|
Plant Physiol, January 2001, Vol. 125, pp. 423-429
Membrane Lipid Biosynthesis in Chlamydomonas
reinhardtii. In Vitro Biosynthesis of
Diacylglyceryltrimethylhomoserine1
Thomas S.
Moore,*
Zhirong
Du, and
Zhi
Chen
Department of Biological Sciences, Louisiana State University,
Baton Rouge, Louisiana 70803
 |
ABSTRACT |
Diacylglyceryltrimethylhomo-Ser (DGTS) is an abundant lipid in the
membranes of many algae, lower plants, and fungi. It commonly has an
inverse concentration relationship with phosphatidylcholine, thus
seemingly capable of replacing this phospholipid in these organisms. In
some places this replacement is complete; Chlamydomonas reinhardtii is such an organism, and was used for these
investigations. We have assayed headgroup incorporation to form DGTS in
vitro. The precursor for both the homo-Ser moiety and the methyl groups was found to be S-adenosyl-L-Met. DGTS
formation was associated with microsomal fractions and is not in
plastids. By analogy with phosphatidylcholine and
phosphatidylethanolamine biosynthesis in higher plants, the microsomal
activity probably is associated with the endoplasmic reticulum. The pH
optimum for the total reaction was between 7.5 and 8.0, and the best
temperature was 30°C. The apparent Km and
Vmax for
S-adenosyl-L-Met in the overall reaction were 74 and 250 µM, respectively.
| |
INTRODUCTION |
Diacylglyceryltrimethylhomo-Ser
[DGTS;
diacylglyceryl-O-4'-(N,N,N-trimethyl)
homo-Ser] is a primary membrane lipid of Chlamydomonas reinhardtii (Giroud et al., 1988 ). Although it is not a
phospholipid, DGTS appears to replace phosphatidylcholine (PtdCho) in
C. reinhardtii; PtdCho has been reported not to occur in
this organism (Giroud et al., 1988). This situation is not unusual,
however, because DGTS is widely distributed, occurring in
pteridophytes, bryophytes, and many algae (Sato, 1992;
Eichenberger, 1993; Dembitsky, 1996; Kato et al., 1996), where its
concentrations relative to PtdCho generally are inversely proportional.
DGTS substitutes for PtdCho more completely than simply as a membrane
component of these organisms because it has been demonstrated that
oleic acid esterified to DGTS might be desaturated to linoleic acid and
an isomer of linolenic acid in C. reinhardtii (Schlapfer and
Eichenberger, 1983; Giroud and Eichenberger, 1989). It has been
proposed, based on the fatty acid composition of DGTS, that this lipid
occurs and is synthesized in membranes outside the plastids
(Eichenberger, 1993). This combination of abundance, broad
distribution, and apparent substitution for PtdCho in lower plants and
algae indicates the importance of this lipid with respect to
understanding membrane lipid function and evolution.
Biosynthesis of the DGTS headgroup has been investigated by in vivo
feeding of radiolabeled methionine to C. reinhardtii (Sato, 1988), Marchantia
polymorpha (Sato and Kato, 1988), Ochromonas danica (Vogel and Eichenberger, 1992),
Cryptomonas sp. CR-1 (Sato and Murata, 1991), and
Rhodobacter sphaeroides (Hofmann and Eichenberger, 1997). In
all cases, Met served as an effective precursor for both the homo-Ser
moiety of the DGTS headgroup and the methyl units bonded to the amine
group of the homo-Ser. Although the methyl units are likely to be
contributed by S-adenosyl-L-Met (SAM) by analogy
with other lipid syntheses (Moore, 1976), neither these
contributions nor a possible role as the precursor to the homo-Ser moiety have been directly demonstrated. We describe here the
first in vitro assays for DGTS biosynthesis and provide evidence that
the form of Met utilized for the synthesis of both the homo-Ser and
methylation contributions is SAM.
| |
RESULTS |
Compartmentalization
Biosynthesis of DGTS was measured predominantly in two fractions
of C. reinhardtii homogenate following differential
centrifugation (Table I). The primary
activity was found in the lowest speed fraction where the plastids and
cell fragments are found. The second highest DGTS synthetic activity
was in the same high-speed microsomal fraction that contained the
majority of PtdCho synthesis, an activity demonstrated to be associated
with the endoplasmic reticulum in plant systems (Moore, 1976). The
microsomal fraction had the highest specific activity, and therefore
the greatest purity, and so was chosen as the enzyme source for most of
the remaining experiments.
View this table:
[in this window]
[in a new window]
|
Table I.
Sub-fractionation of organelles from Chlamydomonas
reinhardtii
Homogenization, organelle fractionation, and enzyme assays were as
described in "Material and Methods." Activities are presented as
percentage of total activity in all fractions. Cyt c, Cytochrome c;
PEP, phosphoenolpyruvate; ppt, precipitate; sup,
supernatant.
|
|
In an effort to further define the location of the activity in the
low-speed fraction, plastids were purified (Mason et al., 1991) and
fractionated (Douce and Joyard, 1980). No activity was found in either
the envelope or inner membrane fractions (data not included).
Therefore, it seems unlikely that the activity is associated with the
chloroplasts. Recentrifugation at 10,000g of
resuspended low-speed fraction resulted in virtually all the activity
appearing in the supernatant, and this activity in turn could be
precipitated by centrifugation at 100,000g.
This result indicates that the activity in the low-speed
precipitate is an artifact resulting from nonspecific trapping or
binding of microsomal membranes.
Source of Homo-Ser
The data presented in Table I provide support for the homo-Ser
moiety of DGTS being derived from SAM. The precursor used was SAM
radiolabeled in the carboxyl group, so the incorporated radioactivity
was not due to methylation. A more detailed examination of this
possibility is presented in Figure 1. In
Figure 1A, the distribution of products of
[14C-carboxyl]-SAM incorporation into the
microsomal fraction is presented. Two obvious peaks are present; the
lower peak cochromatographs with DGTS. The upper spot remains
unidentified because the concentration is low; however, the pulse-chase
data presented in Figure 1, B and C, allow the hypothesis that it is an
intermediate in DGTS synthesis, the radiolabel increasing in the lower
spot while decreasing in the upper during the chase period. The
occurrence of a shoulder above the lower spot following continuous
feeding suggests a second intermediate, but its appearance was variable
under the conditions used and so it probably turns over quickly.
Continuous feeding of SAM to the low-speed and microsomal fractions
resulted in relative proportions of radioactivity in the upper and
lower spots, expressed as both total and specific activities, which
were quite similar in both (Fig. 2),
further supporting them having the same intracellular origin.
View larger version (16K):
[in this window]
[in a new window]
|
Figure 1.
Precursor product relationship of two radiolabeled
spots from microsomes separated on thin-layer chromatography (TLC). A,
The radioactivity in products separated by one-dimensional TLC (see
below for details) after a continuous 1-h assay for DGTS synthesis. B,
The changes in radioactivity in the upper and lower products after the
onset of a chase period. C, The relative activity expressed in percent
remaining in each spot. The pulse-chase procedures involved presenting
microsomal fractions with 130 µM
[14C-carboxyl]- SAM (specific activity = 11 mCi/mmol) for 30 min, followed by the addition of 1.3 mM nonradioactive SAM.
Aliquots were taken at indicated intervals after the latter addition,
the lipids extracted as described in "Materials and Methods," and
the chloroform-soluble fraction was chromatographed on silica gel
G TLC plates using chloroform:methanol:7 N
NH4OH (65:35:4, v/v). Spots were visualized with
I2 vapors and those of interest were scraped from
the plates into scintillation vials for the determination of
radioactivity.
|
|
View larger version (17K):
[in this window]
[in a new window]
|
Figure 2.
Relative total and specific activities for
synthesis of each of the products described in Figure 1 by the active
fractions described in Table I. Note the parallel in relative rates of
synthesis in each of the fractions, thus further supporting a
precursor-product relationship between the upper and lower spots.
|
|
Because the addition of radiolabeled L-Met (Met) to intact
cells leads to incorporation of radioactivity into DGTS in C. reinhardtii (Sato, 1988), and Met might be a degradation
product of SAM, we tested for competition by Met with radiolabel
incorporation from SAM, and also for direct incorporation of Met into
the lipids under the conditions of our standard assay. Met was found to
inhibit the incorporation of radiolabeled SAM; however, the greatest
inhibition in our experiments was with 0.5 mM Met
in the presence of 0.13 mM SAM. The inhibition
under these conditions was about 38%. When radiolabeled Met was used
in place of SAM, no radioactivity was detected in the
chloroform-soluble fraction following the assay.
Substrates and General Assay Requirements
Synthesis of DGTS by the microsomal fraction demonstrates
saturation kinetics with SAM, with an apparent
Km of 74 µM and
Vmax of about 250 µM; SAM was absolutely required for the
synthesis of DGTS (Fig. 3A and
Inset).
View larger version (23K):
[in this window]
[in a new window]
|
Figure 3.
Effects of possible substrates on DGTS synthesis.
A, The effects of increasing SAM concentrations on DGTS synthesis.
Carboxyl-radiolabeled SAM was used and the radioactivity in the DGTS
spot determined after chromatography. The apparent
Km was estimated to be 74 µM and the Vmax to
be about 250 µM. B, Results on DGTS synthesis
by the microsomal fraction resulting from increasing concentrations of
a possible lipid precursor, diacylglycerol (DAG). Egg DAG, derived from
egg PtdCho, was added as a naturally occurring mixture of DAG species.
DAG-16:0, 18:1 is a possible natural precursor of DGTS synthesis.
|
|
The lipid substrate for this reaction remains elusive. Addition of
several molecular species of DAG, the lipid precursor for PtdCho
synthesis (Moore, 1976) and therefore a candidate for DGTS synthesis,
at all concentrations tested resulted in strong inhibition of the
reaction (Fig. 3B). Additions of CDP-DAG or phosphatidate did not
inhibit the reaction at concentrations tested, but also did not appear
to be incorporated into DGTS (data not shown). Further experiments with
possible lipid precursors, perhaps requiring at least partial
solubilization of the enzyme, will be necessary to elucidate the lipid substrate.
The pH optimum was found to be between 7.5 and 8.0 (Fig.
4A), and for temperature the maximum
activity was obtained at about 30°C (Fig. 4B). The addition of
S-adenosylhomo-Cys, an inhibitor of reactions utilizing SAM,
strongly inhibited the activity (Fig. 4C). This inhibition included not
only methylation, but also introduction of the homo-Ser headgroup. This
inhibition was evidenced by the absence of any radioactive peaks
when chloroform-soluble fractions of assays including 0.5 mM of S-adenosylhomo-Cys were
chromatographed.
View larger version (15K):
[in this window]
[in a new window]
|
Figure 4.
Effects of pH (A), temperature (B), and
S-adenosylhomo-Cys (C) on biosynthesis of DGTS by the
microsomal fraction. The inhibitor was added prior to the addition of
radiolabeled SAM. Other conditions were as described in "Materials
and Methods" except as indicated in the figure.
|
|
Methylation
Radiolabeled methyl units of SAM were incorporated into several
chloroform-soluble fractions obtained by two-dimensional chromatography (Fig. 5). One of the more strongly
labeled fractions was DGTS (I2-visualized spot E
in Fig. 5). The carboxyl-labeled precursor was more
specific, the 14C occurring only in DGTS and not
the other visualized spots. It is probable that the precursor
seen in Figure 1 did not occur at a sufficiently strong concentration
to be detected by the iodine vapors.
View larger version (39K):
[in this window]
[in a new window]
|
Figure 5.
Distributions of radiolabel in chloroform-soluble
products following introduction of either
[14C]carboxyl- or
[3H]methyl-labeled SAM. The assays were
performed as described in the materials and methods and the
chloroform-soluble material was chromatographed with the second
two-dimensional system described in "Materials and Methods." The
chloroform-soluble compounds were visualized with iodine vapors after
chromatography (top) and visible spots were scraped into scintillation
vials for determination of radioactivity (bottom).
|
|
CONCLUSIONS AND DISCUSSION
The data presented herein define the in vitro assay of DGTS
synthesis and indicate that SAM is the form that donates both the
homo-Ser and the methyl groups. Thus the role of Met in formation of
the entire trimethylhomo-Ser moiety, as indicated by the results from
the laboratories of Sato (Sato, 1988; Sato and Kato, 1988; Sato, 1992)
and Eichenberger (Vogel and Eichenberger, 1992; Hofmann and
Eichenberger, 1997) is confirmed and expanded to indicate the form of
Met utilized, which is SAM. It is not surprising that multiple products
in addition to DGTS were obtained with both the carboxyl- and
methyl-labeled precursors, and some of them appear to be short-lived
intermediates of DGTS synthesis. In particular, one specific compound
separated from DGTS by one-dimensional TLC (Fig. 1) appears to be a
precursor of DGTS synthesis based on pulse-chase kinetics. The
occurrence of tritium in this compound from methyl-labeled SAM (data
not shown) indicates that at least one methyl group already has been
incorporated, suggesting that the diacylglycerylhomo-Ser intermediate
is short lived. Although other candidates for roles of short-lived
intermediates of DGTS headgroup synthesis occasionally were obtained
upon two-dimensional TLC, no intermediate was found that would qualify
as being diacyglycerylhomo-Ser. After 1 h, most of the
radioactivity from [14C-carboxyl]-SAM occurred
in a spot on the chromatograms that corresponded to DGTS. Thus
methylation occurs rapidly after the homo-Ser addition. We expect that
at least two enzymes are involved in formation of the total headgroup,
one for homo-Ser addition and at least one for methylation, but we are
unable to estimate the total number of enzymes involved in the overall
pathway at this time.
The lipid substrate was anticipated to be DAG, based on its role in
PtdCho synthesis and the fact that DGTS appears to replace multiple
roles of PtdCho in C. reinhardtii (Vogel and
Eichenberger, 1992). However, our repeated efforts to measure
stimulation by various forms of DAG resulted only in inhibition of the
reaction. Such responses to putative lipid precursors in membrane
systems is not unusual and might arise from disruption of the membrane or other perturbations resulting in detrimental environments for the
enzymes (Moore, 1976). Neither phosphatidic acid nor CDP-DAG had any
consistent effect on the synthesis at the concentrations tested,
although they did not inhibit activity. Therefore, partial enzyme
purification might be necessary to elucidate the proper lipid substrate.
The reactions for complete synthesis of DGTS from SAM and a lipid
precursor occur in a microsomal fraction, and by analogy with
biosynthesis of membrane lipids in other systems (Kinney, 1993)
probably are associated with the endoplasmic reticulum. This
compartmentalization would be in agreement with the eukaryotic nature
of DGTS described by Giroud et al. (1988). The release of activity from
the low-speed centrifugation fraction by resuspension followed by
higher speed centrifugation, which packs the larger membranes
more tightly and apparently forces the smaller particles out of an
association with this fraction, results in the activity becoming microsomal.
In summary, the DGTS headgroup is synthesized utilizing SAM for both
the homo-Ser and methyl moieties. The activity is associated with the
microsomal fraction and does not occur in the plastids.
| |
MATERIALS AND METHODS |
Organism and Culture
Chlamydomonas reinhardtii, CC400 cw-15
mt+, a wall-deficient mutant, was originally from
the Duke University (Durham, NC) culture collection and obtained
by us from J. Moroney (Department of Biological Sciences, Louisiana
State University, Baton Rouge). Cells normally were grown in
liquid cultures in a phosphate-buffered medium containing CO2 as the carbon source (minimal medium; Sueoka, 1960).
The cultures were maintained by aeration with 5% (v/v)
CO2 in air with continuous reciprocal shaking, and
illumination was with 300 µE m 2 s1
of white fluorescent light.
Methods
Homogenization and Fractionation
The cells were collected by centrifugation at 2,000 to 3,000 rpm in a Beckman (Fullerton, CA) J2-21 centrifuge using a
JA-10 rotor, then washed with 20 mM of ice-cold HEPES
[4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] buffer (pH 7.5)
and concentrated with a final centrifugation. The concentrated cells
were rapidly diluted to a chlorophyll content of 0.3 mg/mL with a
solution comprised of 300 mM sorbitol, 50 mM
HEPES-KOH (pH 7.5), 2 mM EDTA (pH 7.5), 1 mM
MgCl2, and 1 mM dithiothreitol. The resulting
suspension was placed in a Parr pressure bomb and held at 1500 lbs/in2 pressure for 4 min, after which the contents were
released slowly into a container on ice (Mason et al., 1991).
The resultant homogenate was subjected to sequential centrifugation at
2,500 rpm (760g) for 10 min, 5,000 rpm
(3,000g) for 20 min, 9,500 rpm
(10,000g) for 30 min in a Beckman J2-21
centrifuge with a JA-20 rotor, and finally 37K rpm
(100,000g) for 1 h in a Sorvall
(Newtown, CT) OTD-65B ultracentrifuge with a T-875 rotor. The locations
of cellular components in the precipitates and final supernatant were
identified through the use of marker enzymes for the cytosol (PEP
carboxylase; Belknap and Togasaki, 1981), mitochondria (cytochrome
oxidase; Wigge and Gardestrom, 1987; Eriksson et al., 1995),
endoplasmic reticulum (DAG:CDPcholine cholinephosphotransferase; Moore,
1976), intact chloroplasts (Rubisco; Spreitzer et al., 1988), the
chloroplast envelope (monogalactosyldiacylglycerol synthase; Douce and
Joyard, 1980), and chloroplast thylakoids (chlorophyll; Mason et al.,
1991).
DGTS Assay
DGTS synthesis was routinely assayed with a mixture of 50 mM HEPES (pH 7.5), 5 mM EGTA (pH 7.5), and 130 µM [14C-carboxyl]-SAM in a final volume of
0.5 mL. The assay was conducted for 60 min at 30°C. The reaction was
terminated by the addition of 3.3 mL of chloroform:methanol:water
(10:20:3, v/v), incubated at room temperature for 30 min, and
the lipids partitioned into chloroform following the addition of 1.0 mL
of chloroform, three washes with 1 M KCl and a final wash
with water (based on Bligh and Dyer, 1959, as modified by Bjerve et
al., 1974). The chloroform fraction was either placed into
scintillation vials for direct measurement of radioactivity after
drying, or dried and chromatographed.
Following most assays, DGTS was separated from other radiolabeled
compounds in one dimension on silica gel 60 TLC plates (EM Separations
Technology, Darnstadt, Germany) using chloroform:methanol:7 N NH4OH (65:35:4, v/v). This provided a
relatively rapid and efficient separation of DGTS for measuring
radioactivity (see below).
Chromatography
Two two-dimensional TLC systems were used, both using
silica gel 60 TLC plates. The first solvent system (slightly
modified from Giroud et al., 1988) was: first dimension,
chloroform:methanol:7 N NH4OH (65:35:4, v/v)
and second dimension, chloroform:methanol:acetic acid:water
(170:25:25:2, v/v). The second solvent system (Vogel and Eichenberger,
1992) was: first dimension, chloroform:methanol:water (65:25:4, v/v)
and second dimension,
chloroform:methanol:isopropylamine:concentrated NH4OH (650:350:5:50, v/v).
Measurement of Radioactivity
Radioactivity was measured either after drying the total
chloroform extract in 3.0-mL scintillation vials or after scraping spots visualized with I2 vapors from TLC plates into the
vials. The scintillation cocktail was Cytoscint (ICN, Costa Mesa,
CA) and the scintillation counter was a Beckman
LS-8000.
| |
ACKNOWLEDGMENTS |
The authors would like to thank Dr. James Moroney and Cathy
Mason for valuable assistance during this work. They also would like to
thank Ashley Blouin and Candice Chenier for technical help and
discussions during the progress of the research.
| |
FOOTNOTES |
Received June 21, 2000; modified July 21, 2000; accepted September
18, 2000.
1
This work was supported by the National Science
Foundation (grant no. MCB-9603626 to T.S.M.).
*
Corresponding author; e-mail btmoor{at}lsu.edu; fax
225-388-2597.
| |
LITERATURE CITED |
-
Belknap W, Togasaki R
(1981)
Chlamydomonas reinhardtii cell preparation with altered permeability toward substrates of organellar reactions.
Proc Natl Acad Sci USA
778: 2310-2314
-
Bjerve K, Daae L, Bremer J
(1974)
The selective loss of lysophospholipids in some commonly used lipid-extraction procedures.
Anal Biochem
58: 238-245
[CrossRef][ISI][Medline]
-
Bligh E, Dyer W
(1959)
A rapid method of total lipid extractions and purification.
Can J Biochem Physiol
37: 911-917
-
Dembitsky V
(1996)
Betaine ether-linked glycerolipids: chemistry and biology.
Prog Lipid Res
35: 1-51
[CrossRef][ISI][Medline]
-
Douce R, Joyard J
(1980)
Chloroplast envelope lipids: detection and biosynthesis.
Methods Enzymol
69: 290-301
-
Eichenberger W
(1993)
Betaine lipids in lower plants: distribution of DGTS, DGTA and phospholipids, and the intracellular localization and site of biosynthesis of DGTS.
Plant Physiol Biochem
31: 213-221
-
Eriksson M, Gardestrom P, Samuelsson G
(1995)
Isolation, purification, and characterization of mitochondria from Chlamydomonas reinhardtii.
Plant Physiol
107: 479-483
[Abstract]
-
Giroud C, Eichenberger W
(1989)
Lipids of Chlamydomonas reinhardtii: incorporation of [14C]acetate, [14C]palmitate and [14C]oleate into different lipids and evidence for lipid-linked desaturation of fatty acids.
Plant Cell Physiol
30: 121-128
[Abstract/Free Full Text]
-
Giroud C, Gerber A, Eichenberger W
(1988)
Lipids of Chlamydomonas reinhardtii: analysis of molecular species and intracellular site(s) of biosynthesis.
Plant Cell Physiol
29: 587-595
[Abstract/Free Full Text]
-
Hofmann M, Eichenberger W
(1997)
Biosynthesis of diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) in Rhodobacter sphaeroides and evidence for lipid-linked N-methylation.
In
J Williams, M Khan, N Lem, eds, Physiology, Biochemistry, and Molecular Biology of Plant Lipids. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 116-118
-
Kato M, Sakai S, Adachi K, Ikemoto H, Sano H
(1996)
Distribution of betaine lipids in marine algae.
Phytochemistry
42: 1341-1345
[CrossRef]
-
Kinney A
(1993)
Phospholipid head groups.
In
T Moore, ed, Lipid Metabolism in Plants. CRC Press, Boca Raton, FL, pp 259-284
-
Mason C, Matthews S, Bricker T, Moroney J
(1991)
Simplified procedure for the isolation of intact chloroplasts from Chlamydomonas reinhardtii.
Plant Physiol
97: 1576-1580
[Abstract/Free Full Text]
-
Moore T
(1976)
Phosphatidylcholine synthesis in castor bean endosperm.
Plant Physiol
57: 383-386
-
Sato N
(1988)
Dual role of methionine in the biosynthesis of diacylglyceryltrimethylhomoserine in Chlamydomonas reinhardtii.
Plant Physiol
86: 931-934
[Abstract/Free Full Text]
-
Sato N
(1992)
Betaine lipids.
Bot Mag Tokyo
105: 185-197
[CrossRef]
-
Sato N, Kato K
(1988)
Analysis and biosynthesis of diacylglyceryl-N,N,N-trimethylhomoserine in the cells of Marchantia in suspension culture.
Plant Sci
55: 21-25
[CrossRef]
-
Sato N, Murata N
(1991)
Transition of lipid phase in aqueous dispersions of diacylglyceryltrimethylhomoserine.
Biochim Biophys Acta
1082: 108-111
[Medline]
-
Schlapfer P, Eichenberger W
(1983)
Evidence for the involvement of diacylglyceryl(N,N,N-trimethyl)-homoserine in the desaturation of oleic and linoleic acids in Chlamydomonas reinhardtii (Chlorophyceae).
Plant Sci
32: 243-252
[CrossRef]
-
Spreitzer R, Al-Abed S, Huether M
(1988)
Temperature sensitive photosynthesis-deficient mutants of Chlamydomonas reinhardtii.
Plant Physiol
86: 773-777
[Abstract/Free Full Text]
-
Sueoka N
(1960)
Mitotic replication of deoxyribonucleic acids in Chlamydomonas reinhardtii.
Proc Natl Acad Sci USA
46: 83-91
[Free Full Text]
-
Vogel G, Eichenberger W
(1992)
Betaine lipids in lower plants. Biosynthesis of DGTS and DGTA in Ochromonas danica (Chrysophyceae) and the possible role of DGTS in lipid metabolism.
Plant Cell Physiol
33: 427-436
[Abstract/Free Full Text]
-
Wigge B, Gardestrom P
(1987)
The effects of different ionic conditioning on the activity of cytochrome c oxidase in purified plant mitochondria.
In
A Moore, R Beachy, eds, Plant Mitochondria. Plenum Press, New York, pp 127-130
© 2001 American Society of Plant Physiologists
This article has been cited by other articles:
|
|
|
|
 
W. R. Riekhof, B. B. Sears, and C. Benning
Annotation of Genes Involved in Glycerolipid Biosynthesis in Chlamydomonas reinhardtii: Discovery of the Betaine Lipid Synthase BTA1Cr
Eukaryot. Cell,
February 1, 2005;
4(2):
242 - 252.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION