Lipid composition of subcellular particles of sea urchin eggs Strongylocentrotus intermedius

Comp. Biochem. Physiol.. 1978, Vol. 60B, pp. 99 to 105. Pergamon Press. Printed in Great Britain

LIPID COMPOSITION OF SUBCELLULAR PARTICLES OF SEA URCHIN EGGS S T R O N G Y L O C E N TRO T US I N T E R M E D I US VICTOR P: CHELOMIN and VASILIII. SVETASHEV Far East Research Centre Academy of Sciences of the USSR, Institute of Marine Biology, Laboratory of Comparative Biochemistry, Vladivostok 22, USSR (Received 13 July 1977)

Abstract--1. Nuclei, mitochondria, yolk granules and microsomes isolated from unfertilized eggs of the sea urchin Strongylocentrotus intermedius were studied for lipid composition. 2. All membrane structures studied possessed a specific lipid composition. Considerable differences were detected in the contents of lipids, phospholipids, individual lipid classes and phosphatidylethanolamine-plasmalogen. 3. Polar and neutral lipids of the subcellular particles contained a large amount of unsaturated, mainly polyen¢ fatty acids. Considerable quantitative differences were observed in fatty acid distribution (16:0, 18:0, 20:4 and 20:5) over the organelles. 4. The results obtained were compared with recent data on the lipid composition of subcellular particles of the cells in various animals.

INTRODUCTION An extensive study of lipid composition of the subcellular organelles has been carried out (Fleischer & Rouser, 1965; White, 1973). The results obtained made it possible to formulate a conception on lipid distribution in membrane structures. The conception was principally formulated by Rouser et al. (1968). Each type of subcellular particles of any cell has a diagnostic lipid class composition. The lipid patterns of the same particles isolated from different tissues and species are variable. The variations in lipid composition of plasma membrane and endoplasmic reticulum are more pronounced than those of nuclei and mitochondria. Mitochondria have high levels of diphosphatidylglycerol a n d unsaturated polyene fatty acids, the plasma membrane has high cholesterol and glycolipid contents; compared with other organelles, endoplasmic reticulum has less unsaturated acids. These conclusions were mainly made on the basis of an analysis of intracellular structures of higher animals. There are few works devoted to the study of lipid distribution within the cells of invertebrates (Thompson & Nozawa, 1972; Thompson et al., 1972). Thus, the above conclusions cannot be applied to the membrane structure of the latter group of animals without additional experiments. The marine invertebrates, an extensive group, extremely diverse in habitat, mode of life, mobility and feeding mechanisms, have developed different features in the course of their evolution. A comparative study of a wide range of organisms from different evolutional species help us to understand the n a t u r e of biological membranes. The present paper deals with the distribution of lipids in the subcellular particles of unfertilized eggs of the sea urchin. Sea urchin eggs are a classical subject in embryology and molecular biology, which is also suitable for the study of many problems pertaining membranology (Pasternak, 1973). A detailed study of the cell membrane lipids of the eggs of a sea urchin, 99

an invertebrate, which is taxonomically remote from higher animals, would permit us not only to confirm Rouser's conclusions, but also to broaden our knowledge of functional significance of membrane lipids in embryogenesis. MATERIALS AND METHODS Obtaining of eggs and the homogenate

Mature eggs from the sea urchin (Strongylocentrotus intermedius were used in the study. Adult animals were collected in Posiet Bay, Sea of Japan, during the spawning season (September-October). Eggs were obtained by injecting to the body cavity with a 0.53 M solution of KCl. Prior to homogenization, the eggs were washed with a 0.53 M KCI solution containing 1 mM EDTA. The precipitated eggs were diluted (1:10, v/v) with buffer "A" (50 mM Tris-HCl, pH = 7.5, 0.25 M sucrose, 0.25 M NaCl and 2 mM MgCl2) and homogenized by pressing the eggs suspensions through a hypodermic needle at 0°C. The homogenate was diluted to one half its concentrations by buffer "A" and maintained in the cold for 15-20min or centrifuged at 100-200g for 2rain. Then the homogenate was filtered through four layers of fine-mesh net and subsequently used for isolating organeiles. Fractionation of homogenate An enriched nuclear fraction was obtained by centrifugation of the homogenate at 500-800 g for 5 min in a K-23 centrifuge (Janetzki, GDR). The pellet was reprecipitated three times in buffer "A". Low-speed centrifugation in sucrose was used to obtain a pure nuclear fraction (Hogeboom et al., 1952). Centrifugation at 2500g for 15 min was applied to remove the surface membrane fraction from the homogenate residue. The fraction of mitochondria and yolk granules was then isolated from the supernatant fraction by centrifugation at 10,000g for 15min in a K-24 centrifuge (Janetzki, GDR). The pellet was suspended in buffer "B" (Tris-HCl 50mM, pH = 7.5, 0.25M sucrose, 0.25 M NaC1 and l mM EDTA) (Cantatore et al., 1974), • and the mitochondria and yolk granules were separated by ultracentrifugation in a sucrose gradient (Cantatorc et

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VICTOR P. CHELOMINand VASILllI. SVETASHEV

al., 1974; Maggio, 1959). For further purification, the mitochondrial fraction was additionally centrifuged in a discontinuous gradient of sucrose density (37%, 41% and 44%) in a VAC--601 centrifuge (Janetzki, GDR, rotor 3 x 35 ml) at 20,000rpm for 2 hr, t = 4°C. The yolk granule fraction was additionally centrifuged in a discontinuous sucrose gradient (1.0 M and 0.5 M) at the same speed. The microsome fraction was obtained in the usual way by centrifuging the post-mitochondrial-yolk granules' supernatant fraction at 105,000g for 90min (Nath & Rebhum, 1974). The pellet obtained was suspended in buffer "B" and recentrifuged. Subsequent to microsome precipitation, the supernatant fraction was centrifuged at 150,000g for 30min, and the resulting supernatant was used for further analysis• Marker enzyme assays ATPase activity was determined under conditions described by Stewart (1974). Na+-K+-ATPase activity was estimated by the difference between the total activity of (Na+-K +) Mg2+-ATPase, and Mg2+-ATPase. 5'-nucleotidase activity was tested in accord with Solyom & Trams (1972); glucose-6-phosphatase was assayed by a modified method of Barber & Foy (1973) in an acetate buffer (pH = 5.6) involving addition of 4 mM EDTA and 4 mM KF for inactivating alkaline and acid phosphatases (Sullivan & Volcani, 1974). Acid and alkaline phosphatases were determined under conditions described by Sullivan & Voicani (1974). The activities of the above-cited enzymes were determined by the measurement of liberated inorganic phosphate in accord with Murphy & Riley (1962) as described by Goryushina et al. (1969). The activity of cytochrome C oxidase was determined spectrophotometrically (Barber & Foy, 1973) by the increase in the concentration of the oxidized form of cytochrome C during 1 min incubation at 37°C. Protein concentration in the preparations was determined in accord with Lowry et al. (1951). Human serum albumin was used as a standard. Specific activity of all enzymes was expressed as moles of reaction, products formed at incubation (37°) per milligram of protein fractions in min or hr.

Lip/c/analysis Only distilled solvents were used in the work. The lipids were extracted by a modified technique of Bligh & Dyer (1959). The residue on the filter was additionally extracted using a chlor0form-methano! mixture (2:1 v/v). Lipid concentration was estimated by the bichromate method (Amenta, 1964). The standard curve was plotted for lipids isolated from sea urchin eggs. Lipids were cliromatographed on micro thin-layer plates (Svetashev & Vaskovsky, 1972). Polar lipids were separated by two-dimensional chromatography in solvent systems described by Rouser et al. (1967). Neutral lipids were separated in the system: hexane diethyl-ether acetic acid (85:15:1 v/v). The l]~w~re-dei~'/d~l-~ii ch~0matogi"dhisus;n-g a-io% sblu• non of H2SO4 in methanol with subsequent heating to 180°C and using specific reagents, for phospholipids (Vaskovsky et al., 1975), for phosphorus-containing substances (Vaskovsky & Latyshev, 1975), for amine-containing lipids (2% ninhydrin in acetone). An antron spray (van Gent et al., 1973) modified in our laboratory was used for glycolipids: 0.5% antron solution in benzene and 5% H2SO4 in water. Ubiquinone was detected using leucomethylene blue (Linn et al., 1959). The amounts of cholesterol, free fatty acids and triglycerides were determined after thinlayer chromatography by the bichromate method (Amenta, 1964). Cholesterol, stearic acid and tristearin were used for the standard curves. Ubiquinone was determined quantitatively as described by Linn et al. (1959). The total amount of phospholipids in extracts and phosphorus content in phospholipid spots were determined by the macroand micromethods described by Vaskovsky et al. (1975).

Plasmalogens were determined by reaction microchromatography (Vaskovsky & Dembitsky, 1975). To determine fatty acid composition, the lipids were separated into polar and neutral fractions using column chromatography on silica gel (Rouser et al., 1967). The fractions were subjected to methylation in accord with Christie (1973), and analysed on a glass column (1.5 m x 3.0 mm) with 15% diethylenglycolsuccinate on Chromosorb W-AW (DMCS), 100-120 mesh, at 185°C. The carrier gas was argon. A Shimadzu 5GC (Japan) chromatograph with a flame-ionization detector was used in the work. Faity acids were identified by using gas chromatography and mass spectrometry. The mass spectral data were obtained using an LKB 9000S (Sweden) instrument• RESULTS Enzymatic control of the subcellular fractions

The following marker-enzymes were tested to characterize the purity of the fractions: cytochrome C oxidase, Mg2+-ATPase for mitochondria; glucose-6-phosphatase for microsomes; acid phosphatase for yolk granules; and (Na+-K+)-ATPase, alkaline phosphatase and 5'-nucleotidase for the plasma membrane. The specific activities of the enzymes in the isolated fractions are shown in Table 1. Increased activity of Mg2+-ATPase and cytochrome C oxidase in comparison with the homogenate was observed in the mltochondri~ii fraeti0nl These enzymes displayed slight activity in the nuclear fraction, Cytochrome C oxidase activity was not detected in the other membrane fractions or in the supernatant. Glucose-6phosphatase concentrated primarily in the microsomal fraction, and, to a lesser degree, in the yolk granule fraction. Acid phosphatase, on the contrary, concentrated more in yolk granule fraction and less in microsomes. These enzymes showed low activity in mitochondria. None of the fractions showed any activity of 5'-nueleotidase, (Na+-K+)-ATPase and alkaline phosphatase. Lipid composition of organelles

Table 2 shows the lipid composition of the subcellular particles of an unfertilized sea urchin egg. Yolk granules and microsomes contain the largest amounts of lipids (0.77 and 0.54 mg lipids/mg protein, respectively). A high concentration of lipids is also in the post-microsomal supernatant. The dominant neutral lipids in all the fractions were cholesterol and triglycerides. Free fatty acids were detected in small amounts in nuclear, mitoehondrial and microsomal fractions. Nuclear and microsomal lipids are rich in cholesterol (14.6 and 16.1%, respectively), whereas a major part of yolk granule and supernatant lipids consist of triglycerides (51.3 and 71.8%, respectively). Ubiquinone was detected only in the mitochondrial fraction. Substantial differences are also observed in concentration of phospholipids in the main membrane structures. Phospholipids of mitochondria amount to 86.6%, while in yolk granule and supernatant lipids they amount to only 22.8% and 14.0%, respectively. Microsomes and nuclei are rich in phospholipids (61.7% and 42.6%, respectively). In all the isolated fractions phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the main phospholipids, the PC content being higher

101,

Subeellular particles of sea urchin eggs Table 1. Specitic activity of marker-enzymes in the isolated organelles of sea urchin eggs Enzyme

Cell

Nucleus

Mitochondria

Yolk granules

Microsomes

Supernatant

Mg 2 +-ATPase Cytoehrome C oxidase Acid p-nitrophenylphosphatase Glucose-6-phosphatase

1.45 2.84 0.27 8.6

0.90 1.48 0.10 12.0

6.0 17.6 0.17 6.2

1.70 0.96 25.0

1.08 0.40 60.0

1.30 12.0

Specific activity (SA) of cytochrome C oxidase ~ M oxidized cytochrome C/mg protein/min). SA Mg 2 +-ATPase, p-nitrophenylphosphatase, glucose-6-phosphatase (/tM Pi/mg protein/hr).

Table 2. Lipid composition of subcellular organelles of sea urchin eggs Lipids Lipid/protein (mg/mg) Phospholipids Cholesterol Triglyeerides Free fatty acids Ubiquinone

Cell

Nucleus

Mitochondria

Yolk granules

Microsomes

Supernatant

0.46 33.0 + 0.3 7.1 _ 0.4 42.4 _ 3.0 tr --

0.18 42.6 5:0.5 14.6 4- 1.0 19.5 -I- 0.2 3.9 4- 0.4 --

0.31 86.6 5:2.0 5.0 4- 1.0 3.3 + 0.3 3.0 + 0.4 0.7

0.77 22.8 _ 1.4 7.2 _ 0.6 51.3 + 1.0 tr --

0.54 61.7 _ 0.6 16.1 -I- 0.9 7.9 ___0.5 2.6 + 0.3 --

0.47 14.0 _ 2.7 3.3 _+ 0.1 71.8 + 1.0 tr --

Values are expressed as percentage of total lipids. Results represent means + S.D. (n = 4).

Table 3. Phospholipid composition of sea urchin eggs organelles Phospholipids Phosphatidylcholine diacyl Phesphatidylcholine alkenyl-aeyl Phosphatidylethanolamine diacyl Phosphatidylethanolamine alkenyl-acyl Phosphatidylserine Phosphatidylinositol Diphosphatidylglycerol Lysophosphatidylcholine Phosphatidic acid Phosphatidylglycerol PC/PE ratio

Cell

Nucleus

Mitochondria

Yolk granules

Microsomes

Supernatant

57.5 _ 0.2

36.5 + 2.2

49.9 + 1.0

58.3 + 1.3

49.0 + 0.7

74.9 + 1.2

tr

3.5 5:0.3

2.6 + 0.1

3.0 + 0.2

3.5 5:0.2

6.7 + 0.5

6.5 5:0.3

8.0 5:0.5

13.6 + 0.7

3.3 + 0.2

6.8 + 0.5

0.5 + 0.1

6.2 5:0.3 1.2 + 0.1 6.7 ___0.5 17.5 5:1.4 2.0 ___0.1 -0.6 __ 0.3 2.60

15.6 5:1.0 8.0 + 0.7 9.2 + 0.1 0.8 _ 0.4 1.2 ___0.6 0.9 + 0.4 -3.24

16.1 8.2 7.0 2.1 2.1

5:1.0 _ 0.6 5:0.6 + 0.3 + 0.3 tr -2.37

28.0 13.7 6.5 1.3 2.7

5:1.8 + 0.2 5:0.4 5:0.3 + 0.9 tr -1.11

21.1 11.4 7.4 0.1

_ 0.5 5:0.3 + 0.5 _ 0.1 tr tr -1.88

4.9 + 0.4 3.2 + 0.8 9.7 + 0.3 -tr --15.1

All values are expressed as percentage of total phospholipids. Results represent means + S.D. (n = 4).

t h a n the P E content in all cases (Table 3). The highest P C content was in s u p e r n a t a n t phospholipids (81.6% from the sum of phospholipids), which had the lowest P E content (5.4%). Nuclei contained almost e q u a l a m o u n t s ol" these phospholipidsl C o m p a r e d to the whole cell, nuclear a n d microsomal fractions were characterized by increased phosphatidylserine (PS) content (13.7% a n d 11.4%, respectively), while only a b o u t 1.0°/0 PS was c o n t a i n e d in m i t o c h o n d r i a l phospholipids. Cardiolipin ( D P G ) was concentrated almost solely in m i t o e h o n d r i a , a m o u n t i n g to 17.5% of the total phospholipids; while its content in other subcellular p a r t i c l e s did n o t exceed 1.3%. P h o s p h a t i dylinositol (PI) was distributed quite uniformly in suheellular fractions; only in yolk granule a n d supern a t a n t lipids was its c o n t e n t slightly higher. A n essential p o r t i o n of p h o s p h a t i d y l e t h a n o l a m i n e and a certain a m o u n t of phosphatidylcholine were presented as plasmalogens. The c o n c e n t r a t i o n of the

alkenyl form of P E varies from 31.4% in m i t o c h o n dria to 77-90% in other fractions, wherein cholineplasmalogen is distributed more uniformly. The spot detection on lipid c h r o m a t o g r a m s with an a n t r o n spray showed t h a t glycolipids were presented in all the fractions except mitoehondria. The glycolipids detected were cerebrosides, sulphatides a n d unidentified lipids in the ganglioside zone. Table 4 shows the fatty acid c o m p o s i t i o n of the polar a n d neutral lipids of subcellular particles from sea urchin eggs. T h e lipids of all the organetles were rich in u n s a t u r a t e d fatty acids, especially polar a n d neutral lipids of m i t o e h o n d r i a (83.6% a n d 78.8%, respectively). U n s a t u r a t e d fatty acids of neutral lipids were richer in m o n o e n e acids (16:1, 18:1 a n d 20:1) t h a n in polyene acids in all subcellular fractions except mitochondria. Polyene acids (18:4, 2 0 : 4 a n d 20:5) d o m i n a t e d in polar lipids of all the fractions. It should be noted t h a t a m o n g subcellular particles,

VICTOR P. CHELOMINand VASILUI. SVETASHEV

102

Table 4. Fatty acid composition of lipids in subceilular organelles of sea urchin eggs Fatty acids

polar

14:0 14:0 14:1 15:0 16:0 16:1 16:2 18:0 18:1 18:2 18:4 20:1 20:2 20:3 20:4 w 6 20:4 w 3 20:5 w 3 22:1 Unidentified Unsaturated Polyene

5.8 8.0 3.2 -14.2 3.3 3.0 5.8 4.5 1.8 7.2 11.0 -1.8 11.8 2.0 12.8 -3.8 62.4 40.4

Cell neutral

Nucleus polar neutral

1.2 8.0 2.1 2.3 17.5 15.8 1.6 2.3 9.0 3.7 4.0 10.8 3.8 3.0 -2.1 6.1 5.4 1.3 67.4 24.3

-7.6 . 0.5 12.0 2.8 3.9 5.1 4.2 0.5 10.3 11.6 --17.6 4.0 19.6 -0.3 74.5 55.9

Mitochondria polar neutral

-7.0

0.8 1.8 . . . 2.2 0.3 17.1 7.7 11.1 2.7 0.3 -2.7 5.8 10.3 6.7 3.5 0.7 6.8 8.1 14.7 8.6 -2.1 5.0 0.4 -15.7 2.7 0.5 8.7 38.1 7.6 -0.3 -70.7 83.6 27.0 65.6

. 2.8 . 2.1 9.1 4.4 4.0 7.2 8.6 0.2 5.8 10.1 6.4 --1.8 25.4 12.1 -78.8 43.6

Yolk granules polar neutral .

. 7.3 .

0.6 12.3 1.8 1.7 5.2 6.1 2.4 7.0 12.5 --14.0 1.4 27.3 -0.4 74.2 53.8

Microsomes polar neutral .

7.0 . 1.9 19.4 15.6 1.0 2.2 9.3 3.7 4.8 11.5 4.7 1.7 -1.1 12.0 4.1 . 69.5 29.0

. 15.2 . . 2.6 15.2 0.7 22.5 8.0 1.6 -4.9 5.0 5.1 -9.6 -9.6 -. . 59.0 51.7

. 4.9 . 2.0 28.1 5.2 10.1 20.1 7.3 6.0 -1.7 ----4.4 10.2 . 44.9 20.5

Supernatant polar neutral 6.8

6.8

0.8 16.0 3.0 0.8 9.6 7.8 1.2 4.3 10.4 --13.3 2.1 23.9 --

1.7 17.6 14.1 0.7 1.8 8.5 4.4 4.6 13.9 4.6 1.9 -1.9 10.4 6.9

66.8 45.6

71.9 29.8

.

Values are expressed as the percentage contribution of the peak areas of the methyl esters to the total area of all peaks. mitochondria and microsomes are characterized by a specific fatty acid composition. Thus, in mitochondrial lipids, polyene fatty acids are predominant in both polar and neutral fractions, with the highest content of 20:5 acid (38.1% and 25.4% for the polar and neutral fractions, respectively), the lowest content being 14:0, 16:0 and 18:2 acids. The opposite proved to be the case in microsomal lipids, the main acids were 14:0, 16:0, 17:0-16:2 and the minor ones 18:4, 20:1, 20:4 and 20:5. The distribution of monoene fatty acids in organelles was much more uniform. The only exception was the microsomal polar fraction, wherein only 7.3% of the total acids were monoene acids. In the polar lipids of nuclei, mitochondria, yolk granules, microsomes and the supernatant the polyene acid content was 55.9%, 65.6%, 52.8%, 51.7% and 45.6%, respectively. DISCUSSION

Isolation o f subcellular particles Methods previously used (Maggio, 1959; Cantatore et al., 1974; Nath & Rebhum, 1974) in sea urchin studies were applied in the isolation of membrane structures of unfertilized S. intermedius eggs. However, in isolating nuclei, we were obliged to exclude centrifugation in 2.2 M sucrose (Chaveau et al., 1956), technique successfully used in isolating nuclei from other species of sea urchin (Thaler et al., 1969; Cantatore et al., I974). On centrifugation in viscous sucrose, degradation of nuclei occurred. By using the method of H o g e b o o m et al. (1952), based on centrifugation of low-molar sucrose, we succeeded in decreasing the number of degraded nuclei. In order to characterize the isolated fractions we tested enzyme activities which were markers for t h e subcellular particles of the cells of both higher ani-

mals (Colbeau et al., 1971) and eggs of the sea urchin (Dore & Cousineau, 1967; Schuel et al., 1969; Barber & Foy, 1973). Cytochrome C oxidase was localised in the mitochondrial fraction (Schuel et al., 1969) and acid phosphatase, a marker-enzyme of lysosomes, was associated with the yolk granule fraction in sea urchin eggs (Dore & Cousineau, 1967). According to Barber and Foy (1973), ( N a + - K +) Mg2+-ATPase concentrated in the ghosts of these eggs. We obtained higher specific activities of corresponding marker enzymes in microsomes, mitochondria and yolk granules (Table 1). Nuclear fraction purity was controlled by light microscopy. It had low enzymic activities characteristic also of other organelles. Cytochrome C oxidase and glucose-6phosphatase could be integral parts of nuclear membranes as was shown on cells of higher animals (Kashnig & Kasper, 1969; Kay et al., 1972). ( N a ÷ - K ÷) ATPase was not detected in either of the subcellular particles fractions, this being indicative of the absence of substantial contamination by the surface membrane. Our results on distribution of M g 2 +ATPase correlated with those obtained in fractionating rat thymus cells (Jarasch et al., 1973).

Lipid composition The lipid composition of the plasma membrane of S. intermedius and S. purpuratus eggs has been studied formerly (Romashina et al., 1972, 1973; Barber & Mead, 1975). However, there were virtually no data on lipids of other particles. The subcellular organelles obtained from unfertilized sea urchin eggs differ considerably from each other in respect to lipid composition. Mitochondria contain cardiolipin and ubiquinone, which are either absent or very slightly present in other fractions. In mitochondria, microsomes and nuclei, a major portion of lipids are represented by phospholipids, and in the cell sap and yolk

Subcellular particles granules, by neutral lipids, mainly triglycerides (Table

2). The presence of appreciable amount of lipids in the supernatant fraction is of interest. These lipids are not associated with membrane, structures, since their composition does not resemble any of the organelles. Probably, they are a part of lipoprotein complexes related to those isolated by Marsh (1968) from the eggs of Arbacia punctulata. Egg supcrnatant lipids contained small amount of phospholipids and cholesterol but were rich in triglycerides. A similar pattern was observed in the lipids of the tissue cell sap of higher animals and invertebrates (Getz et al., 1968; Keenan et al,, 1972; Thompson & Nozawa, 1972; Thompson et al., 1972; Okabe & Noma, 1974). In the cell of higher animals the least amount of lipids is associated with nuclei (Levitina, 1975). Sea urchin egg nuclei are poor in lipids, too. The amount of phospholipids in the egg nuclei calculated per milligram of protein, corresponds to the values obtained in the study of lipids in bovine and rat thymus cell nuclei (Jarasch et al., 1973); however, it exceeds the amount of lipids contained in rat liver cell nuclei (Kay et al., 1972). It is possible that despite the precautions taken in the isolation process, we could not fully avoid degradation of nuclei. Partially damaged nuclei could have sorbed cytoplasmatic lipoproteins, rich in triglycerides. The mitochondria and microsomes of the sea urchin eggs differ considerably from other subcellular organelles (Table 2). Mitochondrial have a poor lipid content but contain a large amount of phospholipids (86.6%). In this respect, they resemble the mitochondria of various animal tissues (Fleischer & Rouser, 1965; Fleischer et al., 1967) and differ from the mitochondria isolated from Anthonomus grandis (Thompson et al., 1972) and Tetrahymena pyriformis (Jonah & Erwain, 1971). The mitochondria of sea urchin eggs contained coenzyme Q (ubiquinone), a component of lipid nature characteristic of these subcellular particles, in amount close to those found in the mitochondria of various mammalian tissues (Fleischer et al., 1967). Ubiquinone was not detected in other subcellular organelles of seaurchin eggs, as was detected in rat liver cells (Zambrano et al., 1975). Microsome lipids contain large amounts of phospholipids (61.7%) and cholesterol (16.1%). This is a characteristic feature of microsomes of various mammalian (Glaumann & Daliner, 1968; Lapetina et al., 1968; Gloster & Harris, 1970; Keenan et al., 1972) and crayfish (Mahra & Zachar, 1974) organs. Sea urchin eggs are rich in glycolipids (Vaskovsky et al., 1970) and, as the reaction with the antron spray showed, glycolipids are present in various amounts in all the subcellular fractions, excepting mitochondria. The specificity of the cell organelles in the distribution of phospholipid classes is strongly pronounced (Table 3). In the subcellular particles of the sea urchin, as in the organelles of a wide range of vertebrates (Fleischer & Rouser, 1965; White, 1973) and invertebrates (Thompson & Nozawa, 1972; Thompson et al., 1972), phosphatidylcholine and phosphatidylethanolamine are the main phospholipids. In respect to phospholipid composition, egg mitochondria have much in common with mammalian

of sea urchin eggs

103

mitoehondria (Fleischer et al., 1967; White, 1973). In higher anima'ls the ratio of PC/PE in all tissues is higher in microsomes and nuclei than in mitochondria (Getz et al., 1962; Getz etal., 1968; Thompson et al., 1972), but the reverse is true ~for subcellular particles from sea urchin eggs. This may be connected with the special biological features of Our subject. As studies on mitochondria of tobacco worm tissues showed this ratio is not constant and depends on the development stage of the mitochondrion, changing during pupa metamorphosis ( C h a n & /.,ester, 1970). The phospholipid composition of yolk granules and the supernatant is very specific. Thus, in the lipids of the soluble fraction the PE content is low, phosphatidylinositol rating second in quantity. A low PE content was noted also in the lipids of the rat and sheep liver cell sap (Getz et al., 1968). In sea urchin eggs the distribution of phosphatidylserine, associated mainly with microsomes and nuclei, was not tmiform. In respect to cardiolipin and phosphatidylserine content sea urchin mitochondria have much in common with mitochondria of mammals (Fleischer & Rouser, 1965; Fleischer et al., 1967; Getz et al., 1968; White, 1973) and protozoa (Jakovcic et al., 1971; Thompson & Nozava, 1972). In the lipids of subcellular particles of certain mammalian tissues the principal phospholipids PC and PE are partially in alkenyi form (Getz et al., 1968; White, 1973):The amounts of PC and PE plasmalogens vary to a considerable extent both for tissues and for different subcellular structures. We noted such a specificity also for the lipids of subcellular structures of sea urchin eggs, where the amounts of the PE plasmalogen strongly varies from organelle to organelle (Table 3). Fatty acid composition, expecially the degree of unsaturation of fatty acids, is highly significant in characterizing membrane iipids. The degree of unsaturation of fatty chains essentially affects the liquidcrystalline characteristic of membrane lipids, which control several vitally important cell processes (Emmelot & van Hoeven, 1975; Gribanov, 1975; Watson et al., 1975). The lipids of sea urchin eggs subcellular particles have a set of long-chain polyene acids, characteristic of marine animals (Lovern, 1964). As in the case of mammalian cells (Bartley, 1964; Fleischer & Rouser, 1965), the lipids of sea urchin egg-organelles do not reveal qualitative differences in fatty acid composition and show certain regularities in their quantitative distribution. The level of unsaturated fatty acids is high in both the polar and neutral fractions of organelle lipids. In neutral lipids this is achieved on a c c o u n t of monoene acids (palmitoleic, oleic, eicosenic and docosenic acids), whereas in polar lipids, polyene acids are dominant (18:4, 20:4, 20: 5). An interesting peculiarity is observed in the fatty acid composition of mitochondria and microsomes. In comparison with all the cell organe!les, microsome fatty acids are the most highly saturated; the saturated fatty acid content in neutral and polar lipids is 55.1% and 41.0%, respectively. In the opposite case mitochondrial lipids are the most unsaturated, this being mainly due to a high content of polyene fatty acids. Possibly, this property is specific for mitochondrial lipids, since a similar pattern is observed i n the mitochondrial lipids of higher animals (Fleischer &

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VICTOR P. CHELOMINand VASlLIII. SVETASHEV

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