Geranylgeranyl substituted methylquinols are shown to be precursors of tocopherol biosynthesis in spinach chloroplasts as well as phytyl substituted ones. The geranylgeranyl substituted quinols are methylated even to a greater extent than the phytyl substituted ones. The connection to the so far known biosynthetic origin of -tocopherol is probably -tocotrienol which is hydrogenated to γ-tocopherol and then further methylated to -tocopherol

Chow

Gernot Schultz

Janiszowska

Jensen

Jürgen Soll

Mayer

Soll

Wellburn

Whistance

English

Soll, Jürgen

Schultz, Gernot

Open Access LMU ( Ludwig-Maximilians-Univ. München)

Vol. 91, No. 3, 1979 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS December 14, 1979 Pages 715-720 COMPARISON OF GERANYLGERANYL AND PHYTYL SUBSTITUTED METHYLQUINOLS IN THE TOCOPHEROL SYNTHESIS OF SPINACH CHLOROPLASTS JDrgen Soil’ and Gernot Schultz’ ’ ‘lnstitut fiir Klinische Biochemie und Physiologische Chemie, Medizinische Hochschule Hannover, and ” lnsti tut fijr Tierernahrung, Arbei tsgruppe fiir Phytochemie und Futtermittelkunde, Tierlrztl iche Hochschule Hannover, D-3000 Hannover 1 Received October 8, 1979 SUHHARY: Geranylgeranyl substituted methylquinols are shown to be precursors of tocopherol biosynthesis in spinach chloroplasts as well as phytyl substituted ones. The geranylgeranyl substl- tuted quinols are methylated.even to a greater extent than the phytyl substituted ones. The connection to the so fa’r known biosynthetic origin of a-tocopherol is probably - toco- trienol which is hydrogenated to Y-tocopherol and then further methylated to a-tocopherol. INTRODUCTION: Tocopherols are synthesized from homogentisic acid and a C20 prenol unit to form the 2-methyl-6-prenylquinols (l).Using 2-methyl-6-phytylquinol as precursor for the toco- pherol synthesis by chloroplasts the following sequence of prod- ucts has been found: Me-6-PQH2 - 2,3-He2PQH2 - YT -) af (2). Since tocotrienols have been found in several plant species (3,4) and hydrogenation of tocotrienols to tocopherols by NADPH2 has been shown (5) it seems possible that the precursors of aT are geranylgeranyl and not phytyl substituted methylquinols. So it has been examined whether the enzymatic methylatcon of 2-methylprenylquinols by SAM to form aT exhibit a specifity for the different prenyl side chains. METHODS : The geranylgeranyl and phytyl substituted methylquinols and quinones were prepared from the corresponding methylquinol with trans-geranyllinalool and isophytol by the procedure de- scribed in (2), yT3 was synthesized according to (6). aT3 was T4;;f;A[ry:pDr. F. Weber! Fa. Hoffmann-La Roche, Baste. /He- . act. 50 mCr/mmol) was purchased from Amersham Buchler, Braunschweig. 715 0006-291X/79/23071 5-06$01.00/O c- opyrighr @ 1979 by Academic Press. Inc. AU rights of reproduction in any form reserved. Vol. 91, No. 3, 1979 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Methyl-phytylquinols (PQH~) -geranylger$nylquinols (GGQH~) Y R2 R3 CH3 H H Me-6-PQH2 2-Methyl-6- Me-6-GGQH2 6-Methyl- CH3 CH3 H 2,3-Me2PQH2 2,3-Dimethyl-5- 3 PQH, 2,3-Me2GGQH2 2,3-Dimethyl-5- GGQH2 CH3 CH3 CH3 Me3PQH2 Trimethyl- Me3GGQH2 Trimethyl- The corresponding quinones are signified in the text with Q instead of QH2. R1 R2 R3 CH3 H H CH3 CH3 H CH3 CH3 CH3 Tocopherol s (T) Tococh romanol s Tocotrienol s (T3) 6T 6T3 YT VT3 uT CXT 3 “Broken chloroplasts” were prepared from intact spinach chloro- plasts i.solated according to ref. (7) as modified by Prof. U. Heber (personal communication). Several times intact chloroplasts were used directly with similar results. To get “broken chloro- plasts” the pellet resulting from centrifugation of medium B chloroplasts suspension (7) 10 min at 0’. was suspended with dist. water for The chlorophyll content was at least 2 mg/ml. On- ly nondifferentiated leaves from young plants are able to syn- thesize prenylquinols to a higher extent. The application of substrates to chloroplast suspension for me- thylation and cyclization reaction in tocopherol synthesis was performed as follows: 1000 pl chloroplast suspension in medium C (7) (about 1 mg chlorophyll/ml) or 500 ul “broken chloro- plasts” iluted with 500 ul medium C, 100 Ml 0,2 M NaHCO suspension14 3, 50 cl1 /Me- C/ SAM (= 2,s pCi, soluted in 0,l N H S04), substituted methylprenylquinol as substrate (10 -7 and 10 -a mol., respectively, evaporated from etheral solution) were added in- to a test vial. The rssulting suspension was illu;inatcod by ha- logen lamps (0,l J/cm x s) in a water bath at 20 + 2 for - 30 min. 716 Vol. 91, No. 3, 1979 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS origin yT af 1 1 2,3-Me,P Q Me-6-P Q Me,PQ front 1 1 1 YT, aT:, Me-e-G G Q Me,GGO. Carotine \ 2,3-Me,GGQ I \ I ” detectable with detectable by quenching Emmerie Engel reagent Of fluOreSCence at 254 nm Scheme 1: Sequence of prenylquinones and tocochromanols on TLC system I Isolation and purification of investigated substrates were per- formed as in ref (2). All quinols were oxidized for a more ef- ficient purification by air supply before TLC on system I (for sequence of detectable substances see Scheme 1). All substances were cochromatographed with reference substances. All methyl- prenylquinones could be rechromatographed as single substances on system II, whereas tocopherols and tocotrien 0 1s could be re- chromatographed only in couples of yT l yT3 and aT + aT spectively. 3, re- The following TLC-systems were used: System I - petrol (b.r. 60 - 80°)/Et20 (7 : 1); system I I system (= cellulose lagers impregnated with 7 % petrol (b.r. 100 - 140 )) with Me,CO/H,O (85 : 1 silicagel with reversed phase paraffin in 5). All thin layers (Schleicher & SchUll, Germ’any) 8n glass contained a fluorescent indicator F 254. System I was developed to 20 cm height, system II to 10 cm. RESULTS: Monomethylcompounds as substrates: Studying the in- -- corporation of /He- ‘4;,-f;o; gT4- - - -. C/ SAM into Me-6-GGQH2 and Me-6-PQH2, respectively, a strong preference of the geranyl- geranyl-compound could be observed. The incorporation rates were up to 4,5 times higher as for the phytyl compound. In the case of Me-6-GGH, the total amount of fixed 14 C of applied /Me- 14 C/ SAM was abott 1 % . As described in (2) the first step was the methylation to the 2,3-dimethylprenylquinol (for stereospe- cifity of reaction see 21, the second step was the cyclization to the corresponding d i methyl tocochromanol and the last step was the methylation to the trimethyltocochromanol. The novel point was the hydrogenation of the geranylgeranyl moiety to the phytyl ones. Only traces of 2,3-Me2PQH2 beside yT 3’ yT and aT were found. Thus it might be concluded that hydrogenation occurs on the chromanol stage. Adding 10 -4 14 M of Me-6-GGQH2 the relation of /Me- C/ incorporation in these compounds were as follows: 717 Vol. 91, No. 3, 1979 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 1°0iPrn -u--LAA xE3x 2,3-Me,PQ 2,3+leGGQ YT YT3 I\,, B ~zizrIELlc 2.3~Me,PQ 2.3-MeGGQ VT YT~ . Radioscan of products obtained by incorporation of Wfrom ,“,J4 C/ SAM into Me-6-GGQH2 plus Me-6-PQH2 applied in different concentrations (competition experiment): A. lo-’ M Me-6-GGQH2 plus 10 -4 M He-6-PQH2 as substrate; 6. 10 -4 M He-6-GGQH2 plus lo-’ M Me-6-PQH2 as substrate. The figure shows the re-chromatography of products on system II. For further experimental details see Methods. 2,3-Me2GGQH2 : 2,3-Me2PQH2 : yT3 : yT : aT = 100 : traces : 53 : traces : 18. Using 10 -4 M Me-6-PQH2 as substrate the values were as follows: 2,3-Me PQH 2 2 : yT : aT = 100 : 60 : 30. As it can be seen in Figure 1 a competition of Me-6-GGQH2 and Me-6- PQH, could be observed if these substrates were combined in dif- ferent concentrations in the chloroplast experiment. Dimethyl compounds as substrates: 2,3-Me2GGQH2 and 2,3-Me2PQH2 - -- -------___ were incorporated into aT in similar rates. The optimal concen- tration of 2,3-Me2GGQH2 to form aT is about IO-’ M which fairly agreed with 2,3-Me2PQH2 (2). No radioavtivity could be proved in the trimethylprenylquinones. When 2,3-Me GGQH 2 2 was applied, no aT 3 could be detected. On the other hand, when yT 3 was added, aT 3 was found. The optimal concentrations of yT and yT3 to form trimethyltocochromanols were similar to the above dimethylpre- 718 Vol. 91, No. 3, 1979 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS nylquinols. As could be deduced from ref. (5) additional NADPH2 should result in a more complete hydrogenation of the unsatura- ted prenyl side chain. Under present conditions, however, an addition of 10 -4 M NADPH2 was not effective in case of Me-6- GGQH2, 2,3-Me2GGQH2 and yT3. Obviously NADPH2 supply by photo- synthetic NADP reduction in thylakoids of illuminated chloro- plasts is sufficient for the - presumably saturated - prenyl- hydrogenation system. If no substrate was added, only aT was obtained in reasonable amounts from the endogenous substrate pool.,Application of di- methylprenylquinols increases the amount of labeled aT about twofold. DISCUSSION: The higher methylation rate of He-6-GGQH2 in com- parison to He-6-PQH2 indicates that even in spinach chloro- plasts geranylgeranyl substituted methylquinols can be precur- sors for the tocopherol synthesis. In case of Me-6-GGQH2 the resulting 2,3-Me2GGQH2 is converted to yT3 and then hydrogena- ted to yT. The only yield of aT3 by feeding yT3 and not by 2, 3-MeGGQH2 also points to a saturation reaction on a step bet- ween 2, 3-Me2GGQH2 and yT. This probably represents the connec- tion to tocopherols. The absence of trimethylprenylquinones shows that the cyclization to the chromanol stage is restric- ted to the dimethylprenylquinol stage. The methyltransferase reactions has not only a fairly high specifity for the methyl position at the monomethyl prenylquinol (21, but also for the different prenyl side chains. This might be an indication that the isoprens both phytol and geranylgeraniol (or its pyrophos- Scheme 2: Proposed scheme for the biosynthesis of tocopherols 719 Vol. 91, No. 3, 1979 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS phates) could be substrates for the prenyl-ligase to form 2-me- thyl-6-prenylquinol. From the results two possible pathways of tocopherol synthesis are proposed in Scheme 2. ACKNOWLEDGEMENTS Financial support by the Deutsche Forschungsgemeinschaft is thankfully acknowledged. We thank Mrs. D. Bodenstab for excel- lent technical assistence. References 1. Whi stance 593 - 600 , G.R., and Threlfall, D.R. (1970) Bi ochem .J. 117, 2. Soll, J., and Schultz, G. (1979) Phytochemistry, accepted for pub1 ication. 3. Chow, C.K., Draper, H.H., and Csallany, A.S. (1969) Anal. Biochem. 32, 81 -90. 4. Janiszowska, W., and Pennock, J.F. (1976) Vitam. Horm. (N.Y.) 2, 77 - 105. 5. Wellburn, A.R. (1570) Phytochemistry 743 - 748. 6. Mayer, H., and Isler, 0. 2, (1971) Methods in Enzymology, Vol. 18 C, p. 241 - 348, Academic Press, N.Y. 7. Jensen, R.G.. and Bassham, J.A. (1966) Proc. Natl. Acad. Sci. U.S. 56, 1095. 720 

Comparison of geranylgeranyl and phytyl substituted methylquinols in the tocopherol synthesis of spinach chloroplasts

Universität München: Elektronischen Publikationen

Vol. 91, No. 3, 1979 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 
December 14, 1979 Pages 715-720 
COMPARISON OF GERANYLGERANYL AND PHYTYL SUBSTITUTED 
METHYLQUINOLS IN THE TOCOPHEROL SYNTHESIS OF 
SPINACH CHLOROPLASTS 
JDrgen Soil’ and Gernot Schultz’ ’ 
‘lnstitut fiir Klinische Biochemie und Physiologische Chemie, 
Medizinische Hochschule Hannover, and 
” lnsti tut fijr Tierernahrung, Arbei tsgruppe fiir Phytochemie 
und Futtermittelkunde, Tierlrztl iche Hochschule 
Hannover, D-3000 Hannover 1 
Received October 8, 1979 
SUHHARY: Geranylgeranyl substituted methylquinols are shown to 
be precursors of tocopherol biosynthesis in spinach chloroplasts 
as well as phytyl substituted ones. The geranylgeranyl substl- 
tuted quinols are methylated.even to a greater extent than 
the phytyl substituted ones. The connection to the so fa’r 
known biosynthetic origin of a-tocopherol is probably - toco- 
trienol which is hydrogenated to Y-tocopherol and then further 
methylated to a-tocopherol. 
INTRODUCTION: Tocopherols are synthesized from homogentisic 
acid and a C20 prenol unit to form the 2-methyl-6-prenylquinols 
(l).Using 2-methyl-6-phytylquinol as precursor for the toco- 
pherol synthesis by chloroplasts the following sequence of prod- 
ucts has been found: Me-6-PQH2 - 2,3-He2PQH2 - YT -) af (2). 
Since tocotrienols have been found in several plant species 
(3,4) and hydrogenation of tocotrienols to tocopherols by NADPH2 
has been shown (5) it seems possible that the precursors of aT 
are geranylgeranyl and not phytyl substituted methylquinols. 
So it has been examined whether the enzymatic methylatcon of 
2-methylprenylquinols by SAM to form aT exhibit a specifity for 
the different prenyl side chains. 
METHODS : The geranylgeranyl and phytyl substituted methylquinols 
and quinones were prepared from the corresponding methylquinol 
with trans-geranyllinalool and isophytol by the procedure de- 
scribed in (2), yT3 was synthesized according to (6). aT3 was 
T4;;f;A[ry:pDr. F. Weber! Fa. Hoffmann-La Roche, Baste. /He- 
. act. 50 mCr/mmol) was purchased from Amersham 
Buchler, Braunschweig. 
715 
0006-291X/79/23071 5-06$01.00/O 
c- opyrighr @ 1979 by Academic Press. Inc. 
AU rights of reproduction in any form reserved. 
Vol. 91, No. 3, 1979 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 
Methyl-phytylquinols (PQH~) -geranylger$nylquinols (GGQH~) 
Y R2 R3 
CH3 H H Me-6-PQH2 2-Methyl-6- Me-6-GGQH2 6-Methyl- 
CH3 CH3 H 2,3-Me2PQH2 2,3-Dimethyl-5- 
3 
PQH, 2,3-Me2GGQH2 2,3-Dimethyl-5- GGQH2 
CH3 CH3 CH3 Me3PQH2 Trimethyl- Me3GGQH2 Trimethyl- 
The corresponding quinones are signified in the text with Q instead of QH2. 
R1 R2 R3 
CH3 H H 
CH3 CH3 H 
CH3 CH3 CH3 
Tocopherol s (T) 
Tococh romanol s 
Tocotrienol s (T3) 
6T 
6T3 
YT 
VT3 
uT CXT 
3 
“Broken chloroplasts” were prepared from intact spinach chloro- 
plasts i.solated according to ref. (7) as modified by Prof. U. 
Heber (personal communication). Several times intact chloroplasts 
were used directly with similar results. To get “broken chloro- 
plasts” the pellet resulting from centrifugation of medium B 
chloroplasts suspension (7) 
10 min at 0’. 
was suspended with dist. water for 
The chlorophyll content was at least 2 mg/ml. On- 
ly nondifferentiated leaves from young plants are able to syn- 
thesize prenylquinols to a higher extent. 
The application of substrates to chloroplast suspension for me- 
thylation and cyclization reaction in tocopherol synthesis was 
performed as follows: 1000 pl chloroplast suspension in medium 
C (7) (about 1 mg chlorophyll/ml) or 500 ul “broken chloro- 
plasts” iluted with 500 ul medium C, 100 Ml 0,2 M 
NaHCO 
suspension14 
3, 50 cl1 /Me- C/ SAM (= 2,s pCi, soluted in 0,l N H S04), 
substituted methylprenylquinol as substrate (10 -7 and 10 
-a 
mol., 
respectively, evaporated from etheral solution) were added in- 
to a test vial. The rssulting suspension was illu;inatcod by ha- 
logen lamps (0,l J/cm x s) in a water bath at 20 + 2 for - 
30 min. 
716 
Vol. 91, No. 3, 1979 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 
origin yT af 
1 1 
2,3-Me,P Q 
Me-6-P Q Me,PQ front 
1 1 1 
YT, aT:, Me-e-G G Q Me,GGO. Carotine 
\ 
2,3-Me,GGQ 
I \ I ” 
detectable with detectable by quenching 
Emmerie Engel reagent Of fluOreSCence at 254 nm 
Scheme 1: Sequence of prenylquinones and tocochromanols on 
TLC system I 
Isolation and purification of investigated substrates were per- 
formed as in ref (2). All quinols were oxidized for a more ef- 
ficient purification by air supply before TLC on system I (for 
sequence of detectable substances see Scheme 1). All substances 
were cochromatographed with reference substances. All methyl- 
prenylquinones could be rechromatographed as single substances 
on system II, whereas tocopherols and tocotrien 0 1s could be re- 
chromatographed only in couples of yT l yT3 and aT + aT 
spectively. 
3, re- 
The following TLC-systems were used: System I - 
petrol (b.r. 60 - 80°)/Et20 (7 : 1); system I I 
system (= cellulose lagers impregnated with 7 % 
petrol (b.r. 100 - 140 )) with Me,CO/H,O (85 : 1 
silicagel with 
reversed phase 
paraffin in 
5). All thin 
layers (Schleicher & SchUll, Germ’any) 8n glass contained a 
fluorescent indicator F 254. System I was developed to 20 cm 
height, system II to 10 cm. 
RESULTS: Monomethylcompounds as substrates: Studying the in- -- 
corporation of /He- 
‘4;,-f;o; gT4- - - -. 
C/ SAM into Me-6-GGQH2 and 
Me-6-PQH2, respectively, a strong preference of the geranyl- 
geranyl-compound could be observed. The incorporation rates 
were up to 4,5 times higher as for the phytyl compound. In the 
case of Me-6-GGH, the total amount of fixed 
14 
C of applied /Me- 
14 
C/ SAM was abott 1 % . As described in (2) the first step was 
the methylation to the 2,3-dimethylprenylquinol (for stereospe- 
cifity of reaction see 21, the second step was the cyclization 
to the corresponding d i methyl tocochromanol and the last step 
was the methylation to the trimethyltocochromanol. The novel 
point was the hydrogenation of the geranylgeranyl moiety to the 
phytyl ones. Only traces of 2,3-Me2PQH2 beside yT 
3’ 
yT and aT 
were found. Thus it might be concluded that hydrogenation occurs 
on the chromanol stage. Adding 10 
-4 
14 
M of Me-6-GGQH2 the relation 
of /Me- C/ incorporation in these compounds were as follows: 
717 
Vol. 91, No. 3, 1979 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 
1°0iPrn -u--LA
A 
xE3x 
2,3-Me,PQ 2,3+leGGQ YT YT3 
I\,, 
B 
~zizrIELlc 
2.3~Me,PQ 2.3-MeGGQ VT YT~ 
. Radioscan of products obtained by incorporation of 
Wfrom ,“,J4 C/ SAM into Me-6-GGQH2 plus Me-6-PQH2 
applied in different concentrations (competition experiment): 
A. lo-’ M Me-6-GGQH2 plus 10 
-4 
M He-6-PQH2 as substrate; 
6. 10 
-4 
M He-6-GGQH2 plus lo-’ M Me-6-PQH2 as substrate. 
The figure shows the re-chromatography of products on system II. 
For further experimental details see Methods. 
2,3-Me2GGQH2 : 2,3-Me2PQH2 : yT3 : yT : aT = 100 : traces : 53 : 
traces : 18. Using 10 
-4 
M Me-6-PQH2 as substrate the values 
were as follows: 2,3-Me PQH 2 2 : yT : aT = 100 : 60 : 30. As it 
can be seen in Figure 1 a competition of Me-6-GGQH2 and Me-6- 
PQH, could be observed if these substrates were combined in dif- 
ferent concentrations in the chloroplast experiment. 
Dimethyl compounds as substrates: 2,3-Me2GGQH2 and 2,3-Me2PQH2 - -- -------___ 
were incorporated into aT in similar rates. The optimal concen- 
tration of 2,3-Me2GGQH2 to form aT is about IO-’ M which fairly 
agreed with 2,3-Me2PQH2 (2). No radioavtivity could be proved 
in the trimethylprenylquinones. When 2,3-Me GGQH 
2 
2 was applied, 
no aT 
3 
could be detected. On the other hand, when yT 
3 
was added, 
aT 
3 
was found. The optimal concentrations of yT and yT3 to form 
trimethyltocochromanols were similar to the above dimethylpre- 
718 
Vol. 91, No. 3, 1979 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 
nylquinols. As could be deduced from ref. (5) additional NADPH2 
should result in a more complete hydrogenation of the unsatura- 
ted prenyl side chain. Under present conditions, however, an 
addition of 10 
-4 
M NADPH2 was not effective in case of Me-6- 
GGQH2, 2,3-Me2GGQH2 and yT3. Obviously NADPH2 supply by photo- 
synthetic NADP reduction in thylakoids of illuminated chloro- 
plasts is sufficient for the - presumably saturated - prenyl- 
hydrogenation system. 
If no substrate was added, only aT was obtained in reasonable 
amounts from the endogenous substrate pool.,Application of di- 
methylprenylquinols increases the amount of labeled aT about 
twofold. 
DISCUSSION: The higher methylation rate of He-6-GGQH2 in com- 
parison to He-6-PQH2 indicates that even in spinach chloro- 
plasts geranylgeranyl substituted methylquinols can be precur- 
sors for the tocopherol synthesis. In case of Me-6-GGQH2 the 
resulting 2,3-Me2GGQH2 is converted to yT3 and then hydrogena- 
ted to yT. The only yield of aT3 by feeding yT3 and not by 
2, 3-MeGGQH2 also points to a saturation reaction on a step bet- 
ween 2, 3-Me2GGQH2 and yT. This probably represents the connec- 
tion to tocopherols. The absence of trimethylprenylquinones 
shows that the cyclization to the chromanol stage is restric- 
ted to the dimethylprenylquinol stage. The methyltransferase 
reactions has not only a fairly high specifity for the methyl 
position at the monomethyl prenylquinol (21, but also for the 
different prenyl side chains. This might be an indication that 
the isoprens both phytol and geranylgeraniol (or its pyrophos- 
Scheme 2: Proposed scheme for the biosynthesis of tocopherols 
719 
Vol. 91, No. 3, 1979 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 
phates) could be substrates for the prenyl-ligase to form 2-me- 
thyl-6-prenylquinol. From the results two possible pathways of 
tocopherol synthesis are proposed in Scheme 2. 
ACKNOWLEDGEMENTS 
Financial support by the Deutsche Forschungsgemeinschaft is 
thankfully acknowledged. We thank Mrs. D. Bodenstab for excel- 
lent technical assistence. 
References 
1. Whi stance 
593 - 600 
, G.R., and Threlfall, D.R. (1970) Bi ochem .J. 117, 
2. Soll, J., and Schultz, G. (1979) Phytochemistry, accepted 
for pub1 ication. 
3. Chow, C.K., Draper, H.H., and Csallany, A.S. (1969) Anal. 
Biochem. 32, 81 -90. 
4. Janiszowska, W., and Pennock, J.F. (1976) Vitam. Horm. (N.Y.) 
2, 77 - 105. 
5. Wellburn, A.R. (1570) Phytochemistry 743 - 748. 
6. Mayer, H., and Isler, 0. 
2, 
(1971) Methods in Enzymology, Vol. 
18 C, p. 241 - 348, Academic Press, N.Y. 
7. Jensen, R.G.. and Bassham, J.A. (1966) Proc. Natl. Acad. Sci. 
U.S. 56, 1095. 
720 


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Comparison of geranylgeranyl and phytyl substituted methylquinols in the tocopherol synthesis of spinach chloroplasts

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