Biosynthesis of 2— Aminoethylphosphonolipids
in Fowl Liver Homogenates
Masato TAMARI
Laboratory of Food and Nutrition, Faculty of Education, Nagasaki University, Nagasaki 852, Japan.
(Received March. 14, 1997)
Synopsis
The incorporation of 14C-2-aminoethylphosphonic acid (2-AEP) into phospholipids of liver subcellular fractions were maximum in the unclei lipids, and similar for mitochon- dria and microsome.
Thin-layer chromatography was employed to demonstrate that 14C-2-AEP was incorporated in vitro into phospholipids of the fowl liver. 14C-2-AEP was incorporated into at least three phospholipids of the fowl liver. The formation of nucleotide-bound-2- AEP by liver homogenate was demonstrated. The rate of phosphonolipids formation from free-14C-2-AEP was low compared to that from nucleotide-bound-2-AEP. The results indicate that nucleotide-bound-2-AEP is an intermediate in the synthesis of 2- aminoethylphosphonolipids.
Introduction
The phosphonic acid analog of phosphorylethanolamine, taurine and fi-alanine, 2- AEP (2-aminoethylphosphonic acid) occurs free and as major constituent of the phospholipid of protozoa' -3), marine 4 ) and fresh water invertebrates5' 6) and mammalian tissues?-9). Since the isolation of 2-AEP, many aminophosphonic acids structurrally related to 2-AEP have been discovered in biological materialsm-12) .
Studies on the incorporaton of 2-AEP and related compounds into animal tissues
Abbreviations:
2—AEP: 2—Aminoethylphosphonic acid CMP-2—AEP: Cytidine—mono—phosphate-2—AEP
Key words:
2—aminoethylphosphonic acid;
2—aminoethylphosphonolipid;
cytidine—monophosphate-2—aminoethylphosphonate
were reported in several laboratoriesl3‑19).
Kandatsu and coworkersl3, 14) found that 32P‑2‑AEP administered intraperitoneally was incorporated into rat liver lipids and insoluble residues to the extent of 3. 3 6 and 9. 6
6, respectively.
Krause et al.17) reported that ciliatine was incorporated into lipids of rat liver, that the peak of incorporation was between 3 and '6 hr, and that the half‑1ives of labeled lipids were approximately 4 days. Curley and Hendersonl5) found that 14C‑ 2 ‑AEP administered intravenously was incorporated into rat liver lipids (16 6 of the total injected 2 ‑AEP) .
Rosenthal and Pousoda20) reported that eight synthetic phosphonates containing analogs of lecithin, cephalin, and phosphatidic acid inhibited the hydrolysis by phospholipase C, the licithin analog were the most active. Dana and Douste‑Blazy21) reported that 2 ‑AEP inhibites the utilisation of 32P for the synthesis of phosphatidic acids, phosphatidylethanolamines and phosphatidylcholines, and also observed that 2 ‑ AEP inhibited decarboxylation of phosphatidylserine in the mitochondrial suspension of rat liver22). Bjervel6) demonstrated a low incorporation of trimethylaminoethy‑
lphosphonic acid into phosphonolipids in a rat liver homogenate.
Liang and Rosenberg23) demonstrated the ability of a Tetrahymena homogenate to form the complete lipid from diglyceride and cytidine monophosphate (CMP)‑
aminoethyphosphonate. We reported24. 25) that phosphonolipids were biosynthetised from 2 ‑AEP in rat liver.
The present work aimed to confirm the incorporation of 14C‑ 2 ‑AEP into fowl liver
lipids and to investigate the possibility of its incorporation through a nucleotide‑bound‑intermediate (CMP‑ 2 ‑AEP) .
Materials and Methods
Synthesis of [ I , 2 ‑14C]‑ 2 rminoethylphosphonic acid
The procedure for the synthesis and purification of[ I , 2 ‑ 14C]‑ 2 ‑AEP was car‑
ried out by the method described by previous report25).
Preparation offowl liver homogenate
A fowl weighing approx. I kg was killed by decapitation and liver immersed in cold O. 25M sucrose (pH 7. 4) after perfusion by O. 9 6 NaC1 solution. 10 6 homogenate was per‑
formed in Potter Elvenhjem homogenizer. Nuclei and cell debris were removed by cen‑
trifugation for I , OOO g x 10 min. The incubation medium contained ATP 5 x 10‑ 3 M, CTP 5 x 10‑ 4 M, MgC12 5 x 10‑3M, tris‑HCI buffer(pH 7. 4) 5 x 10‑ 3M and homogenate corresponding to 0.5g of liver in a total volume of 5 . O ml. In same experiments D‑a‑diglyceride ( 5 x 10‑ 3 M) was added in 10 mg of Tween 20. Incubations were perform‑
ed at 37 C in a shaking water bath. The reaction was stopped by the addition of 2. O ml of 5 6 trichloroacetic acid.
Proteins were removed by centrifugations and the supernatant was treated with 5. O ml of a suspension of Charcoal (20mg/ml)26).
After centrifugation the supernatant was sucked off, and the charcoal pellet washed 6 times with 15 ml of O. 9 6 NaC1.
Nucleotide‑bound‑ 2 ‑AEP was eluted from the charcoal by the addition of 10 ml of formic acid. After centrifugation the supernatant was filtered and the charcoal washed 2 times with 10ml of formic acid. The formic acid was evaporated to dryness and I ml of methanol was added. O. 2ml of the nucleotides solution was transferred to a vial for coun‑
ting. Subcellular fractionation of fowl liver homogenate was performed according to Vignais and Nachbaur27) .
Lipid extraction
The extraction chloroform‑methanol ( 2 Folch et al28) .
of lipids from proteins 1 , v/v) and washed with
pellet was carried out using 0.017 6 MgC12 as described by
Thin‑layer chromatography
For the separation of phospholipids and phosphonolipids on Silicagel plate (Merck, 20 x20cm, 2mm thickness)was performed usign following solvent systems ;
1 : Chloroform : Methanol : Acetic acid : Water (75 : 45･ : 12 : 6 ) 29) 2 : Chloroform : Acetic acid : Methanol : Water (75 : 25 : 5 : I .8)30) 3 : Chloroform : Methanol : Water (60 : 35 : 8 ) 31)
4 : Chloroform:Methanol:Acetone:Acetic acid:Water(5 : I : 2 : I : 0.5)32) Radioactive lipids were detected on developed plates with autoradiography.
2 ‑AEP was detected on developed plates with Rosenberg's reagent33) and ninhydrine.
Determination of phosphorus
Determination of phosphrus was performed by the method of Chen et al34) mineralization with HCIO 4 and H 2 SO 4 mixture for 12 hours.
Reagents
A11 reagents used were eithers of analytical grade or of the highest purity.
was prepared by the method described by Kosolapoff35) .
af ter
2 =AEP
ResultS and DiscussiOn
Incorporation in vitro of 14C‑ 2 ‑AEP into phospholipids of subcellular fractions of the fowl
liver .
Results of the incorporation of radioactivity'into phospholipids of liver subcellular fractions of fowl after the incubation of 14C‑ 2 ‑AEP are summarized in Table I . These
results indicated that the specific radioactivity of phospholipids phosphorus was maximum in the nucleic lipid , and this activity was approximately 2 times greater than that of other fractions lipids except that there is no activity in supernatant. In the same experiment, radioactivity in fractions occurred at a higher percentage radioactivity in the nuclei and represented about 75 6 of the total phospholipids of liver. The radioactivity in mitochon‑
dria and microsome indicated the similar incorporation. Microsomes appear to be the primary site for phosphonolipid synthesis36).
Table I . Incorporation of 14 C‑2‑AEP (38690 dpmlpg p)into Phospholipids of Subcellular Frac‑
tions of the Fowl liver Fourty‑eight Hours After an Incubation with Homogenate.
Subcellular fractionation of fowl liver homogenate was performed according to Vignais method27). The extraction of lipid from fractions was carried out using a chloroform:
methanol by the method of Folch et al28).
Fraction Total radioactivity (d pm)
total ( 6)
Specific activity (dpm/pg p)
Nuclei Mitochondria Microsome Su pernatant
358140 55890 57780
75.7
1 1 .8
12.3
63 30 36
However, since the specific activity of the labelled phosphonolipids in this study was approximately the same for the two subcellular fractions after incubation it would appear that there is an exchange of phosphonolipids between the various
subcellular fractions. Our findings support those of other investigators7, 13) that organisms which do not synthesize 2 ‑AEP do incorporate this compound into their tissues lipids. It has been proposed that this synthesis of phosphonolipids. occurs by the phosphono base utilizing the same enzymes which transfer phosphobases from the
cytidine phosphobase into phospholipid.
Figure I shows the distribution of radioactivity on the superimposed thin layer
: ‑
J‑'. T g ;'
x
Fig. I . Autoradiograms of 14C‑ 2‑ AEP Incor‑
‑1hli
porated into Phosphonolipids by Fowl Liver Homogenate.
Solvent systems: No. 3 (First) and No.
4 (Second)
chromatogram. A thin layer plate two‑dimensionally developed with the two solvents revealed three 14C‑ 2 ‑AEP‑containing substances. These results indicate that 2 ‑AEP is incorporated into at least three lipids of the fowl liver.
Tamari et al 14) reported that the rat does not have the ability to decompose the C‑P bond to phosphoric acid. Therefore, these observations indicate that the fowl can incor‑
porate 2 ‑AEP into liver phosphonolipids. But, it was not possible to identify this area with iodine vapor because of the low concentration of phosphonolipids.
Incorporation offree 14C‑ 2 ‑AEP into lipids by fowl liver homogenate.
Table 11 shows that fowl liver homogenates can incorporate 14C‑ 2 ‑AEP ipto phospholipids in vitro. The incorporation was very small, the maximal incorporation was 3 x 10‑ 3 6 at 60 min. of reaction. Bjervel6) reported that 3 ‑trimethylaminopropyl‑
phosphonic acid was incoroporated into phospholipids by rat liver homogenate in vitro, and the maximal incorporation was 2 x 10‑ 6 6 at 30 min. of reaction.
Table IL . Incorporation of 14C‑2‑AEP into Phospholipids by Fowl Liver Homogenate.
A 10 6 fowl liver homogenate was prepared in 0.25 M sucrose. Nuclei and cell debris were removed by centrifugation for 1000g x 10 min. The incubation medium contained ATP : 5, 10‑ 3 M; CTP : 5, 10‑ 4M; MgC12 : 5, 10‑ 3 M; a, p‑
diglyceride : 5, 3 x 10‑3 M; Tween 20 : 10 mg; Tris‑HCI buffer (pH, 7, 4) : 5, 10‑ 3 M; and homogenate corresponding to 0.5g of liver in a total volume of 5, O ml. 14C‑ 2‑ AEP (38690 dpm/pg p)was added as indicated.
Incubations were performed at 37 iC in a shaking water bath. The phospholipids was extracted as described in methods.
Incubation time (min)
Addition 14C‑ 2‑ AEP (pg P/tube)
Total radioactivity
(d pm)
Incorporation ( 6)
15 15 60 60
46 230 46 230
o 38 68 26
O 4 x 10‑4 3 xl0‑3 4 x 10‑4
Conversion of 14C‑ 2 ‑AEP to a charcoal adsorbable compound by fowl liver homogenate.
The formation of nucleotide bound 2 ‑AEP by liver homogenate was then in‑
vestigated. Homogenates was prepared as described in the methods. Reaction mixtures were incubated for 1 5 min. and 60 min. at 37 C and precipitated by the addition of 2. O ml of 5.0 trichloroacetic acid. The protein‑free extracts were treated with charcoal as
described above and the formation of charcoal‑held radioactivity was assayed. The conver‑
sion of 14C‑ 2 ‑AEP to a nucleotide derivative by fowl liver homogenate is shown in Table m.
When the 14C‑ 2 ‑AEP was used as substrate, the formation rate was maximum for 1 5 min., O. 7 6 of nucleotide bound 2 ‑AEP were formed. These results indicate that nucleotide‑bound phosphonate analogs, CMP‑14C‑ 2 ‑AEP had been formed, although these compounds were not isolated, and these results indicate also that CMP‑ 2 ‑AEP is an intermediate in the phosphonolipid synthesis.
Table 111: . Conversion of 14C‑2 AEP to a Charcoal‑adsorbable Compound by Fowl Liver Homogenate.
The preparation of reaction medium and the incubations were performed as described in the Table 11 . The CMP lerivative was separeted as described in the methods.
Incubation time (min)
Addition 14 C‑2‑AEP
( pgP /tube)
Total radioactivity
(d pm)
Specif ic activity (dpm/pg p)
Conversion
(96)
15 15 60 60
46 230 46 230
6648 62675 7296 26016
1 74
1843 38 195
o. 37 o. 70 o. 40 o. 29
Detection of nucleotide‑bound 2 ‑AEPfrom the reaction mixture offowl liver homogenate and 14C‑ 2 ‑AEP.
The filtrate from the charcoal was suspended in 30ml of 6 N HC1, the suspension was refluxed at 120 C for 24 hrs and the HCI was removed under reduced pressure to dryness . The residue was dissolved in water and acid hydrolysate was chromatographed on Kieselgel G plate with solvent system of n‑butanol : acetic acid : water ( 4 : I : 2 , v/v) . Thin‑layer radioautogram showed that the spot contained a radioactive compound which was ninhydrine‑and Rosenberg's reagent positive and migrated as 2 ‑AEP
(Fig. 2 ) .
Since exploratory experiments showed that free 2 ‑AEP was not absorbed on char‑
coal under the condition as described in methods, these results suggested the possibility of a nucleotide‑bound‑ 2 ‑AEP in the original extract.
Incorporation of radioactivity from CMP‑derivative into phospholipids by fowl liver homogenate.
Table IV shows the incorporation of CMP‑derivative into phospholipids by fowl liver homogenate. The rate of incorporation was small, the results was O. 32 6 and
0.14 at 2 hrs and 4 hrs of incubation, respectively. Our results indicate that the rate of phosphonolipids formation from CMP‑derivative is high compared to that from 14C‑ 2 ‑AEP (see Table Il ) .
From these results, there is possibility to synthesize phosphonocephalin from 2 ‑ AEP via the intermediate formation of CMP‑ 2 ‑AEP in animal liver.
These studies are of importance, in that 2 ‑AEP is found in miligram amounts in common human foodstuffs such as edible
shellfish and in smaller amounts in tissues of ruminats such as cattle and goats . Further studies on the in vivo and in vitro metabolism of this and related compounds are progress in our laboratory.
/i ¥"' l
.
1 z
̲,.l
3
Flg 2 Thln layer Chromatogram of
Water‑soluble Phosphate Obtained from a Charcoal‑adsorbable Com‑
pounds by Hydrolysis with 6 N
hydrochloric acid for 24 hrs at 1 20 C . Solvent system : nJbutanol :acetic acid :
water(4 : I : 2) Sample I : Authentic 2‑ AEP
2 : Water soluble phosphates ob‑
tained by hydrolysis 3 : Standard phosphoric acid Spot O : Ninhydrin( and Rosenberg' s‑
reagent positive
e : Radioautogram, ninhydrine‑
and Rosenberg's reagent
positive
L ., ' ..., : Rosenberg's reagent positive
Table W。Incorporation of Radioactivity from CMP−derivative into Phospholipds by Fowl Liver Homogenate．
The preparationofreactionmediumandtheincubationswere perfomed as described inthe Table I［．CMP−derivative（71，
864dpm）was added to incubation mixture．
Incubation time （hr）
Total radioactivity （dpm）
Incorporation （％）
2 4
234 98
0．32 0．14
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