standard reaction condition as indicated. Biosynthesis of both phospholipids was studied in parallel, using the same radioactive r,z-diglyceride emulsion. See Fig. 2 for others. (i-c, lecithins; O-O, phosphatidyl ethanolamines. Fig. 4. Effect of radioactive r,e-diglyceride concentration upon the biosynthesis of lecithins and phosphatidyl ethanolamines. Experiments were performed in the standard reaction condition 0-0, phosphatidyl except that concentration of I,r-diglycerides was varied. r-c , lecithins; ethanolamines.
DGPE
Tween-20
concentrotlon
(mg)
1,2-Dlglyceride
concentration
@moles)
Fig. 5. Effect of Tween-zo concentration on the formation of lecithins and phosphatidyl ethanolamines. Radioactive I,?-diglycerides were obtained from unfractionated rat liver lecithins labeled with [z-3H]g1ycero1 (specific radioactivity: 3.87’ 10~ counts per min/,umole I’). Tween-zo concentration was varied in preparing I,e-diglyceride emulsions as indicated. Other experimental details are the same as given in Fig. 2. O-O, lecithins; O-O, phosphatidyl ethanolamines. Fig. 6. Incorporation of molecular species of r,r-diglycerides into lecithins and phosphatidyl ethanolamines. Subfractions of rat liver lecithins labeled with [z-SH]glycerol were used as sources of radioactive I,e-diglycerides. Specific radioactivities of the original lecithins were as follows: unfractionated, 5.25. IO* counts per min/pmole; dienoic, 5.50’ 10~ counts per min/,umole; tetraenoic, 4.73’10~ counts per min/ymole; hexaenoic lecithin, 5.62.10~ counts per min/,umole. Incubation was performed in the standard reaction condition with each species of I,z-diglycerides at the concentrations stated. o-n, unfractionated : O-O, dienoic ; A-A, tetraenoic and n-n, hexaenoic species of radioactive I ,z-diglycerides. Abbreviations : DGPC, lecithin ; DGPE, phosphatidyl ethanolamine. Biochim.
Biophys.
Acta,
218
(1970)
249-258
BIOSYNTHETIC
0F GLYCER~PHOSPH~LIPIDS
in vitro
255
was necessary to study the biosynthesis of both phospholipids in parallel, using the same emulsion, in order to compare the formations of the two phospholipids. Formation of phospholipids from molecular species of r,a-&glycerides r,z-Diglycerides, obtained from subfractions of labeled rat liver lecithins, were incubated with microsomes and utilization of r,z-diglyceride species in synthesis de novo of the phospholipids was studied. One typical result of two different experiments with similar tendencies are given in Fig. 6. Polyunsaturated r,z-diglycerides, especially tetraenoic species, were found to be utilized relatively more than dienoic species in both phospholipid formations. There is a discrepancy between these results and those reported from other laboratories in the biosynthesis of lecithins13. Possible differences of solubilities of the r,z-diglyceride species must be considered to explain this discrepancy. Experiments
with radioactive I+-diglyceride
mixture obtained from unfractionated
rat
livev lecithins Incorporation of radioactive r,z-diglyceride mixture, obtained from total rat liver lecithins, into subfractions of microsomal phospholipids was studied. After incubation, microsomal lipids were purified, and intact lecithins and phosphatidyl ethanolamines were subfractionated as described before. From the radioactivity of each subfraction, utilization of the r,z-diglyceride species could be studied. Distribution of phosphorus among subfractions was assayed in order to check the reproducibility of the subfractionation. Compositions of microsomal lecithins and phosphatidyl ethanolamines were found to be essentially the same as reported in total rat liver phospholipids12 in preliminary experiments. The results are given in Table I. The ratio of incorporation of [3H]r,2-diglycerides and GDP/Me-%]choline into lecithins was found to be almost I : I, indicating that the influence of endogeneous TABLE
I
INCORPORRTIOK
LECITHINS
OF
INTO
I,2-DIGLYCERIDE
MOLECULAR
MIXTURE
SPECIES
OF
OBTAINED
MICROSOMAL
FROM
LECITHINS
UNFRACTIONATED AND
RAT
PHOSPHATIDYL
LIVER
ETHANOL-
AMINES
I,2-Diglycerides labeled with [z-3H]g1ycerol were obtained from radioactive rat liver lecithins, as described in the text. Mass distribution (mole%) and specific radioactivities of the original rat liver lecithins were as follows: 7.3% monoenoic, 4.00’10~ counts per min/,umole; 24.7ojo dienoic, 4.26. IO* counts per min/pmole; 42.3% tetraenoic, 2.78.10~ counts per min/pmole; 25.70/A hexaenoic, 3.9”. 10~ counts per min/,umole; and total lecithins, 3.60. IO* counts per min/,umole. I,z-Diglycerides, equivalent to 1.0 ymole of the original lecithins, were incubated with 1.0 pmole of CDP (Me-‘Y)choline or with 1.0 pmole of cold CDPethanolamine, and formations of lecithins and phosphatidyl ethanolamines, respectively, were studied in the standard reaction conditions. Abbreviation: DGPE, phosphatidyl ethanolamine.
L&than (Relative 3H
DGPE formed: nmoles (Relative amounts: 96) SH
formed; nmoles amounts: %) -7
_
Total synthesis Subfractions : Monoenoic Dienoic Tetraenoic Hexaenoic Recovery of Radioactivity
263 25.3 (11.2) 76.5 (34.0) 75.4 (33.5) 48.2 (21.3) 88.0%
243 27.9
109 (12.0)
84.0 (36.3) 70.8 (30.6) 48.7 (21.1) 95.0% Biochim.
Biophys.
10.0 (10.7) 21.4 (22.6) 26.9 (28.3) 36.4 (38.4) 87.00/b
Acta,
218
(1970)
249-258
H. K;ZSOH
7.56 TABLE
II
INCORPORATIONOF EQ”IMoLAR MIXTURES OF
MICROSOMAL
OF
RADIO.‘,CTIVE
I,Z-UIGLYCERIDES
IPiT
SUBFRACTIOKS
PHOSPHOLIPInS
Equimolar mixtures of r,a-diglyccrides labeled v&h /2-3H]glycerol were incubated with microsomes at the concentrations stated and the formations of subfractions of phospholipids were studied. Speciilc radioactivities of the r,z-diglycerides were as follows: ,Mixture A: dicnoic, 3.85’ 10“ counts 4.35’ IO* counts per per min/~mole; tetraenoic, 2.98, ro* counts per min/,umole; hcxaenoic, Il~in/~~nole; Mixture I3 and C: dienoic, ~.3r* ro4counts per min/!cmole; hexacnoic, j.35’ IO@counts per min/~mole. Specific radioactivities were determined in the oriejnal rat liver lecithins, as described in the text. Fatty acid composition of Mixture A was as foilows: IO-O, 22.99; ; 1%0, Zj.8~~; IX--I, .+..s:&; 18-r, 17.2”;; 20-1, 17.23;; and 2-6, 12.6”; (Mole:,,). r,z-Diglyccridr A.
Lecithins : Mono- + dienoic Tetraenoic Hexaenoic Phosphatidyl ethanolamines: Mono- + dienoic Tetrsenoic Hexaenoic * Radioactivity
0.16
mk~%~r zncubated
/AWW+&
Dz-
-~
o.r6
,unde
trtva-
-I-
0.16
,~mole
hrxaenoic
B.
o.Izj
/_URO~P
O. rzj
,umolt~
di-
hexa-
l’?WiG
66.5 nmoles 73.3 42.6
9.20
+
0.25 pmole di- or 0.2 j /lP?Wk hrxaescrzc
C.
I r ._jnmolcs
3 5~0 nmoles
14.6*
34.9”
3.00
5.80
12.2
7.00*
22.0
distributed
in polyunsat~lr~lted
subfractions
Ij.Z*
was determined
together.
precursors was negligible and that r,z-diglycerides were as effective as the watersoluble substrate, C~Pcholine. When the modes of utilization of ~,z-diglycerides in the biosynthesis of the two phospholipids were compared, the most remarkable finding was a higher formation of hexaenoic phosphatidyl ethanolamine. Expeyi~le~~ts z&h e~~~~o~~~ ~~i;~~~yeof ~~d~~~~t~v~~,~-d,~~~~~~~~d~s Two kinds of equimolar mixture of I,z-diglycerides were incorporated into microsomal phospholipids at different concentrations, as shown in Table II. When comparing the formations of the two phospholipids, the higher formation of hexaenoic pl~osphatidyl ethanolamine was considered the most significant fact observed. The ratios of formation of mono- plus dienoic and polyunsaturated species were I : I in lecithin formation and I : 2.6 in the biosynthesis of phosphatidyl ethanolamines, as shown in the experiments with Mixture I3 and C in Table II. Incov$wation of double-lab&d r,e-di$yceride lnixture Dienoic r,2-diglyceride labeled with 1r-l*C]glycerol was obtained from biosynthetically labeled rat liver lecithins, and was used in the mixture with hexaenoic -3H]~,2-diglyceride, as well as with tetraenoic j3EIjr,2-diglyceride. As seen in Table III, preferential utilization of hexaenoic r,2-diglyceride in phosphatidyl ethanolamine formation was clearly observed, while the ratio of 3H and l*C incorporated into lecithins remained almost the same as substrate x,2-diglycerides. Tetraenoic z,z-diglyceride seemed more incorporated into both phospholipids than
BIOSYNTHESIS TABLE
OF GLYCEROPHOSPHOLIPIDS
i?&
ZIit3’0
2.57
III
INCORPORATION
OF DOUBLE-LABELED
I,Z-DIGLYCERIDE
MIXTURE
INTO
MICROSOMAL
PHOSPHOLIPIDS
Dienoic i14C]r,z-diglyceride. tetraenoic jJH]r,z-diglyceride and hexaenoic [3H]r,z-diglyceride were incubated with rat liver microsomes in mixtures, as indicated. After incubation microsomal phospholipids were purified and incorporated radioactivities of 3H and i4C were determined. Specific radioactivities of the r,z-diglycerides used were as follows: dienoic [i*C]r,z-diglyceride, 5.zzo*103 counts per min/~mole; tetraenoic [sH]r,z-diglyceride, 2.78.10’ counts per min/pmole: and hexaenoic [WJr,z-diglyceride, 3.90. IO* counts per min~~~mole. Abbreviations : [X$&n, dienoic [r4C]r,z-diglyceride; [3H]d-4, tetraenoic [3H!r.~-digIyceride: [3H]A-h, hexaenoic LSH]r ,z-diglyceride : DGPC, lecithins; DGPE, phosphatidpl ethanolamines. -..__ ______ Radioactive ~,a-lliglycerides Incubated .._...___ ~_
O.~Op%Of~ j% /n-z ro.j2ymole
Ratio
3NjYz:
i”HJd-6
YH
1850 56.3 .._.~_.
__._
[14C]it-2
Ratio =H/%:
cow& per rnin __.-..~._ __ _ DGPC DGPE
o.~gpmole
6.30
Ratio “H/WY
WE
6.85 270 r2.j -l6 ._~________
counts pev n&a 3H w _.____.. .2560
. ___“~
3.95
765
520
i_ o.g6ymole
[3HJll-_f
.-.-...- ~___ Ratio 3HI’“C:
-_
4.92
_.-.-.. “7 ______-._6.54 _-.
the dienoic species. However, the difference in utilization of tetraenoic I+diglyceride in the biosynthesis of the two phospholipids was not as marked as that of hexaenoic ~,a-diglyceride. DISCUSSION
Intact rat liver lecithins biosynthetically labeled with radioactive glycerol were directly subfractionated on AgNO,-impregnated thin-layer plates and were used as sources of radioactive r,z-diglycerides. Subfractions obtained by this method were not pure9,12. Considerable contamination was observed especially between mono- and dienoic species (about IO-15%). However, this incomplete subfractionation did not have significant disadvantages with regard to studying biosynthesis of subfractions of glycerophospholipids in rat liver slices12. As seen in Figs. z-5, remarkable differences were observed in utilization of I,Zdiglycerides between the formations of the two phospholipids. Biosynthesis of lecithins and phosphatidyl ethanolamines were not compared in enzymologically equal conditions in this study, and no further studies were made on this point. Considering the possible variability of solubilities of the r,z-diglyceride species and the variable substrate effectivity of r,a-diglyceride emulsion (see Figs. 225), utilization of r,z-diglycerides in the biosynthesis of the two phospholipids was compared under the same expzrimental conditions. When each species of r,z-diglycerides was incubated separately, no obvious difference of utilization was observed between the two phospholipids. The polyunsaturated I,Ediglycerides seemed incorporated into both phospholipids (Fig. 6) at a slightly higher rate than the dienoic species. The results of experiments with I,a-diglyceride mixtures indicate that hexaenoic 1,2-diglyceride is utilized preferentially, although incompletely, for phosphatidy1 ethanolamine formation. On the other hand, no remarkable difference was detected in utilizing I+diglyceride species for lecithin biosynthesis, in accordance with the results obtained by MUDD et nP. Tetraenoic I,z-diglyceride was incorporated relatively well into both phospholipids (see Fig. 6 and Table III). However, the incorporation of hexaenoic r,z-diglyceride into phosphatidyl ethanolamine was more
258
H. KANOH
marked than the incorporation of tetraenoic r,a-diglyceride, when the biosynthesis of the two phospholipids was compared. The somewhat discrepant results obtained in the present study might suggest that selective utilization of hexaenoic r,z-diglyceride occurs to some extent in synthesis de GOZJO of phosphatidyl ethanolamine through the CDPethanolamine pathway and that this selectivity seems to be rather incomplete and difficult to detect in usual experiments i?z vitro. In the previous paper, the author reported extremely high reactivity of dienoic lecithin and hexaenoic phosphatidyl ethanolamine and low reactivity of tetraenoic species in synthesis de EOVOof gly~erophospholipids in rat liver slices12. Contradictory results were obtained in the present study. Probably, artificial experimental conditions and an impairment of microsomes induced by cell fractionation must be considered to understand this discrepancy. However, the remarkable difference of the mode of utilization of x,z-diglycerides was observed between the biosynthesis of the two pll(~spholipids, especially in utilizing hexaenoic r,z-diglyceride. This result may be helpful in considering the role of hexaenoic phosphatidyl ethanolamine which has been reported to be preferentially converted to lecithin
by stepwise N-methylation
in rat liver*$12.
ACKNOWLEDGEMENTS
The kind advice and continued
encouragement
offered to the author by Prof.
K. Ohno are greatly appreciated. The author wishes to thank Mr. M. Emanuel for his kind help in preparing the manuscript. This work was supported in part by a Scientific Research Grant from the Ministry of Education, Japan (387018). REFERENCES I D. J, HANAHAN, Lip& Chenzist~y. John Wiley, New York, 196o, p. 71. 2 W. E. M. LANDS, Ann. Rev, Biochem., 34 (1965) 313. 3 S. B. WEISS, E. P. KENNEDY AXED J. Y. KIYASU, J.BioZ. Chem., 235 (1960) 40. 4 E. IZ. HILL, D. R. HUSBANDS AND W. E. M. LANDS, J. Biol. Chem., 243 (1968) 4~40. 5 I:. T’OSSMAYER,G. L. SCAERPHOF,T. X.4. R. DUXBELMA;~~, L.M.G.X~AN GOL~E AND L.L.M. VAN DEENEN, Biochim. Biophys. Acta, 176 (1969) 95. 6 IV. E. M. LANIX API‘D1. MERKL, J.Biol. Chem., 238 (1963) HgS. 7 A. K. HAJRA, Bzochem. Biophys. Xes. Common., 33 (1968) gzg. X J. A. BALINT, D. A. BEELBR, D. H. TREBLE AN'D H. L. SPITZER, J. Lipid lies., 8 (1967) 486. g C;. A. E. ARVIDSON, Ewopean J. Biochem., 4 (1968) 478. IO II 12 r3
G. A. l2. ARVIDSO~;,
European
J. Bzochem.,
5 (1968)
415.
D. RYTTER, J. E. MILLER MD IV. E. CORPI~TZER, B&him. Biophys. A&, 152 (1968) 418. H. KANOH, Biochim. Biophys. Acta, 176 (1969) 756. J. B. MUDD, L. hl.G. VAX Gome AND L. L. M. Vnx DEEXES, Biochim.Biophys. Acta, 176
(1969) .517. 14 B. I’. hExxEDY,
J. Binl. Chem., 222 (1950) 185. 15 W. C. SCHNEIDER, W. C;.FISCUS AND J. A. R. LAWLER, Anal. Biochem., rq(~gGG) IZI. 16 G. A. E. ARVIDSON, ,!, Lipid I&s., 6 (1965) 571. 17 0. REXKONEN, Biochznz. Riofihys. Acta, 125 (rg66) 288. 18 L. M. G. VAX GOLDE AXD L. I,.M. VAK DEEKEN, B&him. Bi~pkys. Acta, 125 (1066) 496. 19 D. J. HANAHAN AND R. VERCAMER, J. Am. Chem. Sac., 76 (rgp+) 1804. 20 I;.SXYI~ER, Anal.Biochem., g (1964) 183. 21 A. G. GORNALL, C. S. RARDAWILL AND M. II. DAVID, J. Biol. Chcm., 177 (1949) 751. Hiochim.
Riophys.
Acta,
218
(1970)
249-258
249
**A 55777
BIOSYNTHESIS FROM
VARIOUS
OF LECITHINS
AND
RADIOACTIVE
PHOSPHATIDYL
I,Z-DIGLYCERIDES
ETHANOLAMINES IN RAT
LIVER
MICROSOMES HIDE0
KX?;OH*
Department
(Japan)
of Biochenzistvy,
Sappovo
,Wedical
College, South
I. ITTest I 7, Sappovo.
06 o, Hokkaido
(Received i&y .+th, 1970) (Revised manuscript received August Ioth, 1970)
SUMMARY Various
species of r,n-diglycerides
labeled with [@HIglycerol
or with [I-%]-
glycerol were obtained from biosynthetically labeled rat liver lecithins. The modes of utilization of the radioactive I,z-diglyceride species in the biosynthesis of lecithins and phosphatidyl ethanolamines were compared in rat liver microsomes. When each molecular species of I,z-diglycerides was incubated separately, no obvious difference of utilization was found between the formations of the two phospholipids. However, relatively higher utilization of hexaenoic I,ZZ-diglyceride was found to occur in phosphatidyl ethanolamine formation in contrast to lecithin biosynthesis when incorporation of various mixtures of I,z-diglycerides was studied.
INTRODUCTION Since the finding of characteristic distribution of fatty acids in rat liver glycerophospholipids’y2, the mechanism of formation of specific phospholipids has been studied in many laboratories3-7. In the experiments in vivo, metabolic heterogeneity of molecular species of glycerophospholipids has been reported+rr. In the previous paper, it was suggested the substrate specificity of CDPcholine : I ,a-diglyceride choline phosphotransferase (EC 2.7.8.2.) and CDPethanolamine: r,a-diglyceride ethanolaminephosphotransferase (EC 2.7.8.1.) occur towards different species of r,2-diglycerides, in view of the high reactivities of di- or monoenoic lecithin and hexaenoic phosphatidyl ethanolamine in rat liver slices12. The same findings were also reported by ARVIDSON~~~~,although contradictory results were obtained by HILL et a1.4 in phosphatidyl ethanolamine formation. However, no selectivity of utilization of 1,2diglyceride species was observed in the formation of lecithins in vitro13. In the present experiment, rat liver lecithins biosynthetically labeled with [@HIglycerol or [1-r4C]* Present address : Institute of Animal Physiology, Babraham, Cambridge, ISngland. Biochim.
Biophys.
Acta,
218
(1970)
z4g-258
H. KANOH
250
glycerol were directly subfractionated by argentation thin-layer chromatography and were used as sources of the radioactive r,z-diglycerides. It became possible to obtain specific and highly unsaturated radioactive r,z-diglycerides, such as r-saturated, z-arachidonoyl or r-saturated, a-hexaenoyl, with known specific radioactivities minimizing the chance of isomerization or oxidation of fatty acids. To consider the possible differences of solubilities of r,a-diglyceride species, the incorporations of r,z-diglycerides into lecithins and phosphatidyl ethanolamines were compared, substrates being taken from the same emulsion, and remarkable differences of utilization of the substrates were observed between the formations of the two phospholipids. MATERIALS Wistar
female
rats
of rso-zoog
body
weight,
maintained
with
commercial
experimental diets, were used after overnight fasting. Lz-3H]glycerol, [I-Xlglycerol and [Me-%]choline were obtained from the Radiochemical Centre (Amersham) and were used without further purification. CDP [Me-%]choline (specific activity : 20500 counts per min/pmole) and cold CDPethanolamine were prepared from phosphoryl[Me-%]choline and phosphorylethanolamine, respectively, by KENNEDY’S methodr4. Phosphoryl[Me-14C]choline was prepared by the method of SCHNEIDER et a1.15 and phosphorylethanolamine was purchased from Sigma (U.S.A.). CDP[Me-14C]choline and CDPethanolamine were purified by column chromatography on Dowex-r formate by the method of KENNEDY~~, and their purity was confirmed by paper chromatography by the method of SCHNEIDER et a1.15. Paper chromatographically pure, nonlabeled CDPcholine was a generous gift of Takeda Chemical Industries (Japan). Phospholipase C (Clostridium welchii) was obtained from Sigma. Solvents of analytical grade were distilled and deaerated with N, gas before use. No antioxidants were used. METHODS Preparation of biosynthetically labeled rat liver lecithins and subfractionation according to degree of unsaturation Rat liver lecithins were purified by thin-layer chromatography on Kiesel gel G (E. Merk A.G.), developed with chloroform-methanol-water (70: 30: 5, v/v/v), after removal of acidic lipids through DEAE-cellulose column, as described in the previous pape?. Lecithins were directly subfractionated according to the degree of unsaturation by ARVIDSON’S methodic. The results of the subfractionation were essentially the same as reported previouslylz. Mono-, di-, tetra- and hexaenoic species were the 4 major components and other minor species could not be detected clearly. Considerable contamination was found between mono- and dienoic species separated by this procedure12, but no correction was made in presenting the data. Although more strict subfractionation of r,a-diglycerides released from glycerophospholipids was reported 17,18, direct subfractionation of intact lecithins was performed, because of its rapidity in minimizing transacylation and oxidation of fatty acids in the r,z-diglyceride unit of the phospholipid. Biochim.
Biophys.
Ada,
218 (1970)
z4g-258
BIOSYNTHESIS
OF GLYCEROPHOSPHOLIPIDS
ifavitro
2sr
Subfractionation of lecithins and phosphatidyl ethanolamines, purified from rat liver microsomes after incubation, was also performed to study the formations of the subfractions of the phospholipids. Fig. I shows changes of specific radioactivities of molecular species of rat liver
13
6
12
Time after administratlon
24 (h)
TimeCmln)
Fig. r. Changes of specific radioactivities of subfractions of rat liver lecithins after intraperitoneal administration of [GWjglycerol in long-term experiments. 5 female rats of r5og body weight were injected intraperitoneaily with 1.0 mC of rz-*Hlglyeerol after overnight fasting. Lecithins __were purified from iivers at the time indicated and further subfractionated-into 4 major species. C-U, total lecithins: O-O, monoenoic lecithin; O-O, dienoic lecithin; A-A,, tetraenoic lecithin; n-A ; hexaenoic lecithin. Fig. 2. Effect of incubation time on the formation of lecithins and nhosnhatidvl ethanolamines from radioactive r,z-diglyceride mixture. r,z-Diglyceride mixture eqkvafent to’r pmole of original rat liver lecithins labeled with [z-3HS]glycerol (specific radioactivity: 4.52. ro4 counts per min/ jcmole P) was incubated in the standard reaction mixture. See text for details. O-O, lecithins; O-O, phosphatidyl ethano~amines.
lecithins, in intraperitoneal administration of I mC [z?H)glycerol in a preliminary experiment. Dienoic species had the highest reactivity, and the teraenoic subfraction was found to have a directly opposite metabolic behavior. These findings are consistent with the results of the previous studyl2. To obtain satisfactory radioactivity in tetraenoic species, rats were sacrificed 6 h after administration of 2.5 mC of [2-3H]glycerol in standard experiments. About 0.7% of radioactivity administered was recovered in rat liver lecithins. Throughout these experiments, specific radioactivities of unfractionated lecithins were in the range of 3.5-5.5 .104 counts per min/pmole and the lowest specific radioactivity was constantly found in the tetraenoic subfraction. In some experiments, ~o,LI,Cof [r-l*C]glycerol were injected into rats, and labeled rat liver lecithins were obtained after I h, as described before. Lecithins were stored in chloroform under nitrogen at -20~ and were used within a week after purification. Preparation of radioactive I,2diglycerides 3-5 pmoles of total or subfractionated
lecithins with known specific radiowere hydrolyzed with Fhospholipase C by the method of HANAHAN AND VERCAMER". After a 3 h incubation with vigorous stirring at room temperature, J,Zdiglycerides were extracted with ether and washed twice with water. An ether layer was evaporated under nitrogen, dissolved in IO ml of chloroform-methanol (98: 2, v/v) activities
Eiochfm.
Biofihys.
Acta, 218 (197”)
249-258
H. KANOH
252
and applied on a silicic acid column (I I 10 cm) with successive elution with 50 ml of the same solvent. Sg-zoo~~ of the radioactivity was recovered in this eluate. Aliquots were loaded on a thin-layer plate with carrier djpalmitin and cold rat liver lecithins, as described before. Over 95% of the loaded dipalmitin which moves with the solvent front, the lecithin area. On gas-liquid chromatographic analysis, the same fatty acid composition as the original glycerides are the representatives of the original
radioactivity was found with carrier and no radioactivity was detected in I,z-diglycerides
thus
obtained
had
lecithins, indicating that the r,z-dilecithins. In some experiments, slight
loss of hexaenoic fatty acids was detected in r,a-diglycerides. Although stereochemical analysis of r,a-diglycerides was not performed, diglycerides were prepared at the day of experimentation and used immediately.
I,Z-
Ether solution of radioactive r,z-diglycerides was dried under a nitrogen Bow in small tubes made of cellulose nitrate. Appropriate amounts of 19; Tween-2o and solutions of chemicals other than enzymes were added, and the tubes were sonicated twice for 3 min each time a.t o” with a Kubota Sonifier (IO ks. IOO IV, Kubota Co., Japan). The sonicated volumes were made constant (2.0 ml). The emulsion obtained was slightly tur-bid and radioacti~Iities of the aliquots were measured to determine the amounts of I+diglycerides actually incubated. Preparation of rat liver ~microsomes Rats were sacrificed by decapitation after overnight fasting. Livers were homogenized in 9 volumes of 0.25 M sucrose-r m&L EDTA in a teflon pestle--glass homogenizer. The homogenate was centrifuged at ~oooxg for IO min to remove cell debris and nuclei. The supernatant was centrifuged at IOOOO~,a for 30 min and the pellets were discarded. The ~ooooxg supernatant was centrifuged for 60 min at ~ogoooxg by a Hitachi Ultracentrifuge Model 65-P (Hitachi Co., Japan). The pellet was resuspended in the same solution at the concentration of 50 mg protein/ml and was used as enzymes.
The following reaction conditions were used in the standard experiments unless otherwise indicated : I+‘or the biosynthesis of lecithins, the reaction mixture contained radioactive x,2-diglycerides (equivalent to 1.0 ,umole of the original lecithins), 1.0 ,umole of CDPcholine (free form), 20 pmoles of MgCI,, 50 pmoles of Tris-HCl (pH T.j), I mg of Tween-2o and IO mg protein microsomes in a final volume of 1.0 ml. Incubations were performed in glass test tubes (1.5 .IO cm) at 37O for 30 min. The formation of phosphatidyl ethanolamines from the r,a-diglycerides was studied in parallel to lecithin biosynthesis. Experimental details were the same as in lecithin biosynthesis except that 1.0 ymole of CDPethanolamine was used instead of CDPcholine. The reactions were stopped by the addition of 5 ml of chloroformmethanol (I : 2, v/v). After standing overnight at -4O, microsomal lipids were further extracted with 4 ml of the same solvent and finally with IO ml of chloroform. The extracts were washed with z ml of 0.9 9,4 saline. The chloroform layer was evaporated Biochim.
Bin~hys.
.4&z,
218
(1970) 249-258
BIOSYNTHESIS OF GLY~EROPHOSPHO~~FIDS i?Z 7&O to dryness under nitrogen quots was fractionated
and redissolved
on a thin-layer
253
in 1.0 ml of chloroform.
0.5 ml of the ali-
plate of Kiesel gel G as described
before.
Radioactivity of phospholipid-containing silica gel was measured to calculate the amounts of phospholipids synthesized. In some experiments, two incubations were combined and phospholipids were quantitatively recovered from the gel by ARVIDSON’S method8. Lecithins and phosphatidyl ethanolamines obtained were further subfractionated by argentation thin-layer chromatography, and the radioactivity of each subfraction was determined. Although the data are not presented, additions of IO @moles of reduced glutathione, IO ,umoles of ATP and I mg of bovine serum albumin (Fraction V) were not markedly effective in the formation of phospholipids in this experiment. The amounts of newly formed phospholipids were calculated from the specific radioactivities of r,z-diglycerides (or the original rat liver lecithins) incubated. Retermi~zation of Ya~~o~ct~~ity Radioactivity was measured in dioxane-water scintillatorzO, using a liquid scintillation counter model LS-500, Horiba, Japan. Quenching was monitored by the external standard method and counting efficiency was 20% for %I and 55% for 14C, being fairly constant through the experiments. In the experiments shown in Fig. I, radioactivity of purified lecithins was determined in the toluene scintillator, as described elsewhere12. In some experiments, phospholipid-containing silica gel was scraped carefully into counting vials after detection with iodine vapor, and the radioactivity was determined with a good recovery. In double label experiments, radioactivity was determined by the channels ratio method.
0th.m Protein was determined by the biuret method Z*. Fatty acids were analyzed by gas-liquid chromatography as described in the previous paper. Other methods were the same as described in the previous report12. RESULTS Studies of the conditions of incubations The influence of incubation time on the formation of phospholipids is shown in Fig. 2. x,2-Diglycerides were incorporated linearly into lecithins during the period studied, while maximum incorporation was reached in 30 min in the case of phosphatidy1 ethanolamine formation. In Fig. 3, the effects of enzyme concentrations were presented. The reason for the slight decrease observed in the formations of both phospholipids in higher enzyme concentration is unknown. The biosynthesis of lecithins and phosphatidyl ethanolamines were also shown to be different in the study of the effects of r,a-diglyceride concentrations, as given in Fig. 4. The effect of Tween-zo concentration in the reaction mixture was studied and the results are presented in Fig. 5. In these experiments, radioactive r,2-diglycerides were obtained from unfractionated rat liver lecithins. The amounts of r,2-diglycerides and newly formed phospholipids were calculated from the specific radioactivity of the original lecithins. As seen in Figs. 2-5, considerable variability of substrate effectivity of the r,2-diglyceride emulsion was observed in different experiments. It B&whim.
Biophys.
A&,
218 (1970)
249-258
H. KANOH
254
;i
s!
E
e 400 E 6 3 3 E 2oc % a 0c IOC
f
a
Mlcrosomal
protein
hg)
/,.’
05 10 25 20 1,2-Dlglycerlde concentmtlon(wnOie equwalent to orlglnal rat liver leclthlns)
Fig. 3. Effect of enzyme concentration on the formations of lecithins and phosphatidyl ethanolamines. Concentrations of microsomal protein were changed in the standard reaction condition as indicated. Biosynthesis of both phospholipids was studied in parallel, using the same radioactive r,z-diglyceride emulsion. See Fig. 2 for others. (i-c, lecithins; O-O, phosphatidyl ethanolamines. Fig. 4. Effect of radioactive r,e-diglyceride concentration upon the biosynthesis of lecithins and phosphatidyl ethanolamines. Experiments were performed in the standard reaction condition 0-0, phosphatidyl except that concentration of I,r-diglycerides was varied. r-c , lecithins; ethanolamines.
DGPE
Tween-20
concentrotlon
(mg)
1,2-Dlglyceride
concentration
@moles)
Fig. 5. Effect of Tween-zo concentration on the formation of lecithins and phosphatidyl ethanolamines. Radioactive I,?-diglycerides were obtained from unfractionated rat liver lecithins labeled with [z-3H]g1ycero1 (specific radioactivity: 3.87’ 10~ counts per min/,umole I’). Tween-zo concentration was varied in preparing I,e-diglyceride emulsions as indicated. Other experimental details are the same as given in Fig. 2. O-O, lecithins; O-O, phosphatidyl ethanolamines. Fig. 6. Incorporation of molecular species of r,r-diglycerides into lecithins and phosphatidyl ethanolamines. Subfractions of rat liver lecithins labeled with [z-SH]glycerol were used as sources of radioactive I,e-diglycerides. Specific radioactivities of the original lecithins were as follows: unfractionated, 5.25. IO* counts per min/pmole; dienoic, 5.50’ 10~ counts per min/,umole; tetraenoic, 4.73’10~ counts per min/ymole; hexaenoic lecithin, 5.62.10~ counts per min/,umole. Incubation was performed in the standard reaction condition with each species of I,z-diglycerides at the concentrations stated. o-n, unfractionated : O-O, dienoic ; A-A, tetraenoic and n-n, hexaenoic species of radioactive I ,z-diglycerides. Abbreviations : DGPC, lecithin ; DGPE, phosphatidyl ethanolamine. Biochim.
Biophys.
Acta,
218
(1970)
249-258
BIOSYNTHETIC
0F GLYCER~PHOSPH~LIPIDS
in vitro
255
was necessary to study the biosynthesis of both phospholipids in parallel, using the same emulsion, in order to compare the formations of the two phospholipids. Formation of phospholipids from molecular species of r,a-&glycerides r,z-Diglycerides, obtained from subfractions of labeled rat liver lecithins, were incubated with microsomes and utilization of r,z-diglyceride species in synthesis de novo of the phospholipids was studied. One typical result of two different experiments with similar tendencies are given in Fig. 6. Polyunsaturated r,z-diglycerides, especially tetraenoic species, were found to be utilized relatively more than dienoic species in both phospholipid formations. There is a discrepancy between these results and those reported from other laboratories in the biosynthesis of lecithins13. Possible differences of solubilities of the r,z-diglyceride species must be considered to explain this discrepancy. Experiments
with radioactive I+-diglyceride
mixture obtained from unfractionated
rat
livev lecithins Incorporation of radioactive r,z-diglyceride mixture, obtained from total rat liver lecithins, into subfractions of microsomal phospholipids was studied. After incubation, microsomal lipids were purified, and intact lecithins and phosphatidyl ethanolamines were subfractionated as described before. From the radioactivity of each subfraction, utilization of the r,z-diglyceride species could be studied. Distribution of phosphorus among subfractions was assayed in order to check the reproducibility of the subfractionation. Compositions of microsomal lecithins and phosphatidyl ethanolamines were found to be essentially the same as reported in total rat liver phospholipids12 in preliminary experiments. The results are given in Table I. The ratio of incorporation of [3H]r,2-diglycerides and GDP/Me-%]choline into lecithins was found to be almost I : I, indicating that the influence of endogeneous TABLE
I
INCORPORRTIOK
LECITHINS
OF
INTO
I,2-DIGLYCERIDE
MOLECULAR
MIXTURE
SPECIES
OF
OBTAINED
MICROSOMAL
FROM
LECITHINS
UNFRACTIONATED AND
RAT
PHOSPHATIDYL
LIVER
ETHANOL-
AMINES
I,2-Diglycerides labeled with [z-3H]g1ycerol were obtained from radioactive rat liver lecithins, as described in the text. Mass distribution (mole%) and specific radioactivities of the original rat liver lecithins were as follows: 7.3% monoenoic, 4.00’10~ counts per min/,umole; 24.7ojo dienoic, 4.26. IO* counts per min/pmole; 42.3% tetraenoic, 2.78.10~ counts per min/pmole; 25.70/A hexaenoic, 3.9”. 10~ counts per min/,umole; and total lecithins, 3.60. IO* counts per min/,umole. I,z-Diglycerides, equivalent to 1.0 ymole of the original lecithins, were incubated with 1.0 pmole of CDP (Me-‘Y)choline or with 1.0 pmole of cold CDPethanolamine, and formations of lecithins and phosphatidyl ethanolamines, respectively, were studied in the standard reaction conditions. Abbreviation: DGPE, phosphatidyl ethanolamine.
L&than (Relative 3H
DGPE formed: nmoles (Relative amounts: 96) SH
formed; nmoles amounts: %) -7
_
Total synthesis Subfractions : Monoenoic Dienoic Tetraenoic Hexaenoic Recovery of Radioactivity
263 25.3 (11.2) 76.5 (34.0) 75.4 (33.5) 48.2 (21.3) 88.0%
243 27.9
109 (12.0)
84.0 (36.3) 70.8 (30.6) 48.7 (21.1) 95.0% Biochim.
Biophys.
10.0 (10.7) 21.4 (22.6) 26.9 (28.3) 36.4 (38.4) 87.00/b
Acta,
218
(1970)
249-258
H. K;ZSOH
7.56 TABLE
II
INCORPORATIONOF EQ”IMoLAR MIXTURES OF
MICROSOMAL
OF
RADIO.‘,CTIVE
I,Z-UIGLYCERIDES
IPiT
SUBFRACTIOKS
PHOSPHOLIPInS
Equimolar mixtures of r,a-diglyccrides labeled v&h /2-3H]glycerol were incubated with microsomes at the concentrations stated and the formations of subfractions of phospholipids were studied. Speciilc radioactivities of the r,z-diglycerides were as follows: ,Mixture A: dicnoic, 3.85’ 10“ counts 4.35’ IO* counts per per min/~mole; tetraenoic, 2.98, ro* counts per min/,umole; hcxaenoic, Il~in/~~nole; Mixture I3 and C: dienoic, ~.3r* ro4counts per min/!cmole; hexacnoic, j.35’ IO@counts per min/~mole. Specific radioactivities were determined in the oriejnal rat liver lecithins, as described in the text. Fatty acid composition of Mixture A was as foilows: IO-O, 22.99; ; 1%0, Zj.8~~; IX--I, .+..s:&; 18-r, 17.2”;; 20-1, 17.23;; and 2-6, 12.6”; (Mole:,,). r,z-Diglyccridr A.
Lecithins : Mono- + dienoic Tetraenoic Hexaenoic Phosphatidyl ethanolamines: Mono- + dienoic Tetrsenoic Hexaenoic * Radioactivity
0.16
mk~%~r zncubated
/AWW+&
Dz-
-~
o.r6
,unde
trtva-
-I-
0.16
,~mole
hrxaenoic
B.
o.Izj
/_URO~P
O. rzj
,umolt~
di-
hexa-
l’?WiG
66.5 nmoles 73.3 42.6
9.20
+
0.25 pmole di- or 0.2 j /lP?Wk hrxaescrzc
C.
I r ._jnmolcs
3 5~0 nmoles
14.6*
34.9”
3.00
5.80
12.2
7.00*
22.0
distributed
in polyunsat~lr~lted
subfractions
Ij.Z*
was determined
together.
precursors was negligible and that r,z-diglycerides were as effective as the watersoluble substrate, C~Pcholine. When the modes of utilization of ~,z-diglycerides in the biosynthesis of the two phospholipids were compared, the most remarkable finding was a higher formation of hexaenoic phosphatidyl ethanolamine. Expeyi~le~~ts z&h e~~~~o~~~ ~~i;~~~yeof ~~d~~~~t~v~~,~-d,~~~~~~~~d~s Two kinds of equimolar mixture of I,z-diglycerides were incorporated into microsomal phospholipids at different concentrations, as shown in Table II. When comparing the formations of the two phospholipids, the higher formation of hexaenoic pl~osphatidyl ethanolamine was considered the most significant fact observed. The ratios of formation of mono- plus dienoic and polyunsaturated species were I : I in lecithin formation and I : 2.6 in the biosynthesis of phosphatidyl ethanolamines, as shown in the experiments with Mixture I3 and C in Table II. Incov$wation of double-lab&d r,e-di$yceride lnixture Dienoic r,2-diglyceride labeled with 1r-l*C]glycerol was obtained from biosynthetically labeled rat liver lecithins, and was used in the mixture with hexaenoic -3H]~,2-diglyceride, as well as with tetraenoic j3EIjr,2-diglyceride. As seen in Table III, preferential utilization of hexaenoic r,2-diglyceride in phosphatidyl ethanolamine formation was clearly observed, while the ratio of 3H and l*C incorporated into lecithins remained almost the same as substrate x,2-diglycerides. Tetraenoic z,z-diglyceride seemed more incorporated into both phospholipids than
BIOSYNTHESIS TABLE
OF GLYCEROPHOSPHOLIPIDS
i?&
ZIit3’0
2.57
III
INCORPORATION
OF DOUBLE-LABELED
I,Z-DIGLYCERIDE
MIXTURE
INTO
MICROSOMAL
PHOSPHOLIPIDS
Dienoic i14C]r,z-diglyceride. tetraenoic jJH]r,z-diglyceride and hexaenoic [3H]r,z-diglyceride were incubated with rat liver microsomes in mixtures, as indicated. After incubation microsomal phospholipids were purified and incorporated radioactivities of 3H and i4C were determined. Specific radioactivities of the r,z-diglycerides used were as follows: dienoic [i*C]r,z-diglyceride, 5.zzo*103 counts per min/~mole; tetraenoic [sH]r,z-diglyceride, 2.78.10’ counts per min/pmole: and hexaenoic [WJr,z-diglyceride, 3.90. IO* counts per min~~~mole. Abbreviations : [X$&n, dienoic [r4C]r,z-diglyceride; [3H]d-4, tetraenoic [3H!r.~-digIyceride: [3H]A-h, hexaenoic LSH]r ,z-diglyceride : DGPC, lecithins; DGPE, phosphatidpl ethanolamines. -..__ ______ Radioactive ~,a-lliglycerides Incubated .._...___ ~_
O.~Op%Of~ j% /n-z ro.j2ymole
Ratio
3NjYz:
i”HJd-6
YH
1850 56.3 .._.~_.
__._
[14C]it-2
Ratio =H/%:
cow& per rnin __.-..~._ __ _ DGPC DGPE
o.~gpmole
6.30
Ratio “H/WY
WE
6.85 270 r2.j -l6 ._~________
counts pev n&a 3H w _.____.. .2560
. ___“~
3.95
765
520
i_ o.g6ymole
[3HJll-_f
.-.-...- ~___ Ratio 3HI’“C:
-_
4.92
_.-.-.. “7 ______-._6.54 _-.
the dienoic species. However, the difference in utilization of tetraenoic I+diglyceride in the biosynthesis of the two phospholipids was not as marked as that of hexaenoic ~,a-diglyceride. DISCUSSION
Intact rat liver lecithins biosynthetically labeled with radioactive glycerol were directly subfractionated on AgNO,-impregnated thin-layer plates and were used as sources of radioactive r,z-diglycerides. Subfractions obtained by this method were not pure9,12. Considerable contamination was observed especially between mono- and dienoic species (about IO-15%). However, this incomplete subfractionation did not have significant disadvantages with regard to studying biosynthesis of subfractions of glycerophospholipids in rat liver slices12. As seen in Figs. z-5, remarkable differences were observed in utilization of I,Zdiglycerides between the formations of the two phospholipids. Biosynthesis of lecithins and phosphatidyl ethanolamines were not compared in enzymologically equal conditions in this study, and no further studies were made on this point. Considering the possible variability of solubilities of the r,z-diglyceride species and the variable substrate effectivity of r,a-diglyceride emulsion (see Figs. 225), utilization of r,z-diglycerides in the biosynthesis of the two phospholipids was compared under the same expzrimental conditions. When each species of r,z-diglycerides was incubated separately, no obvious difference of utilization was observed between the two phospholipids. The polyunsaturated I,Ediglycerides seemed incorporated into both phospholipids (Fig. 6) at a slightly higher rate than the dienoic species. The results of experiments with I,a-diglyceride mixtures indicate that hexaenoic 1,2-diglyceride is utilized preferentially, although incompletely, for phosphatidy1 ethanolamine formation. On the other hand, no remarkable difference was detected in utilizing I+diglyceride species for lecithin biosynthesis, in accordance with the results obtained by MUDD et nP. Tetraenoic I,z-diglyceride was incorporated relatively well into both phospholipids (see Fig. 6 and Table III). However, the incorporation of hexaenoic r,z-diglyceride into phosphatidyl ethanolamine was more
258
H. KANOH
marked than the incorporation of tetraenoic r,a-diglyceride, when the biosynthesis of the two phospholipids was compared. The somewhat discrepant results obtained in the present study might suggest that selective utilization of hexaenoic r,z-diglyceride occurs to some extent in synthesis de GOZJO of phosphatidyl ethanolamine through the CDPethanolamine pathway and that this selectivity seems to be rather incomplete and difficult to detect in usual experiments i?z vitro. In the previous paper, the author reported extremely high reactivity of dienoic lecithin and hexaenoic phosphatidyl ethanolamine and low reactivity of tetraenoic species in synthesis de EOVOof gly~erophospholipids in rat liver slices12. Contradictory results were obtained in the present study. Probably, artificial experimental conditions and an impairment of microsomes induced by cell fractionation must be considered to understand this discrepancy. However, the remarkable difference of the mode of utilization of x,z-diglycerides was observed between the biosynthesis of the two pll(~spholipids, especially in utilizing hexaenoic r,z-diglyceride. This result may be helpful in considering the role of hexaenoic phosphatidyl ethanolamine which has been reported to be preferentially converted to lecithin
by stepwise N-methylation
in rat liver*$12.
ACKNOWLEDGEMENTS
The kind advice and continued
encouragement
offered to the author by Prof.
K. Ohno are greatly appreciated. The author wishes to thank Mr. M. Emanuel for his kind help in preparing the manuscript. This work was supported in part by a Scientific Research Grant from the Ministry of Education, Japan (387018). REFERENCES I D. J, HANAHAN, Lip& Chenzist~y. John Wiley, New York, 196o, p. 71. 2 W. E. M. LANDS, Ann. Rev, Biochem., 34 (1965) 313. 3 S. B. WEISS, E. P. KENNEDY AXED J. Y. KIYASU, J.BioZ. Chem., 235 (1960) 40. 4 E. IZ. HILL, D. R. HUSBANDS AND W. E. M. LANDS, J. Biol. Chem., 243 (1968) 4~40. 5 I:. T’OSSMAYER,G. L. SCAERPHOF,T. X.4. R. DUXBELMA;~~, L.M.G.X~AN GOL~E AND L.L.M. VAN DEENEN, Biochim. Biophys. Acta, 176 (1969) 95. 6 IV. E. M. LANIX API‘D1. MERKL, J.Biol. Chem., 238 (1963) HgS. 7 A. K. HAJRA, Bzochem. Biophys. Xes. Common., 33 (1968) gzg. X J. A. BALINT, D. A. BEELBR, D. H. TREBLE AN'D H. L. SPITZER, J. Lipid lies., 8 (1967) 486. g C;. A. E. ARVIDSON, Ewopean J. Biochem., 4 (1968) 478. IO II 12 r3
G. A. l2. ARVIDSO~;,
European
J. Bzochem.,
5 (1968)
415.
D. RYTTER, J. E. MILLER MD IV. E. CORPI~TZER, B&him. Biophys. A&, 152 (1968) 418. H. KANOH, Biochim. Biophys. Acta, 176 (1969) 756. J. B. MUDD, L. hl.G. VAX Gome AND L. L. M. Vnx DEEXES, Biochim.Biophys. Acta, 176
(1969) .517. 14 B. I’. hExxEDY,
J. Binl. Chem., 222 (1950) 185. 15 W. C. SCHNEIDER, W. C;.FISCUS AND J. A. R. LAWLER, Anal. Biochem., rq(~gGG) IZI. 16 G. A. E. ARVIDSON, ,!, Lipid I&s., 6 (1965) 571. 17 0. REXKONEN, Biochznz. Riofihys. Acta, 125 (rg66) 288. 18 L. M. G. VAX GOLDE AXD L. I,.M. VAK DEEKEN, B&him. Bi~pkys. Acta, 125 (1066) 496. 19 D. J. HANAHAN AND R. VERCAMER, J. Am. Chem. Sac., 76 (rgp+) 1804. 20 I;.SXYI~ER, Anal.Biochem., g (1964) 183. 21 A. G. GORNALL, C. S. RARDAWILL AND M. II. DAVID, J. Biol. Chcm., 177 (1949) 751. Hiochim.
Riophys.
Acta,
218
(1970)
249-258