Phospholipid composition of subcellular fractions and phospholipid-exchange activity in chicken liver and MC-29 hepatoma

23

Biochimica et Biophysics Acta, 713 (1982) 23-28 Elsevier Biomedical Press

BBA 51195

PHOSPHOLIPID COMPOSITION OF SUBCELLULAR FRACTIONS AND PHOSPHOLIPID-EXCHANGE ACTIVITV IN CHICKEN LIVER AND MC-29 HEPATOMA KAMEN ALBENA

KOUMANOV =, ATANAS BOYANOV ‘, TANIA NEICHEVA ‘, TANIA MARKOVSKA MOMCHILOVA a, EKATERINA GAVAZOVA b and HEN1 CHELIBONOVA-LORER

a, h

u Central Laboratory of Biophysics and h Institute of General and Comparative Pathology, Bulgarian Academy of Sciences, Sofia I I1 3 (Bulgaria) (Received December (Revised manuscript

24th, 1981) received May 17th, 1982)

Key words: Phospholipid composition; Phospholipid exchange; (Chicken liver, Hepatoma)

The phospholipid composition of mitochondria, microsomes and plasma membranes from liver and MC-29 hepatoma from White Leghorn chickens has been investigated. It was established that all mit~hondria and microsome phospholipid fractions obtained from MC-29 hepatoma are increased strongly compared to those from liver. The sphingomyelin augmentation was particularly great. In hepatoma plasma membranes only the sphingomyelin quantity was increased. Sphingomyelin- and phosphatidylcholine-exchange activities were observed in avian liver for the first time. These two activities were increased in MC-29 hepatoma cells. Three phospholipid-exch~ge proteins have been established in chicken liver 105000 X g supernatant. One of them s~ific~ly transports phos~atidylcholine, the second one is non-s~ific for p~phati~lc~li~ and sphingomyelin, and the third one is specific only for sphingomyelin. In hepatoma cells only a non-specific phosphatidylcholine- and sphingomyelin-exchange protein was found.

the activity of these proteins during malignant cell growth as well as their responsibility for the phospholipid composition changes of cell membranes observed during malignant processes. A protein capable of sphingomyelin transfer between membranes has been found in the highspeed postmicrosomal supernatant (105~ X g) from rat hepatoma [lo]. Such a protein has not been detected in normal rat liver. The present paper provides some results concerning the phospholipid composition and the activity of phospholipid-exch~ge proteins in chicken liver and MC-29 virus-induced hepatoma.

Investigations on tumour cell membrane composition and structural organisation are very important for clarification of the origin of malignant processes. Recent studies have revealed considerable alterations in the phospholipid composition of various cellular structures (mitochondria, microsomes, plasma membranes) from different rat and mouse hepatomas, compared to that from liver [l-6]. These alterations in the phospholipid content lead to changes in the activity of some membrane-bound enzymes [5-9] and interfere with the normal behaviour of the tumour cells. The role of phospholipid-exchange proteins in membrane genesis and in the maintenance of the membrane structure has recently been widely discussed. However, there are very scarce data about 0005-2760/82/0000-OCOO/$O2.75

0 1982 Elsevier Biomedical

Materials and Methods Chickens. 2-3-day-old White Leghorn chickens were subcutaneously transplanted with lo6 tumour Press

24

cells provoked by MC-29 leukemic virus. The tumours formed 8-10 days after transplantation and the livers of normal chickens of the same age, used as control, were submitted to fractionation and investigation separately. Preparation of subcellular fractions from MC-29 hepatoma and liver. Mitochondria and microsomes were prepared by differential centrifugation in 0.2 M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4 (buffer 1). The postmicrosomal 105000 X g supernatant was used for further investigations of phospholipid-exchange proteins. Plasma membranes were isolated by a method described in Ref. 11. The purity of the various membrane fractions has been checked by electron microscopy and by determination of the activity of some marker enzymes and the results were published in a previous paper [ 121. Partial purification of phospholipid-exchange proteins from MC-29 hepatoma and liver. The postmicrosomal 105000 X g supernatant was adjusted to pH 5.1 with 3 N HCI. After 1 h the precipitate was sedimented by centrifugation for 15 min at 10000 X g in a Janetzki K-24 (G.D.R.) centrifuge and discarded. The pH of the supernatant was readjusted to 7.4 with 5 N NaOH. Solid ammonium sulphate was slowly added to the supernatant to 90% saturation and the mixture was stirred overnight. The precipitate was sedimented by centrifugation for 1 h at 10000 X g and the pellet was suspended in water. The fraction was dialyzed for 24 h against water with at least two changes of medium. The dialyzed protein was loaded onto a Sephadex G-75 column (2 X 80 cm) and was eluted at 3 ml/cm* per h with 5 mM (NaH,HPO,/ NaH,PO,)/lO mM 2-mercaptoethanol, pH 7.4. 2.8-ml fractions were pooled and used for determination of the phospholipid-exchange activity. Preparation of the liposomes. Rats were injected intravenously with 25 pCi/rat [ 1-‘4C]palmitic acid (Amersham), spec. act., 59 mCi/mmol. The rats were killed 1 h after intravenous injection and their liver phospholipids were extracted as described in Ref. 13. [ I4 ClPhosphatidylcholine and [ “C]sphingomyelin were isolated by thin-layer chromatography on silica gel 60 thin-layer plates chloroform/methanol/acetic (Merck) in acid/water (70 : 35 : 8 : 4).

Liposomes were prepared from rat liver [ I4 Clphosphatidylcholine or [ I4 Clsphingomyelin and a trace (1%) of [ 3H]trioleoylglycerol (Amersham) as nonexchangeable marker. The lipids were dissolved in diethyl ether and the solvent was reevaporated under nitrogen. The lipids were suspended in buffer 1 (pH 7.4) and sonicated in a UZDN-1 (U.S.S.R.) sonifier at 25°C for 10 min. Assay of phospholipid-exchange protein activity. Liposomes (75 pg phospholipid/ml incubation medium) were incubated with rat liver mitochondria (375 pg phospholipid phosphorus/ ml incubation medium) and an appropriate aliquot of the exchange protein in a total volume of 4 ml of buffer 1. Incubations of liposomes and mitochondria were performed in parallel in the absence of phospholipid-exchange proteins; these served as controls and for correction of the transfer activity due to nonspecific exchange. Incubations were carried out for 15 min at 37°C in a water bath with constant agitation. At the end of the incubation, exchange was stopped by chilling the mixture in an ice-bath and sedimenting the mitochondria at 12000 X g for 15 min in a refrigerated Janetzki K-24 centrifuge (G.D.R.). The aliquots of the liposome - containing supernatants were counted in a scintillation medium for aqueous samples (Unisolve- 1, Koch-Light) and the “C/3H ratio was used for calculation of the phospholipid-exchange activity as described in Ref. 14. The exchange activity was expressed in terms of nmol of phospholipid exchanged/min per mg protein. Analysis. Phospholipids were extracted from chicken hepatoma and liver mitochondria, microsomes and plasma membranes by he method in Ref. 13 and subsequently chromatographed on silica gel 60 thin-layer plates (Merck). Chloroform/methanol/acetic acid/water (70 : 35 : 8 : 4, by vol.) and chloroform/methanol/iso-propanol/ 0.25 N KCl/triethylamine (30: 9 : 25 : 6 : 18, by vol.) were used as developing solvents. The amounts of phospholipid phosphorus were estimated as described in Ref. 15. Protein determination was carried out by the method described in Ref. 16.

I

Ratio phosphatidylcholine/ sphingomyelin

Phosphatidylcholine Phosphatidylserine Phosphatidylinositol Phosphatidylethanolamine Phosphatidylglyccrol Diphosphatidylglyccrol

sphingomyelin

protein;

5.31

8.5* 0.7 * 49.9* 5.8 265.4k21.0 78.5-t 6.5 7.8* 0.6 177.0” 9.4 25.6-c 3.0 21.6* 1.9

3.41

24.1* 3.1 250.0123.3 853.2e36.1 273.8*24.1 100.3* 13.4 632.9* 38.5 95.2* 8.0 77.7* 5.0

* ** *** l * l ** ** l * **

4.88

8.6% 0.9 45.0* 5.0 219.9120.1 85.0-t 7.1 IO.22 0.9 143.52 12.1 10.0* 1.1 9.6~ 0.8

Liver

according

AND r-test:

MC-29 * P ~0.05,

HEPATOMA

3.81

30.3? 4.2 * 187.7* 13.4 ** 716.2-t31.0 *** 238.4* 16.4 l * 45.9k3.1 l* 451.2k28.1 * 26.3* 1.8 ** 12.4* 1.1

Hepatoma

to Student’s

LIVER

Liver

significant

OF CHICKEN

Microsomes Hepatoma

are statistically

FRACTIONS

Mitochondria

by asterisks

OF SUBCELLULAR

those marked

COMPOSITION

Lysophosphatidylcholine

Phospholipids

Values are nmol/mg

PHOSPHOLIPID

TABLE

10.76

42.72

4.43

126.2’11.5

2.1 10.0

22.3% 115.0* _ _

3.1 *

0.9* 3.8 ** 16.4

Hepatoma 18.72 42.0* 186.2-

membranes

26.0* 1.8 16.3* 1.4 176.3* 15.8

Liver

Plasma

** P cO.01,***P
26

TABLE

II

PHOSPHOLIPID EXCHANGE ACTIVITY OF THE pH 5.1 FRACTION FROM 105000X R SUPERNATANT Liposomes (75 pg phospholipid phosphorus/ml incubation medium) were incubated with rat liver mitochondria (375 pg phospholipid phosphorus/ml incubation medium) and 50 pg exchange protein (fraction pH 5.1) in a total volume of 4 ml of buffer 1. Incubations were carried out for 15 min at 37°C. Values are nmol phospholipids exchanged/min per mg, those for hepatoma are statistically significant according to Student’s I-test: P
Liver tested

Hepatoma

Sphingomyelin Phosphatidylcholine

15.94-‘0.21 14.34kO.83

23.65 + 1.76 28.67 i- 0. IO

Results The phospholipid composition of chicken liver and MC-29 hepatoma mitochondria. microsomes and plasma membranes shows interesting differences (Table I). The phospholipid content in hepatoma mitochondria and microsomes, compared to that in liver, was strongly augmented. In plasma membranes the changes were less pronounced. The individual phospholipids changed at a different rate: the sphingomyelin level was augmented five times in mitochondria four times in microsomes and about three times in plasma membranes. The phosphatidylcholine quantity was increased three times only in mitochondria and microsomes. As a result of these changes the phosphatidylcholine/ sphingomyelin molar ratio in hepatoma subcellular fractions was strongly decreased, which was most severely expressed in plasma membranes. The changes in phosphatidylethanolamine quantity were identical to the changes in phosphatidylcholine - there was an increase only in mitochondria and microsomes. The content of phosphatidylserine and phosphatidylinositol was augmented in all hepatoma subcellular fractions. in phosphatidylinositol in The increase mitochondria was particularly strong. The quantity of phosphatidylglycerol and diphosphatidylglycerol was also enhanced in MC-29 hepatoma mitochondria and microsomes. In order to elucidate the reasons for the changes

Fraction

number

Fig. 1. Elution profile of 105000X R postmicrosomal supernatant proteins from chicken liver on Sephadex G-75. The dialyzed protein was loaded onto a Sephadex G-75 column (2X80 cm) and was eluted at 3 ml/cm*) per h with 5 mM (Na,HPO,/NaH,PO,)/lO mM 2-mercaptoethanol. Fractions of 2.8 ml were pooled. The phosphatidylcholine (0) and sphingomyelin (0) exchange activities were measured.

in the phospholipid composition of hepatoma subcellular fractions, we investigated the phospholipid-exchange proteins in 105000 X g postmicrosomal supernatant from both tissues (Table II). First of all, the comparatively high phosphatidylcholine- and sphingomyelin-exchange activity of the proteins in both tissues should be noted. Very interesting is the 40% augmentation of phosphati-

b 2200

.__ E

1800 1400

35

45

55

65

75

<

1000

x 2 2 2

600

5 ;;

200

Fraction

a E

j

85

number

Fig. 2. Elution profile of 105000X g postmicrosomal supernatant proteins from chicken MC-29 hepatoma on Sephadex G-75. The purification of the phospholipid-exchange proteins was performed under the same experimental conditions as explained for Fig. 1,

27

dylcholineand sphingomyelin-exchange activities in tumour cells. The results of a partial purification of the phospholipid-exchange proteins on a Sephadex G-75 column are of a particular interest (Fig. 1). The phosphatidylcholine-exchange activity in liver was eluted in fractions 45-80, the main activity being in fractions 65-75, with a maximum at fraction 70 (1282 nmol/min per mg). The elution profile of the phosphatidylcholine-exchange activity suggests the probable existance of a second maximum at fraction 60. The sphingomyelin-exchange activity profile showed two peaks - the first one coinciding with the phosphatidylcholine-exchange activity peak, with a maximum at fraction 55, and the second one at fraction 80 (250 and 679 nmol/min per mg, respectively). Quite different was the elution profile of the phospholipid-exchange proteins in MC-29 hepatoma 105 000 X g postmicrosomal supernatant (Fig. 2). In this case, the phosphatidylcholine-exchange activity was eluted mainly between fractions 40 and 65, with a maximum at fraction 50 (2276 nmol/min per mg). The sphingomyelin-exchange activity gave one peak only with a maximum at fraction 50 as well. Discussion Our investigations show that malignant processes influence strongly the phospholipid composition of subcellular membrane structures. Especially in chicken hepatoma provoked by MC-29 the phospholipid content of leukemic virus, mitochondria and microsomes is strongly enhanced. Similar results on rat and mouse hepatoma have been published by some other authors [1,2,5,10]. They establish an increase in the quantity of sphingomyelin, phosphatidylserine, and phosphatidylinositol, and a decrease in the phosphatidylcholine content. According to our data, the sphingomyelin augmentation in MC-29 hepatoma subcellular fractions, which is typical for most hepatomas, is accompanied by an augmentation of all other phospholipids. The relatively low phosphatidylcholine/sphingomyelin ratio is due to the stronger augmentation of sphingomyelin compared to that of phosphatidylcholine in tumour cells. Selkirk and Elwood [5]

found a 2-fold increase of sphingomyelin content in H-35 hepatoma plasma membranes in comparison with liver plasma membranes. An increase of mitochondrial and microsomal sphingomyelin quantity has also been described in Zajdela ascite hepatoma and Jensen sarcoma [7-lo]. In a previous paper [ 171 we established that the biosynthesis of the sphingomyelin in MC-29 hepatoma was more active compared to its biosynthesis in chicken liver and also compared to the other phospholipid classes in MC-29 hepatoma. The enhanced biosynthesis of the phospholipids in hepatomas is accompanied by their decreased degradation. Recent data [ 181 show that the activity of both phospholipases A, and A, in plasma membranes from ascite tumour cells is considerably decreased. In the present investigation, a phosphatidylcholineand sphingomyelin-exchange activity in the 105 000 X g postmicrosomal supernatant from chicken liver and MC-29 hepatoma was established for the first time. The results show that both activities are much higher in MC-29 hepatoma than in liver. Furthermore, these enhanced transport activities correspond to an enhanced quantity of the respective phospholipids in the subcellular membrane fractions. The sphingomyelin-exchange proteins in liver 105 000 X g postrnicrosomal supernatant, which were partially purified on a Sephadex G-75 column, show two activity maximums - the first one being eluted together with the phosphatidyl-exchange activity and the second one being eluted by itself much later than the first one. This shows that there are two or three exchange proteins in the 105 000 X g supernatant from normal chicken liver. One of them transports phosphatidylcholine specifically while the second one transports phosphatidylcholine and sphingomyelin nonspecifically (as well as phosphatidylethanolamine and phosphatidylinositol - unpublished data) and its properties resemble strongly the protein found in rat hepatoma [IO]. The third protein transports specifically only sphingomyelin and it seems probable that this very protein is typical only for chickens (or for birds in general), since it has not been detected in mammalian liver. Perhaps the relatively high sphingomyelin content in chickens membrane cell structures requires the presence of

28

a specific sphingomyelin-exchange protein, which should provide the sphingomyelin transport from its biosynthesis sites up to where it is being utilized. In the 105000 X g postmicrosomal supernatant from MC-29 hepatoma, the sphingomyelinand phosphatidylcholine-exchange activities seem to be due to a nonspecific phospholipid-exchange protein. The elution profile from Sephadex G-75 column shows that these activities are being eluted together and have a common maximum at fraction 50. An interesting fact in this case is the absence of the specific sphingomyelin-exchange protein which can be found in the 105000 X g postmicrosomal supernatant from chicken liver. However, the nonspecific phosphatidyl-exchange protein in hepatoma is much more active than the one found in liver, and apparently it is responsible for the generally higher exchange activity which we determined in the 105 000 X g postmicrosomal MC-29 hepatoma supernatant. It seems that the dedifferentiation of the phospholipid synthesis which was established for different hepatomas is accompanied by an activation of the transport proteins, which leads to very serious changes in the phospholipid composition of tumour cells membrane fractions. These changes probably determine various functional disorders which are typical for neoplastic cells. References 1 Bergelson, L.D., Dyatlovitskaya, E.V., Sorokina, LB. and Gorkova, N.P. (1974) Biochim. Biophys. Acta 360, 361-365

2 Bergelson, L.D., Dyatlovitskaya, E.V., Torkhovskaya, T.I., Sorokina, I.B. and Gorkova, N.P. (1970) Biochim. Biophys. Acta 210, 287-298 3 Feo, F., Canuto, R.A., Bertone, (1973) FEBS Lett. 33, 229-232

G., Garcea,

R. and Pani, P.

4 Hostetler, K.Y., Zenner, D.B. and Morris, H.P. (1976) Biochim. Biophys. Acta 398, 231-238 5 Selkirk, J.K. and Elwood. J.,C. (1971) Cancer Res. 31, 27-31 6 Van Hoeven, R.P. and Emmelot, P. (1972) J. Membr. Biol. 9, 105-126 7 Dyatlovitskaya, E.V., Valdnietse, A.T. and Bergelson, L.D. (1977) Biokhimiya 42, 2039-2043 8 Dyatlovitskaya, E.V., Lemenovskaya, A.F. and Bergelson, L.D. (1979) Biochimiya 44.498-503 9 Dyatlovitskaya, E.V., Sinitsina, E.M., Lemenovskaya. A.F. and Bergelson, L.D. (1978). Biochimiya 43, 420-423 10 Diatlovitskaya, E.V., Timofeeva, N.G. and Bergelson, L.D. (1978) Eur. J. Biochem. 82, 463-471 11 Bachmann. W., Harms, E., Hassels, B., Henninger, H. and Reuter. W. (1977) B&hem. J. 166, 455-462 12 Gavazova, E.. Ivanov. S. and Chelibonova-Lorer, H. (1980) Neoplasma 27, 4. 399-408 13 Folch, J., Lees, M. and Sloane-Stanley, G.H. (1957). J. Biol. Chem. 226. 497-503 14 Zilversmit, D.B., Hughes, M.E. (1976) in Methods in Membrane Biology. Vol. 7 (Korn, E.D., ed.), p. 211. Plenum Press, New York 15 Kahovcova. J. and Odavic. R. (1969). J. Chromatogr. 40. 90-96 16 Lowry. O.H.. Rosebrough. N.J.. Farr. A.L. and Randall, R.J. (1951) J. Biol. Chem. 193. 265-275 17 Koumanov, K., Gavazova, E. and Chelibonova-Lorer. H. (1981) Gen. Comp. Pathophysiol. 1 I, 5-10 18 Koizumi, K., Iti, Y., Okuda. J., Fujii. T., Tamiya-Koizumi, K. and Kojima, K. (1980). J. Biochem. 88, 949-954