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Plant Physiol. (1985) 77, 296-299
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Characterization of Peroxisomes from the Alga Bumilleriopsis
fiiformis1
Received for publication August 17, 1984 and in revised form October 17, 1984
WOLFGANG GROSS, UWE WINKLER, AND HELMUT STABENAU*
Universitat Oldenburg, Fachbereich Biologie, Postfach 2503, D-2900 Oldenburg, West Germany
ABSTRACT
The Xa"thophycean alp Bumilkropsis filiformis possesses peroxisomes which on electron micrographs show a mostly spherical or ovoid
shape with a diameter in the range of 03 micrometer. Their granular
matrix is usually of moderate electron density and in a very few cases
contains amorphous inclusions. No associations with other organelles
could be observed.
During separation in a sucrose gradient, the peroxisomes from Bumilkeiopsis equilibrate at a density of 1.22 grams per cubic centimeter.
Glycolate oxidase and glyoxylate-glutamate aminotransferase were found
in the isolated organelles along with catalase and uricase. However, no
further leaf peroxisomal enzymes were detected. This is the first time
that an ala of the group of Xaathophyceae has been demonstrated to
possess a glycolate oxidase.
The organelles from Bumileriopsis differ from leaf peroxisomes also
by the absence of enzymes of the a-oxidation pathway. All enzymes for
the degradation of fatty acids which were tested are located solely in the
mitochondria.
Microbodies have been demonstrated by electron microscopy
to be present in many different algae (18), but only from a few
of those organisms could the microbodies be isolated for biochemical characterization. Nevertheless, it has already become
evident that there are different types of algal microbodies (7, 23).
In Spirogyra and Mougeotia the peroxisomes are of the leaf
type and are therefore involved in glycolate metabolism (22, 25,
30). On the other hand, the peroxisomes of the unicellular algae
Chlorogonium, Chlamydomonas, ,Eremosphaera, and Polytomella contain neither enzymes of the glycolate metabolism nor
enzymes of the glyoxylic acid cycle (6, 20, 21, 24, 27). Thus, they
have been regarded as unspecialized organelles.
Until now algal microbodies of the glyoxysomal type have
been isolated only from heterotrophically grown Euglena (1 1).
Under autotrophic conditions, the corresponding organelles of
this alga like leaf peroxisomes contain enzymes of glycolate
metabolism (5). But these microbodies differ from leaf peroxisomes in that they contain a glycolate dehydrogenase rather than
a glycolate oxidase (4, 16). Furthermore, there are also contrary
reports whether catalase is a constituent of the organelles in
Euglena (3, 14, 29).
Recently it was reported that the unspecialized peroxisomes
from Eremosphaera and the leaftype organelles from Mougeotia
both contain enzymes of the #-oxidation pathway (28). Considering also the data for higher plants (8, 9), it appeared that the
possession of enzymes for the a-oxidation is a characteristic of
' Supported by the Deutsche Forschungsgemeinschaft.
all different types of microbodies. This assumption now seems
to be questionable since we have isolated peroxisomes from the
Xanthophycean alga Bumilleriopsis which could not be demonstrated to contain enzymes for degradation of fatty acids.
MATERIALS AND METHODS
Algal Material and Growth Conditions. Bumilleriopsisfiliformis (Xanthophyceae), strain 802-2, was obtained from the algae
collection of the Institute for Plant Physiology, University of
Gottingen, W. Germany.
Cultures were grown autotrophically in glass tubes at 23YC in
continuous light of either 3,000 or 20,000 lux in nutrient medium
(12). The cultures were aerated with air plus 2% CO2 or air only.
Preparation of Cell Homogenates and Separation of Organelles. Cells of 1 L of suspension were collected by low speed
centrifugation and washed with grinding medium (28). For homogenization, the cells were transferred to 20 ml of the same
medium containing 100 mg Polyclar AT, 20 mg cystein, 15 mg
DTT, 70 mg BSA, and 15% (w/w) sucrose. Cells were broken in
a Virtis homogenizer after adding 30 ml glass beads; the homogenate was centrifuged at 5OOg for 10 min. The resulting supernatant was used for sucrose density centrifugation (28).
Assays. All assays for measuring the activities ofenzymes were
carried out at 25°C in 1 ml total volume. Spectrophotometric
measurements were made on a Uvikon 810 photometer.
Glycolate oxidase activity was measured by the reaction of
glyoxylate with phenylhydrazine and by 02 consumption assayed
with a Rank electrode (26). The H202 formed by the glycolate
oxidase was measured by the peroxidase-aminoantipyrine reaction (13). The activity of catalase in the test medium was inhibited by addition of 0.1 mM NaN3. All other enzymes were
measured as already described: catalase, Cyt c oxidase, hydroxypyruvate reductase, and malate dehydrogenase (21); uricase (24);
acyl-CoA dehydrogenase, enoyl-CoA hydratase, and hydroxyacyl-CoA dehydrogenase (28); acyl-CoA oxidase (8); glutamateglyoxylate aminotransferase and serinq-glyoxylate aminotransferase (30). The amino acids formed during the aminotransferase
reactions were determined by HPLC analysis (10).
The protein was measured by the Folin reagent method (15).
The content of Chl was measured as described by B6ger (2).
Electron Microscopy. Whole cells were fixed with 4% glutaraldehyde in K-phosphate, 0.05 M (pH 8) for 1.5 h at room
temperature. Then the cells were collected by low speed centrifugation and fixed for 1 h at room temperature in 2% osmium
tetraoxide.
Pooled peroxisomal fractions from sucrose gradients were fixed
with 4% glutaraldehyde (flnal concentration) for 1 h at 4°C (1).
The glutaraldehyde solution used was prepared in 45% (w/w)
sucrose. Then the suspension was diluted with an equal volume
of 40% (w/w) sucrose in gradient buffer and the organelles were
pelleted by centrifugation for 45 min at 40,000g. After washing
with the same medium the pellet was fixed for 1 h in 2.0%
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Plant Biologists. All rights reserved.
297
MICROBODIES FROM BUMILLERIOPSIS
osmium tetraoxide in K-phosphate, 0.05 M (pH 8) containing
40% (w/w) sucrose.
Fixed organelles or whole cells were dehydrated in a graded
ethanol-water series and in 100% acetone. The material was
embedded using a three-step infiltration in low viscosity resin
(19). Sections were cut and stained with lead citrate (17). The
sections then were examined in a ZEISS EM 109 electron microscope.
Chemicals. All chemicals for electron microscopy and the
Polyclar AT were obtained from Serva (Heidelberg, W. Germany). Glass beads (diameter: 0.25-0.30 mm) were purchased
from Braun (Melsungen, W. Germany). All other chemicals were
provided by Sigma (St. Louis).
-
c
0
0.
E
io
RESULTS
Organelles from the -crude homogenate of autotrophically
grown cells were separated in a linear sucrose gradient. As
indicated by the distribution of Chl and Cyt oxidase, chloroplasts
and mitochondria were clearly separated equilibrating at densities of 1.18 and 1.20 g. cm-3, respectively (Fig. la, b). Since the
fraction with the density of 1.20 g- cm-3 contains a relatively
high activity of malate dehydrogenase, most of the mitochondria
are believed to be unbroken.
Microbodies in the gradient were identified by electron microscopy as well as by the distribution of catalase and uricase.
Both enzymes show a main peak at a density of 1.22 g cm3 (Fig.
lc). The smaller peak at a density of 1.16 g- cm-3 may possibly
be due to some microbodies trapped in the chloroplast fraction.
About 70% of the total activities of catalase and uricase were
found in the particulate fraction. Therefore, most of the microbodies were not disrupted during homogenization and gradient
centrifugation. No peak of protein was seen in the microbody
fraction (density 1.22 g cm-3, Fig. la), indicating that the numUrote oxidase
30 ber of microbodies is relatively low.
The microbodies from Bumilleriopsis contain a glycolate oxidizing enzyme capable of transferring electrons directly to oxygen. As shown in Table I, during this reaction, the oxygen is
N
reduced to H202 as in higher plants. Similar to the corresponding
C
w
oxidase from Mougeotia (26), the enzyme shows some activity
with L-lactate but not with 1-lactate as the substrates (Table I).
10
Using glycolate, a 20-fold higher activity was measured compared
with L-lactate, and under this condition the Km, value determined
by a Lineweaver-Burk plot was found to be 250 gM.
In addition to glycolate oxidase, the microbodies from Buma Hydnocyl-CoA DH
illeriopsis contain a glyoxylate-glutamate aminotransferase (Fig.
o Enoyl.CoA hydr.
lc). However, no aminotransferase capable of converting seine
o Acyl.CoA DH(d
to hydroxypyruvate was found in these organelles. For the latter
X.10I
Desty(203
2012
test, either glyoxylate, a-ketoglutarate, or pyruvate were used as
the amino acceptors. No activity of hydroxypyruvate reductase
was detected at a density of 1.22 g- cm-3 or in any other fraction
of the gradient, using either NADH or NADPH.
To demonstrate the presence of the fl-oxidation pathway in
Bumilleriopsis the enzymes enoyl-CoA hydratase, hydroxyacylCoA dehydrogenase, acyl-CoA dehydrogenase, and acyl-CoA
oxidase were assayed. No activity of acyl-CoA oxidase could be
found in the fractions of the gradient measuring either the
palmitoyl-CoA dependent 02 consumption or H202 production.
The activity of the acyl-CoA dehydrogenase was relatively low
Density (glcm3)
1.20 1.22
as compared to the other two enzymes.
FIG. 1. Separation of organelles from Bumilleriopsis in a linear suAll three enzymes of the fl-oxidation demonstrated to be crose
gradient. Units of enzymes per ml fraction per min: catalase, Mmol
present in Bumilleriopsis were found to be constituents of the substrate;
urate oxidase, nmol substrate (ordinate values [ord.v.] x 0.07
mitochondria but not of the microbodies from Bumilleriopsis = actual values
[act. v.j); glutamate-glyoxylate aminotransferase, nmol
(Fig. ld).
substrate (ord.v. x 2.5 = act.v.); glycolate oxidase, nmol substrate (ord.v.
x 0.45 = act.v.); malate dehydrogenase, 0.1 mol substrate; Cyt oxidase,
DISCUSSION
nmol substrate (ord.v. x 5 = act.v.); enoyl-CoA hydratase, 0.01 grmol;
The microbodies from Bumilleriopsis are spheroid or ovoid in hydroxyacyl-CoA dehydrogenase, nmol substrate (ord.v. x 7.5 = act.v.);
shape in most cases and their average diameter was determined acyl-CoA dehydrogenase, nmol substrate.
d
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Plant Physiol. Vol. 77, 1985
GROSS ET AL.
298
Table I. Activity of Glycolate Oxidasefrom Bumilleriopsis
The tests were performed with 50 mm lactate or glycolate. The enzyme
was partially purified by precipitation with (NH4kSO4 (50-55% of satu-
ration).
D-Lactate
L-Lactate
Product Formed or 02 Consumed
nmol/mg protein. min
0 Pyruvate
6.3 Pyruvate
Glycolate
Glycolate
Glycolate
114.2 Glyoxylate
109.5 H202
106.5 02
Substrate
A,
Fijito be 0.3 ;tm (Fig. 2a). They sometimes appear somewhat elongated. The granular matrix has a higher density than that of the
surrounding cytoplasm and contains amorphous inclusions in
only a very few cases (Fig. 2d). No associations with other
organelles could be observed as in other algae (1 8).
When centrifuged to equilibrium in a sucrose density gradient,
the microbodies from Bumilleriopsis were recovered at a density
of 1.22 g- cm-3. On electron micrographs of the fraction 1.22
most of them appeared undamaged (Fig. 2, b-d). As demonb
strated in Figure lc, the isolated organelles contain catalase and
two oxidases forming the substrate for the catalase reaction.
The glycolate oxidase is similar to the enzyme found in the
microbodies from the Chlorophycean alga Mougeotia in that it
is able to oxidize L-lactate to a small extent. The K,,, value (250
i iM) is somewhat lower than that of the enzyme from Mougeotia
(415 jiM) (26) and almost the same as for the enzyme in leaf
peroxisomes of higher plants (7). Uricase, the other oxidase in
the peroxisomes from Bumilleriopsis, has been demonstrated to
be present also in the microbodies of some unicellular algae (6,
7, 24), but not in the organelles from Mougeotia.
Beside glycolate oxidase, the peroxisomes from Bumilleriopsis
contain a glutamate-glyoxylate aminotransferase, and in that
respect they are similar to leaf peroxisomes. However, no other
leaf peroxisomal enzymes could be detected. No enzyme activity
was found for formation of hydroxypyruvate from serine using
different a-keto acids as the amino acceptors. In agreement with
this, no activity of hydroxypyruvate reductase could be measured
with either NADH or NADPH as the coenzyme. From these
results it may be concluded that the physiological role of the
peroxisomes in Bumilleriopsis is not the same as that of leaf
s,
ukilia
d
FIG. 2. Electron micrographs of microbodies from Bumilleriopsis. a,
in situ (x 35,000); b and c, isolated organelles in the fraction with a
density of 1.22 g-cm-3 (x 35,000); d, isolated organelles, one with an
amorphous inclusion (x 70,000). Chl, chloroplast; M, mitochondrion;
Mb, microbody; N, nucleus.
peroxisomes.
The peroxisomes from Bumilleriopsis are different from the
leaf type organelles of Mougeotia not only by the absence of
hydroxypyruvate reductase and a serine transforming aminotransferase but also by the absence of enzymes for degradation
of fatty acids. In Mougeotia and Eremosphaera, three enzymes
of the ,8-oxidation were found to be constituents of the microbodies and partially of the mitochondria, too (28). However, in
Bumilleriopsis these enzymes are located only in the mitochondria. Therefore, in contrast to our former assumption (28), we
have to conclude that the capability for degradation of fatty acids
is not a common feature of all types of microbodies.
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MICROBODIES FROM BUMILLERIOPSIS
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