The effect of atp and Mg2+ on the synthesis of phosphatidylglycerol in Escherichia coli preparations

Biochitt7ictr et Biopl7ysicu Actrr. 337 ( I 974) 3 I 8-324 c Elsevier Scientific Publishing Company, Amsterdam

- Printed

in The Netherlands

BBA 56406

THE

EFFECT

OF ATP

AND

PHOSPHATIDYLGLYCEROL

Mg2+ ON THE IN ESCHERICHIA

G. BENNS

and

Deprrrtuwf7t

qf Biocl7et77istr.v, F~irmlt,r of Medicine,

(Received

SYNTHESIS COLI

OF PREPARATIONS

I’. PROULX

September

zath,

Univrrsitj~ of’ Otttrwtr. Ottuww (Ctrr7rrtkr 1

1973)

SUMMARY

The incorporation of srr-[U-‘4C]glycero-3-phosphate into lipids of Esclwricl~ia was studied. Addition of MgZf and ATP caused a marked increase in the incorporation of 14C label into phosphatidylglycerol which was not abolished by the presence of added palmitoyl-CoA or CTP. Degradation of phosphatidylglycerol with phospholipase C or by acetolysis revealed that most of the labelled precursor incorporated itito the unacylated glycerol moiety, the radioactivity of which substantially increased with addition of Mg 2+ , ATP and/or CTP. The results are discussed in terms of a partial synthesis of phosphoglycerides and the involvement of endogenous phosphatidyl precursors. coli preparations

INTRODUCTION

Recently, we reported on the distribution of label found in phosphoglycerides of Escherichia co/i homogenates incubated with sn-[U-‘4C]glycero-3-phosphate as precursor [I]. Mild alkaline hydrolysis and phospholipase C degradation of the phosphatides indicated that most of the radioactive substrate was incorporated directly. In the process, the unacylated glycerol moiety of phosphatidylglycerol became labelled to an appreciably greater extent than the phosphatidyl moiety. It was apparent from this that synthesis of phosphatidylglycerol was not completely de novo and proceeded partly from an endogenous pool of phosphatidyl precursor(s). Such a precursor pool was not formed, under the conditions specified, by the acyl dihydroxyacetone pathway [2]. The incubation media for these experiments usually contained ATP and Mg’+. These additives were later found to greatly influence the incorporation of sn-[U-‘4C]glycero-j-phosphate into phosphatidylglycerol as well as the distribution of label within this lipid. We now report these effects in detail and present evidence for an ATP plus Mg2+ dependent partial synthesis of phosphoglycerides in E. co/i. METHODS

E.coli

015 cells cultured

15 g bactopeptone,

to the late log phase in a medium containing per I, I g yeast extract, 20 g glucose and 5 g NaCl were harvested by

319

centrifugation at 5000 x g for I 5 min suspended in 0.07 M phosphate buffer (pH 7.3), and sonicated IO min in ice at 125 W with a Biosonik Jl (Bronwill Scientific Co.) cell disruptor. The homogenate was centrifuged at 3000 x g for IO min and the resulting cell-free supernatant was diluted with buffer to a required protein concentration determined by the method of Lowry et al. [3]. Particulate fractions were prepared by centrifuging the cell-free homogenate for 40 min at 34000 x g and washed once by suspension of the pellet in buffer followed by centrifugation. Incubation conditions were varied as specified in the text. Lipids were extracted by the method of Bligh and Dyer [4] and separated on plates of silica gel H containing (NH,),SO, and activated 90 min at I IO “C just before use. The solvent used in this case was a mixture of chloroform-methanol-water (65: 25:4, by vol.) [I 1. Alternatively lipids were separated on activated silica gel G plates with chloroform-methanol-acetic acid (65:25:8, by vol.) as solvent [5]. Both systems gave good separations for phosphatidylglycerol, phosphatidylethanolamine and cardiolipin which were revealed by the periodate-Schiff reagent, ninhydrin and/or iodine as well as by scanning each chromatoplate with an Actigraph III (Nuclear Chicago Corp.). The labelled fractions were further identified by mild alkaline hydrolysis [6] followed by chromatography of the deacylated products on Whatman No. I with butanol-acetic acid-water (5:4: I, by vol.) or propanol-ammonia-water (6: 3 : I, by vol.) as solvents. The thin-layer chromatography systems usually employed were not suitable for complete separation of cardiolipin and phosphatidic acid, however, in most experiments the latter substance was only a very minor component as could be ascertained by analyses of mild alkaline hydrolysis products. When incubation conditions were such that phosphatidic acid production was predictably large, the area of the chromatogram corresponding to phosphatidic acid and cardiolipin was removed and eluted. The components were then separated on silica gel G with chloroform-methanol-ammonia-water (70: 30: 4: 2, by vol.) as solvent. In this system phosphatidic acid remained close to the origin whereas cardiolipin had a R, value of 0.40-0.50. Phosphatidic acid isolated in this manner was characterized by mild alkaline hydrolysis and paper chromatography. Lipids and water-soluble products were counted as previously described [I]. Hydrolysis with phospholipase C obtained from Bacillus cereus [7] or acetolysis for 5 h as described by Renkonen [8] served to analyze the distribution of label in phosphatidylglycerol. Diglycerides were separated by thin-layer chromatography on silica gel G with light petroleum (b.p. 60-90 ‘C-diethyl ether-formic acid (55 : 45 : I .5, by vol.) as solvent. The water-soluble product, glycerophosphate, was identified by paper chromatography. In the case of acetolysis, the products were separated into triacetin and diacylmonoacetin as described by Renkonen [8]. sn-[LJ-‘4C]gIycero-3-phosphate was purchased from International Chemical and Nuclear Corp. and from New England Nuclear Corp. ATP, CTP, CoA and palmitoyl-CoA were purchased from Sigma Chemicals. t-[ i-‘“C]palmitoyl-snI ,2diglyceride (spec. act. 0.055 Ci/mole) was prepared from hepatic lecithin as previously described [I I]. Data presented are the average of two or more experiments yielding similar results.

320 TABLE

I

THE EFFECT OF Mg2+ IN THE srr-[U-‘4C]GLYCERO-3-PHOSPHATE

PRESENCE OF INTO LIPIDS

ATP ON THE OF E. COLl

INCORPORATION

OF

The incubation medium contained in 2 ml, 0.07 M phosphate buffer (pH 7.3) as solvent and diluent, mM CoA, 2.8 mM ATP, o. I mM palmitic acid sonicated in buffer, 0.2 PCi of sn-[U-“‘C]glycero-3phosphate-i”C (spec. act. 16 Ci/mole) 0.4 mM CTP, IO mM MgCI, when specified and E.cvli homogenate 1.1 mg protein, I ml. Incubations were for I h at 25 ‘C.

0.2

dpm recovered

Conditions

Phosphatidylethanolamine __~~~~ .~~ No Mg*+ IO mM Mg*+

RESULTS

AND

3 9oo 7 5oo

in each fraction Phosphatidylglycerol ~~. 2 400 1I 900

Cardiolipin

I 600 3 100

DISCUSSION

Results illustrated in Table I, indicate the stimulatory effect of Mg2+, in the presence of IO mM ATP, on the incorporation of sn-[U-‘4C]glycero-3-phosphate into lipids by E. coli homogenates. was increased to IO mM or greater, a marked When the Mg2+ concentration stimulation of phosphatidylglycerol synthesis occurred concurrent with only a moderate increase in the labelling of phosphatidylethanolamine. This result was tentatively explained on the basis of the known cation requirement of phosphatidylglycerophosphate synthetase and phosphatase reactions. Phospholipase C degradation of the phosphatidylglycerol formed indicated however that Mgzc stimulated incorporation mainly into the unacylated glycerol moiety. There was comparatively little increase in the labelling of the phosphatidyl residue such that the glycerophosphate/diglyceride ratio of counts augmented 2-j-fold. The results suggested one additional effect of Mg2+ under these conditions, namely that this cation caused an enlargement of a pool of endogenous phosphatidyl group precursors; otherwise, Mg2+ TABLE

II

THE EFFECT OF Mg2+ IN THE PRESENCE OF ATP ON THE DISTRIBUTION OF LABEL WITHIN PHOSPHATIDYLGLYCEROL FORMED FROM sn-[U-“CIGLYCERO-3-PHOSPHATE Labelled phosphatidylglycerol was formed under conditions similar to those described for Table 1 except that the protein concentration was increased to I I mg/ml and the ATP concentration was 2.8 mM. Expts I and 2 were performed in triplicate at 25 and 37 ‘C, respectively. .~.____ -__ ___ __ _~ Expt. Conditions for Counts recovered in products obtained after phospholipase C No. phosphatidylglycerol hydrolysis of phosphatidylglycerol synthesis 5) GlyceroB/A ratio ~_.__~___ ~_ _ (A) DiMgz+ Phosphatidylglyceride phosphate glycerol formed (mM)

(dpm) I

0 10

2

0 10

I 32

800 240 900 123 300 23 I 900

~____ 39 go0 43 3oo 41900 30 100

.-__. 92 9oo I 92 700 80 100 176 200

~~~~~~~ ~~ _ . 2.4 4.4 I .')

5.9

~

Y,, pM

Palmltoyl

I

0.5

OS

Co,4

Fig. I. The effect of palmitoyl-CoA on the incorporation of sn-[U-“Clglycero-3-phosphate into lipids of E. coli in the presence or absence of ATP. The incubation mixture contained in 2 ml, 0.07 M phosphate buffer (pH 7.3) as solvent and diluent, IO mM MgCI,, 0.44 mM CTP, various concentrations of palmitoyl-CoA, 0.2 &i of sn-[U-‘4C]glycero-3-phosphate (spec. act. 16 Ci/mole) E. co/i sonicate (1.2 mg protein/ml) and when specified, 2.8 mM ATP. Incubations were for I h at 25 “C. A. phosphatidylethanolamine. B. phosphatidylglycerol. C. cardiolipin: IT --3. +ATP; l -- -0, -ATP.

should not have affected the relative distribution of label within phosphatidylglycerol (cf. Table II). ATP also selectively stimulated incorporation of s,z-[14C]glycero-j-phosphate into phosphatidylglycerol provided MgZf was added at a concentration equal to or exceeding that of nucleotide. Inhibitory effects of ATP noticed at high concentrations (results not shown) were attributed to the known ability of this nucleotide, to chelate Mg 2f . Results in Fig. I indicate that even in the presence of optimal concentrations of palmitoyl-CoA and Mgzf, ATP greatly stimulated the labelling of phosphatidylglycerol without significantly affecting incorporation into phosphatidylethanolamine or cardiolipin. The combined effect of ATP and Mg2+ could not therefore be explained on the basis of an increase in acyl donors resulting in a greater production of labelled phosphatidyl precursors. We deduce that these additives did however increase the pool size of unlabelled phosphatidyl precursors. At this point attempts to directly implicate diglyceride kinase [9, IO] with the ATP plus Mg2’ effect were unsuccessful. Results in Table III indicate that 14Clabelled diglyceride could be converted readily by particulate fractions into labelled phosphatidic acid, which, however, accumulated as the end product under the conditions used for assay of diglyceride kinase. In the presence of detergent, s~-[‘~Clglycero-3-phosphate incorporation into phosphatides other than phosphatidic acid did occur but to a lesser extent than when no detergent was added. Detergent therefore inhibited the conversion of phosphatidic acid to more complex phosphatides but the extent of inhibition depended on the course of phosphatidic acid formation. Previous results by Pieringer and Kunnes [9] indicated that diglyceride kinase activity in vitro is completely dependent on an added source of diglyceride. The endogenous levels of this lipid must therefore be negligible and on this basis involvement of diglyceride kinase in the Mg2+ plus ATP effect did not seem a likely possibility.

322

TABLE

III

INCORPORATION J-PHOSPHATE

OF “C-LABELLED ~~1.2 INTO LIPIDS OF E. COLf

DIGLYCERIDE

AND

sn-[U-“C]GLYCERO-

The incubation medium contained in 2 ml, 0.07 M phosphate buffer (pH 7.3), Triton X-100. 14 mg/‘ml when specified. 2.5 mM ATP, 1.0 mM CTP, IO mM MgCII, 0.2 ,Ki sn-[U-“Clglycero-3-phosphate (spec. act. 16 Ci/mole) or 0.4 PCi ~w[‘~C]I.L diglyceride (spec. act. 0.055 Cijmole) and E.co/i particulate fraction, 0.6 mg/ml. Incubations were for I h at 37 C. _ dpm recovered in each fraction Phosphatidic acid

Phosphatidylglycerol

Phosphatidyl-

Cardiolipin

Fatty acids

ethanol-

amine [l”C]DiglyceridemtmTritonX~oo sn-[‘JC]Glycero-3-phosphate sw[1”C]Glycero-3-phosphate-I

TABLE

TritonX~oo

18000

600

II00

14400 9 3oo

5 100

600 2500

,100

,100

1300

5000

IV

THE EFFECT 3-PHOSPHATE

OF ATP AND Mg ‘+ ON THE INTO LIPIDS OF E. COLl

INCORPORATION

OF

sn-[U-“C]GLYCERO-

The incubation medium contained in 2 ml, 0.07 M phosphate buffer pH 7.3 as solvent and dlluent, 2.5 mM ATP and IO mM MgClz when specified. 0.2 /Ki of .s~~-[U-‘1C]glycero-3-pllosphatc (spcc. act. 16 Ci//rmolc) and E.co/i particulate fraction 0.4 mg protcin:ml. Incubations \+cre for I 11at 37 C. Additions

_~

-~ Counts

recovered

~~~

Phosphatidic

in each lipid ~~~~ Phosphatidyl-

Phosphatidyl-

acid

ethanolamine

glycerol

~_. Cardiolipin

None

600

M$+

410

1430 I550

2100

225

1050

37.50

9400

420

Mg*+ ATP __ .~

600

430

Results in Table IV show that in the absence of Mg” incorporation of labelled sn-glycero-J-phosphate into phosphatidylglycerol was small and accounted for only 2X ‘;;, of the total lipid counts. Addition of either Mg*+ alone or Mg’+ plus ATP greatly stimulated the incorporation and increased the proportion of label in polyglycerophosphatides to 65-70 7,: of the total lipid counts. It was reasoned that if the effect of ATP plus Mg” was mainly to increase the pool of endogenous phosphatidyl precursor, any CTP effect on incorporation should be additive to that of the other nucleotide. If ATP served as energy source for formation or maintenance of CTP levels, addition of the latter nucleotide in optimal concentrations should abolish any ATP effect. Results not shown indicated the stimulatory effect of CTP on the incorporation of labelled precursor into phosphatidylglycerol which consistently accounted for 70-75 “/i of the total lipid counts. The optimal CTP concentration was I .o-I .5 mM. Results in Table V reveal that addition of CTP to a mixturecontaining ATP plus Mg2+ significantly increased 14C incorporation into the combined phosphatidyl groups of phosphatidic acid, phosphatidylethanolamine and phosphatidylglycerol but a much greater stimulation was noticed in the labelling of the unacylated glycerol moiety of

323 TABLE

V

THE EFFECT OF CTP, ATP, ~~z-[U-‘~C]~LYCERO~-FH~SPHATE

AND

Mg2+ ON THE [NT0 LIPIDS OF

INCORPORATION E. COLI

OF

The incubation medium contained in z ml, 0.07 M phosphate buffer (pH 7.3), 2.5 mM ATP. 1.0 mM CTP, 10 mM MgCl,, each when specified, 0.2 /*Ci sn-[U-“YZ]glycero-3-phosphate (spec. act. J6 Ci/ mole) and E. co/i particulate fraction 0.6 mg protein/ml. Incubations were for I h at 37 “C. Phosphoglycerides were extracted and separated as stated in the text. The phosphatidylglycerol fraction was eluted, subjected to complete acetolysis and the products were separated [8]. The counts recovered in diacyt monoacetin were added to those of phosphatidic acid and phosi~hatidyiethanolamine (total phosphatidyl groups) and the counts recovered in triacetin were attributed to the unacylated glycerol moiety of phosphatidylglycerol.

~~.----

.____._-__

Additions

~~____

I__ _

Counts recovered in each _~~. --- ._._ -._ _ . fraction Unacylated Total phosphatidyl groups of phosphatidic acid, glycerol phosphatidyiethanolamine and phosphatidyl~lyccrol

ATP -C MgZ + (A) CT-P -I--ATP -I- Mg’+ CTP + Mgz+ (C)

6 680 (B) _._.~.

Jo 530 8930

~_i-_~

glycerol

JJ 570 40 370 19500

~~~~~__~~.

of phosphatidyl-

~_.

~_._.

phosphat~dylg~y~ero~ (A and B compared). Likewise addition of ATP to a mixture containing CTP and Mg*+ caused a marked increase in the r4C labelling of the unacylated glycerol of phosphatidylglycerol but only a small rise in the radioactivity of the combined phosphatidyl groups (B and C compared). From these results it was concluded that CTP and ATP could act at different sites of a partial synthesis pathway. By its involvement in CDP-diglyceride formation, CTP enhances the utilization of unlabelled phosphatidyl precursor, the supply of which becomes increased in the presence of ATP. Since digiyceride is a very minor pool in E. coli and since in any case its involvement in the synthesis of complex phosphatides could not be readily shown under our conditions, phosphoglycerides appeared as a more likely source of endogenous phosphatidyl groups. To further support this possibility the effects of added cardiolipin and other phosphoglycerides on the incorporation of sn-glycero-3-phosphate are being further investigated. Results to date have indicated that ~ardio~ipin causes a slight stimulation frg-25 “/‘,) on the incorporation of this precursor into the unacylated glycerol moiety of phosphatidylglycerol. The endogenous phosphatidyl precursor could not be identified using the present approach but further studies have indicated that it is likely a phosphatidic acid pool, at least partly derived from an ATP plus Mg2+ stimulated hydrolysis of cardiolipin [I 21. ACKNOWLEDGEMENT

This work was supported

by a grant

from the Medical

Research

Council

of

Canada. REFERENCES

I Benns, G. and Proulx, P. (1972) Can. J. Biochem. 50. 16-19 z Agranoff, B. W. and Hajra, A. R. (1971) Proc. Natl. Acad. Sci. U.S. 68. 4JJ-415 3 Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem.

193,265~275

324 4 5 6 7 8 9 IO 11 12

Bligh, E. G. and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 91 1-917 Ames, G. F. (1968) J. Bacterial. 95. 833-843 Ferrari, R. A. and Benson, A. A. (1961) Arch. Biochem. Biophys. 93, 1X5-192 Chu, H. P. (1949) J. Gen. Microbial. 3. 255-272 Renkonen, 0. (1965) J. Am. Oil Chem. Sot. 42. 298-304 Pieringer, P. A. and Kunnes, R. S. (1963) J. Biol. Chem. 240, 2833-2838 Chang, Y. Y. and Kennedy, E. P. (1967) J. Biol. Chem. 242, 516~519 Nantel, G. and Proulx, P. (1973) Biochim. Biophys. Acta 316 156-161 Cole. R., Berms, G. and Proulx, P. (1974) Biochim. Biophys. Acta 337, 325-332