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Phospholipids of subcellular organelles isolated from cultured BHK cells
243
Biochimica et Biophysics @ Elsevier/North-Holland
Acta,
486
Biomedical
(1977)
243-253
Press
BBA 56939
PHOSPHOLIPIDS OF SUBCELLULAR CULTURED BHK CELLS *
JAAKKO
BROTHERUS
July 22nd,
ISOLATED
FROM
and OSSI RENKONEN
Laboratory of Lipid Research, Department SF-00290 Helsinki 29 (Finland)
(Received
ORGANELLES
of Biochemistry,
University
of Helsinki,
1976)
Summary
Mitochondria and nuclei were purified from cultured hamster fibroblasts (BHKZl cells) by centrifugation in sucrose gradients. The phospholipid compositions of the preparations were compared to those of the previously purified plasma membranes, endoplasmic reticulum and lysosomes. The mitochondria had a characteristically high content (approx. 16% of lipid phosphorus) of cardiolipin, which was practically absent from the other purified organelles. The nuclei were enriched in phosphatidylcholine and phosphatidylinositol (approx. 68% and 5% of lipid phosphorus, respectively). Lysobisphosphatidic acid was almost absent from the mitochondria and nuclei, as well as from the plasma membrane and endoplasmic reticulum, which suggests that this phospholipid is confined to the lysosomes of the BHK cell. The nuclei and the mitochondria contained relatively little sphingomyelin, a characteristic lipid of the plasma membrane. The distributions of the total cellular phospholipid and protein between the various organelles were calculated and compared to the corresponding data estimated for the rat liver. The BHK cell contained relatively more phospholipids in the nucleus and the lysosomes than the liver. All the organelles of the BHK cell contained less protein per phospholipid than the equivalent organelles of the liver. _. ._ Introduction
Cultured mammalian cells are frequently used in studies of the structure, function and biogenesis of lipids in cell membranes. These cells offer a better * part of the data has been published in a preliminary form (Renkkonen, 0.. Luukonen. A., Brothems, J. and KGiritinen, L. (1974) in Control of Proliferation in Animal Cells (Clarkson, B. and Baserga. R., eds.). pp. 495-504, Cold Spring Harbor Laboratory, Cold Spring Harbor).
244
system for many types of experiments than e.g. intact animals, tissue slices or isolated cells. The lipids of purified plasma membranes of several cultured cell lines (Z-4), including baby hamster kidney cells (BHK cells) [5,6], have been studied rather extensively in connection with recent work on the assembly of certain enveloped viruses. It has become evident in these studies that the composition of the plasma membrane lipids in the cultured cells is similar to that found in plasma membranes of mammalian cells in general; sphingomyelin, sphingoglycolipids and free cholesterol are enriched in this organelle. Even the lysosomal lipids of cultured BHK cells appear to have some resemblance to those found in mammalian lysosomes in general. The lysosomes of the BHK cell contain large amounts of lysobisphosphatidic acid [7], a characteristic phospholipid of the lysosomes of many mammalian systems [ 8-111. The present report shows that also the mitochondria and the nuclei of the BHK cell have phospholipid patterns similar to those of the homologous organelles from other sources. Taken together, the data on BHK cell indicate that this cell possesses the mechanisms which create and maintain the characteristic phospholipid compositions of the subcellular membranes in mammals. This appears not to be the case in all cells [ 12,131. The present data allow also an estimation of the fractions amounts of total cellular phospholipid belonging to the different organelles of the BHK cell. Materials and Methods Organelle ~r~c~~ona~~o~. The cultivation of the BHK21 cells (strain Wi-2) and the prelimin~y organelle fractionation by differential cent~fugation were described previously [ 71. The crude mitochondrial fraction, suspended in 6 ml of cold 0.3 M sucrose which was buffered with 5 mM Tris . HCl, 0.5 mM EDTA, pH 7.4 (Tris/EDTA buffer), was pipetted on top of a gradient [14] consisting of a 2 ml cushion of 52% (1.88 M) sucrose overlaid by a 30 ml linear gradient from 45% (1.58 M) to 31% (1.02 M) sucrose; both solutions were buffered with the Tris/EDTA. The gradient was centrifuged for 60 min at 25 000 rev./min (82 000 X g,“) in A Beckmann L3-50 ultracentrifuge, rotor SW 27. Fractions of 2 ml were collected from below. The most turbid fractions of the mitochondrial peak were pooled, after taking samples for analysis, diluted to 6 ml by the Tris/EDTA buffer and recentrifuged in a similar manner as above. The crude nuclear fraction in 5 ml of 0.3 M sucrose, buffered with the Tris/ EDTA, was diluted with 30 ml of 2.3 M sucrose buffered with the Tris/EDTA, and centrifuged in the same conditions as above. The packed layer on the surface was collected with a spatula and the sucrose solution was removed with a Pasteur pipette. The nuclear pellet was suspended in 0.3 M sucrose in the Tris/ EDTA buffer. The subcellular fractions were stored at -20°C before analysis. AnalyticaE methods. Succinate dehydrogenase (E.C. 1.3.99.1), P-glucuronidase (E.C. 3.2.1.31), glucose-6-phosphatase (E.C. 3.1.3.9), phospholipids and protein were analyzed as previously [7]. The protein standards contained the same amount of sucrose and Tris/EDTA as the actual samples. DNA was
245
determined [15] using calf thymus DNA (Worthington Biochemical Corp., Freehold, N.J.) as a standard. Electron microscopy. The cell fractions were diluted to approx. 0.3 M sucrose concentration with the Tris/EDTA buffer and pelleted by centrifugation. The pellets were fixed and processed for microscopy as earlier [ 71. Results Purification of the mitochondria The activity of succinate dehydrogenase appeared as a relatively symmetric peak around the density of approx. 1.17 g/ml after centrifugation of the crude mitochondrial fraction in a sucrose gradient (Fig. 1). Some activity remained in the interphase between the gradient and the sample zone; electron micrographs showed that this fraction also contained some mitochondria. Glucose-6-phosphatase and acid fl-glucuronidase had broad peaks at somewhat lower densities than succinate dehydrogenase. These activities probably originated from lysosomes. A repeated gradient centrifugation reduced the specific activities of these enzymes in the pooled mitochondrial fractions by almost 50 per cent (Table I). h glucose-6-phosphatase and /3-glucuronidase had another major peak at
’ 5
FRACTION
15
IO
20
NUMBER
Fig. 1. Purification of the crude mitochondrial fraction by density gradient centrifugation. The enzyme activities and protein concentrations are expressed as per cent of the total amounts recovered from the succinate dehydrol----*, gradient. Fraction Nos. 7-10 (black bar) were pooled for repurification. n -.-.--a , @-glucuronidase. glucos&Sphosphatase; protein; A-, genase ; 04,
246
TABLE I PURIFICATION
OF THE BHK CELL MITOCHONDRIA
The specific activities were normalized to the specific activities of the crude mitocbmdrial the individual experiments. Vatues are mean + S.D. (n = 3). ___._.______._._. -. ..__-_.--.--~---I------. -__-
fractions
of
~ -
Relative specific activity Succinate dehydrogenase
Glucose-6phosphatase
Crude mitochondria
1.00
1.00
1.00
Pooled peak from the 1st gradient
1.40 + 0.22
0.86 F 0.16
0.74 * 0.09
Pooled peak fram the 2nd gradient
1.65 f: 0.30
0.47 i 0.29
0.45 t 0.16
the ~ple-~adient i~~~hase (Fig, l), which was rich in membrane fragments. We propose that the main source of the activities in the top fractions was the fragmented endoplasmic reticulum. This interpretation is consistent with OUKearlier evidence, which suggested the presence of glucose-6-phosphatase and /3-glucuronidase both in the lysosomes and in the endoplasmic reticulum of the BHK cell [‘7,16].
The matrix of the purified mitochondria was contracted and densely stained; the outer membrane was swollen and frequently disrupted. This state probably results from the consecutive hypo- and hyperosmotic conditions during the purification process. The most frequent contaminant consisted of membranelimited vacuoles containing small ribosome-like particles, The subcellular origin of these structures is not known. Particles that could be identified as Iysosomes were infrequent. The purified nuclei were electron-lucent and the nucleoli had disappeared, probably due to the presence of EDTA in the preparation media. The outer nuclear membrane was usually preserved although often partially detached from the inner membrane. The preparations were contaminated by some mitochondria, and oceasion&ly by whole cells.
Enzymic characteristics of the mitochandria and nuclei Table II summarizes the specific activities of the enzymes and the phospholipid contents of the purified nuclei and mitochondria. The table also includes our earlier data on the crude microsomal and fight mitochodrial fractions [7], for comparison, Succinate dehydrogenase was enriched in the mitachondria, on the average, 11-fold relative to its specific activity in the cell homogenate. The average specific activities of jocose-6-phosphatase and ~-glucuronid~e in the mitochondria were 10 and 20%, respectively, of their activities in the microsomal and light mitochondrial fractions. Deoxyribonucleic acid was enriched over 6-fold in the purified nuclei (Table II). The specific activity of succinate dehydrogenase in the preparations was only 4 per cent of the activity in the purified mitochondria. The activities of ~ucose-6-phosphat~e and ~-glucuronid~e in the nuclei were high. On phos-
247 TABLE II ENZYME
ACTIVITIES
TIONS ISOLATED
AND CONTENTS
OF DNA AND PHOSPHOLIPID
IN SUBCELLULAR
FRAC-
FROM THE BHK CELLS
Specific activities and concentrations are expressed per mg of protein. mean f S.D. (number of determinations in brackets).
Whole cell
Succinate dehydrog.
p-glucuronidase
Glucose-6phosphatase
DNA
Phospholipid
WmoI/h)
(I.rmoIlh)
@moI/h)
(mg)
(mg)
0.04 * 0.01
0.4 f 0.1
0.095 f 0.014
0.16 f 0.02 ’
(10)
(9)
(12)
0.04 * 0.02
0.5 f 0.1
0.59 (3)
3.5 f 0.4 (19)
Nuclei
1.5 f 0.7 (5)
Mitochondria
39
+6
(3) Microsomal fraction
0.3 f 0.2 (5)
Light mitochondrial fraction
11
+3
(5)
(4)
(4)
0.03 + 0.03
0.15 ? 0.01
(14) + 0.04
0.15 + 0.01 (4) 0.39 r 0.03
(3)
(3)
(3)
0.15 + 0.05
1.4 f 0.3
0.72 + 0.08
(5)
(5)
(5)
0.16 t 0.06
1.3 + 0.6
0.55 f 0.10
(5)
(5)
(5)
* Data taken from reference 7.
pholipid basis (obtained phospholipid to protein cose8-phosphatase was mitochondrial fractions; fractions. This indicates nuclear envelope. Phospholipids
dividing the specific activities on protein basis by the ratio of the preparations) the specific activity of gluhigher in the nuclei than in the microsomal or light that of fl-glucuronidase was almost equal in all three that the two enzymes were true components of the
of the BHK cell organelles
The phospholipid compositions of the purified mitochondria and nuclei are shown in Table III. For comparison, the earlier data on the floating lysosomes [ 71, plasma membranes and endoplasmic reticulum [ 61 are also included. Cardiolipin was present in appreciable amounts only in the mitochondria, where it was enriched (relative to phospholipid) approximately 5-fold over the average cellular content. The cardiolipin content of the various subcellular fractions was linearily correlated to the specific activity of succinate dehydrogenase (Fig. Z), which indicates that the small amounts of cardiolipin in the other organelle preparations were due to contaminating mitochondria. The amount of phosphatidylethanolamine in the mitochondria was higher than in most other organelles whereas the percentage of phosphatidylcholine was lower than in the nuclei and endoplasmic reticulum, but higher than in the plasma membrane and lysosomes. The nuclei were characterized by a very high content of phosphatidylcholine, which amounted to 68% of the total phospholipids. Phosphatidylinositol was also quite specifically enriched in the nuclei; the other organelles, with the exception of the endoplasmic reticulum, contained far lesser amounts of this lipid. Only minute amounts of lysobisphosphatidic acid were found in most organelle preparations except in the floating lysosomes, where it was strongly
’ 1
248
TABLE
III
PHOSPHOLIPID
COMPOSITIONS
0F ORGANELLES
The values are mole per cent of lipid phosphorus, Whole cell a
Mitnchondria
PURIFIED
FROM THE BHK CELLS
mean + S.D. Nuclei
(%f
(%)
(W)
Phosphatidylcholine c
55.7 2 2.9
47.5 i 3.2 f
673
Phosphatidylethanolamine
25.1 + 1.2
26.9 i 1.5
19.6 f 1.8 f
Sphingomyelin
6.5 * 0.7
+ 3.6 f
4.2 t 4.8 g
2.1 i 1.2 f
Fioating lysoSomeB a w+
brane b (9%)
40i4f
41.6 + 2.6 f
59.6 + 6.0
30i:Gd
20.2 f. 1.9 f
16.2 t 2.4 f
7+3
20.0 ?:3.5 *
11.5 + 5.7 e
Plasma mem-
EndopiasTc reticulum .-
(%I
Phosphatidylserine
4.3 4 1.0
1.9 i 1.4 e
2.2 + 0.9 f
2+2@
9.2 + 1.4 f
3.3 i 1.2 d
Cardiolipin
3.2 t f.1
15.6 t 4.7 f
1.6 i 0.6 f
Oilf
0.3 i 0.3 f
1.8 t 1.6 d
Pbosphatidylin&tot
2.3 A 0.9
3.0 * 2.3
4.9 + 2.Se
223
3.6 * 3.3
5.0 r 1.8 f
1.7 + 0.6
0.3 * 0.3 f
0.5 ?:0.8 e
19t6f
0.8 f 0.6 '
0.9 + 0.7
0.6 + 0.5
0.7 L 0.3
0.5 * 0.4
4.1 ?:1.6 f
1.7 It1.1 d
Lysobisphosphatidic acid Phosphatidic Number of anaty%%
acid
0
(7)
a Taken from reference 7. b Data of Renkonen et al [6]; the figures for cardiolipin and lysobisphosphatidic acid have not been published previously. CIncluding lysophosphatidylchaiine. d,e,f Difference to the whole cell value significant at the confidence levels of 0.90, 0.95 and 0.99. respectively. g The high mean and large S.D. for the mitochondriai sphingomyelin were caused by the apparent content of 9.8% in one sampie. The mean i half range of the other two analyses Was 1.31 * 0.03.
enriched [7]. The enrichment of sphingomyelin and phosphatidylserine in the plasma membrane was noted previously 163 ; the mitochondria and nuclei contained only low amounts of these lipids, Protein and phospholipid pools of the BHK cell organelles Table IV presents the estimation of the fractional amount.s of the total cellular protein and phospholipid in the different organetles. The eaiculations rely on the ~surnp~o~ that the observed enrichments of the best markers corresponded to pure and representative organefle samples. Approximately 16% of the cellular protein was localized in the nuclei. The actual figure may be still higher; some of the nuclear protein may have been extracted during the purification in the absence of divalent cations. The protein pools of the other organehes were less than 10% of total. The phospholip~d pool sizes are of interest because they represent roughly the relative surface areas of the organelle membranes. Mitoehondrial phospholipids represent about one fifth of the total phospholipids in BHK cells. The same result is obtained by using succinate dehydrogenase and cardiolipin as the mitochondrial marker. Plasma membrane phospholipids are quantitatively even more important, and the nuclei, too, contribute about one sixth of the cellular
e
249
20-
a 15CT 1 0 0 \ a IO%
I
0.5
1.0
1.5
2.0
2.5
3.0
DEHYDROGENASE ( pmolx h-’ x )Jg P-’ )
SUCCINATE
Fig. 2. Correlation between the cardiolipin content and succinate dehydrogenase activity in BHK cell fractions. The data are expressed in terms of lipid phosphorus. The use of protein as a basis preserves the linear relationship but shifts the organelles up or down the line depending on their phospholipid contents. Correlation coefficient r = 0.972. A, purified mitochondria; a. crude mitochondrial fraction; +, whole cell: l_ light mitochondrial fraction; 0, crude nuclear fraction; n, purified nuclei; C. microsomal fraction.
TABLE
IV
THE SIZES
OF THE PROTEIN
AND PHOSPHOLIPID
POOLS
IN SOME BHK CELL ORGANELLES
The fractional amounts of cellular protein in nuclei, mitochondria and plasma membrane were obtained as the inverse values of the protein-based enrichments of the enzyme markers and DNA. The fractional amounts of cellular phospholipids in mitchondria and lysosomes were obtained as the inverse values of the phospholipid-based enrichments of the marker lipids. Multiplication of the relative pool size of proteins by a factor, which is obtained by dividing the weight ratio of phospholipid/protein in the organelle by the weight ratio of phospholipid/protein in the cell, gives the relative pool size of phospholipid in all organelles. Standard deviations were calculated by the usual propagation-of-error formulae [231. organe11e
Marker
Enrichment
of marker
Per protein
Nuclei
DNA
Mitochondria
Succinate
Per phospholipid
Phospholipid (g)
Relative Protein
Protein
(g)
(%)
pool size Phospholipid (%)
6.2 + 1.0
0.15
+ 0.01
15i
3
11.2 * 2.2
0.39
f 0.03
9+
2
21*
5
4.9 * 2.2
0.39
+ 0.03
9f
4
20+
9
11.4 * 5.3
0.88
*
2kl
9*
4
16 f 3
dehydrogenase Mitochondria
Cardiolipin
Lysosomes
Lysobisphosphatidic acid
Plasma membrane
(Na+ + K+)-ATPase
a From ref. 7. b From ref. 6. c From ref. 16.
15.3 * 7.8 ***
0.77 + 0.08
**
7f3
31 * 17
250
phospholipids. The relative size of the lysosomal phospholipid pool appeared to be about 9%, but this value is likely to be quite variable [l]. Put together, these data leave 24% of the cellular phospholipids unaccounted for. Most of the missing material is likely to represent phospholipids of the endoplasmic reticulum and the Golgi apparatus. The relative pool sizes of the individual phospholipid classes, phosphatidylcholine, sphingomyelin etc., in the different organelles can be obtained from the data of Tables III and IV. This information is necessary for the understanding of the equilibration of newly synthetized phospholipid molecules among the different cellular membranes. Discussion Phospholipids of the subcellular organelles Mitochondria purified from a number of mammalian tissues contain fairly constant amounts of cardiolipin, 15-20% of lipid phosphorus [17]. They generally have slightly higher amounts of phosphatidylethanolamine and less phosphatidylcholine than the whole homogenate, and they contain very little of the other phospholipids. The phospholipid composition of the mitochondria purified from the BHK cells showed all these characteristics. Cardiolipin is normally absent from the other subcellular membranes [ 171; this was the case also in the BHK cell. This effective regulation of the location of cardiolipin seems to be disturbed in some malignant hepatomas, where many membrane classes were reported to contain considerable amounts of cardiolipin [ 12,131. The membranes of the mammalian nuclei are characterized by a high content of phosphatidylcholine and an elevated amount of phosphatidylinositol [17, 181. The concentrations of all the other lipids, except phosphatidylethanolamine, are usually low. The BHK cell nuclei were typical in all these features; their content of phosphatidylcholine was among the highest so far observed. On the basis of the similarities in the enzyme activities and lipid compositions of the nuclear membrane and endoplasmic reticulum of rat liver, Kartenbeck et al. [19] concluded that the two membranes belong to a single membrane class (see also ref. 18). Our observations suggest that this is the case also in the BHK cell. The nuclear preparations contained high activities of glucose-6phosphatase and P-glucuronidase, which were also enriched in the microsomal fraction. The purified endoplasmic reticulum of the BHK cell also contains /3glucuronidase [16]. The phospholipid compositions of the nuclei and endoplasmic reticulum also had similarities: both were rich in phosphatidylcholine and phosphatidylinositol. The major distinctive feature in the composition of the endoplasmic reticulum, the high content of sphingomyelin, may be at least partially due to a heavy contamination of the preparations by plasma membrane and the membranes of the Golgi apparatus, which also are rich in sphingomyelin [20,21]. High concentrations of lysobisphosphatidic acid have been repeatedly observed in lysosomes [8-111 including the floating neutral lipid-rich lysosomes isolated from the BHK cells [7]. The present work demonstrated that lysobisphosphatidic acid is virtually absent from all the other major organelles of the BHK cell. A similar conclusion may be drawn from the data of Fleischer
251
and Fleischer [20] on the membranes af the bovine liver. The enrichment of sphingomyel~ in the BHK cell plasma membrane was discussed earlier [S] . Its concentration in the purified mitochondria and nuclei of the BHK cell was low {and variable), as is the case in other tissues [17,18]. Phosphatidylserine also was specifically concentrated in the plasma membrane of the BHK cell; this seems to be the case also in the rat liver [22], but not in all cells [Z--4]. In conclusion, it is evident that the membranes of the major organefles of the BHK cell have characteristic phospholi~id compositions similar to those of the homologous organelles isolated from the tissues of intact animals, This indicates that the mechanisms of phospholipid metabolism, which control the membrane compositions, are fully functional in the BHK cell. While this manuscript was in preparation, Micklem et al. [49], also reported that the phospholipid compositions of the plasma membrane, nuclei, mitochondria and endoplasmic reticulum purified from BHK21 cells were similar to those of homologous organelles from other sources.
Relative abundances of the BHK cell organelles: comparison to the rat liver In Table V we have calculated the relative abundances of the major organelles of the rat liver on the basis of the wealth of analytical data reported in recent publications= This allows a comparison of the cultured BHK cell to the highly differentiated hepatocyte. In the BHK cell the proportion of the cellular phospholipid in the nucleus is ten times higher than in the liver. The relative abundances of the mitochondrial membranes are quite similar in the two cells as judged by almost identical
TABLE V THE SIZES OF THE PROTEIN AND PHOSPHOLIPID POOLS OF THE RAT LIVER OREANELLES The phospholipid content of the whole liver was taken as 0.16 g/g protein I241 and the DNA content as 0.013 gJg protein 1251. The protein pools of the mitoehondria, lysosomes and endoplasmic reticulum were taken directly from Leighton et al. tttil. The enrichment of markers for the nucleus (DNA) and plasma membrane (enzymesf, as w&l as the phospholipid contents of all organ&es were taken from recent literature and expressed as mean + S.D. of the reported vaIues. The calcutations were analogous to those of Table V. Organelle
Nucleus
Enrichment of markers
Phospholipid (g) -___-
Relative pool size, per cent of total
Protein (gj
Protein
0.07 * 0.02 b
32t7a
3.z + 0.1
Mitochondria
0.15 f 0.04 c
20.2
Lysosomes
0.19 + 0.03 d
2.0
0.30 rt 0.05 e
21.5
Endoplasmic reticulum Plasma membrane
0.61 f 0.35 g
26*qf
3.9 t 0.6 50.1
Sum a Based on the DNA content
of the liver nucieus of 0.42 t 0.10
b Data from references 19, 27, 30-33. c Data from references 33-39. d Only tritasomes are included [ 22.36.401. e Data from references 29. 34, 36-38.41. f Data from references 24,33,38,40.42--44, g Data from references 33,36,38,40,42,4547,
Phospboiipid 1.3 L 0.5 19
1:5
2.3 ?z0.3 40
+I
13
+s
15.6
(S.D.) g/g protein [19,27-311.
252
phospholipid pool sizes. The BHK cell contains approximately 3 times more lysosomal phospholipids than the liver. The phospholipid pool of the BHK cell plasma membrane seems to be larger than that of the rat liver, but the data is somewhat inconclusive because of the large variations in the phospholi~id~~rotein ratios reported for the liver plasma membrane. This may be partly due to the enrichment of chemically different regions of the plasma membrane [45] in the preparations of different laboratories. In the BHK cell the nuclei, mitochundria, lysosomes and plasma membrane together contain 76% of the cellular phosph~lipid. In the liver these organelles contain only 35% of the tatal cellular phospholipid. Thus the relative amount of phospholipid belonging to other pools (mostly membranes and soluble lipoproteins of endoplasmic reticulum) is likely to be higher in the liver than in the BHK cell. Indeed, Table V shows that endoplasmic reticulum of liver contains as much as 40% of the total phospholipid. In terms of protein, too, the relative size of the BHK cell nucleus is several times higher than that of the liver. In contrast, the two cells reveal protein fractions of the same size in their lysosomes. Also the protein pools of the two plasma membranes are rather similar in size, but the mitochondria of the BHK cell contain a much smaller fraction of the cellular protein than do the mitochondria of the Ever. A similar dis~ibution of protein has been reported also for cultured mouse fibroblasts [ 481. Tables IV and V show that the BHK cell organelles have lower protein/phospholipid ratios than the corresponding organelles of liver. This may be a sign of a comparatively low activity in many of the metabolic functions of the BHK cell organelles and membranes. Acknowledgement We thank Dr. Carl Gahmberg for the permission to use the data on the plasma membrane and endoplasmic reticulum, Dr. Ismo Vi&men for two of the nucIear preparations and instruction in electron microscopy, and Mrs. Anneli Asikainen, Mrs. Satu Cankar, Miss Pirkko Leikas and lS&ss Ritva Saarlemo for assistance. The financial support of the Ju&lius foundation, &omen Kulttuurirahasto and Kordelin foundation is gratefully acknowledged. References 1
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Bergelson. L.D., Dyatlovitskaya, E.V.. Torkhovskaya, T.I., Sorokina. I.B. and Gorkova, N.P. (1970) Biochim. Biophys. Acta 210. 287-298 Bergelson, L.D., Dyatlovitskaya. E.V.. Sorokina, I.B. and Gorkova, N.P. (1974) Biochim. Biophys. Acta 360.361-365 Nass. M.M.K. (1969) J. Mol. Biol. 42. 521-528 Burton, K. (1956) Biochem. J. 62. 315-323 Gahmberg. C.G. and Simons, K. (1970) Acta Pathol. Microbial. &and. B78. 176-182 McMurray, W.C. (1973) in Form and Function of Phospholipids (Ansell, G.B., Hawthorne, J.N. and Dawson, R.M.C., eds.), 2nd Edn., pp. 205-251, Elsevier, Amsterdam Wunderlich, F., Berezney, R. and Kleining, H. (1976) in Biological Membranes, (Chapman, D. and Wallach. D.F.H.. eds.). Vol. 3, pp. 241-333. Academic Press. New York Kartenbeck. J., Jarasch. E.D. and Franke, W.W. (1973) Exp. Cell Res. 81. 175-194 Fleischer, B. and Fleischer, S. (1971) in Biomembranes (Manson, L., ed.), Vo1.2, pp. 75-95, Academic Press, New York Keenan. T.W. and MorrB, D.J. (1970) Biochemistry 9,19-25 Thines-Sempoux, D. (1972) in Lysosomes in Biology and Pathology (Dingle. J.T., ed.), Vol. 3. pp. 278-299. North-Holland. Amsterdam Parratt. L.G. (1966) Probability and Experimental Errors in Science. John Wiley & Sons, New York Thin&Sempoux. D. (1974) in Methodological Developments in Biochemistry, Subcellular Studies (Reid, E., ed.), Vol. 4. pp. 157-166 Leslie, I. (1955) in The Nucleic Acids (Chargaff, E. and Davidson, J.N.. eds.), Vol. 2, pp. l-50, Academic Press, New York Leighton, F., Poole, B., Beaufay, H., Baudhuin. P., Coffey, J.W.. Fowler. S. and DeDuve. C. (1968) J. Cell Biol. 37, 482-513 Kashning, D.M. and Kasper, C.B. (1969) J. Biol. Chem. 244, 3786-3792 Zentgraf, H., Deumling, B. and Franke. W.W. (1969) Exp. Cell Res. 56, 333-337 Franke, W.W., Deumling. B., Ermen. B., Jarasch, E.-D. and Kleining. H. (1970) J. Cell Biol. 46. 379395 Kay, R.R., Fraser. D. and Johnston, I.R. (1972) Eur. J. Biochem. 30, 145-154 Monneron. A., Blobel, G. and Palade, G.E. (1972) J. Cell Biol. 55. 104-125 Khandwala, AS. and Kasper. C.B. (1971) J. Biol. Chem. 246.62424246 Fleischer. S. and Kervina, M. (1974) in Methods in Enzymology, Biomembranes Part A (Fleischer. S. and Packer, L., eds.). Vol. 31. pp. 6-41. Academic Press, New York, San Francisco, London Stoffel, W. and Schieffer, H.-G. (1968) Hoppe-Seyler’s Z. Physiol. Chem. 349, 1017-1026 Newman, H.A.I.. Gordesky. S.E., Hoppel. C. and Cooper, C. (1968) Biochem. J. 107, 381-385 Colbeau, A., Nachbaur. J. and Vignais, P.M. (1971) Biochim. Biophys. Acta 249.462492 Feo, F., Canuto, R.A.. Bertone. G.. Carcea, R. and Pani, P. (1973) FEBS Lett. 33. 229-232 Arborgh, B.#.M. Glaumann. H. and Ericsson, J.L.E. (1974) Lab. Invest. 30. 664-673 Guerra, F.C. (1974) in Methods in Enzymology, Vol. 31, Biomembranes Part A (Fleischer. S. and Packer. L., eds.), pp. 299-305, Academic Press. New York Henning, R.. Kaulen, H.D. and Stoffer, W. (1970) Hoppe-Seyler’s Z. Physiol. Chem. 351, 1191-1199 Glaumann, H. and Dallner, G. (1968) J. Lipid Res. 9, 720-729 Touster, 0.. Aronson. N.N., Dulaney, J.T. and Hendrickson, H. (1970) J. Cell. Biol. 47, 604-618 MorrB. D.J. (1971) in Methods in Enzymology, Vol. 22. Enzyme Purification and Related Techniques (Jakoby, W.B., ed. ), pp. 130-148, Academic Press. New York Morrb, D.J. (1973) In Molecular Techniques and Approaches in Developmental Biology (Chrispeels, M.J., ed.), pp. 1-27. John Wiley &Sons. New York Evans, W.H. (1969) FEBS L&t. 3, 237-241 Emmelot, P. and Bos, C.J. (1972) J. Membr. Biol. 9, 83-104 Emmelot, P., Bos, C.J.. van Hoeven, R.P. and van Blitterswijk, W.J. (1974) in Methods in Enzymology, Vol. 31, Biomembranes Part A (Fleischer, S. and Packer, L., eds.), PP. 75-102, Academic Press, New York Glick, M.C., Comstock, C.A.. Cohen, M.A. and Warren, L. (1971) Biochim. Biophys. Acta 233, 247257 Micklem, K.J., Abra. R.M., Knutton, S., Graham. J.M. and Pasternak. C.A. (1976) Biochem. J. 154. 561-566
Biochimica et Biophysics @ Elsevier/North-Holland
Acta,
486
Biomedical
(1977)
243-253
Press
BBA 56939
PHOSPHOLIPIDS OF SUBCELLULAR CULTURED BHK CELLS *
JAAKKO
BROTHERUS
July 22nd,
ISOLATED
FROM
and OSSI RENKONEN
Laboratory of Lipid Research, Department SF-00290 Helsinki 29 (Finland)
(Received
ORGANELLES
of Biochemistry,
University
of Helsinki,
1976)
Summary
Mitochondria and nuclei were purified from cultured hamster fibroblasts (BHKZl cells) by centrifugation in sucrose gradients. The phospholipid compositions of the preparations were compared to those of the previously purified plasma membranes, endoplasmic reticulum and lysosomes. The mitochondria had a characteristically high content (approx. 16% of lipid phosphorus) of cardiolipin, which was practically absent from the other purified organelles. The nuclei were enriched in phosphatidylcholine and phosphatidylinositol (approx. 68% and 5% of lipid phosphorus, respectively). Lysobisphosphatidic acid was almost absent from the mitochondria and nuclei, as well as from the plasma membrane and endoplasmic reticulum, which suggests that this phospholipid is confined to the lysosomes of the BHK cell. The nuclei and the mitochondria contained relatively little sphingomyelin, a characteristic lipid of the plasma membrane. The distributions of the total cellular phospholipid and protein between the various organelles were calculated and compared to the corresponding data estimated for the rat liver. The BHK cell contained relatively more phospholipids in the nucleus and the lysosomes than the liver. All the organelles of the BHK cell contained less protein per phospholipid than the equivalent organelles of the liver. _. ._ Introduction
Cultured mammalian cells are frequently used in studies of the structure, function and biogenesis of lipids in cell membranes. These cells offer a better * part of the data has been published in a preliminary form (Renkkonen, 0.. Luukonen. A., Brothems, J. and KGiritinen, L. (1974) in Control of Proliferation in Animal Cells (Clarkson, B. and Baserga. R., eds.). pp. 495-504, Cold Spring Harbor Laboratory, Cold Spring Harbor).
244
system for many types of experiments than e.g. intact animals, tissue slices or isolated cells. The lipids of purified plasma membranes of several cultured cell lines (Z-4), including baby hamster kidney cells (BHK cells) [5,6], have been studied rather extensively in connection with recent work on the assembly of certain enveloped viruses. It has become evident in these studies that the composition of the plasma membrane lipids in the cultured cells is similar to that found in plasma membranes of mammalian cells in general; sphingomyelin, sphingoglycolipids and free cholesterol are enriched in this organelle. Even the lysosomal lipids of cultured BHK cells appear to have some resemblance to those found in mammalian lysosomes in general. The lysosomes of the BHK cell contain large amounts of lysobisphosphatidic acid [7], a characteristic phospholipid of the lysosomes of many mammalian systems [ 8-111. The present report shows that also the mitochondria and the nuclei of the BHK cell have phospholipid patterns similar to those of the homologous organelles from other sources. Taken together, the data on BHK cell indicate that this cell possesses the mechanisms which create and maintain the characteristic phospholipid compositions of the subcellular membranes in mammals. This appears not to be the case in all cells [ 12,131. The present data allow also an estimation of the fractions amounts of total cellular phospholipid belonging to the different organelles of the BHK cell. Materials and Methods Organelle ~r~c~~ona~~o~. The cultivation of the BHK21 cells (strain Wi-2) and the prelimin~y organelle fractionation by differential cent~fugation were described previously [ 71. The crude mitochondrial fraction, suspended in 6 ml of cold 0.3 M sucrose which was buffered with 5 mM Tris . HCl, 0.5 mM EDTA, pH 7.4 (Tris/EDTA buffer), was pipetted on top of a gradient [14] consisting of a 2 ml cushion of 52% (1.88 M) sucrose overlaid by a 30 ml linear gradient from 45% (1.58 M) to 31% (1.02 M) sucrose; both solutions were buffered with the Tris/EDTA. The gradient was centrifuged for 60 min at 25 000 rev./min (82 000 X g,“) in A Beckmann L3-50 ultracentrifuge, rotor SW 27. Fractions of 2 ml were collected from below. The most turbid fractions of the mitochondrial peak were pooled, after taking samples for analysis, diluted to 6 ml by the Tris/EDTA buffer and recentrifuged in a similar manner as above. The crude nuclear fraction in 5 ml of 0.3 M sucrose, buffered with the Tris/ EDTA, was diluted with 30 ml of 2.3 M sucrose buffered with the Tris/EDTA, and centrifuged in the same conditions as above. The packed layer on the surface was collected with a spatula and the sucrose solution was removed with a Pasteur pipette. The nuclear pellet was suspended in 0.3 M sucrose in the Tris/ EDTA buffer. The subcellular fractions were stored at -20°C before analysis. AnalyticaE methods. Succinate dehydrogenase (E.C. 1.3.99.1), P-glucuronidase (E.C. 3.2.1.31), glucose-6-phosphatase (E.C. 3.1.3.9), phospholipids and protein were analyzed as previously [7]. The protein standards contained the same amount of sucrose and Tris/EDTA as the actual samples. DNA was
245
determined [15] using calf thymus DNA (Worthington Biochemical Corp., Freehold, N.J.) as a standard. Electron microscopy. The cell fractions were diluted to approx. 0.3 M sucrose concentration with the Tris/EDTA buffer and pelleted by centrifugation. The pellets were fixed and processed for microscopy as earlier [ 71. Results Purification of the mitochondria The activity of succinate dehydrogenase appeared as a relatively symmetric peak around the density of approx. 1.17 g/ml after centrifugation of the crude mitochondrial fraction in a sucrose gradient (Fig. 1). Some activity remained in the interphase between the gradient and the sample zone; electron micrographs showed that this fraction also contained some mitochondria. Glucose-6-phosphatase and acid fl-glucuronidase had broad peaks at somewhat lower densities than succinate dehydrogenase. These activities probably originated from lysosomes. A repeated gradient centrifugation reduced the specific activities of these enzymes in the pooled mitochondrial fractions by almost 50 per cent (Table I). h glucose-6-phosphatase and /3-glucuronidase had another major peak at
’ 5
FRACTION
15
IO
20
NUMBER
Fig. 1. Purification of the crude mitochondrial fraction by density gradient centrifugation. The enzyme activities and protein concentrations are expressed as per cent of the total amounts recovered from the succinate dehydrol----*, gradient. Fraction Nos. 7-10 (black bar) were pooled for repurification. n -.-.--a , @-glucuronidase. glucos&Sphosphatase; protein; A-, genase ; 04,
246
TABLE I PURIFICATION
OF THE BHK CELL MITOCHONDRIA
The specific activities were normalized to the specific activities of the crude mitocbmdrial the individual experiments. Vatues are mean + S.D. (n = 3). ___._.______._._. -. ..__-_.--.--~---I------. -__-
fractions
of
~ -
Relative specific activity Succinate dehydrogenase
Glucose-6phosphatase
Crude mitochondria
1.00
1.00
1.00
Pooled peak from the 1st gradient
1.40 + 0.22
0.86 F 0.16
0.74 * 0.09
Pooled peak fram the 2nd gradient
1.65 f: 0.30
0.47 i 0.29
0.45 t 0.16
the ~ple-~adient i~~~hase (Fig, l), which was rich in membrane fragments. We propose that the main source of the activities in the top fractions was the fragmented endoplasmic reticulum. This interpretation is consistent with OUKearlier evidence, which suggested the presence of glucose-6-phosphatase and /3-glucuronidase both in the lysosomes and in the endoplasmic reticulum of the BHK cell [‘7,16].
The matrix of the purified mitochondria was contracted and densely stained; the outer membrane was swollen and frequently disrupted. This state probably results from the consecutive hypo- and hyperosmotic conditions during the purification process. The most frequent contaminant consisted of membranelimited vacuoles containing small ribosome-like particles, The subcellular origin of these structures is not known. Particles that could be identified as Iysosomes were infrequent. The purified nuclei were electron-lucent and the nucleoli had disappeared, probably due to the presence of EDTA in the preparation media. The outer nuclear membrane was usually preserved although often partially detached from the inner membrane. The preparations were contaminated by some mitochondria, and oceasion&ly by whole cells.
Enzymic characteristics of the mitochandria and nuclei Table II summarizes the specific activities of the enzymes and the phospholipid contents of the purified nuclei and mitochondria. The table also includes our earlier data on the crude microsomal and fight mitochodrial fractions [7], for comparison, Succinate dehydrogenase was enriched in the mitachondria, on the average, 11-fold relative to its specific activity in the cell homogenate. The average specific activities of jocose-6-phosphatase and ~-glucuronid~e in the mitochondria were 10 and 20%, respectively, of their activities in the microsomal and light mitochondrial fractions. Deoxyribonucleic acid was enriched over 6-fold in the purified nuclei (Table II). The specific activity of succinate dehydrogenase in the preparations was only 4 per cent of the activity in the purified mitochondria. The activities of ~ucose-6-phosphat~e and ~-glucuronid~e in the nuclei were high. On phos-
247 TABLE II ENZYME
ACTIVITIES
TIONS ISOLATED
AND CONTENTS
OF DNA AND PHOSPHOLIPID
IN SUBCELLULAR
FRAC-
FROM THE BHK CELLS
Specific activities and concentrations are expressed per mg of protein. mean f S.D. (number of determinations in brackets).
Whole cell
Succinate dehydrog.
p-glucuronidase
Glucose-6phosphatase
DNA
Phospholipid
WmoI/h)
(I.rmoIlh)
@moI/h)
(mg)
(mg)
0.04 * 0.01
0.4 f 0.1
0.095 f 0.014
0.16 f 0.02 ’
(10)
(9)
(12)
0.04 * 0.02
0.5 f 0.1
0.59 (3)
3.5 f 0.4 (19)
Nuclei
1.5 f 0.7 (5)
Mitochondria
39
+6
(3) Microsomal fraction
0.3 f 0.2 (5)
Light mitochondrial fraction
11
+3
(5)
(4)
(4)
0.03 + 0.03
0.15 ? 0.01
(14) + 0.04
0.15 + 0.01 (4) 0.39 r 0.03
(3)
(3)
(3)
0.15 + 0.05
1.4 f 0.3
0.72 + 0.08
(5)
(5)
(5)
0.16 t 0.06
1.3 + 0.6
0.55 f 0.10
(5)
(5)
(5)
* Data taken from reference 7.
pholipid basis (obtained phospholipid to protein cose8-phosphatase was mitochondrial fractions; fractions. This indicates nuclear envelope. Phospholipids
dividing the specific activities on protein basis by the ratio of the preparations) the specific activity of gluhigher in the nuclei than in the microsomal or light that of fl-glucuronidase was almost equal in all three that the two enzymes were true components of the
of the BHK cell organelles
The phospholipid compositions of the purified mitochondria and nuclei are shown in Table III. For comparison, the earlier data on the floating lysosomes [ 71, plasma membranes and endoplasmic reticulum [ 61 are also included. Cardiolipin was present in appreciable amounts only in the mitochondria, where it was enriched (relative to phospholipid) approximately 5-fold over the average cellular content. The cardiolipin content of the various subcellular fractions was linearily correlated to the specific activity of succinate dehydrogenase (Fig. Z), which indicates that the small amounts of cardiolipin in the other organelle preparations were due to contaminating mitochondria. The amount of phosphatidylethanolamine in the mitochondria was higher than in most other organelles whereas the percentage of phosphatidylcholine was lower than in the nuclei and endoplasmic reticulum, but higher than in the plasma membrane and lysosomes. The nuclei were characterized by a very high content of phosphatidylcholine, which amounted to 68% of the total phospholipids. Phosphatidylinositol was also quite specifically enriched in the nuclei; the other organelles, with the exception of the endoplasmic reticulum, contained far lesser amounts of this lipid. Only minute amounts of lysobisphosphatidic acid were found in most organelle preparations except in the floating lysosomes, where it was strongly
’ 1
248
TABLE
III
PHOSPHOLIPID
COMPOSITIONS
0F ORGANELLES
The values are mole per cent of lipid phosphorus, Whole cell a
Mitnchondria
PURIFIED
FROM THE BHK CELLS
mean + S.D. Nuclei
(%f
(%)
(W)
Phosphatidylcholine c
55.7 2 2.9
47.5 i 3.2 f
673
Phosphatidylethanolamine
25.1 + 1.2
26.9 i 1.5
19.6 f 1.8 f
Sphingomyelin
6.5 * 0.7
+ 3.6 f
4.2 t 4.8 g
2.1 i 1.2 f
Fioating lysoSomeB a w+
brane b (9%)
40i4f
41.6 + 2.6 f
59.6 + 6.0
30i:Gd
20.2 f. 1.9 f
16.2 t 2.4 f
7+3
20.0 ?:3.5 *
11.5 + 5.7 e
Plasma mem-
EndopiasTc reticulum .-
(%I
Phosphatidylserine
4.3 4 1.0
1.9 i 1.4 e
2.2 + 0.9 f
2+2@
9.2 + 1.4 f
3.3 i 1.2 d
Cardiolipin
3.2 t f.1
15.6 t 4.7 f
1.6 i 0.6 f
Oilf
0.3 i 0.3 f
1.8 t 1.6 d
Pbosphatidylin&tot
2.3 A 0.9
3.0 * 2.3
4.9 + 2.Se
223
3.6 * 3.3
5.0 r 1.8 f
1.7 + 0.6
0.3 * 0.3 f
0.5 ?:0.8 e
19t6f
0.8 f 0.6 '
0.9 + 0.7
0.6 + 0.5
0.7 L 0.3
0.5 * 0.4
4.1 ?:1.6 f
1.7 It1.1 d
Lysobisphosphatidic acid Phosphatidic Number of anaty%%
acid
0
(7)
a Taken from reference 7. b Data of Renkonen et al [6]; the figures for cardiolipin and lysobisphosphatidic acid have not been published previously. CIncluding lysophosphatidylchaiine. d,e,f Difference to the whole cell value significant at the confidence levels of 0.90, 0.95 and 0.99. respectively. g The high mean and large S.D. for the mitochondriai sphingomyelin were caused by the apparent content of 9.8% in one sampie. The mean i half range of the other two analyses Was 1.31 * 0.03.
enriched [7]. The enrichment of sphingomyelin and phosphatidylserine in the plasma membrane was noted previously 163 ; the mitochondria and nuclei contained only low amounts of these lipids, Protein and phospholipid pools of the BHK cell organelles Table IV presents the estimation of the fractional amount.s of the total cellular protein and phospholipid in the different organetles. The eaiculations rely on the ~surnp~o~ that the observed enrichments of the best markers corresponded to pure and representative organefle samples. Approximately 16% of the cellular protein was localized in the nuclei. The actual figure may be still higher; some of the nuclear protein may have been extracted during the purification in the absence of divalent cations. The protein pools of the other organehes were less than 10% of total. The phospholip~d pool sizes are of interest because they represent roughly the relative surface areas of the organelle membranes. Mitoehondrial phospholipids represent about one fifth of the total phospholipids in BHK cells. The same result is obtained by using succinate dehydrogenase and cardiolipin as the mitochondrial marker. Plasma membrane phospholipids are quantitatively even more important, and the nuclei, too, contribute about one sixth of the cellular
e
249
20-
a 15CT 1 0 0 \ a IO%
I
0.5
1.0
1.5
2.0
2.5
3.0
DEHYDROGENASE ( pmolx h-’ x )Jg P-’ )
SUCCINATE
Fig. 2. Correlation between the cardiolipin content and succinate dehydrogenase activity in BHK cell fractions. The data are expressed in terms of lipid phosphorus. The use of protein as a basis preserves the linear relationship but shifts the organelles up or down the line depending on their phospholipid contents. Correlation coefficient r = 0.972. A, purified mitochondria; a. crude mitochondrial fraction; +, whole cell: l_ light mitochondrial fraction; 0, crude nuclear fraction; n, purified nuclei; C. microsomal fraction.
TABLE
IV
THE SIZES
OF THE PROTEIN
AND PHOSPHOLIPID
POOLS
IN SOME BHK CELL ORGANELLES
The fractional amounts of cellular protein in nuclei, mitochondria and plasma membrane were obtained as the inverse values of the protein-based enrichments of the enzyme markers and DNA. The fractional amounts of cellular phospholipids in mitchondria and lysosomes were obtained as the inverse values of the phospholipid-based enrichments of the marker lipids. Multiplication of the relative pool size of proteins by a factor, which is obtained by dividing the weight ratio of phospholipid/protein in the organelle by the weight ratio of phospholipid/protein in the cell, gives the relative pool size of phospholipid in all organelles. Standard deviations were calculated by the usual propagation-of-error formulae [231. organe11e
Marker
Enrichment
of marker
Per protein
Nuclei
DNA
Mitochondria
Succinate
Per phospholipid
Phospholipid (g)
Relative Protein
Protein
(g)
(%)
pool size Phospholipid (%)
6.2 + 1.0
0.15
+ 0.01
15i
3
11.2 * 2.2
0.39
f 0.03
9+
2
21*
5
4.9 * 2.2
0.39
+ 0.03
9f
4
20+
9
11.4 * 5.3
0.88
*
2kl
9*
4
16 f 3
dehydrogenase Mitochondria
Cardiolipin
Lysosomes
Lysobisphosphatidic acid
Plasma membrane
(Na+ + K+)-ATPase
a From ref. 7. b From ref. 6. c From ref. 16.
15.3 * 7.8 ***
0.77 + 0.08
**
7f3
31 * 17
250
phospholipids. The relative size of the lysosomal phospholipid pool appeared to be about 9%, but this value is likely to be quite variable [l]. Put together, these data leave 24% of the cellular phospholipids unaccounted for. Most of the missing material is likely to represent phospholipids of the endoplasmic reticulum and the Golgi apparatus. The relative pool sizes of the individual phospholipid classes, phosphatidylcholine, sphingomyelin etc., in the different organelles can be obtained from the data of Tables III and IV. This information is necessary for the understanding of the equilibration of newly synthetized phospholipid molecules among the different cellular membranes. Discussion Phospholipids of the subcellular organelles Mitochondria purified from a number of mammalian tissues contain fairly constant amounts of cardiolipin, 15-20% of lipid phosphorus [17]. They generally have slightly higher amounts of phosphatidylethanolamine and less phosphatidylcholine than the whole homogenate, and they contain very little of the other phospholipids. The phospholipid composition of the mitochondria purified from the BHK cells showed all these characteristics. Cardiolipin is normally absent from the other subcellular membranes [ 171; this was the case also in the BHK cell. This effective regulation of the location of cardiolipin seems to be disturbed in some malignant hepatomas, where many membrane classes were reported to contain considerable amounts of cardiolipin [ 12,131. The membranes of the mammalian nuclei are characterized by a high content of phosphatidylcholine and an elevated amount of phosphatidylinositol [17, 181. The concentrations of all the other lipids, except phosphatidylethanolamine, are usually low. The BHK cell nuclei were typical in all these features; their content of phosphatidylcholine was among the highest so far observed. On the basis of the similarities in the enzyme activities and lipid compositions of the nuclear membrane and endoplasmic reticulum of rat liver, Kartenbeck et al. [19] concluded that the two membranes belong to a single membrane class (see also ref. 18). Our observations suggest that this is the case also in the BHK cell. The nuclear preparations contained high activities of glucose-6phosphatase and P-glucuronidase, which were also enriched in the microsomal fraction. The purified endoplasmic reticulum of the BHK cell also contains /3glucuronidase [16]. The phospholipid compositions of the nuclei and endoplasmic reticulum also had similarities: both were rich in phosphatidylcholine and phosphatidylinositol. The major distinctive feature in the composition of the endoplasmic reticulum, the high content of sphingomyelin, may be at least partially due to a heavy contamination of the preparations by plasma membrane and the membranes of the Golgi apparatus, which also are rich in sphingomyelin [20,21]. High concentrations of lysobisphosphatidic acid have been repeatedly observed in lysosomes [8-111 including the floating neutral lipid-rich lysosomes isolated from the BHK cells [7]. The present work demonstrated that lysobisphosphatidic acid is virtually absent from all the other major organelles of the BHK cell. A similar conclusion may be drawn from the data of Fleischer
251
and Fleischer [20] on the membranes af the bovine liver. The enrichment of sphingomyel~ in the BHK cell plasma membrane was discussed earlier [S] . Its concentration in the purified mitochondria and nuclei of the BHK cell was low {and variable), as is the case in other tissues [17,18]. Phosphatidylserine also was specifically concentrated in the plasma membrane of the BHK cell; this seems to be the case also in the rat liver [22], but not in all cells [Z--4]. In conclusion, it is evident that the membranes of the major organefles of the BHK cell have characteristic phospholi~id compositions similar to those of the homologous organelles isolated from the tissues of intact animals, This indicates that the mechanisms of phospholipid metabolism, which control the membrane compositions, are fully functional in the BHK cell. While this manuscript was in preparation, Micklem et al. [49], also reported that the phospholipid compositions of the plasma membrane, nuclei, mitochondria and endoplasmic reticulum purified from BHK21 cells were similar to those of homologous organelles from other sources.
Relative abundances of the BHK cell organelles: comparison to the rat liver In Table V we have calculated the relative abundances of the major organelles of the rat liver on the basis of the wealth of analytical data reported in recent publications= This allows a comparison of the cultured BHK cell to the highly differentiated hepatocyte. In the BHK cell the proportion of the cellular phospholipid in the nucleus is ten times higher than in the liver. The relative abundances of the mitochondrial membranes are quite similar in the two cells as judged by almost identical
TABLE V THE SIZES OF THE PROTEIN AND PHOSPHOLIPID POOLS OF THE RAT LIVER OREANELLES The phospholipid content of the whole liver was taken as 0.16 g/g protein I241 and the DNA content as 0.013 gJg protein 1251. The protein pools of the mitoehondria, lysosomes and endoplasmic reticulum were taken directly from Leighton et al. tttil. The enrichment of markers for the nucleus (DNA) and plasma membrane (enzymesf, as w&l as the phospholipid contents of all organ&es were taken from recent literature and expressed as mean + S.D. of the reported vaIues. The calcutations were analogous to those of Table V. Organelle
Nucleus
Enrichment of markers
Phospholipid (g) -___-
Relative pool size, per cent of total
Protein (gj
Protein
0.07 * 0.02 b
32t7a
3.z + 0.1
Mitochondria
0.15 f 0.04 c
20.2
Lysosomes
0.19 + 0.03 d
2.0
0.30 rt 0.05 e
21.5
Endoplasmic reticulum Plasma membrane
0.61 f 0.35 g
26*qf
3.9 t 0.6 50.1
Sum a Based on the DNA content
of the liver nucieus of 0.42 t 0.10
b Data from references 19, 27, 30-33. c Data from references 33-39. d Only tritasomes are included [ 22.36.401. e Data from references 29. 34, 36-38.41. f Data from references 24,33,38,40.42--44, g Data from references 33,36,38,40,42,4547,
Phospboiipid 1.3 L 0.5 19
1:5
2.3 ?z0.3 40
+I
13
+s
15.6
(S.D.) g/g protein [19,27-311.
252
phospholipid pool sizes. The BHK cell contains approximately 3 times more lysosomal phospholipids than the liver. The phospholipid pool of the BHK cell plasma membrane seems to be larger than that of the rat liver, but the data is somewhat inconclusive because of the large variations in the phospholi~id~~rotein ratios reported for the liver plasma membrane. This may be partly due to the enrichment of chemically different regions of the plasma membrane [45] in the preparations of different laboratories. In the BHK cell the nuclei, mitochundria, lysosomes and plasma membrane together contain 76% of the cellular phosph~lipid. In the liver these organelles contain only 35% of the tatal cellular phospholipid. Thus the relative amount of phospholipid belonging to other pools (mostly membranes and soluble lipoproteins of endoplasmic reticulum) is likely to be higher in the liver than in the BHK cell. Indeed, Table V shows that endoplasmic reticulum of liver contains as much as 40% of the total phospholipid. In terms of protein, too, the relative size of the BHK cell nucleus is several times higher than that of the liver. In contrast, the two cells reveal protein fractions of the same size in their lysosomes. Also the protein pools of the two plasma membranes are rather similar in size, but the mitochondria of the BHK cell contain a much smaller fraction of the cellular protein than do the mitochondria of the Ever. A similar dis~ibution of protein has been reported also for cultured mouse fibroblasts [ 481. Tables IV and V show that the BHK cell organelles have lower protein/phospholipid ratios than the corresponding organelles of liver. This may be a sign of a comparatively low activity in many of the metabolic functions of the BHK cell organelles and membranes. Acknowledgement We thank Dr. Carl Gahmberg for the permission to use the data on the plasma membrane and endoplasmic reticulum, Dr. Ismo Vi&men for two of the nucIear preparations and instruction in electron microscopy, and Mrs. Anneli Asikainen, Mrs. Satu Cankar, Miss Pirkko Leikas and lS&ss Ritva Saarlemo for assistance. The financial support of the Ju&lius foundation, &omen Kulttuurirahasto and Kordelin foundation is gratefully acknowledged. References 1
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