Neutron activation paper chromatographic analysis of phosphatides in mammalian cell fractions

ARCHIVES

OF

BIOCHEMISTRY

Neutron

AND

BIOPHYSICS

Activation Phosphatides

344-348 (1960)

88,

Paper

Chromatographic

in Mammalian

E. H. STRICKLAND2 From

the Department of Physics The Pennsylvania

MATERIALS

OF CELL

and Biological Pennsylvania

Chemistry,

16, 1959

was found in the mitochondria of a number of estimated by neutron activation chromatoderivative, 1,3-diglyeerophosphoryl-glycerol. of this lipid. The distributions of the other and microsomes were similar. A possible funcis discussed.

In a recent publication, Marinetti, Erbland, and Stotz (1) reported a difference in the distribution of phosphatides in pig heart mitochondria and microsomes. A lipid which was tentatively identified as a polyglycerol phosphatide was found in much higher concentration in mitochondria than in microsomes. In the present study the method of neutron activation chromatography was used to determine whether similar differences in phosphatide composition occur in other tissue cell fractions. It was found that mitochondria characteristically contained diphosphatidylglycerol. This lipid is probably the same as the one found in pig heart mitochondria and identical with “cardiolipin” (2).

ISOLATION

AND A. A. BENSON

November

INTRODUCTION

Mitochondria, isolated from

of

Fractions’

Department of Agricultural University, University Park,

Diphosphatidylglycerol (cardiolipin) cell fractions. Its concentration was graphic analysis of the deacylated Microsomes contained little or none glycerol phosphatides in mitochondria tional role for diphosphatidylglycerol

AND

Cell

and the State

Received

METHODS

Analysis

FRACTIONS

lysosomes, and microsomes were rat liver according to the method of

1 This work was supported by the U. S. Atomic Energy Commission, Research Grant A-2567 from the Institute for Arthritis and Metabolic Diseases of the Public Health Service, the National Science Foundation, and the Pennsylvania Agricultural Experiment Station. 2 Predoctoral Fellow of the National Science Foundation.

de Duve et al. (3). A similar procedure was used for other tissues except that the 250,000 X g-min. fraction, which probably consisted of a mixture of mitochondria and microsomes, was discarded.

EXTRACTION CELL

OF LIPIDS Z~CTIONS

FROM

The cell fractions were resuspended in 2 ml. of 0.25 M sucrose and pipetted into 300 ml. of boiling 80% ethanol to lyse the particles and extract the lipids. After separation of the particle fragments, they were re-extracted with two 3-ml. portions of absolute ethanol, 3 ml. methanol-toluene (75:25, v/v), and five 3-ml. portions of chloroform. The combined extracts were concentrated in vacua to 2 ml. The lipids were extracted with chloroform and washed with water. The chloroform extract was evaporated to incipient dryness and taken up in absolute ethanol. The ethanol was evaporated with added toluene to remove the traces of water and chloroform. The concentrate was taken up in absolute methanol. Methanolysis of phosphatides by the method of Dawson (4) was carried out in 0.1 N methanolic potassium hydroxide at 37” for 15 min. (5). After neutralization with Dowex 50H+ resin, the phosphate diesters were chromatographed on Whatman No. 4 paper in phenolwater (100:38), PW, and in butanol-propionic acid-water (142:71:100), BPAW.

NEUTRON Prior monium

344

ACTIVilTION CHROMATOGRAPHIC ANALYSIS

to activation, phosphate)

1.0~pg. spots of P31 (as amwere applied along the edges

Ah-ALYSIS

OF

of the ordinary or silicic acid-impregnated paper chromatograms as standards. The chromatograms were rolled and sealed in 2.5-cm. diameter polyethylene tubes. After irradiation at the face of the swimming pool-type reactor for 15 hr. in a flux of 5 X 10” neutrons/sq. cm./sec., the short-lived radioisotopes were allowed to decay for 5-7 days. Radiograms were prepared using a l%mg./sq. cm. thick plastic sheet as a filter for the soft background radiation to discriminate against lowenergy beta-emitting impurities. Exposure times were from 3 to 14 days. The induced act,ivity in the phosphate diesters, as defined by the radiograms was measured with a large diameter GeigerWilier tube, corrected for background in adjacent areas of the paper, and compared with the activity of the 1.0~pg. P spots subjected to identical neuTABLE PHOSPHATIDE MAMMALIAN

CELL Micrograms

Cell

I

CONCENTRATIONS

IN

FRACTIONS P/ml.

packed

cell fraction

i-ion-

fraction GPS

$;yd lipids

--

Rat liver Mitochondria Lysosomes Microsomes Beef heart mite. chondriah

370 300

224 165

86 138

57 57

10 16

a

100 130 80

a u 5

a Trace. b The authors appreciate the generosity D. E. Green and R. L. Lester in providing ple of beef heart mitochondria.

of Drs. a sam-

tron flux. The nonuniformity in the neutron flux and neutron-activated paper impurities might result in errors of as much as &loo]0 in determining the phosphorus content of chromatographic spots. EXPERIMENTAL

DISTRIBUTION

OF

II

PHOSPHATIDES

WITHIN

TISSUE

Per cent of total Source

Rat liver mitochondria Rat liver lysosomes Rat liver microsomes Rat kidney mitochondria Rat kidney microsomes Sheep heart mitochondria Beef heart mitochondria Rat brain mitochondria amount;

P from

FRACTIONS deacylated

lipids”

of lipid GPC

a f, trace than 0.5%.

RESULTS

E’or many elements, neutron act,ivation analysis has a simplicity, convenience, and sensit.ivity that, can rarely be obt,ained by classical chemical techniques. Activation of chromatograms has proved less successful because of the high level of background activity induced in the impurities in the paper. However, the combination of the relatively high neutron activat.ion cross section of 1’31, the 14day half-life and the high-energy betas emitted byP allows a yuantit’ive determination of trace amounts of phosphorus-containing compounds on chromatograms (e-10). The sensitivity can be increased somewhat, by washing the paper with nitric acid, followed by oxalic acid and water to remove some of the impurities. Because of the phosphorus content of natural cellulose and enhanced radiation decomposition of the paper, t,he washing procedures have proved t,o be of lit,tle value. Nevertheless, as little as 0.05 pg. P/q. cm. can be easily detected in chromatographic spots. Neutron activation paper chromatographic analysis was carried out on the deacylated phosphatides of several tissue fractions listed in Tables I and II. The methanolyzat’es cont’ained the phosphate diesters,

TABLE RELATIVE

345

PHOSPHATIDES

++,

GPE

49 45 68 41 +++ 46 38 55 approximately

5-2570;

GPGPG

GPI

GPG

12 20 9 10 16 2

8 8 12 14 +t 5 6 5

1 2

30 24 18, 12 30 ++ 35 37 32 +++,

more

than

25%;

---,

2,5

GPS

-

3 -

1 +

4 11

2 none

detected;

+

4 less

346

STRICKLAND

AND

BENSON

FIG. 1. Neutron radiogram of deacylated lipids of sheep heart mitochondria. Chromatogram was developed in x-direction with phenol-water, in y-direction with butanol-propionic acid-water. Control samples of 1.0 pg. P3i, as ammonium phosphate, were applied at left edge of paper prior to activation with 2.7 X 10’6 neutrons/sq. cm.

GP13 GPG, GPC, and GPE, which were readily identified from Rf values (4, 11). In addition to these compounds, the mitochrondrial and lysosomal methanolyzates (Figs. 1 and 2) reveal a phosphorus-containing compound not present in the microsomal methanolyzates (Fig. 3). This compound cochromatographed precisely with P32-labeled GPGPG from Chlorella (12). Silicic acid chromatograms prepared according to the method of Marinetti et al. (13) showed that GPGPG lipid had the same R, as the fastmoving polyglycerol phosphatide found in pig heart mitochondria (1). GPGPG lipid appears to have a structure identical with that proposed by Macfarlane (12, 14) for cardiolipin (diphosphntidylglycerol). The high concentration of GPGPG lipid in sheep heart and beef heart mitochondria (see below) strongly suggest’s that it is identical with cardiolipin. 3 The following abbreviations are used in this article: GP, glycerophosphate (n-glycerol l-phosphate) ; GPI, glycerophosphorylinositol; GPG, glycerophosphorylglycerol; GPC, glycerophosphorylcholine; GPS, glycerophosphorylserine; GPE, glycerophosphorylethanolamine ; GPGPG, 1,3-diglycerophosphoryl-glycerol.

Concentrations of phosphatides in rat liver mitochondria, lysosomes and microsomes are given in Table I. The relative distribution of phosphatides within several ot’her tissue fractions is presented in Table II. In general, the phospholipids are remarkably similar in all t,issue fractions investigated, even though the microsomes and mitochondria have much different chemical properties and came from different types of tissues. Phosphatidylcholine accounts for about one half or more of the phosphatides; phosphatidylethanolamine about one fourth to one third; and phosphatidylinositol about a tenth to a twentieth. Trace amounts of phosphatidylglycerol were detected. In some tissues, trace amounts of phosphatidylserine were present. Only GPGPG lipid shows any striking difference in distribution among the cell fractions. This lipid is much more concentrated in lysosomes and all mitochondria analyzed than in microsomes. In t’he two microsomal fractions investigated, those from rat kidney and liver, no GPGPG lipid was detected. DISCUSSION

The cellular distribution of the individual phosphatides may offer some clues as to their

AKALYSIS

OF

PROSPI1ATIDES

FIG.

2.

Neutron

radiogram

of deacylated

lipids of rat liver lysosomes.

FIG.

3. Neutron

radiogram

of deacylated

lipids of rat liver microsomes.

function in the cell. In general, phospholipids in the cell seem to be concentrated in membranes and have been considered import.ant structural elements. Electron microscopic investigations have revealed that cytoplasmic membranes are remarkably similar (I 5). Probably t>he great similarit’y of t,he phospha-

tides in the various cell fractions may reflect t,heir structural function in these membranes. The unusual distribut,ion of GPGPG lipid suggests that it may perform a different function from t,he other phosphntides. GPGPG lipid was associated wit.h the mitochondrial fraction of the cells in all t,isslles investigat.ed

348

STRICKLAND

AND

BENSON

sible model of an enzyme-GPGPG lipid complex is shown in Fig. 4. The hydrogen-bonding and ionic-bonding potent’ialities of GPGPG lipid are especially relevant since it can form two bonds, one with each of the two phosphate diesters. The four fatty acid residues of GPGPG lipid, which extend into the center of the membrane, would seriously restrict movement’ of t’he phosphate groups. By forming hydrogen bonds with t’he side chains of a pair of amino acid residues, such as lysine or arginine, GPGPG lipid could influence an enzyme’s configuration in such a way as to affect its activity or specificity.

PROTEIN

REFEREKCES

FIG. 4. Proposed by diphosphatidylglycerol.

mode

of enzyme

activation

by us* and by Marinetti et al. (1). It is most interesting that a purified cytochrome b-cytochrome cl preparation has a still higher concenkation of GPGPG lipid (16). It is one of the three major lipids found in the chromatophores of the photosynthetic bacterium Rhodospirillum rubrum (12). Thus GPGPG lipid appears to be concentrated in cell structures which have membranes considered to have high enzymic activity. Cardiolipin and other anionic surfactants activate certain in vitro enzyme systems by adsorption on lipid substrates (17). The resulting anionic micellar surface appears to be effective in facilitating approach of the enzyme to the substrate (18). We have observed that GPGPG is particularly strongly adsorbed on a variety of surfaces. GPGPG lipid might be bonded to a membrane enzyme in such a way as to alter its structure and enzymic properties. A pos4 Footnote Added in olated corn coleoptile contain relatively high phatidylglycerol. The parable concentration

Proof: Mitochondria of etitissue have been found to concentrations of diphosother phospholipid in comwas phosphatidylglycerol.

1. MARINETTI, G. V., ERBLAND, J., AND STOTZ, E., J. Biol. Chem. 233, 562 (1958). M. C., J. Biol. Chem. 168, 351 2. PANGBORN, (1947). B. C., GIANETTO, R., 3. DE DUVE, C., PRESSMAN, WATTIAUX, R., ANL) APPELMANS, F., I3iothem. J. 60, 604 (1955). R. M. C., Biochim. et Biophys. Acta 4. DAWSON, 14, 374 (1954). B., AND BENSON, A. A., J. Biol. Chem. 5. MARUO, 234, 254 (1959). A. A., MARUO, B., FLIPSE, R. J., 6. BENSON, YUROW, H. W., MILLER, W. W., Proc. Intern. Conf. Peaceful Uses Atomic Energy, 2nd Conf., Geneva, 1968, Vol. 24, part I, pp. 289. F. P. W., HARRISON, A., AND 7. WINTERINGHAM, BRIDGES, R. C., Nucleonics 10, 52 (1952). K., AND JERCHEL, D., Angew. 8. SCHMEISER, Chem. 66, 366, 490 (1953). 9. SELLERS, P. A., SATO, T. R., AND STRAIN, H. H., J. 1norg. &Nuclear Chem. 6.31 (1957). 10. SATO, T. R., Anal. Chem. 31, 841 (1959). A. A., AND MARUO, B., Biochim. et 11. BENSON, Biophys. Acta 27, 189 (1958). A. A., AND STRICKLAND, E. H., Bio12. BENSON, chim. et Biophys. Acta, in press. G. V., ERBLAND, J., AND KOCHEN, 13. MARINETTI, J., Federation Proc. 16, 837 (1957). 14. MACFARLANE, M. G., Nature 162, 946 (1958); MACFARLANE, M. G. AND WHEELDON, L. W., Nature 183, 1808 (1959). F. S., Revs. Modern Phys. 31, 301 15. SJ~STRAND, (1959). G. V., KOCHEN, J., ERBLAND, J., 16. MARINETTI, AND STOTZ, E., J. Biol. Chem. 229, 1067 (1957). J. 70, 559 (1958). 17. DAWSON, R. M. C., Biochem. 18. BANGHAM, A. D., AND DAWSON, R, M. C., Biothem. J. 72, 486 (1959).