Phosphatidylcholine biosynthesis in Tetrahymena pyriformis

r4*

BIOCHIMICA ER BIOPHYSICA ACTA

BBA 55654

PHOSPHATIDYLCHOLINE

BIOSYNTHESIS

IN

TETRAHYMENA

of Chicago, Chicago,

Ill. 60637 (U.S.A.)

PYRI-

FORMIS

JOSEPH Department

DONALD

SMITH

of Biochemistry.

AND

JOHN

The University

H. LAW

(Received September znd, 1969)

SUMMARY I. Phosphatidylcholine biosynthesis in Tetrahymena pyriformis was investigated. Experiments in vivo using [Me-W]methionine showed incorporation of radioactivity into the choline portion of phosphatidylcholine. No methylation of z-aminoethylphosphonic acid was detected. Incorporation of the intact choline molecule into lecithin was demonstrated when the cells were grown on [Me-14C]choline. 2. The transfer of the methyl group of [Me-14C]S-adenosyl-L-methionine to form lecithin was demonstrated in cell-free systems. The activity was localized in the microsomal cell fraction. The enzyme system accepted exogenous phosphatidylmonomethylethanolamine as substrate but not phosphatidylethanolamine or phosphatidyldimethylethanolamine. 3. CDP-choline : diglyceride phosphocholinetransferase activity was localized in the mitochondria. The enzyme was twice as active with the optimal concentration of Mna+ as Mg2+; the enzymatic activity was inhibited by low concentrations of Ca2+ or Hg2+.

INTRODUCTION

Phosphatidylcholine biosynthesis is known to occur by two different pathways in a wide variety of organisms. KENNEDY AND WEISS~~~ and WEISS et aL3 showed that incorporation of the intact choline molecule in rat liver and chicken liver, proceeds through phosphorylcholine and CDP-choline, the final step being the transfer of the phosphocholine of CDP-choline to a r,a-diglyceride. This pathway has been studied by other workers and has been shown to occur in other organisms, including frogs?, snails4, and several insect+“. The CDP-choline : diglyceride phosphocholinetransferase was demonstrated by SCHNEIDER~ to be localized in the microsomal fraction of rat liver and by CHOJNACKI AND KORZYRSKI~ in the mitochondrial fraction of chicken liver. The second pathway involves the conversion of phosphatidylethanolamine to phosphatidylcholine by three sequential methylation reactions utilizing the methyl Biochim.

Biophys.

Acta,

202 (1970) 141-152

J. D. SMITH, J. H. LAW

142 group of S-adenosyl-L-methionine. conversion, the first responsible phatidylmonomethylethanolamine,

In Neuros$ora

cyassa two enzymes

catalyze

this

for the transfer of one methyl group to form phosand the second for the formation of phosphatidyl-

choline7p8. The methylation system has also been demonstrated in rat livers-14, Agrobacterium tumefacie&5, dog lungr6, Ochromonas malhanaemsis17, and Euglena gracilis (ref. 18). E. gracilis lacks the phosphocholinetransferase the

genus

bacteria

Agrobacterium

lack

the

complete

neither possess phosphatidylcholine

incorporate

la. Most bacteria except those of methylation pathwaylO. In general,

nor are they able to take up choline and

it into their lipidsZO. The Agrobacteria,

while able to methylate

tidylethanolamine to lecithin are apparently unable to incorporate into lecithin; SHERR AED LAW~Ohave shown that these organisms

phospha-

preformed choline lack the phospho-

cholinetransferase. Recently, various investigators have shown that the phospholipids of Tetrahymena pyriformis, a free-living protozoan, possess in addition to ethanolamine phosphate, its phosphonic acid analogue z-aminoethylphosphonic acid2r-2*. LIANG AND ROSENBERG~~ showed aminoethylphosphonic

that cell-free extracts of the organism could incorporate 2acid into a phospholipid by the Kennedy pathway. THOMPSON

(ref. 24) has presented evidence that lecithin is involved in the formation of the carbon-phosphorus bond. Earlier he mentioned that the methylated derivatives of 2-aminoethylphosphonic

acid do not occur in Tetrahymena,

but presented

no experi-

mental

evidence for this contentionzl. We felt that a more detailed knowledge of lecithin metabolism in Tetrahymena would provide a better basis for study of the phosphonic acid metabolism in this organism.

The results of these studies are presented

in this paper.

ExPERIMENTAL PROCEDURE Materials Organism. Tetrahymena pyviformis obtained from Dr. Hewson Swift.

Mating

Type

II variety

I (WH

14) was

Radioactive compounds. [Me-14C]methionine, [Me-l*C]S-adenosyl-r.-methionine and [Me-l*C]choline were obtained from New England Nuclear Corporation, Boston, Mass. [r,z-14C,]CDP-choline was obtained from Tracerlab, Waltham, Mass. Phospholipids. Lecithin was purified from crude soybean phospholipid (asolectin) by the method of CHUNG AND LAWNS.Escherichia coli PE was the gift of Dr. T. 0. Henderson. Phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine from N. uassa were the gift of Dr. J. F. Nyc. Thin-layer silica gel plates were the product of Brinkmann Instruments, Westbury, N.Y. Phosphonic acids were purchased from Calbiochem, Los Angeles, Calif. Unisil silicic acid was obtained from Clarkson Chemical Corporation, Williamsport, Pa, All other chemicals and solvents were commercial products. Methods Culturing Tetrahymena was maintained on a medium consisting of 2% proteose-peptone Stock cultures (Difco), 0.2% g lucose, 0.1% yeast extract and 0.003% iron-EDTA. Biochim.

Biophys.

Acta,

202

(1970)

141-152

PHOSPHATIDYLCHOLINE

BIOSYNTHESIS IN TETRAHYMENA

I43

were kept at room temperature in IO ml of medium in screw-cap culture tubes and transfered every 2-3 weeks or as needed. For large cultures 500 ml of fresh medium in a z-1 erlenmeyer flask was inoculated with a ro-ml culture and grown on a shaker at 37’. When radioactive compounds were included for growth of the organism, the medium was autoclaved with the labeled compounds present. After 24 h (log phase) or 48 h (stationary phase) of growth, cells were harvested in a Servall Refrigerated Centrifuge type RCZ-B, at 500 xg. Lipids were extracted by the method of FoLCH et CZ~.~~. Phospholipids were separated on a column of Unisil. The total lipids were dissolved in benzene and placed on the column. The neutral lipids were eluted with chloroform, phosphohpids with methanol, and more polar lipids with 1074 acetic acid in methanol. Recovery was greater than 99% by weight and radioactivity. ~~YomatogYa~~y T%?z &EY. The system of ARTOI@~, chloroform-methanol- 7M NH,OH (60:35 : 5, by vol.) was used for the separation of phospholipids on silica gel G. Detection of spots was by iodine vapors and by comparison with authentic phospholipids run in parallel with the sample. Paper. For the separation of phospholipid bases the system of BREMER AND GREENBERG~~ was employed. Standard bases were added to each sample before application to Whatman No. I paper. Detection was by a combination of the quinonez8 and Dragendorff reagents 29, The paper was sprayed with the quinone reagent for ethanolamine, monomethylethanolamine and dimethylethanolamine. After color development (about z h) the strip was sprayed with the Dragendorff reagent for choline. Paper strips were counted in the Nuclear Chicago Actigraph III Model 1002 strip counter and thin-layer plates in the Model 1006 attachment. Detection reagents were not used until after the plates and strips had been counted. The radioactive compounds on thin-layer chromatograms were scraped off the plates into screw-cap test tubes and hydrolyzed in 6 M HCl in the autoclave for 5 h (ref. 7). The lipid components were extracted with chloroform. The aqueous phase was concentrated and applied to paper. Mild alkaline hydrolysis was performed by the method of DAWSON~~. Enzyme assays The preparation of the subcellular fractions used in the enzyme assay is given in Scheme 2. Protein concentration was determined by the biuret methodsI. Usually the filter-paper-disc assay method of GOLDFINE~~ was used. Elowever when product characterization was needed, the method of KANESHIRO AND LAW'" was employed. Lipids added to the reaction system were suspended in the appropriate buffer by sonic oscillation. Scintillation counting was performed in a Packard Tri-Carb using a dioxane scintiliation system for aqueous samples and a toluene system for the filter paper discs33.

Biochim. Biophys. A&z,

202 (1970) 141-152

J. Il. SMITH,

I44

J. H. LAW

RESULTS

Cells growz. on jMe-14C]nzetCzionine

(I)

Cells grown to stationary phase on a medium containing nine (16,L/pmole) were harvested and the lipids were isolated

3 ,LK [Me-14C]methioas shown in Scheme I.

Cells ,

(Ce”trifurd500X

g,IO,n,a,P

Cells

Medium

I

Washedl9% KCI, 10 ml/g cells. centrifuged 500 X g, IO mm

Suspended same volumeP9% KCl. extracted 2.5 vol. chloroformethanol

(2.1. v/vi

I Aqueous phase

I Grganic phase

1

I

Extracted I vol. chloroform 1

Pooled I Washed. 0.1 vol.

I

“pure solvents upper

Orgmc

“i”““‘I phase

Pooled aqueous

(lipIds)

Scheme

I.

Procedure

for the isolation

phase

of lipids from T. pyvifo_;mis.

The distribution of radioactivity in the various fractions is shown in Table I. A portion of the lipid fraction was further fractionated on a column of Unisil as described in Methods (Table II). Fractions I and III were not investigated further. Part of the material was subjected to thin-layer chromatography; the remainder of the material was hydrolyzed and chromatographed on paper. The only radioactive phospholipid was lecithin and the only labeled base was choline. There was no indication of phosphatidylmonomethylethanolamine or phosphatidyldimethylethanolamine. Nor was there any indication of the n’-methyl derivatives of z-aminoethylphosphonic acid, all of which remain at the origin in this chromatographic system. TABLE

1

DISTRIBUTION OF RADIOACTIVE LABEL IX

THE

VARIOUS

FRACTIONS

OF CELLS

GROWN

ON

[Me-‘“Cl-

METHIONISE

Tetrahymena was grown on a medium containing 3 $2 [Me-14C]methionine (16 pC/ymole). Cells were harvested after 48 h of growth at 37” and fractionated according to the scheme presented in Scheme I. ____ - ~____ Counts/min x roA5 yO Fraction Growth medium KC1 wash Lipids Aqueous phase and residue

56.5 1.02

5.99 2.23 65.75

B&him.

Biophys. Ada,

202 (1970)

141-152

85.9 1.6 9.1 3.4 100

PHOSPHATIDYLCHOLINE TABLE

A

OF RADIOACTIVITY

IN

THE

of of

the

lipid

(I

Unisil

from

the

experiment

cm x 20 cm)

LIPID

presented

as described

in the

FRACTIONS

FROM

[Me-%]METHIONINE

in

I. Chloroform

Neutral

hlethanol 10% Acetic

acid

in

I was

further

fractionated

Counts/min x Io-3 lipids

on

a

“/o

8.4

Phospholipids “Polar lipids”

methanol

Table

text.

Identity

Eluting solvent

II. III.

SEPARATED

CELLS

portion

column

I45

IN TETRAHYMENA

II

DISTRIBUTION GROWN

BIOSYNTHESIS

8.1

92.25

89.3 2.6

2.7

(II) Cells grown on [MeJ4C]choline

Cells were grown on a medium containing 1.3,uC [Me-Wlcholine (47 &/~mole) and harvested after 48 h of growth by the procedure shown in Scheme I. The distribution of label among the isolated fractions is shown in Table III. Further fractionaTABLE

III

DISTRIBUTION

OF RADIOACTIVE

Tetrahymena

was

cells

were

in Scheme

harvested

grown after

KC1

medium

wash

Lipids Aqueous

LABEL a

medium

48 h of growth

IN

CELLS

GROWN

containing at 37” and

I.3

ON

[Me-%]CHOLINE

&

[Me-%]choline

fractionated

according

(47 to the

,uC/,umole). procedure

The given

I.

Fraction Growth

on

phase

and

residue

Counts/min x 10-j

oh

24.9

83.3

1.67

5.6

0.81

2.7

2.51 28.89

8.4 100

tion of the lipids demonstrated that all of the radioactivity was associated with the lecithin. Paper chromatography of each of the fractions before and after acid hydrolysis showed that all of the radioactivity was present as free choline. No evidence of choline oxidation or redistribution of the methyl groups was observed. (III)

Phospholipid N-methyltransferase(s) The microsomal fraction (see V) was used for assays of this enzyme system. The general properties of this system are shown in Table IV. The enzyme required phosphatidylmonomethylethanolamine for maximum activity, but was not stimulated by either phosphatidyldimethylethanolamine or phosphatidylethanolamine. The slight inhibition by EDTA is difficult to explain since the system is not dependent on any divalent cations for its activity. Probably it is due to disruption of the microsomal particle, since attempted solubilization of the enzyme by sonication or treatment with detergents resulted in complete loss of activity. Product identity is shown in Figs. Ia and Ib. The thin-layer chromatogram (ra) demonstrates that the major portion of the radioactivity was associated with the lecithin peak. The peaks of greater RF were non-enzymatic degradation products of S-adenosyl-I,-methionine. Standard methylthioadenoisne and methylthioribose were prepared according to the method of SMITH AND SCHLENK~~. Samples of S-adenosylB&him.

Bioflhys. Acta,

202

(1970)

141-152

J. D. SXITH, J. H. LAW

146

EFFECT OF

VARIOUS

LIPIIJS

AND

POSSIBLE

COFACTORS

On- THE

hlETHYLATION

SYSTEM

The enzymatic reaction was measured by the filter-paper-disc assay methodS2. The incubation system consisted of 20 ,ul enzyme, 0.1 mM jJ~e-‘*C,!S-adenosyl-L-methionine, and enough 0.1 12 Tris-HCl (~15 8.5) to bring volume to 0.2 ml. The reaction proceeded for 30 min at 37’; aliquots wre withdrawn with ~1 pipettes and the liquid placed on zz-mm discs of Q’hatman No. 3 filter paper. The discs were dried in a hot air stream, placed in ION’, trichloroacetic acid for 30 min, then successively washed in 5 “,b trichloroacetic acid, and two changes of water for 15 min each. Thz discs were air dried and counted. __ _.._ ,____. .~ ~~ __ Addifiom

lncoyboratiolz (nmoles of ‘&&HI grozlp

PPVl’ag +wteis)

-~None 0.2 p&f phosphatidylethanolan~ille

0.5 & 0.1*

0.2 @I

phosphatidylmonomethylethanolaminc phospha~id~ldinlethylethanolamine I .o ,uM lecithin + phosphatidylmonomcthylethanolamine i- phosphatidylmonomethylethanolamine +phosphatidylmonomethylethanolamine + phosphatidylmonomethylethanolamine + phosphatidylmonomethylethanolamine S-adenosyl-homocysteinc

0.2

p&I

$ + + -t +

o. I mM EDTA o. I mM Hg”+ 8.5 mM cysteine o. I M 1\Ig2-i40 PM

Standard

deviation

27 TOO

0.7 o I.4

40 o 7s

0.8

43

27

I .5

83

I.7

94 ~

~_.__

53

of the mean for three determinations

5 IN

0

0.5 I.8

0.8 ~-

*

0

Maxivzwz 4 cliaitv

DISTANCE

km)

IO

/

F

Fig. I. Product characterization of the methyltransferase reaction. a. Thin-layer chromatogram. b. Paper chromatogram of the water-soluble portion of the lipid hydrolyzate. LYSOLEC, Iysolecithin; LEG, lecithin ; PE, phosphatidylethanolamine: FM, phosphatid~rlmonomethylethanolamine PD, phosphatidyldimethylethanolamine. Biochim.

Bio$hys.

Acta,

202 (Ig7o)

141-152

PHOSPHATIDYLCHOLINE

BIOSYNTHESIS IN TETRAHYMENA

I47

L-methionine incubated in the system without enzymegave these breakdown products. Fig. Ib is a paper chromatogram of the water-soluble radioactivity of the sample after acid hydrolysis. As with the incorporation in viva of methionine, there was no evidence of the intermediates monomethylethanolamine or dimethylethanolamine, or the methylated phosphonic acids. The products were the same whether only the endogeneous lipids were present or if phospbatidylethanolamine, phosphatidylmonomethylethanolamine or phosphatidyldimethylethanolamine were added to the incubation system. Addedlipids did not cause a~~mu~ation of label in possible intern~ediates. Some of the characteristics of the enzyme system are presented in Figs. 2 and 3.

3.0

10 20 30 40

”

60

10 20

PI enzyme Kl?jJg/J_a

.: $ I .$ 2.0,L h E \ 9 g

PI

40

PM(2mM)

0 (I

1.0

‘-----:

iIc_

E

I

~ 60

7.0

6.0

9.0

10.0

PH

Fig. 2. pH-activity profile of the methyl transferase o-o, 0.1 &!ITris-HCl buffer.

10

25

81 SAM

system.

50

10

@m&i1

O-0,

20

30

timeimm) 0.1

M phosphate buffer:

Fig. 3. Characteristics of the methyltransferase system. The activity was assayed as described in the text. P&I, phosphatidylmonomcthylethanolamine; SAM, S-adenosyl -L-methioninc.

The pH optimum was 8.5. Saturation of the system by either of the substrates phosphatidylmonomethylethanolamine or S-adenosyl-L-methionine could not be achieved under the conditions employed. The reaction was linear for 30 min after which it levelled off. The presence of 40 (uM S-adenosylhomocysteine in the incubation mixture caused 60% inhibition of the reaction. (IV) CDP-choline:diglycerillle phosphocholinetransferase The mitochondrial fraction was used as a source for this enzyme. The requirements for the reaction and some inhibitors are presented in Table V. The enzyme required 5 mM Mna+ or IO mM Co 2+ for maximum activity. The concentration of Mg2+ at which the system was most active (IO mM) gave only half the activity of MrP or Co2+. Dependence of the enzyme on added diglyceride could not be demonstrated. Use of detergents produced inhibition of the enzyme; again, no stimulation by added diglyceride was observed. The enzyme was sensitive to Ca’J+ (707/oinhibition at 5 mM) and to Hga+ (IOOO~ B&xhina. Biophys.

A&a, ax

(1970) rq-r5z

148

J. D. SMITH, J. H. LAW

TABLE

V

REQUIREMENTS

OF

THE

PHOSPHOCHOLINETRANSFERASE

REACTION

o.e-ml incubations for z min at 37’; 0.1 M Tris-HCl (pH 8.5), 20 pl enzyme; ro ~1 [I,z-‘%,]CDPcholine, 9.3 &/pmole. The filter paper-disc assay used is described in the legend of Table IV. Incovpovation (nmoles/mg protein)

Additions

None

5 ro ro 5 5 5 5 5 5 5 5

mM mM mM mM mM mM mM mM mM mM mM

o/6Maximum activity 0

0

Mn2+ Mg2+ Co2f Mn2++o.z mg diglyceride Mn2+ + 5 mM Ca2+ Mn2++o.5 mM EDTA Mn2++o.r M KC1 Mn2+ + I o PM Hg2-C Mn2++8.5 mM cysteine Mn2++ IO ,uM CMP Mn2+ + I mM lecithin

* Standard

deviation

1.2

&

0.1%

IO0

0.6 1.3 1.I 0.4 0.7

50 IO0 100

33 62 83

I.0 0

8; 33

I.0 0.1

100

1.2

of the mean for three determinations.

at IO PM). 0.5 mM EDTA inhibited the reaction 40%, probably by removing some of the metal ion required for maximum activity. Some of the other properties of the enzyme are presented in Fig. 5. The pH optimum of the enzyme was 8.0-8.5 (Fig. 4). At lower pH values the enzyme was less active in phosphate buffer than in Tris or imidazole buffers. The product characterization of this reaction is shown in Fig. 6. The scan of the thin-layer chromatograph shows that only the lecithin fraction was radioactive. Upon acid hydrolysis and paper chromatography, free choline was the only radioactive compound. Mild alkaline hydrolysis gave a water-soluble radioactive compound which migrated with authentic glycerylphosphorylcholine. The radioactivity remaining in the organic phase (about zoo/O)coincided with lysolecithin on thin-layer chromato-

6.0

7.0

6.0

9.0

10.0

PH

Fig. 4. pH-activity profile of the CDP-choline:diglyceride 0.1 M imidazole buffer; 0.1 M phosphate buffer; O---Y, Fig. 5. Characterististics BiOChim. Biophys.

ACta,

of the phosphocholinetransferase. 202

(1970)

141-152

phosphocholinetransferase. O---O, 0.1 M Tris-HCl buffer.

O---O,

c.

O--O,

Mn2+; O--O,

Mg2+.

PHOSPHATIDYLCHOLINE

BIOSYNTHESIS IN TETRAHYMENA

I49

Fig. 6. Product characterization of the phosphocholinetransferase. the isolated lipids. LEC, lecithin.

Thin-layer

chromatogram

of

Fig. 7. Thin-layer chromatogram of the chloroform-soluble products of the mild alkaline hydrolysis of the lipids formed in the phosphocholinetransferase reaction. LEC, lecithin; LYSOLEC, lysolecithin.

graphy (Fig. 7). Th is was to be expected glycerylether phospholipidzl.

since the lecithin

of Tetrahymena

contains

(V) Localization of enzyme activities The subcellular fractionation of cells was carried out by the method presented in Scheme 2. The mitochondrial and microsomal fractions were examined by electron Washed

cells

Suspended

,

in 10 vol.

blended

30 sec.centrifuged

Pellet

500

X

g ,or

10 min

/y:y;y

Pellet

“cell

M BUCPOSP-

supernatant

,

pAzT---unbroken cells debris”

0.25

/o.j MhiS.lCI buffer, p” 7.4

z3upernatant

,

~z?yiY

30min

and supernatant

Pellet

,

~:;zFy:;~o

Pellet

rni”

supernatant Centrifuged l00000 x g, 60 min

4 Pooled mitochondria suspended in fresh buffer. centrifuged 10000 x g, 15 min Pe~“rta”t washed mitochondria _-L Pellet microsamcs

supernatnnt

Scheme 2. Preparation of subcellular fractions. All operations were carried out at 4’. The pellets were resuspended in fresh sucrose buffer with a Potter-Elvehjem homogenizer. Biochzruz. Biophys.

Ada,

202

(1970)

141-152

J. D. SMITH, J. H. LAW

150

microscopy to establish their identities. The microsomal fraction was essentially free of intact mitochondria, but the mitochondrial fraction contained considerable nonmitochondrial contamination. This could be reduced somewhat by resuspending the pellet in the sucrose-Tris buffer and resedimenting several times. The specific activities of the two enzyme systems for lecithin biosynthesis are shown in Table VI. The phosphocholinetransferase was predominantly localized in the mitochondrial fraction and the methylation system predominantly in the microsomes. There was a difference in the specific activities of the enzymes isolated from stationary or log phase cells. T_%BLE

VI

LOCALIZATION

OF THE

ENZYMES

OF LECITHIN

BIOSYNTHESIS

Incorporation of W label into lipids by each subcellular fraction as prepared according to Scheme 2. The enzymatic reactions were carried out as described in the text. Incor$oration (nmdeslmg Methvltransfevase , , Homogenate “Cell debris” Mitochondria Microsomes Supernatant

protein pev 15 win) Phos~hocholinetransfevase

Log phase

Stationary phase

Log phase

-.-Stationary phase

1.2

4.0 2.7 0.3

0.3 0.6 0.6 0.3 o

0.9 I.4 I.9 0.7 0 -~

1.2

0.4 0.8 0.4

I.2

0.3

------

.--

Both enzymes lost about ~0% activity in z4 h when stored at pH 7.4, either frozen or at 0’. If storage was at pH 8.5, the pH optimum of both enzymes, loss of activity was only 20% in several weeks at oO. Again, inactivation was more rapid if the systems were frozen. If isolation of the organelles was carried out in 0.1 M Tris-HCl (pH 8.5) without sucrose, the specific activities of each fraction were not as reproducible but the stability of the enzyme systems was better. DISCUSSION

The experiments presented in this paper indicate that lecithin biosynthesis in Tetrah~ena is similar to that in other organisms. The protozoan possesses a phospho~pid ~-methylation system which is mierosomal as is that of rat liver+x4, dog lungl*, 0. ~a~~a~e~s~s~~,and N. cyassa7*8.The Tetrahymena system could only be stimulated by exogenous phosphatidylmonomethylethanolamine but not phosphatidylethanolamine or phosphatidyldimethylethanolamine. While neither of the two intermediates (phosphatidylmonomethylethanolamine or phosphatidyldimethylethanolamine) could be detected in methylation studies either ilz viva or in vitro, the stimulation by added phosphatidylmonomethylethanolamine indicates that the formation of lecithin proceeds from phosphatidylethanolamine through phosphatidylmonomethyletl~anolamine and phosphatidyldimethylethanolamine as in the other systems studied. The CDP-choline : diglyceride phosphocholinetransferase is located in the mitochondria in Tetr~~ena. Thus the subcellular localization of this enzyme appears to differ among various organisms. The original work of WEISS et aL3 was done using Biochim.

Biophys. Acta,

202

(1970) 141-152

PHOSPHATIDYLCHOLINE

BIOSYNTHESIS

IN TETRAHYMENA

151

preparations from rat liver and chicken liver which contained both mitochondria and microsomes. Subsequent work has shown that in rat liver the phosphocholinetransferase is localized in the microsomess and in chicken liver in the mitochondria4. Two photosynthetic protozoa, E. gracilis and 0. malhamensis both apparently lack the phosphocholinetransferase17tl*. The lack of methylated phosphonic acids in the lipids of Tetrahymena has been mentioned beforeZ1 but without any presentation of experimental detail. This work has confirmed the observation. We were unable to observe an incorporation of choline into sphingomyelin either in vivo or in vitro even though Tetrahymena is reported to contain this comderivatives of z-aminoethylphospound?3>35. Similarly, the lack of the methylated phonic acid in Tetrahymena and their presence in other organisms36-39, poses some intriguing questions as to biosynthesis of the carbon-phosphorus bond. Thompson suggests that the phosphorus of lecithin becomes part of the phosphonic acidZ4. Certainly the presence of the methylated derivatives of 2-aminoethylphosphonic acid, in the phospholipids of Tetrahymena would probably interfere with carbon-phosphorus biosynthesis. On the other hand, since other organisms possess these methylated compounds, is their absence in Tetrahymena due to inability of the methylating enzymes to react with diacylglyceryl-z-aminoethylphosphonic acid or are the phosphonolipids and the enzymes compartmentalized in the organism ? ACKNOWLEDGMENTS

We are grateful to Dr. Hewson Swift for the electron micrographs of the subcellular fractions of Tetrahymena. J.D.S. was supported by a Predoctoral Fellowship (ES35485) from the Division of Environmental Health Sciences of the National Institute of Health. These studies were supported by a grant from the U.S. Public Health Service, National Institute of General Medical Sciences, No. GM 13863. REFERENCES I E. P. KENNEDY AND S. B. WEISS, 1. Am. Chem. SOL. 77 (1955) 240. 2 E. P. KENNEDY AND S. B. WEISS] 1. Bid. Chem., 22; i&56) 193. 3 S. B. WEISS, S. W. SMITH AND E. P. KENNEDY, J. Bid. Chem., 231 (1958) 53. 4 T. CHOJNACKI AND T. KORZYBSKI, Acta Biochem. Polon.. X (1963) 455. 5 H. D. CRONE, R. W. NEWBURGH AND C. MEZEI, J, Insect Physiol., 12 (1966) 6x9. 6 W. C. SCHNEIDER, J. Bid. Chem., 238 (1963) 3572. 7 G. A. SCARBOROUGHAND 1. F. NYC, I. Biol. Chem., 242 (1967) ., 238, & G. A. SCARBOROUGH AND j. F. NYC, kiochim. Biophys: Acta, r46-(1967) III. 9 C. ARTOM AND H. B. LOFLAND, Jr.,B&hem. Biophys. Res. Commun., 3 (1960) 244. IO C. ARTOM, B&hem. Biophys. Res. Commun., 15 (1964) 201. II J. BREMER AND D. M. GREENBERG, B&him. Biophys. Ada, 35 (1959) 287. 12 J. BREMER AND D. M. GREENBERG, Biochim. Biophys. Acta, 46 (1961) 205. 13 K. E. COOKSEY AND D. M. GREENBERG, Biochem. Biophys. Res. Commun., 6 (1961) 256. 14 D. REHBINDER AND D. M. GREENBERG. Arch. Bzochem.Biobhvs.. 108 f1065) IIO. I; T. KANESHIRO AND J. H. LAW, J. Biol. Chem., 239 (1964) ;7d5. ’ _ I’ 16 T. E. MORGAN, Biochim. Biophvs. Acta, 178 (1969) 21. 17 G. LUST AND L. J. DANIEL, *A&h. BiocheA. Biophys., 113 (1966) 603. 18 C. L. TIPTON AND M D. SWORDS, J. Protozool., 13 (1966) 469. Ig H. GOLDFINE AND M. E. ELLIS, J. Bacterial., 87 (1964) 8. 20 S. I. SHERR AND J. H. LAW, J. Bid. Chew, 240 (1965) 3760. 21 G. A. THOMPSON, Jr,,Biochemistry, 6 (1967) 2015. 22 C. R. LI~NG AND H. ROSENBERG, Biochim. Biophys. Acta, 156 (1968) 437. I

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