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Enhancement of chlorophyll a fluorescence yield, low-temperature F685F730 fluorescence emission ratio, and electron transport rate by ether phospholipids (platelet activating factor and analogs) in isolated chloroplasts
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 275, No. 1, November 15, pp. 2’71-279,1989
Enhancement
of Chlorophyll a Fluorescence Yield, Low-Temperature F685/F730 Fluorescence Emission Ratio, and Electron Transport Rate by Ether Phospholipids (Platelet Activating Factor and Analogs) in Isolated Chloroplasts J. H. ARGYROUDI-AKOYUNOGLOU1 Institute
AND
C. VAKIRTZI-LEMONIAS
of Biology, NRCPS “‘Demokritos, “Aghia Paraskevi, 15510 Athens, Greece Received April
13,1989, and in revised form July 21,1989
Acetyl glyceryl ether phosphorylcholine (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine; or platelet activating factor (PAF)), when incubated with chloroplasts or subchloroplast fractions derived from stroma or grana lamellae, induces a drastic increase in the low-temperature fluorescence emission ratio F6a5/F7a0 (7’7°K). The molecular structure requirement for the effect to be elicited is the ether bond and a long C chain at the C-l position of glycerol, a short C chain at C-2 (or the lyso form), and a large polar head at C-3, the most potent effector being PAF C-16. The effect is more pronounced in grana-derived fractions. PAF also induces an increase in the chlorophyll a fluorescence yield, enhances the association of chlorophyll in the supramolecular pigment-protein complexes of the thylakoid (especially those of Photosystem II), and enhances electron transfer from 1,5-diphenyl carbazide to 2,6-dichlorophenol indophenol. These effects are attributed to alteration of the Photosystem II unit organization via the incorporation/ intercalation in the grana of the wedge-shaped PAF. 0 19X9AcademicPress,Inr.
Acetyl glyceryl ether phosphorylcholine (AGEPC),2 formerly known as platelet activating factor, PAF, is a potent lipid mediator with multiple functions in mammals (l-5). Apart from its ability to induce aggregation of platelets, and a number of other physiological responses, it stimulates influx of calcium into rabbit platelets and breakdown of phosphoinositides, suggesting a hormone-like action (l-5). In addition, PAF has been found to stimulate
ATP-dependent H+ transport in plant microsomes (6). Recently we reported that PAF induces an increase in the low-temperature Fss/ FTsofluorescence emission ratio in isolated thylakoids washed and suspended in Tritine (7), which was considered to reflect alteration of the photosynthetic unit organization via its incorporation in the thylakoid. Since phosphatidylcholine (PC) with similar charge properties was ineffective, it was proposed that the changes observed were not related to charge effects (7), that might lead to lateral movement of complexes in the thylakoid affecting spillover (as earlier proposed for cation action (8)). Since PAF may serve as a probe for studying the thylakoid organization in relation to the photosynthetic reaction rate, and in order to understand what the molecular structure requirement for the
1 To whom correspondence should be addressed. ’ Abbreviations used: AGEPC, acetyl glyceryl ether phosphorylcholine; PAF, platelet activating factor; Tricine, N-[Z-hydroxy-1,1-bis(hydroxymethyl)ethyl glycine; PC, phosphatidylcholine; DPC, 1,5-diphenyl carbazide; DCIP, 2,6-dichlorophenol indophenol; Chl, chlorophyll; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PS, Photosystem; CMC, critical micellar concentration; DCMU, 3(3’,4’dichlorophenyl)-1,1-dimethylurea. 271
0003-9861/89 $3.00 Copyright@1989by AcademicPress,Inc. All rights of reproductionin any form reserved.
272
ARGYROUDI-AKOYUNOGLOU
AND TABLE
VAKIRTZI-LEMONIAS
I
EFFECT OF PHOSPHOLIPID MOLECULAR STRUCTURE ON THE F6,/F7m RATIO AT 77°K AND THE Chla FLUORESCENCE YIELD (F,,,,,/Chl) IN PEA PLASTIDS Fs85/Fmo ratio” Plastids in Tricine-NaOH Plastids in phosphate-KC1 Plastids in Tricine-NaOH + Phospholipid 1-0-C-16-2-Acetyl-glycero-3-PC (PAF C-16) 1-0-C-1%2-Acetyl-glycero-3-PC (PAF C-18) 1-0-C-16-Glycero-3-PC (L-PAF C-16) I-0-C-1%Glycero-3-PC (L-PAF C-18) 1,2-Di-C-16-glycero-3-PC (PC C-16) 1,2-Di-C-1%glycero-3-PC (PC C-18) 1-0-C-16-2-Acetyl-glycerol 1-O-C-16-glycerol 1-0-C-16-2-Acetyl-glycero-3-phospho N(CH,)-hexanolamine (PAF antagonist) 1-C-16-Glycero-3-PC (L-PC C-16) 1,2-O-0-Di-C-16-glycero-3-PC 1,2-0-O-Di-C-16-glycerol
F,,,,,/Chl” (arbitrary
0.36 1.10
48 100
3.00 0.45 1.37 0.60 0.43 0.37 0.36 0.40
130 43 160 80 42 52 -
2.05 0.53 0.41 0.37
59” 60 46 -
units)
a Thylakoid aliquots of 50 ~1, washed and suspended in Tricine (30 pg Chl/ml), were incubated with 0.5 rmol of each phospholipid. * Thylakoid aliquots of 50 ~1, washed and suspended in Tricine (13 fig Ch1/50 rl), were incubated with 0.25 hrnol of each phospholipid, except in (c) where 0.05 Fmol was used.
effect to be elicited is, we have tested a number of PAF derivatives and analogs as to their effectiveness in inducing changes in the fluorescence parameters of the thylakoids. We found that the most effective molecule is PAF C-16, and that the crucial requirements for the molecular structure is the ether bond and C-16 chain length at Cl of the glycerol moiety, a short C chain at C-2 (or the lyso form), and a large polar head at C-3. We also found that PAF C-16, in addition to stimulating the chlorophyll a fluorescence yield, stimulates electron transfer from DPC to DCIP and sustains the organization of chlorophyll in the pigment-protein complexes of the thylakoid, and especially in those of Photosystem II. MATERIALS
AND
METHODS
Chloroplasts were isolated from bean or pea leaves by homogenizing 4 g fresh weight tissue with 40 ml homogenization buffer (0.3 M sucrose-O.01 M KCI-0.05 M phosphate buffer, pH 7.2). The chloroplast pellet (10008 for 10 min) was washed twice with 0.05 M Tri-
tine-NaOH, pH 7.2, and suspended in Tricine to yield 30 mg Chl/ml. Fifty-microliter aliquots (1.5 pg Chl) were added to freshly prepared films of PAF or analogs, and incubated for 30 min in an ice bath. The thylakoid suspension was removed into a capillary tube and frozen in liquid nitrogen and its fluorescence emission spectrum recorded at 77°K. Fluorescence measurements were done as previously described in a Dewar cuvette, filled with liquid nitrogen, made to fit the cuvette compartment (9). For Chl a fluorescence yield measurements, 50-~1 aliquots of thylakoids, containing 13 pg Chl were incubated with the phospholipid film as above in the dark; after a 30-min incubation with the film of phospholipid, the suspension was removed with the aid of a Hamilton microsyringe and transferred into a 2.5ml Tricine-containing cuvette; the F,,,,, was recorded thereafter at 685 nm, as in (10). Photosystem II activity was determined as the DPC-DCIP photoreduction (ll), in samples of thylakoids prior to or after their incubation with the film of phospholipid as above (50 ~1 thylakoid suspension = 1.5 pg Chl + phospholipid film for 30 min in an ice bath). SDS-PAGE for pigment-protein complex separation was done as previously described (12) in thylakoid samples prior to or after incubation with the phospholipid film, as above.
CHLOROPHYLL
a FLUORESCENCE IN CHLOROPLASTS
273
3.6 3.2
.
2.8 2tb - I ‘“‘“i”_‘“‘~__,
2.~ -j 1.6:
,,.-
-a
-----*
-x
lamcllae
Stromo lomellae
0
1
2
3
L PAF
5
6
7
6
9
10
1 20
imM)
0.0
I
I
I
I
I
I
I
I
I
I
:’
0123L5679910
c 20
PAF
tmM1
FIG. 1. Fs8JFTN increase in chloroplasts, grana, and stroma lamellae of bean (right) or pea (left) leaves, after their incubation with PAF C-16. All thylakoid fractions were washed twice with 50 mM Tricine-NaOH, pH 7.2, and were suspended in Tricine to yield 30 pg Chl/ml. Fifty-microliter aliquots were added to the freshly prepared phospholipid film, incubated for 30 min at 4”C, transferred to a capillary tube, and immersed in liquid nitrogen, and their fluorescence emission spectra recorded at -196°C. Grana and stroma lamellae were isolated by differential centrifugation of French press disrupted chloroplasts suspended in 0.05 M phosphate-O.15 M KCl, pH 7.2, at 400 pg ChWml(l3). The 10K and 240K pellet fractions (grana and stroma lamellae, respectively) were washed and suspended in Tricine to yield 30 pg Chl/ml, and aliquots of 50 ~1 were incubated with the phospholipid film, as above. All phospholipids were products of NOVA, Switzerland, unless otherwise specified. They were dissolved in chloroform:methanol (1:2), and aliquots were completely dried under a stream of nitrogen, just before use. RESULTS
Incubation of thylakoids, washed and suspended in Tricine-NaOH, with a film of PAF (with a 4:l ratio of C-16:C-18 in l-Oalkyl chain) has been found to induce a drastic increase in the F685/F730 ratio at 77”K, which depends on PAF concentration and PAF/Chl ratio (7). PC, on the other hand, even though it has similar electrical properties, is completely ineffective (7). In addition, the effect of pure
PAF 1-O-C-18 is far less than that of PAF 1-O-C-16. Out of a wide variety of PAF or PC compounds tested, with shorter or longer C chains at C-l and/or C-2, or a larger or smaller polar head at C-3, only PAF C-16 and its lyso analog were found to be effective (Table I). As deduced from Table I, the molecular structure requirement for the effect to be elicited is the ether bond and a long C chain (preferably a C-16 chain) at the C-l position of the glycerol moiety, a short C tail (or the lyso form) at C-2, and a large polar head at C3. Since the active compounds are zwitterionic, their effect cannot be attributed to thylakoid surface charge neutralization, in a way similar to cation action (8); thus lateral movement of complexes leading to segregation of PSI and PSI1 (in stroma and grana lamellae, respectively), and concomitant inhibition of spillover, is not expected. The finding that PAF C-16 can also induce enhancement of the F685/F,30 ratio in isolated subchloroplast fractions of grana and stroma lamellae leads to the
274
ARGYROUDI-AKOYUNOGLOU
AND
VAKIRTZI-LEMONIAS
a 1.5
BEAN
t
Chl
10 K Igrand alb=2,5
4 ,OmM / /=b
PAF:
PAF-0
0
1
780
700
620
WAVELENGTH
(nml
FIG. 2. The low-temperature fluorescence spectrum of grana and stroma thylakoids (10K and 240K, respectively) isolated from bean (a, b) or pea (c) leaves, as affected by incubation with increasing concentrations of PAF C-16. For experimental details see Fig. 1 and Materials and Methods.
same conclusion. Figure 1 shows the concentration dependence of the effect in isolated chloroplasts and their grana and stroma lamellar fractions from pea or bean. As shown the effect is pronounced in all thylakoid fractions; however, it is most pronounced in grana fractions. In addition, while in pea the effect is more drastic in grana than in chloroplasts or stroma lamellae, in bean it is equally pronounced in chloroplasts and grana. Figure 2 shows the low-temperature fluorescence emission spectra obtained from grana and stroma lamellar fractions of bean and pea chloroplasts, prior to or after incubation with PAF C-16. As shown, the change is dependent on PAF concentration, and much more drastic in the PSII-rich grana than in the PSI-rich
stroma lamellae fractions. This suggests that the effect of PAF is specifically directed to the PSI1 unit. Indeed, we found that, in addition to the increase in the F,& FTso ratio at 77”K, the Chl a fluorescence yield (F,,JChl at room temperature) is similarly affected (Tables I and II). The increase in F,,,/Chl is similar to that observed upon cation addition (MgCl,) to Tricine-unstacked (agranal) chloroplasts. In addition, and in agreement with the findings on the effect of various phospholipids on the F685/F730 ratio, the most potent effector in this case is also the PAF C16 and its lyso analog. To check whether PAF C-16 affects these parameters via alteration of the pigmentprotein complex organization in the photosynthetic units (14-16), we further studied
b 1.5 - Bean t
240K [ st roma
Chl
a/b=
4.6
‘;i
.c3
-----
1OmMPAF
-
OPAF
:
\
I
\ t
620
I
I
700
780
WAVE LENGTH :
in m)
680 Pea
240 K Chla/b=4.3
*-..e***
tOmMPAF
1
-.-.-2,5mMPAF
.ir
--me
O,GmMPAF!
-
OPAF
;I
620
700 WAVELENGTH
(nm)
FIG. 2-Continued 275
780
276
ARGYROUDI-AKOYUNOGLOU
AND TABLE
EFFECT OF Me,
VAKIRTZI-LEMONIAS
II
PAF, PC, AND THEIR DERIVATIVES ON THE CHLOROPHYLLS FLUORESCENCE YIELD (F,,,.,/Chl) AT ROOM TEMPERATURE OF PEA THYLAKOIDS
Phospholipid A. No phospholipid Chloroplasts in sue-phos-KC1 Chloroplasts in Tricine + Mga+ (1 mM) + MgZ+ (2 mM) + Mg2+ (4 mM) B. Chloroplasts in sue-phos-KC1 Chloroplasts in Tricine + 0.25 pmol PAF C-16” + 0.50 pmol PAF C-16” C. Chloroplasts in sue-phos-KC1 Chloroplasts in Tricine +0.25 pmol PAF C-16 PAF C-18 L-PAF C-16 PAF C-16 (-polar group) PAF 1,2-0-0-Di-C-16 PC C-16 L-PC C-16 L-PAF C-18 0.05 amol PAF C-16 (+larger polar group)
F,,,,,/Chl (arbitrary units)
% Increase
75 38 73 90 85
97 0 92 136 124
68
121
28 54 100
0 93 257
100
108
48
0
130
170
43
80
0 233 8 0 0 25 67
59
23
160
52 46 42 60
Note. Pea plastids, isolated in sue-phos-KC1 were washed twice in Tricine and then suspended in Tricine to yield 12.5 pg Chl/ZO rl (B) or 13 pg Ch1/50 ~1 (C). Aliquots containing 13 pg Chl were either added to a cuvette containing 2.5 ml Tricine and the F,., determined (A) or incubated in an ice bath with the freshly dried phospholipid film for 30 min and thereafter added to the Tricine-containing cuvette for determination of the fluorescence yield (B, C). Mg2f was added directly to the Tricine-containing cuvette, and after an incubation at room temperature for 5 min in the dark the F,, was determined. All samples were kept in the dark prior to F,,,., determination to have the electron acceptor Q in the reduced state. The light admitted to the cuvette compartment was adequate for full reduction. All phospholipid compounds were products of NOVA, except for (a) which were products of Sigma.
the distribution of Chl among the pigment-protein complexes of the thylakoid. Figure 3 and Table III show representative results, obtained with thylakoids incubated in the presence or absence of PAF C16 and then dissociated with and electrophoresed in SDS. As indicated, incubation with PAF reduces the amount of Chl running as free pigment upon electrophoresis, suggesting that PAF promotes chlorophyll binding in the complexes. Thus, the CPIa/ CPI and LHCPVLHCP3 ratios are drastically increased, suggesting that PAF surpresses dissociation of the supramolecular structures by SDS. Assuming that the
LHCPB complex originates in the LHC-I, or at least represents part of the LHC-I (1’7,18), the Chl distribution between PSI1 and PSI complexes (LHCPl + CPa + LHCP3 and CPIa + CPI + LHCPB, respectively) changes after incubation with PAF in favor of PSI1 as seen from the data in Table III. We do not know whether this effect of PAF in suppressing the dissociation of oligomeric forms is in any way related to the changes observed in the fluorescence parameters. However, it is possible that the enhanced Chl binding, especially in the pigment-protein complexes of the PSI1 unit induced by incubation with
CHLOROPHYLL EA.
thylakoids
a FLUORESCENCE
277
IN CHLOROPLASTS
ma ”
EAN
thylokoids
1 .o W isi Q ,”
0.5
:: m Q
0 .o
-0
1
3
2 DISTANCE
[cm)
0
1
2
3
DISTANCElcm)
FIG. 3. Densitograms of mild SDS-PAGE resolution patterns obtained from SDS-solubilized bean (right) or pea (left) cbloroplasts washed twice and suspended in Tricine-NaOH (50 mM, pH 7.2) to yield 25 Fg Ch1/50 ~1. Fifty-microliter aliquots were added to a film of 1 pmol PAF C-16 or PC C-16 and incubated at 4°C for 30 min. SDS was then added to have an SDS/Chl ratio equal to 10, and electrophoretic analyses followed immediately at 4°C.
PAF, reflects the association of Chl in a ids, PAF C-16 and its lyso analog are the PSI1 unit of larger absorption cross sec- most potent effecters in photosynthetic tion, leading to increased F685/F730ratio. membranes. The molecular structure/ Finally, incubation of thylakoids with shape of the active molecules suggests that PAF C-16 induces an increase in the elec- for a compound to be active, the capacity tron transfer rate via PSI1 (in a DPC- to be incorporated in the phospholipid biDCIP photoreduction) (Fig. 4). Such an ac- layer of the thylakoid membrane is crucial; tivation in the Hz0 splitting activity rate, only the cone-like wedge shaped PAFs are however, was not observed; in addition, we active, but not the rod-like PCs. In addifound that PAF C-16 mediates electron tion, the difference in activity between transport from DPC to DCIP even in the PAF C-16 and PAF C-18 may be attributed absence of thylakoids, or in the presence of to the easier incorporation of the former DCMU, as well, suggesting that PAF C-16 into the thylakoid, as compared to the latmay enhance electron transfer activity ter, because of its larger critical micellar after its incorporation in the thylakoid concentration on one hand (an increase by by a strictly chemical reaction/mediation, one methylene group of the alkyl chain in without the involvement of the Photosys- PAF or lyso-PAF results in a 3-fold or lotern II unit itself. PC, on the other hand, fold decrease in CMC, respectively (19)) is completely ineffective in this respect and its apparently smaller micellar vol(Fig. 4). ume, as suggested by the data for l-O-C16- and 1-0-C-18-2-deoxy PC, which give 140 and 4900 molecules per micelle, respecDISCUSSION tively (20). This reasoning, however, does The results presented above suggest that not seem to hold in the case of PAF C-16 and lyso-PAF C-16; the latter is less effecout of a wide variety of ether phospholip-
ARGYROUDI-AKOYUNOGLOU
AND
VAKIRTZI-LEMONIAS
tive, even though its CMC is three times higher than that of the former (19). This may, therefore, suggest that the acetyl group in C-2 of PAF C-16 is crucial for activity. The zwitterionic character of all these compounds speaks against their involvement in the neutralization of thylakoid surface charges, in a way similar to that of cations (known to affect the Fm/FTm ratio in a similar way); thus, their action in enhancing the F685/F730ratio cannot be attributed to such processes leading to lateral movement of complexes from stroma to grana lamellae, blocking spillover of excitation energy from Photosystem II to Photosystem I (8). Isolated grana or stroma lamellae, when incubated with PAF, behave in a similar way. However, the effect of PAF on grana J’,&FTsO ratio is more pronounced than that on stroma lamellae (see Fig. l), suggesting that PAF mainly acts by affecting the Photosystem II unit. This is also suggested by the strong mediation of Photosystem II activity induced by PAF C-16. This effect on electron transport rate, induced by PAF (DPC-DCIP reduction), has been found also in the absence of Chl, or in DCMU-poisoned chloroplasts. This indicates, therefore, that PAF itself enhances electron transport from DPC to DCIP; actually PAF can stimulate the DPC-DCIP electron transport in a buffer system and in the dark as well (C. Vakirtzi-Lemonias and J. H. ArgyroudiAkoyunoglou, in preparation). We do not know how this mediation takes place; PAF might offer the necessary surface organization via micelle formation when incubated with DPC and DCIP in the absence of thylakoids. In the presence of thylakoids, however, PAF appears to act possibly by intercalating in the membrane. The action of PAF on the fluorescence parameters seems to be due to its incorporation into the thylakoid phospholipid bilayer, resulting in alteration of the organization of the photosynthetic units. The results of SDS-PAGE analyses of pigmentprotein complexes in thylakoids, prior to or after incubation with PAF or PC, support this conclusion. As shown, Chl bind-
CHLOROPHYLL
a FLUORESCENCE
1500 -
I-Chl
ol
1
1
1
1
0
1
9
PAF C-16 or + DCMU)
3
’
1
Phospholipid
’
1.0
0,5 (pmoles150h)
FIG. 4. PAF C-16-induced mediation of electron transport through PSII. Chloroplasts washed and suspended in 50 mM Tricine-NaOH, pH 7.2, yielding 1.5 pg Ch1/50 ~1, were used. PSI1 activity was determined as the DPC-DCIP reduction at saturating light intensity. Incubation with the phospholipid film (PAF C-16 or PC) was for 30 min at 0°C prior to PSI1 activity determination.
ing in Photosystem I and Photosystem II complexes is greatly increased in the presence of PAF but not in the presence of PC. The increase seems to be larger in the case of the Photosystem II complexes than in those of Photosystem I (hence the PSII/ PSI ratio, as monitored by the percentage of Chl bound in PSI1 complexes to that in PSI, is enhanced). This may be reflected by the increased F685/F730 ratio found under these conditions: upon PAF incorporation into the thylakoid, the effective absorption cross section of Photosystem II may be increased (via enhanced Chl binding), so that the distribution of absorbed photons may favor the Photosystem II unit. REFERENCES 1. VARGAFTIG, B. B., CHIGNARD, M., BENVENISTE, J., LEFORT, J., AND WAL, F. (1981) Ann. N. Y. Acad sci. 370,119-137.
IN CHLOROPLASTS
279
2. HANAHAN, D. J. (1986) Annu. Rev. B&hem 55, 483-509. 3. LEE, T. C., AND SNYDER, F. (1986) in Phospholipids and Cellular Regulation (Kuo, J. K., Ed.), Vol. II, pp. l-39 CRC Press, Boca Raton, FL. 4. SHUKLA, S. D., AND HANAHAN, D. J. (1982) Biochem Biophys. Res. Commun, 106, 697703. 5. BILLAH, M. M., AND LAPETINA, E. G. (1983) PTOC. Natl Acad. Sci. USA 80,965-968. 6. SCHERER, G. F. E. (1985) B&hem. Biophys. Res. Commun 133,1160-1167. 7. ARGYROUDI-AKOYUNOGLOU, J. H., AND VAKIRTZILEMONIAS, C. (1987) Arch. Biochem Biophys. 253.38-45. 8. BARBER, J. (1980) FEBSLett. 11&l-10. 9. ARGYROUDI-AKOYUNOGLOU, J. H., AND AKOYUNOGLOU, G. (1983) Arch. Biochem. Biophys. 227, 469-477. 10. ARGYROUDI-AKOYUNOGLOU, J. H., AND AKOYUNOGLOU, G. (1977) Arch. Biochem Biophys. 179, 370-377. 11. VERNON, L. P., AND SHAW, E. R. (1969) Plant Physiol. 44,1645-1649. 12. ANDERSON, J. M., WALDRON, J. C., AND THORNE, S. W. (1978) FEBS Lett. 99,227-233. 13. SANE, P. V., GOODCHILD, D. J., AND PARK, R. B. (1970) Biochim. Biophys. Acta 216,162-178. 14. ARGYROUDI-AKOYUNOCLOU, J. H., CASTORINIS, A., AND AKOYUNOGLOU, G. (1982) Photobicthem. Photobiophys. 4,201-210. 15. AKOYUNOGLOU, G., AND ARGYROUDI-AKOYUNOGLOU, J. H. (1986) in Ion Interactions in Energy Transfer Biomembranes (Papageorgiou, G., andBarber, J., Eds.), pp. 211-222, Plenum, New York. 16. ARGYROUDI-AKOYUNOGLOU, J. H., AND AKOYUNOGLOU, G. (1986) in Regulation of Chloroplast Differentiation (Akoyunoglou, G., and Senger, H., Eds.), pp. 329-338, A. R. Liss, New York. 17. ARGYROUDI-AKOYUNOGLOU, J. H. (1984) FEBS Lett. 171,47-53. 18. KUANG, T. Y., ARGYROUDI-AKOYUNOGLOU, J. H., NAKATANI, H., WATSON, J., AND ARNTZEN, C. J. (1984) Arch. B&hem. Biophys. 235,618627. 19. KRAMP, W., PIERONI, G., PINCKARD, R. N., AND HANAHAN, D. J. (1984) Chem. Phys. Lipids 35, 49-62. 20. WELTZIEN, H. U., ARNOLD, B., AND REUTHER, R. (1977) B&him Biophys. Acta 466,411-421.
Enhancement
of Chlorophyll a Fluorescence Yield, Low-Temperature F685/F730 Fluorescence Emission Ratio, and Electron Transport Rate by Ether Phospholipids (Platelet Activating Factor and Analogs) in Isolated Chloroplasts J. H. ARGYROUDI-AKOYUNOGLOU1 Institute
AND
C. VAKIRTZI-LEMONIAS
of Biology, NRCPS “‘Demokritos, “Aghia Paraskevi, 15510 Athens, Greece Received April
13,1989, and in revised form July 21,1989
Acetyl glyceryl ether phosphorylcholine (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine; or platelet activating factor (PAF)), when incubated with chloroplasts or subchloroplast fractions derived from stroma or grana lamellae, induces a drastic increase in the low-temperature fluorescence emission ratio F6a5/F7a0 (7’7°K). The molecular structure requirement for the effect to be elicited is the ether bond and a long C chain at the C-l position of glycerol, a short C chain at C-2 (or the lyso form), and a large polar head at C-3, the most potent effector being PAF C-16. The effect is more pronounced in grana-derived fractions. PAF also induces an increase in the chlorophyll a fluorescence yield, enhances the association of chlorophyll in the supramolecular pigment-protein complexes of the thylakoid (especially those of Photosystem II), and enhances electron transfer from 1,5-diphenyl carbazide to 2,6-dichlorophenol indophenol. These effects are attributed to alteration of the Photosystem II unit organization via the incorporation/ intercalation in the grana of the wedge-shaped PAF. 0 19X9AcademicPress,Inr.
Acetyl glyceryl ether phosphorylcholine (AGEPC),2 formerly known as platelet activating factor, PAF, is a potent lipid mediator with multiple functions in mammals (l-5). Apart from its ability to induce aggregation of platelets, and a number of other physiological responses, it stimulates influx of calcium into rabbit platelets and breakdown of phosphoinositides, suggesting a hormone-like action (l-5). In addition, PAF has been found to stimulate
ATP-dependent H+ transport in plant microsomes (6). Recently we reported that PAF induces an increase in the low-temperature Fss/ FTsofluorescence emission ratio in isolated thylakoids washed and suspended in Tritine (7), which was considered to reflect alteration of the photosynthetic unit organization via its incorporation in the thylakoid. Since phosphatidylcholine (PC) with similar charge properties was ineffective, it was proposed that the changes observed were not related to charge effects (7), that might lead to lateral movement of complexes in the thylakoid affecting spillover (as earlier proposed for cation action (8)). Since PAF may serve as a probe for studying the thylakoid organization in relation to the photosynthetic reaction rate, and in order to understand what the molecular structure requirement for the
1 To whom correspondence should be addressed. ’ Abbreviations used: AGEPC, acetyl glyceryl ether phosphorylcholine; PAF, platelet activating factor; Tricine, N-[Z-hydroxy-1,1-bis(hydroxymethyl)ethyl glycine; PC, phosphatidylcholine; DPC, 1,5-diphenyl carbazide; DCIP, 2,6-dichlorophenol indophenol; Chl, chlorophyll; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PS, Photosystem; CMC, critical micellar concentration; DCMU, 3(3’,4’dichlorophenyl)-1,1-dimethylurea. 271
0003-9861/89 $3.00 Copyright@1989by AcademicPress,Inc. All rights of reproductionin any form reserved.
272
ARGYROUDI-AKOYUNOGLOU
AND TABLE
VAKIRTZI-LEMONIAS
I
EFFECT OF PHOSPHOLIPID MOLECULAR STRUCTURE ON THE F6,/F7m RATIO AT 77°K AND THE Chla FLUORESCENCE YIELD (F,,,,,/Chl) IN PEA PLASTIDS Fs85/Fmo ratio” Plastids in Tricine-NaOH Plastids in phosphate-KC1 Plastids in Tricine-NaOH + Phospholipid 1-0-C-16-2-Acetyl-glycero-3-PC (PAF C-16) 1-0-C-1%2-Acetyl-glycero-3-PC (PAF C-18) 1-0-C-16-Glycero-3-PC (L-PAF C-16) I-0-C-1%Glycero-3-PC (L-PAF C-18) 1,2-Di-C-16-glycero-3-PC (PC C-16) 1,2-Di-C-1%glycero-3-PC (PC C-18) 1-0-C-16-2-Acetyl-glycerol 1-O-C-16-glycerol 1-0-C-16-2-Acetyl-glycero-3-phospho N(CH,)-hexanolamine (PAF antagonist) 1-C-16-Glycero-3-PC (L-PC C-16) 1,2-O-0-Di-C-16-glycero-3-PC 1,2-0-O-Di-C-16-glycerol
F,,,,,/Chl” (arbitrary
0.36 1.10
48 100
3.00 0.45 1.37 0.60 0.43 0.37 0.36 0.40
130 43 160 80 42 52 -
2.05 0.53 0.41 0.37
59” 60 46 -
units)
a Thylakoid aliquots of 50 ~1, washed and suspended in Tricine (30 pg Chl/ml), were incubated with 0.5 rmol of each phospholipid. * Thylakoid aliquots of 50 ~1, washed and suspended in Tricine (13 fig Ch1/50 rl), were incubated with 0.25 hrnol of each phospholipid, except in (c) where 0.05 Fmol was used.
effect to be elicited is, we have tested a number of PAF derivatives and analogs as to their effectiveness in inducing changes in the fluorescence parameters of the thylakoids. We found that the most effective molecule is PAF C-16, and that the crucial requirements for the molecular structure is the ether bond and C-16 chain length at Cl of the glycerol moiety, a short C chain at C-2 (or the lyso form), and a large polar head at C-3. We also found that PAF C-16, in addition to stimulating the chlorophyll a fluorescence yield, stimulates electron transfer from DPC to DCIP and sustains the organization of chlorophyll in the pigment-protein complexes of the thylakoid, and especially in those of Photosystem II. MATERIALS
AND
METHODS
Chloroplasts were isolated from bean or pea leaves by homogenizing 4 g fresh weight tissue with 40 ml homogenization buffer (0.3 M sucrose-O.01 M KCI-0.05 M phosphate buffer, pH 7.2). The chloroplast pellet (10008 for 10 min) was washed twice with 0.05 M Tri-
tine-NaOH, pH 7.2, and suspended in Tricine to yield 30 mg Chl/ml. Fifty-microliter aliquots (1.5 pg Chl) were added to freshly prepared films of PAF or analogs, and incubated for 30 min in an ice bath. The thylakoid suspension was removed into a capillary tube and frozen in liquid nitrogen and its fluorescence emission spectrum recorded at 77°K. Fluorescence measurements were done as previously described in a Dewar cuvette, filled with liquid nitrogen, made to fit the cuvette compartment (9). For Chl a fluorescence yield measurements, 50-~1 aliquots of thylakoids, containing 13 pg Chl were incubated with the phospholipid film as above in the dark; after a 30-min incubation with the film of phospholipid, the suspension was removed with the aid of a Hamilton microsyringe and transferred into a 2.5ml Tricine-containing cuvette; the F,,,,, was recorded thereafter at 685 nm, as in (10). Photosystem II activity was determined as the DPC-DCIP photoreduction (ll), in samples of thylakoids prior to or after their incubation with the film of phospholipid as above (50 ~1 thylakoid suspension = 1.5 pg Chl + phospholipid film for 30 min in an ice bath). SDS-PAGE for pigment-protein complex separation was done as previously described (12) in thylakoid samples prior to or after incubation with the phospholipid film, as above.
CHLOROPHYLL
a FLUORESCENCE IN CHLOROPLASTS
273
3.6 3.2
.
2.8 2tb - I ‘“‘“i”_‘“‘~__,
2.~ -j 1.6:
,,.-
-a
-----*
-x
lamcllae
Stromo lomellae
0
1
2
3
L PAF
5
6
7
6
9
10
1 20
imM)
0.0
I
I
I
I
I
I
I
I
I
I
:’
0123L5679910
c 20
PAF
tmM1
FIG. 1. Fs8JFTN increase in chloroplasts, grana, and stroma lamellae of bean (right) or pea (left) leaves, after their incubation with PAF C-16. All thylakoid fractions were washed twice with 50 mM Tricine-NaOH, pH 7.2, and were suspended in Tricine to yield 30 pg Chl/ml. Fifty-microliter aliquots were added to the freshly prepared phospholipid film, incubated for 30 min at 4”C, transferred to a capillary tube, and immersed in liquid nitrogen, and their fluorescence emission spectra recorded at -196°C. Grana and stroma lamellae were isolated by differential centrifugation of French press disrupted chloroplasts suspended in 0.05 M phosphate-O.15 M KCl, pH 7.2, at 400 pg ChWml(l3). The 10K and 240K pellet fractions (grana and stroma lamellae, respectively) were washed and suspended in Tricine to yield 30 pg Chl/ml, and aliquots of 50 ~1 were incubated with the phospholipid film, as above. All phospholipids were products of NOVA, Switzerland, unless otherwise specified. They were dissolved in chloroform:methanol (1:2), and aliquots were completely dried under a stream of nitrogen, just before use. RESULTS
Incubation of thylakoids, washed and suspended in Tricine-NaOH, with a film of PAF (with a 4:l ratio of C-16:C-18 in l-Oalkyl chain) has been found to induce a drastic increase in the F685/F730 ratio at 77”K, which depends on PAF concentration and PAF/Chl ratio (7). PC, on the other hand, even though it has similar electrical properties, is completely ineffective (7). In addition, the effect of pure
PAF 1-O-C-18 is far less than that of PAF 1-O-C-16. Out of a wide variety of PAF or PC compounds tested, with shorter or longer C chains at C-l and/or C-2, or a larger or smaller polar head at C-3, only PAF C-16 and its lyso analog were found to be effective (Table I). As deduced from Table I, the molecular structure requirement for the effect to be elicited is the ether bond and a long C chain (preferably a C-16 chain) at the C-l position of the glycerol moiety, a short C tail (or the lyso form) at C-2, and a large polar head at C3. Since the active compounds are zwitterionic, their effect cannot be attributed to thylakoid surface charge neutralization, in a way similar to cation action (8); thus lateral movement of complexes leading to segregation of PSI and PSI1 (in stroma and grana lamellae, respectively), and concomitant inhibition of spillover, is not expected. The finding that PAF C-16 can also induce enhancement of the F685/F,30 ratio in isolated subchloroplast fractions of grana and stroma lamellae leads to the
274
ARGYROUDI-AKOYUNOGLOU
AND
VAKIRTZI-LEMONIAS
a 1.5
BEAN
t
Chl
10 K Igrand alb=2,5
4 ,OmM / /=b
PAF:
PAF-0
0
1
780
700
620
WAVELENGTH
(nml
FIG. 2. The low-temperature fluorescence spectrum of grana and stroma thylakoids (10K and 240K, respectively) isolated from bean (a, b) or pea (c) leaves, as affected by incubation with increasing concentrations of PAF C-16. For experimental details see Fig. 1 and Materials and Methods.
same conclusion. Figure 1 shows the concentration dependence of the effect in isolated chloroplasts and their grana and stroma lamellar fractions from pea or bean. As shown the effect is pronounced in all thylakoid fractions; however, it is most pronounced in grana fractions. In addition, while in pea the effect is more drastic in grana than in chloroplasts or stroma lamellae, in bean it is equally pronounced in chloroplasts and grana. Figure 2 shows the low-temperature fluorescence emission spectra obtained from grana and stroma lamellar fractions of bean and pea chloroplasts, prior to or after incubation with PAF C-16. As shown, the change is dependent on PAF concentration, and much more drastic in the PSII-rich grana than in the PSI-rich
stroma lamellae fractions. This suggests that the effect of PAF is specifically directed to the PSI1 unit. Indeed, we found that, in addition to the increase in the F,& FTso ratio at 77”K, the Chl a fluorescence yield (F,,JChl at room temperature) is similarly affected (Tables I and II). The increase in F,,,/Chl is similar to that observed upon cation addition (MgCl,) to Tricine-unstacked (agranal) chloroplasts. In addition, and in agreement with the findings on the effect of various phospholipids on the F685/F730 ratio, the most potent effector in this case is also the PAF C16 and its lyso analog. To check whether PAF C-16 affects these parameters via alteration of the pigmentprotein complex organization in the photosynthetic units (14-16), we further studied
b 1.5 - Bean t
240K [ st roma
Chl
a/b=
4.6
‘;i
.c3
-----
1OmMPAF
-
OPAF
:
\
I
\ t
620
I
I
700
780
WAVE LENGTH :
in m)
680 Pea
240 K Chla/b=4.3
*-..e***
tOmMPAF
1
-.-.-2,5mMPAF
.ir
--me
O,GmMPAF!
-
OPAF
;I
620
700 WAVELENGTH
(nm)
FIG. 2-Continued 275
780
276
ARGYROUDI-AKOYUNOGLOU
AND TABLE
EFFECT OF Me,
VAKIRTZI-LEMONIAS
II
PAF, PC, AND THEIR DERIVATIVES ON THE CHLOROPHYLLS FLUORESCENCE YIELD (F,,,.,/Chl) AT ROOM TEMPERATURE OF PEA THYLAKOIDS
Phospholipid A. No phospholipid Chloroplasts in sue-phos-KC1 Chloroplasts in Tricine + Mga+ (1 mM) + MgZ+ (2 mM) + Mg2+ (4 mM) B. Chloroplasts in sue-phos-KC1 Chloroplasts in Tricine + 0.25 pmol PAF C-16” + 0.50 pmol PAF C-16” C. Chloroplasts in sue-phos-KC1 Chloroplasts in Tricine +0.25 pmol PAF C-16 PAF C-18 L-PAF C-16 PAF C-16 (-polar group) PAF 1,2-0-0-Di-C-16 PC C-16 L-PC C-16 L-PAF C-18 0.05 amol PAF C-16 (+larger polar group)
F,,,,,/Chl (arbitrary units)
% Increase
75 38 73 90 85
97 0 92 136 124
68
121
28 54 100
0 93 257
100
108
48
0
130
170
43
80
0 233 8 0 0 25 67
59
23
160
52 46 42 60
Note. Pea plastids, isolated in sue-phos-KC1 were washed twice in Tricine and then suspended in Tricine to yield 12.5 pg Chl/ZO rl (B) or 13 pg Ch1/50 ~1 (C). Aliquots containing 13 pg Chl were either added to a cuvette containing 2.5 ml Tricine and the F,., determined (A) or incubated in an ice bath with the freshly dried phospholipid film for 30 min and thereafter added to the Tricine-containing cuvette for determination of the fluorescence yield (B, C). Mg2f was added directly to the Tricine-containing cuvette, and after an incubation at room temperature for 5 min in the dark the F,, was determined. All samples were kept in the dark prior to F,,,., determination to have the electron acceptor Q in the reduced state. The light admitted to the cuvette compartment was adequate for full reduction. All phospholipid compounds were products of NOVA, except for (a) which were products of Sigma.
the distribution of Chl among the pigment-protein complexes of the thylakoid. Figure 3 and Table III show representative results, obtained with thylakoids incubated in the presence or absence of PAF C16 and then dissociated with and electrophoresed in SDS. As indicated, incubation with PAF reduces the amount of Chl running as free pigment upon electrophoresis, suggesting that PAF promotes chlorophyll binding in the complexes. Thus, the CPIa/ CPI and LHCPVLHCP3 ratios are drastically increased, suggesting that PAF surpresses dissociation of the supramolecular structures by SDS. Assuming that the
LHCPB complex originates in the LHC-I, or at least represents part of the LHC-I (1’7,18), the Chl distribution between PSI1 and PSI complexes (LHCPl + CPa + LHCP3 and CPIa + CPI + LHCPB, respectively) changes after incubation with PAF in favor of PSI1 as seen from the data in Table III. We do not know whether this effect of PAF in suppressing the dissociation of oligomeric forms is in any way related to the changes observed in the fluorescence parameters. However, it is possible that the enhanced Chl binding, especially in the pigment-protein complexes of the PSI1 unit induced by incubation with
CHLOROPHYLL EA.
thylakoids
a FLUORESCENCE
277
IN CHLOROPLASTS
ma ”
EAN
thylokoids
1 .o W isi Q ,”
0.5
:: m Q
0 .o
-0
1
3
2 DISTANCE
[cm)
0
1
2
3
DISTANCElcm)
FIG. 3. Densitograms of mild SDS-PAGE resolution patterns obtained from SDS-solubilized bean (right) or pea (left) cbloroplasts washed twice and suspended in Tricine-NaOH (50 mM, pH 7.2) to yield 25 Fg Ch1/50 ~1. Fifty-microliter aliquots were added to a film of 1 pmol PAF C-16 or PC C-16 and incubated at 4°C for 30 min. SDS was then added to have an SDS/Chl ratio equal to 10, and electrophoretic analyses followed immediately at 4°C.
PAF, reflects the association of Chl in a ids, PAF C-16 and its lyso analog are the PSI1 unit of larger absorption cross sec- most potent effecters in photosynthetic tion, leading to increased F685/F730ratio. membranes. The molecular structure/ Finally, incubation of thylakoids with shape of the active molecules suggests that PAF C-16 induces an increase in the elec- for a compound to be active, the capacity tron transfer rate via PSI1 (in a DPC- to be incorporated in the phospholipid biDCIP photoreduction) (Fig. 4). Such an ac- layer of the thylakoid membrane is crucial; tivation in the Hz0 splitting activity rate, only the cone-like wedge shaped PAFs are however, was not observed; in addition, we active, but not the rod-like PCs. In addifound that PAF C-16 mediates electron tion, the difference in activity between transport from DPC to DCIP even in the PAF C-16 and PAF C-18 may be attributed absence of thylakoids, or in the presence of to the easier incorporation of the former DCMU, as well, suggesting that PAF C-16 into the thylakoid, as compared to the latmay enhance electron transfer activity ter, because of its larger critical micellar after its incorporation in the thylakoid concentration on one hand (an increase by by a strictly chemical reaction/mediation, one methylene group of the alkyl chain in without the involvement of the Photosys- PAF or lyso-PAF results in a 3-fold or lotern II unit itself. PC, on the other hand, fold decrease in CMC, respectively (19)) is completely ineffective in this respect and its apparently smaller micellar vol(Fig. 4). ume, as suggested by the data for l-O-C16- and 1-0-C-18-2-deoxy PC, which give 140 and 4900 molecules per micelle, respecDISCUSSION tively (20). This reasoning, however, does The results presented above suggest that not seem to hold in the case of PAF C-16 and lyso-PAF C-16; the latter is less effecout of a wide variety of ether phospholip-
ARGYROUDI-AKOYUNOGLOU
AND
VAKIRTZI-LEMONIAS
tive, even though its CMC is three times higher than that of the former (19). This may, therefore, suggest that the acetyl group in C-2 of PAF C-16 is crucial for activity. The zwitterionic character of all these compounds speaks against their involvement in the neutralization of thylakoid surface charges, in a way similar to that of cations (known to affect the Fm/FTm ratio in a similar way); thus, their action in enhancing the F685/F730ratio cannot be attributed to such processes leading to lateral movement of complexes from stroma to grana lamellae, blocking spillover of excitation energy from Photosystem II to Photosystem I (8). Isolated grana or stroma lamellae, when incubated with PAF, behave in a similar way. However, the effect of PAF on grana J’,&FTsO ratio is more pronounced than that on stroma lamellae (see Fig. l), suggesting that PAF mainly acts by affecting the Photosystem II unit. This is also suggested by the strong mediation of Photosystem II activity induced by PAF C-16. This effect on electron transport rate, induced by PAF (DPC-DCIP reduction), has been found also in the absence of Chl, or in DCMU-poisoned chloroplasts. This indicates, therefore, that PAF itself enhances electron transport from DPC to DCIP; actually PAF can stimulate the DPC-DCIP electron transport in a buffer system and in the dark as well (C. Vakirtzi-Lemonias and J. H. ArgyroudiAkoyunoglou, in preparation). We do not know how this mediation takes place; PAF might offer the necessary surface organization via micelle formation when incubated with DPC and DCIP in the absence of thylakoids. In the presence of thylakoids, however, PAF appears to act possibly by intercalating in the membrane. The action of PAF on the fluorescence parameters seems to be due to its incorporation into the thylakoid phospholipid bilayer, resulting in alteration of the organization of the photosynthetic units. The results of SDS-PAGE analyses of pigmentprotein complexes in thylakoids, prior to or after incubation with PAF or PC, support this conclusion. As shown, Chl bind-
CHLOROPHYLL
a FLUORESCENCE
1500 -
I-Chl
ol
1
1
1
1
0
1
9
PAF C-16 or + DCMU)
3
’
1
Phospholipid
’
1.0
0,5 (pmoles150h)
FIG. 4. PAF C-16-induced mediation of electron transport through PSII. Chloroplasts washed and suspended in 50 mM Tricine-NaOH, pH 7.2, yielding 1.5 pg Ch1/50 ~1, were used. PSI1 activity was determined as the DPC-DCIP reduction at saturating light intensity. Incubation with the phospholipid film (PAF C-16 or PC) was for 30 min at 0°C prior to PSI1 activity determination.
ing in Photosystem I and Photosystem II complexes is greatly increased in the presence of PAF but not in the presence of PC. The increase seems to be larger in the case of the Photosystem II complexes than in those of Photosystem I (hence the PSII/ PSI ratio, as monitored by the percentage of Chl bound in PSI1 complexes to that in PSI, is enhanced). This may be reflected by the increased F685/F730 ratio found under these conditions: upon PAF incorporation into the thylakoid, the effective absorption cross section of Photosystem II may be increased (via enhanced Chl binding), so that the distribution of absorbed photons may favor the Photosystem II unit. REFERENCES 1. VARGAFTIG, B. B., CHIGNARD, M., BENVENISTE, J., LEFORT, J., AND WAL, F. (1981) Ann. N. Y. Acad sci. 370,119-137.
IN CHLOROPLASTS
279
2. HANAHAN, D. J. (1986) Annu. Rev. B&hem 55, 483-509. 3. LEE, T. C., AND SNYDER, F. (1986) in Phospholipids and Cellular Regulation (Kuo, J. K., Ed.), Vol. II, pp. l-39 CRC Press, Boca Raton, FL. 4. SHUKLA, S. D., AND HANAHAN, D. J. (1982) Biochem Biophys. Res. Commun, 106, 697703. 5. BILLAH, M. M., AND LAPETINA, E. G. (1983) PTOC. Natl Acad. Sci. USA 80,965-968. 6. SCHERER, G. F. E. (1985) B&hem. Biophys. Res. Commun 133,1160-1167. 7. ARGYROUDI-AKOYUNOGLOU, J. H., AND VAKIRTZILEMONIAS, C. (1987) Arch. Biochem Biophys. 253.38-45. 8. BARBER, J. (1980) FEBSLett. 11&l-10. 9. ARGYROUDI-AKOYUNOGLOU, J. H., AND AKOYUNOGLOU, G. (1983) Arch. Biochem. Biophys. 227, 469-477. 10. ARGYROUDI-AKOYUNOGLOU, J. H., AND AKOYUNOGLOU, G. (1977) Arch. Biochem Biophys. 179, 370-377. 11. VERNON, L. P., AND SHAW, E. R. (1969) Plant Physiol. 44,1645-1649. 12. ANDERSON, J. M., WALDRON, J. C., AND THORNE, S. W. (1978) FEBS Lett. 99,227-233. 13. SANE, P. V., GOODCHILD, D. J., AND PARK, R. B. (1970) Biochim. Biophys. Acta 216,162-178. 14. ARGYROUDI-AKOYUNOCLOU, J. H., CASTORINIS, A., AND AKOYUNOGLOU, G. (1982) Photobicthem. Photobiophys. 4,201-210. 15. AKOYUNOGLOU, G., AND ARGYROUDI-AKOYUNOGLOU, J. H. (1986) in Ion Interactions in Energy Transfer Biomembranes (Papageorgiou, G., andBarber, J., Eds.), pp. 211-222, Plenum, New York. 16. ARGYROUDI-AKOYUNOGLOU, J. H., AND AKOYUNOGLOU, G. (1986) in Regulation of Chloroplast Differentiation (Akoyunoglou, G., and Senger, H., Eds.), pp. 329-338, A. R. Liss, New York. 17. ARGYROUDI-AKOYUNOGLOU, J. H. (1984) FEBS Lett. 171,47-53. 18. KUANG, T. Y., ARGYROUDI-AKOYUNOGLOU, J. H., NAKATANI, H., WATSON, J., AND ARNTZEN, C. J. (1984) Arch. B&hem. Biophys. 235,618627. 19. KRAMP, W., PIERONI, G., PINCKARD, R. N., AND HANAHAN, D. J. (1984) Chem. Phys. Lipids 35, 49-62. 20. WELTZIEN, H. U., ARNOLD, B., AND REUTHER, R. (1977) B&him Biophys. Acta 466,411-421.