129

Biochimica et Eiophysica Acta, 1043 (1990) 129-133 Elsevier

BBALIP 53355

Changes in phospholipid composition and phospholipase D activity during the differentiation of Physarum polycephalum Akiko Minowa I, Tetsuyuki Kobayashi 2, Yukiko Shimada *, Harumi Maeda I, Kimiko Murakami-Murofushi ‘, Jiro Ohta ’ and Keizo Inoue 2 ’ Department of Biology, Faculty of Science, Ochanomitu Faculty of Pharmaceutical

University and 2 Department of Health Chemistry, Science, The University of Tokyo, Tokyo (Japan)

(Received 15 August 1989) (Revised manuscript received 21 November 1989)

Key words: Phospholipid composition; Phospholipase D; Differentiation; (Physarum

polycephalum)

Changes in phospholipid composition and phospholipase D activity were observed during a differentiation from haploid myxoamoebae to diploid plasmodia of a true slime mold, Physarum polycephulum. In the amoeboid stage, the main components of phospholipid fraction were phosphatidylethanolamine (PE, 43.3’S), phosphatidylcholine (PC, 28.8%) and phosphatidylinositol (PI, Ml%), but in the plasmodial stage, PC was dominant (40.7%) and other main components were PE (31.5%) and phosphatidic acid (PA, 11.0%). The specific activity of phospholipase D in the plasmodia was 5.7-times higher than that in the myxoamoebae when measured in the presence of Ca*’ at the alkaline pH. In the amoeboid stage, phospholipase A activity (A, or A,) was detected at the alkaline pH with Ca*+. Phospholipase D activity in the plasmodia was characterized: pH optimum was 6.0; Ca*’ was required for the reaction and Ba*’ could substitute partly for Ca*‘* PE was the best substrate for the hydrolytic activity and PC and PI were not appreciably hydrolyzed; and all detergenk tested inhibited the enzyme activity.

Introduction In myxomycete, Physarum polycephalum, the haploid myxoamoebae of different mating types conjugate in pairs and the zygotes formed fuse each other and differentiate to multinuclear plasmodia. Mating compatibility and zygote differentiation are genetically determined [l-5] and mannosyl glycoprotein(s) located on the surface of the myxoamoebae may play important role(s) in the recognition during zygote formation [6]. Tiny plasmodia grow up and fuse each other easily without any artificial treatments. During the course of differentiation from myxoamoebae to plasmodia, some changes in membrane characteristics might occur. Recently, we found that UDP-glucose : poriferasterol glucosyltransferase activity was expressed in the course of

Abbreviations: PC, phosphatidylcholine; mine; PI, phosphatidylinositol.

PE, phosphatidylethanola-

Correspondence: K. Murakami-Murofushi, Department of Biology, Faculty of Science, Ochanomizu University, 2-1-1, Ohtsuka, Bunkyoku, Tokyo 112, Japan. 0005-2760/90/$03.50

the differentiation from myxoamoebae into plasmodia and the synthesis of poriferasterol monoglucoside was started [7,8]. The change of sterol contents in the cells at both stages was also examined [9]. Phospholipids are major and important membrane components and they are supposed to function as enzyme activators [lO,ll], or as precursors of bioactive substances [12,13]. The involvement of the degradation product of phosphatidylinositol bisphosphate (PIP,) in signal-transfer system is established [14,15]. Comes and Kleinig [16] reported the phospholipid composition and a potent phospholipase D activity in the plasmodia of P. polycephalum , and Schrauwen [17] showed the quantitative change of phospholipids after the fusion of sensitive and killer plasmodia. We have been interested in the changes in phospholipids and their turnover during the differentiation from myxoamoebae to plasmodia in the relation to the change of membrane characteristics and examined phospholipid composition of the cells at both stages or during the differentiation. It was found that the quantitative and qualitative changes in phospholipids as well as change of enzyme activity related to their degradation occurred during differentiation.

0 1990 Elsevier Science Publishers B.V. (Biomedical Division)

130 Materials and Methods

Preparation

Organisms

Washed cells were suspended in 20 mM Tris-HCl (pH 7.4) and disrupted by sonication, followed by centrifugation at 2000 X g for 5 min at 0 o C. The resultant supernatant was used as the enzyme source.

Haploid myxoamoebae of P. polycephalum, two compatible strains F and J, were cultured in the dark at 24’ C on a lawn of the bacteria, Aerobacter aerogenes, on agar medium as described [18,19]. To allow differentiation into plasmodia, the myxoamoebae of strain F and J, which were washed several times with distilled water to remove bacteria, were suspended separately in 0.1 M potassium phosphate buffer (pH 6.0) with a density of 3.5 . lo8 cells/ml and equal volumes of the suspensions were mixed, then 0.4 ml aliquots were placed on non-nutrient agar plates (10 cm diameter) and the plates were kept in the dark at 24OC [6]. Diploid plasmodia were submerged and agitated in a sterile semi-defined culture medium according to the method of Daniel and Rusch [20]. Apogamic myxoamoebae of strain Colonia [21], kindly provided by Dr. S. Kawano (The University of Tokyo) were cultured at 30 o C and allowed to differentiate into plasmodia when the temperature was reduced to 25 o C [21,22]. The cultured myxoamoebae and plasmodia were harvested and washed several times with 10 mM EDTA/20 mM TrisHCl (pH 7.4).

Chemicals

[l-‘4C]Palmitic acid and [1-‘4C]linoleic acid were obtained from Amersham International and phosphatidylcholine was purified from egg yolk by chromatographies on aluminium oxide and silicic acid.

Extraction

and separation

of phospholipids

The washed cells were heated at 100 o C for 10 n-tin in 20 mM Tris-HCl (pH 7.4) containing 10 mM EDTA and sonicated, then the lipids were extracted according to the procedure of Bligh and Dyer [23]. The extracts were washed once with distilled water and separated by two-dimensional thin-layer chromatography on silica-gel 60 plates (E. Merck, Darmstadt) with the solvent systems I and II: I, chloroform/methanol/28% aq. ammonia (65 : 35 : 5, v/v) and chloroform/methanol/ acetone/acetic acid/water (10 : 2 : 4 : 2 : 1, v/v); II, chloroform/methanol/acetic acid (65 : 25 : 10, v/v) and chloroform/methanol/88% aq. formic acid (65 : 25 : 10, v/v). The spots of phospholipids were detected by spraying with Dittmer reagent [24], ninhydrin reagent [25] and Dragendorf reagent [25] and identified by co-chromatography with standard phospholipids. The individual phospholipids to be quantified were visualized with iodine vapor to identify them and were scraped from the thin-layer plates and extracted. Phosphate determination was then performed by the method of Bartlett [26].

of crude enzyme fraction

Assay of hydrolytic activities of phospholipases

Radioactive substrates, 1-[1-‘4C]palmitoyl-2-acylglycerophosphocholine, l-[l-‘4C]palmitoyl-2-acyl-glycerophosphoethanolamine, 1-[1-‘4C]palmitoyl-2-acylglycerophosphoinositol, l-[l-‘4C]palmitoyl-2-acyl-glycerophosphoserine, 1-acyl-2-[1-‘4C]linoleoyl-glycerophosphocholine, 1-acyl-2-[ l-l4 Cllinoleoyl-glycerophosphoethanolamine, 1-acyl-2[1-‘4C]linoleoyl-glycerophosphoinositol and 1-acyl-2-[‘4C]linoleoyl-glycerophosphoserine were prepared according to Lands and Merck1 [27] with a slight modification [28]. Hydrolytic activities were assayed essentially according to Arai et al. [29] using the mixture of each substrate labeled at two different acyl groups. The standard reaction mixture (total volume of 0.1 ml) consisted of 20 mM Bistris buffer (pH 6.0), 1 mM CaCl,, 0.2 mM radioactive substrate and the crude enzyme fraction containing 50 pg protein. After incubation at 37” C for 10 min, the reaction was stopped by heating at 1OO’C for 1 n-tin followed by the addition of 0.6 ml of chloroform/ methanol (2 : 1, v/v) and the reaction products were extracted by the procedure of Bligh and Dyer described above [23]. The chloroform phase was evaporated to dryness under vacua, dissolved in a small amount of chloroform/methanol (2 : 1, v/v) and subjected to thin-layer chromatography (TLC) in a solvent system of chloroform/ methanol/ acetic acid (65 : 25 : 10, v/v). The spots were visualized with iodine vapor and each spot was scraped off from the plate and the radioactivity was measured in a liquid scintillation fluid. Results and Discussion Change in phospholipid composition associated with differentiation from myxoamoebae to plasmodia

The phospholipid compositions in the different stages of P. polycephalum are shown in Table I. The main components of amoeboid phospholipid were PE, PC and PI. But in the plasmodia, PC was dominant, the content of PE decreased and the PA content increased significantly. The amounts of total lipids per g dry wt. of the cells were 120 mg in the myxoamoebae and 94 mg in the plasmodia, and those of total phospholipids per g dry wt. were 54.0 pmol in the myxoamoebae and 38.9 pmol in the plasmodia, respectively. The change in the phospholipid composition roughly paralleled the degree of differentiation from myxoamoebae to plasmodia monitored by the conjugation rate (Fig. 1). After the zygote formation, phospholipid composition seemed to begin to change and PE content

TABLE

I

Phospholipid

composition

Values represent Compound

a

at different stages

Myxoamoebae

Total PL PE PC PI PS PA CL” Lyso PE b Others

b

of Physarum

M yxoamoebae

the mean f S.D. (n = 3). Plasmodia

Z-days

p mol/g dry cells

mol% of total PL

p mol/g dry cells

mol% of total PL

54.0+ 1.9 23.4+ 1.2 15.6kO.5 4.3 + 0.2 2.0 f 0.05 1.8kO.03 2.6 f 0.05 3.2 f 0.03 1.1 f 0.03

100 43.3 28.8 8.0 3.7 3.3 4.8 6.0 2.1

38.9f 1.5 12.3 f 0.7 15.8 f 0.5 1.6 + 0.05 1.5 f 0.02 4.3 * 0.07 1.5+0.03 0.4+0.01 1.6 f 0.06

100 31.5 40.7 4.0 3.8 11.0 3.9 1.0 4.1

after

LCGtdJUGiTION)

3-days after mating

4-days after

Fig. 1. Change of phospholipid composition during the differentiation from myxoamoebae to plasmodia. Myxoamoebae of different mating types, F and J, were mated and the lipids were extracted every 24 h and analyzed by two-dimensional TLC. The conjugation rate was determined under microscopic observation. LPE, lysophosphatidylethanolamine; others, other unidentified phospholipids.

II

Phospholipid

composition

Values represent Compound

and PA content increased significantly. PC was dominant through two different phases and absolute value of this phospholipid was unchanged (Table I). These changes of phospholipid composition may be due to a different expression of phospholipases or synthesizing enzymes in two different stages. The changes could also be partly due to different membranes being produced. With the mutant, apogamic strain Colonia, which lacks zygote formation and differentiates from myxoamoebae into plasmodia without nuclear DNA change, almost the same phospholipid composition as that observed with wild-typed strain were obtained; the increase in PC content and in PA content and the decrease in PE content and in lyso PE were also apparent upon differentiation (Table II).

of Colonia strain of Physarum

decreased

the mean f S.D. (n = 3). Plasmodia

Myxoamoebae

Total PL PE PC PI PS PA CL” Lyso PE b Others

pmol/g dry cells

mol% of total PL

pmol/g dry cells

molX of total PL

56.1 k 2.3 26.3 f 1.4 13.6kO.9 2.9kO.l 2.0 f 0.05 1.2*0.04 3.OrtO.l 4.1 f 0.3 3.1 f 0.2

100 46.9 24.2 5.1 3.6 2.2 5.3 7.3 5.5

43.3 f 2.0 16.0 f 0.5 17.4*0.7 1.6kO.05 1.4 f 0.03 3.2 + 0.06 1.9kO.06 0.9 f 0.02 1 .o f 0.03

100 37.0 40.2 3.7 3.2 7.4 4.3 2.0 2.3

a Cardiolipin. b Lysophosphatidylethanolamine.

TABLE

mating

Plasmodia

Cardiolipin. Lysophosphatidylethanolamine.

TABLE

mating

III

Actioities

of phospholipases Activity

(nmol/mg

protein

per min)

Reaction product Relating enzyme

Lyso PE PLase A a

PH

5.0

Ca’+

+

-

+

_

n.d. b n.d.

n.d. n.d.

0.09 n.d.

n.d. n.d.

n.d. n.d.

0.08 n.d.

PA PLase D 8.0

5.0

8.0

+

_

+

-

0.01 n.d.

1.34 4.90

0.33 0.21

0.44 2.52

0.19 0.16

0.04 n.d.

1.42 6.26

0.32 0.21

0.31 3.28

0.17 0.15

Wild type Myxoamoebae Plasmodia Mutant Myxoamoebae Plasmodia a Phospholipase b Not detected.

A, or A,

(not identified).

132 The values obtained with plasmodia in the present study were very similar to those by Comes and Kleinig [16] except for the relatively high PA value. The remarkable change of phospholipid composition during the differentiation of Physarum cells may reflect some important change of membrane characteristics. Activities of phospholipases in amoeboid and plasmodial stages In order to determine the reason for the difference in lipid composition in two different stages, the activities of phospholipases were measured under acidic, or alkaline conditions with or without Ca2+ ion (Table III). The crude enzyme fractions were incubated with radioactive PE at 37” C for 10 min, then the reaction products were analyzed (see Materials and Methods for details). When the enzyme fraction from myxoamoebae was incubated, the production of PA and lysophosphatidylethanolamine (lyso PE) was observed, which suggests the existence of phospholipases D and A activities. These activities were dependent on Ca2+ ion. But this phospholipase A activity was not identified as A, or A,. In plasmodial cells, high Ca’+-dependent phospholipase D activity was detected, but phospholipase A activity was not observed. In the wild-typed cells, phospholipase D activity in the plasmodia was 3.7-times higher at the acidic pH and 5.7-times higher at the alkaline pH than that in the myxoamoebae. In Colonia strain, increase of the enzyme activity was more significant and the activity in the plasmodia was 4.4-times and 10.6-times higher in each case. Such a high activity of phospholipase D may be responsible for high PA content observed in the plasmodial cells. The relatively high amount of lyso PE in the myxoamoebae may reflect the enhancement of phospho lipase A.

Characterization of plasmodial phospholipase D activity Using the crude enzyme fraction from plasmodia, the characteristics of phospholipase D activity were examined. The linearity of the reaction was obtained at least for 10 min at pH 6.0 when 1 mM CaZf was present. In the presence of Ca2+, the activity was highest at pH 6.0. When the reaction mixture contained 1 mM EDTA instead of 1 mM CaCl,, no appreciable activity was observed at any pH ranges tested (pH 3-10). of 1 mM gave the Ca 2+ ion at the concentration maximum value as shown in Fig. 2. Ba2+ ion at 1 mM could substitute partly (78%) for Ca2+, but other divalent cations tested in the concentration of 1 mM could not (Mg’+, 19%; Zr?+, 5%). Substrate specificity was next examined; instead of PE, the substrates, PI, PS, or PC were reacted under the

0

1

2

345

10

20

50

Ca*+( mM ) on the enzyme activity. Values Fig. 2. Effect of Ca’+ concentrations are expressed as the percentage of the maximum activity (at 1 mM Ca2+ ).

TABLE Suhstraie

IV

ofphospholipase D

specificity

Substrate

Specific activity

PE PS PI PC

4.9 1.9 n.d. a nd. a

nmol/mg

protein

per min

% (100) (39) (-) (-)

a Not detected.

standard conditions. The enzyme could hydrolyze PS but not PI and PC at all (Table IV). All detergents tested showed the inhibitory effects on the enzyme activity at low concentrations (Fig. 3). Transphosphatidylation occurred when high concentration of serine or glycerol was present in the reaction mixture (10 in the molar ratio to PE). The

LO 0 0.1 0.2 CTAB

Ia

0

0.005 SDS

( %)

“0.05

( mM

1.0 1

0

(102 Tn ton

5

X-100 (%)

Fig. 3. Effects of detergents on the phospholipase D activity. Anionic detergents, deoxycholate (0) and sodium dodecyl sulfate (SDS, 0). cationic detergent, cetyltrimethyl ammonium bromide (CTAB), and non-ionic detergent, Triton X-100 were tested. Values are expressed as the percentages of the activity without detergents.

133 reaction was very slow comparing to the hydrolyzing reaction and initial velocity of the PS formation was about one tenth of that of the hydrolysis of PE. The speed of formation of phosphatidylglycerol (PG) was about one third of that observed with PE hydrolysis. Further investigations were needed for discussing the involvement of these activities in the change of the phospholipids during the differentiation. The characteristics described above are very similar to those of phospholipase D widely distributed in higher plants [30-331 except for the high substrate specificity of the present enzyme. As Physarum enzyme could not hydrolyze PC, which is a good substrate for other phospholipases D, it may recognize the amino groups of phospholipids. The biological significance of phospholipase D has not yet been clarified. The progress of our investigation on the change in phospholipid composition and phospholipase D activity associated with the differentiation of P. polycephalum may give a valuable clue to resolve this issue in near future. Acknowledgment This work was supported in part by Grant-in-Aid from the Ministry of Education, Science and Culture of Japan. References 1 2 3 4

Collins, O.R. (1961) Am. J. Bot. 48, 674-683. Dee, J. (1966) J. Protozool. 13, 610-616. Dee, J. (1978) Genet. Res. 31, 85-92. Dee, J. (1982) in Cell biology of Physarum and Didymium (Aldrich, H.C. and Daniel, J.W., eds.), Vol. I, pp. 211-251, Academic Press, New York. 5 Kirouac-Brunet, J., Masson, S. and Pallotta, D. (1980) Can. J. Genet. Cytol. 23, 9-16. 6 Murakami-Murofushi, K., Minowa, Y., Yamada, R. and Ohta, J. (1986) Cell Struct. Funct. 11, 219-225.

7 Murakami-Murofushi, K., Nakamura, K., Ohta, J., Suzuki, M., Suzuki, A., Murofushi, H. and Yokota, T. (1987) J. Biol. Chem. 262, 16719-16723. 8 Murakami-Murofushi, K. and Ohta, J. (1989) Biochim. Biophys. Acta 992, 412-415. 9 Murakami-Murofushi, K., Nakamura, K., Ohta. J. and Yokota, T. (1987) Cell Struct. Funct. 12, 519-524. 10 Spatz, L., Strittmatter, P. (1971) Proc. Nat]. Acad. Sci. USA 68, 1042-1046. 11 Nelson, N. (1976) Biochim. Biophys. Acta 456, 314-338. 12 Chilton, F.H., O’FIaherty, J.T., Ellis, J.M., Swendsen, C.L. and WykIe, R.L. (1983) J. Biol. Chem. 258, 7268-7271. 13 Kramer, R.N., Patton, G.M., Pritzker, C.R. and Deykin, D. (1984) J. Biol. Chem. 259, 13316-13320. 14 Kaibuchi, K., Matsumoto, K. and Tamanoi, F. (1987) Saibokohgaku 6, 971-981 (in Japanese). 15 Berridge, M.J. (1984) B&hem. J. 220, 345-360. 16 Comes. P. and Klein& H. (1973) Biochim. Biophys. Acta 316, 13-18. 17 Schrauwen, J.A.M. (1985) Can. J. Microbial. 31, 782-785. 18 Murakami-Murofushi, K., Hiratsuka, A. and Ohta, J. (1984) Cell Struct. Funct. 9, 311-315. 19 Taniguchi, M., Yamazaki, K. and Ohta, J. (1978) Cell Struct. Funct. 3, 181-190. 20 Daniel, J.W. and Rusch, H.P. (1961) J. Gen. Microbial. 25, 47-59. 21 Cooke, D.J. and Dee, J. (1974) Gen. Res. 23, 307-317. 22 WheaIs, A.E. (1970) Genetics 66, 623-633. 23 Bligh, E.G. and Dyer, W.J. (1959) Can. J. B&hem. Physiol. 37, 911-917. 24 Dittmer, J.C. and Lester, R.L. (1964) J. Lipid Res. 5, 126-127. 25 Wagner, H., Hoernhammer, L. and Wolff, P. (1961) B&hem. Z. 334, 175-184. 26 Bartlett, G.R. (1959) J. Biol. Chem. 234, 466-468. 27 Lands, W.E.M. and MerckI, I. (1963) J. Biol. Chem. 238,898-904. 28 Arai, H., Inoue, K., Natori-Tamori, Y., Banno, Y., Nozawa, Y. and Nojima, S., (1985) J. B&hem. (Tokyo) 97, 1525-1532. 29 Arai, H., Inoue, K., Nishikawa, K., Banno, Y., Nozawa, Y. and Nojima, S. (1986) J. Biochem. (Tokyo) 99, 125-133. 30 Kates, M. and Sastry, P.S. (1969) Methods Enzymology, 14, pp. 197-203. 31 Davidson, F.M. and Long, C. (1958) Biochem. J. 69, 458-466. 32 Quarles, R.H. and Dawson, R.M.C. (1969) Biochem. J. 112, 787-794. 33 Vaskovsky, V.E., Gorovot, P.G. and Suppes, Z.S. (1972) Int. J. Biochem. 647-650.

Changes in phospholipid composition and phospholipase D activity during the differentiation of Physarum polycephalum.

Changes in phospholipid composition and phospholipase D activity were observed during a differentiation from haploid myxoamoebae to diploid plasmodia ...
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