Plant Physiol. (1989) 91, 1393-1401
0032-0889/89/91/1 393/09/$01 .00/0
Received for publication April 18, 1989
and in revised form July 7, 1989
Structural Similarities between Spinach Chloroplast and
Cytosolic Class I Fructose 1,6-Bisphosphate Aldolases'
Immunochemical and Amino-Terminal Amino Acid Sequence Analysis
James J. Marsh, Kenneth J. Wilson, and Herbert G. Lebherz*
Department of Chemistry, San Diego State University, San Diego, California 92182 (J.J.M., H.G.L.), and Applied
Biosystems Inc., 850 Lincoln Center Drive, Foster City, California 94404 (K.J.W.)
ABSTRACT
structural features of a number of these plant isoenzymes have
been recently reviewed (21).
We have been using the glycolytic enzyme fructose bisphosphate aldolase as a model for studies on protein evolution. Early studies by Anderson indicate that although the
two forms of pea leaf aldolase differ somewhat in charge (4)
and amino acid composition (1), they possess very similar
catalytic (4) and structural (2) properties. Results of genetic
experiments have shown that the pea chloroplast aldolase is
inherited in a Mendelian (nonmaternal) fashion (3, 28), suggesting that this form of aldolase is coded for by a nuclear
gene. Previous work by Kruger and Schnarrenberger (12, 22)
and ourselves (14) showed that the cytosolic and chloroplast
forms of spinach leaf aldolase exhibit similar specific catalytic
activities and appear to possess functional carboxy-terminal
structures. However, they are distinctive on the basis of
charge, subunit molecular weight, thermal stability, amino
acid composition, tryptic peptide patterns and immunological
properties. Hence, the two spinach leaf enzymes are presumably the products of different structural genes.
All aldolases are further categorized as being either class I
or class II enzymes depending on the mechanism they utilize
for the cleavage of fructose bisphosphate to triose phosphates
(19), and the two classes of fructose bisphosphate aldolase are
known to have a somewhat restricted phylogenetic distribution (19). For example, all fructose bisphosphate aldolases of
higher eukaryotes, including those of plant chloroplasts, are
class I (Schiff's base) enzymes while, most procaryotic cells
contain class II (metallo) aldolases. More recently, it has been
demonstrated that some procaryotic organisms express a class
I aldolase (6, 9), which is often induced under nutritional
conditions that require the synthesis of hexoses via the gluconeogenic pathway.
The present studies were undertaken to better clarify the
structural relationships which may exist between the spinach
(Spinacia oleracea) chloroplast aldolase and the procaryotic
and eucaryotic forms of class I fructose bisphosphate aldolase.
In this report, we present the results of immunological experiments, NH2-terminal amino acid sequence analysis and comparisons of the amino acid compositions of procaryotic and
eucaryotic aldolases. Our findings suggest that the chloroplast
aldolase is structurally much more similar to the class I
eucaryotic aldolases than it is to the procaryotic class I en-
Immunochemical studies using polyclonal antisera prepared
individually against highly purified cytosolic and chloroplast spinach leaf (Spinacia oleracea) fructose bisphosphate aldolases
showed significant cross reaction between both forms of spinach
aldolase and their heterologous antisera. The individual cross
reactions were estimated to be approximately 50% in both cases
under conditions of antibody saturation using a highly sensitive
enzyme-linked immunosorbent assay. In contrast, the class I
procaryotic aldolase from Mycobacterium smegmatis and the
class 11 aldolase from yeast (Saccharomyces cerevisiae) did not
cross-react with either type of antiserum. The 29 residue long
amino-terminal amino acid sequences of the procaryotic M. smegmatis and the spinach chloroplast aldolases were determined.
Comparisons of these sequences with those of other aldolases
showed that the amino-terminal primary structure of the chloroplast aldolase is much more similar to the amino-terminal structures of class I cytosolic eucaryotic aldolases than it is to the
corresponding region of the M. smegmatis enzyme, especially in
that region which forms the first "beta sheet" in the secondary
structure of the eucaryotic aldolases. Moreover, results of a
systematic comparison of the amino acid compositions of a
number of diverse eucaryotic and procaryotic fructose bisphosphate aldolases further suggest that the chloroplast aldolase
belongs to the eucaryotic rather than the procaryotic "family" of
class I aldolases.
One of the most striking distinctions between eucaryotic
and procaryotic life forms is the complexity of their intracellular architectures. In contrast to procaryotes, most eucaryotic
cells contain specialized membrane-limited intracellular organelles in which specific biochemical pathways are compartmentalized. For example, reactions of the Krebs cycle and
those of the oxidative phosphorylation pathway occur in
mitochondria while the "light" and "dark" reactions of photosynthesis take place within chloroplast organelles of photosynthetic eucaryotes. Many glycolytic enzymes in the leaves
of higher plants exist as enzyme "pairs," one member of the
pair residing in the cytosol and the other member sequestered
within the chloroplast organelle (8, 26). The regulation and
'This work was supported, in part, by Research Grant GM 23045
from the National Institutes of Health.
1393
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MARSH ET AL.
1 394
zymes. The implications of these findings concerning the
probable evolutionary origin of the chloroplast aldolase are
discussed.
MATERIALS AND METHODS
Materials
Mycobacterium smegmatis (CDC 46) was grown on Youmans and Carlson medium as previously described (9) using
glycerol as the primary carbon source. Bacteria were harvested
by centrifugation at I0,000g, washed twice with ice-cold
water, and then were stored frozen at -20°C until used. Cubes
of Fleishmann's cake yeast (Saccharomyces cerevisiae) and
fresh spinach leaves (Spinacia oleracea) were purchased locally. The Bradford Protein Assay reagent was obtained from
Pierce Chemical Co. (Rockford, IL). Nitrocellulose paper, the
protein A-horseradish peroxidase conjugate and the peroxidase activity color development reagent (4-chloro-1-napthol)
were purchased from Bio-Rad (Richmond, CA). Polystyrene
assay plates (Falcon 3912) used for quantitative ELISA were
obtained from Becton Dickinson Labware Co. (Oxnard, CA).
All biochemicals, other than the aldolases, were purchased
from Sigma Chemical Co. Other chemicals used were of
reagent grade.
Purification of Fructose Bisphosphate Aldolases
The cytosolic and chloroplast forms of spinach leaf aldolase
were purified from fresh leaf tissue as previously described
(14). Purified preparations of both types of spinach aldolase
were judged to be at least 95% pure as determined by electrophoretic analysis of enzyme preparations in SDS polyacrylamide slab gels followed by densitometric analysis of gels
stained for protein with Coomassie blue.
The M. smegmatis class I aldolase was purified 150-fold
from 100 g of bacteria by the method of Jayanthi Bai et al.
(9), with the following modifications. (a) Instead of using
sonication, cells were mechanically disrupted at 4°C with glass
beads in a Bead-Beater apparatus (Biospec Products, Bartlesville, OK) according to the manufacturer's instructions using
three, 1 min pulses. (b) Two, instead of one, DEAE-cellulose
chromatography steps were performed, the first on a Whatman DE-52 cellulose column (2 x 20 cm) equilibrated in 50
mM Tris-HCl, 1 mm 2-mercaptoethanol, 0.5 mm EDTA (pH
8.0). The column was developed with a 0 to 0.5 M NaCl
gradient (400 ml total). The second DEAE step was performed
on a 1.4 x 16 cm DE-52 column equilibrated in 10 mM Mes,
1 mm 2-mercaptoethanol, 0.5 mM EDTA (pH 6.0) and this
column was developed with a 0 to 0.3 M NaCl gradient (200
mL total). (c) We omitted the gel filtration step of the original
procedure. The M. smegmatis aldolase preparation obtained
in this fashion contained a major 39 kD protein (presumed
to be the aldolase subunit) plus several minor lower molecular
mass (<35 kD) polypeptides as revealed by electrophoretic
analysis. We positively identified the major 39 kD protein as
the fructose bisphosphate aldolase subunit in the following
way. An aliquot of the M. smegmatis aldolase preparation
was subjected to cellulose polyacetate strip electrophoresis
(see ref. 14) and briefly (<5 min) stained for aldolase activity
Plant Physiol. Vol. 91, 1989
in order to visualize the position of the aldolase band. This
band (approximately 2 mm wide) was carefully excised from
each of four identical strips and placed in a minimal volume
of SDS sample buffer. After soaking for 2 h, the entire mixture
was heated at 100°C for 2 min and the resulting solution
subjected to SDS gel electrophoresis. Only the 39 kD protein
was present following this separation.
The class II (metallo) aldolase derived from yeast was
purified about 10-fold by ammonium sulfate fractionation
using a procedure similar to that originally described by Rutter
and co-workers (20). Yeast cakes were suspended in 50 mM
2-mercaptoethanol, 0.1 mm ZnSO4, 0.2 mM PMSF, and the
cells were harvested by centrifugation at 10,000g for 10 min.
After washing the cells two more times, the final cell paste
was resuspended in two volumes of the same solution and the
cells were disrupted in the "Bead-Beater" apparatus as above.
After centrifugation at 1 5,000g for 20 min to remove particulate material, the supernate was adjusted to 80% ammonium
sulfate saturation by the slow addition of the solid salt. After
stirring for 1 h, precipitated proteins were collected by centrifugation and were discarded. The supernate was then adjusted to 95% saturation by addition of solid ammonium
sulfate and, after stirring, this aldolase-enriched protein fraction was collected by centrifugation. The class II yeast aldolase
represented about 50% of the total protein in this fraction as
judged by electrophoretic analysis.
All aldolase preparations were stored at 4°C as precipitates
in 95% saturated ammonium sulfate prepared in 10 mM TrisHCI and 1 mM 2-mercaptoethanol (pH 7.6) until used.
Electrophoretic Procedures and Protein Determinations
Crude extracts and purified aldolase preparations were analyzed by SDS-PAGE in 9% polyacrylamide slab gels using the
reagents and buffer system suggested by Laemmli (see ref. 14).
Gels were fixed in 10% (v/v) acetic acid, 45% (v/v) methanol
and were then stained for protein with 0.2% (w/v) Coomassie
blue R250 prepared in fixing solution. Gels were destained in
fixing solution and densitometric analysis of stained gels was
performed using the Zehneih "soft-laser" scanner. Protein
determinations were performed by the method of Bradford
(see ref. 14), using BSA as the standard.
Immunological Methods
Mono-specific antisera were raised in rabbits against pure
preparations of the chloroplast and cytosolic spinach leaf
aldolases using native protein emulsified in Freund's adjuvant
by the procedure recently described by us (14). More than
one rabbit was used for production of each type of antiserum
and, after initial screening for specificity, antisera raised
against each type of spinach aldolase were pooled separately
and stored frozen in small aliquots.
The abilities of these antibody preparations to recognize
aldolases from various sources was assessed on a qualitative
basis by probing the enzymes, which were previously bound
to nitrocellulose paper, using the procedure recommended by
Bio-Rad Laboratories. Antigen-antibody complexes were visualized using the protein A-horseradish peroxidase conjugate
followed by histochemical staining of the paper using the
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SIMILARITIES BETWEEN CHLOROPLAST AND CYTOSOLIC ALDOLASE
method and peroxidase color development reagent supplied
by Bio-Rad.
Quantitative estimates of the degrees of immunological
recognition of various aldolases by antisera directed against
the two spinach aldolases were determined by quantitative
ELISA as follows: Samples of aldolase preparations were
diluted to 50 ng aldolase/ml in 50 mm sodium bicarbonate
(pH 9.6) and these samples were incubated in wells (200 ,uL/
well) of microtiter plates overnight at 25°C. After washing to
remove any unbound aldolase, remaining protein binding
sites in the wells were saturated by blocking with a 0.1 % (w/
v) solution of BSA prepared in the same bicarbonate buffer;
after the blocking step, wells of microtiter plates were washed
with "antibody binding" buffer (20 mM NaPO4,150 mM NaCl
[pH 7.4] which also contained freshly added Tween-20 and
BSA, final concentrations, 0.05% [v/v] and 0.1% [w/v], respectively). Increasing concentrations ofantisera (in triplicate)
were then added to these wells and, after 2.5 h, unbound
protein was removed from the wells by washing with buffer.
Finally, the relative amounts of rabbit immunoglobulin G
bound to each well was determined by incubating the wells
for 1 h with a 1:5000 dilution of the protein A-horseradish
peroxidase conjugate and, after washing, each well was treated
with 250 uL of peroxidase substrate solution (50 mM
Na2HPO4, 25 mm citric acid [pH 5.0] containing 5 mM 0phenylenediamine and 1 mM H202, both added immediately
before use). The reaction was monitored by measuring the
absorbance of each well at 490 nm 20 min after the addition
of substrate in an automatic Bio-Tek Model EL-3 10 ELISA
reader. In all cases, a linear increase in absorbance was observed throughout the 20 min incubation period. A control
consisting of all reagents except antigen was performed at
each antibody dilution to assess, and correct for, any nonspecific binding of immunoglobulin G to the wells.
Amino Acid Sequence Analysis
NH2-terminal amino acid sequence analysis was performed
on SDS polyacrylamide gel electroblotted samples of chloroplast and M. smegmatis aldolase according to the method of
Matsudaira (17) using a model 477A gas-phase sequencer
with an on-line model 120A PTH analyzer (Applied Biosystems, Foster, City, CA). Chemicals from the same source were
used in each instrument. Data reduction/quantitation was
carried out using the software developed for sequence calling
by the same company.
See the legends of specific figures or tables for any additional
methods or procedures.
RESULTS
Immunological Cross-Reactivity Exhibited by the
Cytosolic and Chloroplast Forms of Spinach Leaf
Aldolase
Results of an electrophoretic analysis of the chloroplast and
cytosolic aldolase preparations used in this study are shown
in Figure 1. The subunit molecular masses were estimated to
be 38 kD and 40 kD, respectively, as previously described
(14). The two forms of aldolase did not cross-react in Ouch-
1 395
1 2
38kD- _
m -4OkD
Figure 1. Electrophoretic analysis of spinach leaf aldolases. Purified
preparations of spinach chloroplast (4.8 jig, lane 1) and cytosolic (6.3
jig, lane 2) aldolases were electrophoresed on an SDS-polyacrylamide
slab gel and stained for protein with Coomassie blue as described in
"Materials and Methods." Subunit molecular masses were determined
previously describedi (1 4).
as
terlony double-diffusion tests (14), a procedure which, however, only detects insoluble (precipitating) immune complexes. In contrast, some cross-reactivity between the two
spinach aldolases was reported by Kruger and Schnarrenberger (12) using a more sensitive immunological technique.
Consequently, we investigated in more detail the immunological relationships which may exist between the two spinach
leaf aldolases and included the procaryotic class I aldolase
derived from M. smegmatis and the prototypic class II yeast
aldolase in these analyses for comparative purposes. We chose
the M. smegmatis aldolase since, unlike other class I procaryotic aldolases, the M. smegmatis enzyme is similar to class I
eucaryotic aldolases in terms of its subunit molecular mass
(about 40 kD) and its tetrameric structure (9).
As shown in Figure 2, both spinach leaf aldolases tested
positive in qualitative "dot blot" experiments when probed
with antisera raised against pure preparations of the chloroplast and cytosolic aldolases. In contrast, neither type of
antiserum recognized the class I procaryotic aldolase, nor the
structurally and catalytically distinct class II yeast aldolase.
Similar results were obtained when several different, independently generated, antisera were used in dot blot analysis
(data not shown).
More quantitative estimates of the degrees of immunological cross-reactivity exhibited by the two spinach aldolases
were obtained using a quantitative ELISA. In contrast to
Ouchterlony analysis, this technique (as well as the dot blot
procedure) will detect all types of immune complexes, including nonprecipitating ones. Equal amounts of each spinach
aldolase, as well as the procaryotic class I enzyme and the
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1396
MARSH ET AL.
1
2
3
4
Figure 2. Qualitative "dot blot" analysis of various aldolases probed
with antisera raised against the cytosolic and chloroplast spinach leaf
aldolases. Samples (about 2 gsg) of spinach chloroplast (lane 1),
spinach cytosolic (lane 2), M. smegmatis (lane 3), and yeast (lane 4)
aldolases were spotted onto nitrocellulose paper and, after the blocking reaction, each section of the nitrocellulose paper containing an
individual aldolase was incubated independently in 16 mm plastic
wells with 0.5 ml of a 1:50 dilution of either antichloroplast (row A) or
anticytosolic (row B) antisera. Finally, the blots were developed as
described under "Materials and Methods."
class II yeast aldolase, were incubated with increasing levels
of each antiserum and the relative amounts of antigen-antibody complexes formed were quantitated using the protein
A-horseradish peroxidase conjugate system as described under
"Materials and Methods." Typical immunotitration curves
obtained in these experiments are presented in Figure 3. As
shown, the curves obtained with both spinach aldolases, titrated with each type of antiserum, were hyperbolic and
reached saturation at high levels of antisera. As expected,
neither type of antiserum was able to bind to microtiter wells
containing either the class I procaryotic aldolase or the class
II yeast enzyme. Double-reciprocal plots of the immunotitration data (Fig. 4) were then constructed to calculate the
theoretical maximum degrees of immunological cross-reactivity expressed by the two spinach aldolases. Comparisons of
the "y-intercepts" in these double-reciprocal plots compiled
from a number of independent immunotitration experiments
(Table I) showed that about 60% of the antibody populations
raised against the chloroplast aldolase cross-reacted with the
cytosolic enzyme and that a similar percentage (about 50%)
of the antibody populations raised against the cytosolic aldolase cross-reacted with the chloroplast enzyme. Thus, the two
antisera exhibit the immunological phenomenon known as
reciprocity (25), a property which is taken as evidence that
the measured cross-reactivity is actually due to the presence
of homologous antibody binding sites on the cross-reacting
proteins.
Finally, we ruled out the unlikely possibility that the observed immunological cross-reactivity exhibited by the two
spinach aldolases could have been due to "cross-contamination" of antibody preparations resulting, for instance, by the
immunization of rabbits with impure preparations of the
spinach aldolases. This was done in "competition" experiments in which increasing amounts of the two pure aldolases
were preincubated with a constant amount of their respective
homologous antisera and then, these preincubated samples
were tested for the presence of "free antibody" in our standard
ELISA system. Results obtained in one of these control experiments are presented in Figure 5. When antiserum directed
Plant Physiol. Vol. 91, 1989
against the cytosolic aldolase was incubated with increasing
amounts of pure cytosolic aldolase, the level of free antibody
that could subsequently bind to either aldolase adsorbed onto
microtiter wells was proportionately decreased. If specific
antibody to the chiloroplast aldolase was present in this anticytosolic antiserum, then preincubation ofthe antiserum with
cytosolic aldolase would have had no effect on the ability of
the "chloroplast-specific" antibody (if present) to subsequently bind to the chloroplast aldolase on the microtiter
plate, and no competition would have been observed. Thus,
the cytosolic aldolase is capable of "neutralizing" antibody
populations in the anticytosolic antiserum that recognize both
the cytosolic and chloroplast forms of aldolase in a similar
dose-dependent manner. Similar results were obtained in
competition experiments in which antiserum raised against
the chloroplast enzyme was preincubated with increasing
amounts of pure chloroplast aldolase (data not shown). Results of these competition experiments show that the observed
immunological cross-reactivity is due to the presence of a
homologous antigenic determinant(s) on both the chloroplast
and cytosolic aldolases.
Structural Similarities between the Cytosolic and
Chloroplast Forms of Spinach Leaf Aldolase
It is known that some proteins can cross-react immunologically, even though they may be quite different in terms of
their primary structures (11). Consequently, we wanted to
obtain structural data to supplement results of our comparative immunological analysis. We employed micro-amino acid
sequencing techniques to determine the NH2-terminal sequences of the class I chloroplast and procaryotic M. smegmatis aldolases (a total of 29 residues each). Amino terminal
sequence analysis of the cytosolic spinach aldolase was not
feasible because, like other class I eucaryotic aldolases, it
apparently has a "blocked" NH2 terminus (14). We then
compared the NH2-terminal sequences of the chloroplast and
M. smegmatis aldolases with the full length sequences of
eucaryotic class I aldolases obtained by a computer assisted
search of the National Biomedical Research Foundation protein sequence databases; at present, there is no sequence
information available on any class I procaryotic fructose
bisphosphate aldolase in these databases. Sequence alignments of the various aldolases compared were performed
using the Pearson Fast-P program (15). In all cases, optimal
alignment of the chloroplast and M. smegmatis aldolase sequences occurred within the NH2-terminal regions of the
aldolase sequences extracted from the database (Fig. 6). It is
apparent that the NH2-terminal sequence of the chloroplast
aldolase is much more similar to sequences near the NH2
terminus of the eucaryotic cytosolic aldolases (52% identity
to the maize cytosolic aldolase) than it is to the NH2-terminal
sequence of the M. smegmatis aldolase ( 13% identity). Particularily striking was the observation that the chloroplast aldolase contains a 10 amino acid long sequence which is very
highly conserved in the class I cytosolic eucaryotic aldolases
(see boxed region, Fig. 6). Only two differences were observed
in this region; namely a functionally conservative lysine/
arginine difference at position 19 and an alanine/methionine
substitution at position 24 of the chloroplast aldolase se-
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SIMILARITIES BETWEEN CHLOROPLAST AND CYTOSOLIC ALDOLASE
1 397
7
=2.0
0
0
CYT
0
-11.0
0
0
100
200
300
RELATIVE [Ab]
ANTI CYTOSOLIC
YEAST/ M. smeg
400
500
-
CYT
2.0
0~~~~
CHL
°21
2.0 p
O
0~~~~
M.smeg
~~~~~~~~~~~YEAST/
/
100
200
300
RELATIVE [At
400
500
Figure 3. Typical ELISA immunotitration curves obtained when spinach chloroplast (CHL), spinach cytosolic (CYT), M. smegmatis and yeast
aldolases were probed with antibody preparations against the chloroplast (top) or cytosolic (bottom) forms of spinach aldolase. The ordinate
refers to the rate (velocity) of the peroxidase reaction in terms of the change in absorbance at 490 nm per 20 min. During this incubation period,
the change in absorbance with time was found to be linear in all cases. The antiserum dilution used for each point has been normalized such
that a relative antibody concentration (abscissa) of 512 corresponds to a 1 :10 dilution of antiserum.
quence. Recent high resolution x-ray crystallographic analysis
shows that the first beta sheet in the secondary structure of
the prototypic class I rabbit muscle aldolase (24) is contained
within this highly conserved region. Also, a 13 residue long
alpha helical segment is known to precede the first beta sheet
in the secondary structure of rabbit muscle aldolase (Fig. 6).
Interestingly, the chloroplast aldolase sequence starts precisely
where this alpha helical region begins in rabbit muscle aldolase. In contrast, no clear-cut relationship between the NH2terminal region of the procaryotic M. smegmatis aldolase and
the corresponding segments of the chloroplast or cytosolic
eucaryotic aldolases was observed (Fig. 6).
To verify the observed similarities in NH2-terminal se-
quences of the chloroplast enzyme and other eucaryotic class
I aldolases, we utilized the method described by Lipman and
Pearson ( 15) to assess the statistical significance of the alignments by comparing the chloroplast sequence with randomly
permuted versions of the entire sequence of each potentially
related aldolase. Results of this type of analysis are expressed
as a "Z" value. The relationship between two sequences
yielding Z values greater than six is regarded as significant,
and when Z is found to be greater than 15, the sequences are
considered to be genuinely related, while unrelated sequences
will generally have Z values less than three. As shown in
Figure 6, the cytosolic maize and trypanosome aldolases
received the highest Z value ranking (both greater than 15)
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Plant Physiol. Vol. 91, 1989
MARSH ET AL.
1 398
ANTI - CHLOROPLAST
yYV
4
8
12
Figure 4. Double reciprocal plots of the immunotitration data presented in Figure 3. Linear
regression analysis of the experimental data
points was performed using the Enzpack computer software and an Apple 2E computer. Theoretical antibody saturation levels for each immunotitration curve were obtained from the inverse of the y-intercepts.
ANTI - CYTOSOLIC
_V
N--
1
[b]xl
02
when compared to the chloroplast sequence. The enzyme
from Trypanosoma brucei is known to be posttranslationally
sequestered in a glycolytic organelle (5) and provides an
interesting parallel to the chloroplast aldolase which is assumed to be posttranslationally processed as it is transported
into the chloroplast organelle (14). The muscle aldolases (type
A, Fig. 6) form a group, with corresponding Z values of about
seven, followed by the liver aldolases (type B, Fig. 6) which
are of borderline significance (Z < 6). Finally, the low Z value
obtained for the M. smegmatis aldolase (Z = 0.7) suggests
that this procaryotic enzyme is unrelated to the chloroplast
aldolase, at least at the NH2-terminal region of the molecule.
A final comparative study was performed to determine if
the dissimilarities observed between the chloroplast and M.
smegmatis class I aldolases could be generalized to other class
I procaryotic aldolases. Since no amino acid sequence information on any class I procaryotic fructose bisphosphate aldolase could be found by computer search, we attempted to
estimate the possible similarities between the chloroplast and
procaryotic class I aldolases by making comparisons of their
amino acid compositions utilizing the method of Marchalonis
and Weltman (16). Although not as sensitive as comparative
amino acid sequence analysis, this method can be used to
make reasonable predictions concerning the relative similarities in the primary structures of different proteins. Marchalonis and Weltman found that comparisons ofthe amino acid
compositions of proteins which have similar primary structures generated "similarity indices" (referred to as SAQ values)
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SIMILARITIES BETWEEN CHLOROPLAST AND CYTOSOLIC ALDOLASE
Table I. Saturation Levels and Cross-Reactivities of Aldolases Using
Antisera Prepared against Spinach Cytosolic and Chloroplast
Aldolases
Serum
Saturation
a
Level"
100
Percent
Cross Reaction
at Saturationb
Antichloroplast aldolase serum
100
2.15 ± 0.17
Chloroplast
62
1.34 ± 0.03
Cytosolic
0
<0.05
M. smegmatis
0
<0-05
Yeast
Anticytosolic aldolase serum
100
2.52 ± 0.14
Cytosolic
48
1.22 ± 0.15
Chloroplast
0
<0.05
M. smegmatis
0
<0.05
Yeast
a Saturation levels are defined as the change in absorbance at 490
nm in 20 min observed under conditions of antibody saturation. These
maximum levels of antibody binding were obtained from doublereciprocal plots of ELISA data (see Fig. 4). Saturation levels are
b Ratio of
averages ±SD for four independent determinations.
saturation levels (expressed as a percentage) of the potentially crossreacting protein relative to the protein used as the immunizing agent.
of less than 50, with the theoretical minimum value being
for a comparison of identical proteins. Comparisons of
amino acid compositions of proteins which are structurally
unrelated generated SAQ values of greater than 100. Using
this approach, very low SAQ values (less than 15) were
observed when the amino acid composition of the chloroplast
aldolase was compared with those of both the cytosolic spinach aldolase and the prototypic class I rabbit muscle aldolase.
In contrast, we observed relatively high SAQ values (70-100)
when the amino acid composition of the chloroplast aldolase
was compared with those of the class I aldolases derived from
diverse procaryotes including Staphylococcus aureus, Lactobacillus casei, Escherichia coli, and Micrococcus aerogenes.
These latter, rather high SAQ values are similar to those
observed when the amino acid composition of the chloroplast
aldolase was compared with those of a number of structurally
unrelated proteins, including two class II (metallo) aldolases
(data not shown).
zero
DISCUSSION
Results of the present studies suggest that the chloroplast
form of spinach leaf fructose bisphosphate aldolase is structurally more related to the class I eucaryotic aldolases than it
is to aldolases derived from procaryotic origin. This contention is based on our demonstration that: (a) approximately
50% of the populations of polyclonal antibodies in antiserum
raised against the chloroplast aldolase recognized a homologous antigenic determinant(s) on the cytosolic enzyme (and
vice versa) while neither type of antiserum recognized the
procaryotic class I aldolase derived from M. smegmatis nor
the class II (metallo) yeast aldolase; (b) The NH2-terminal
amino acid sequence of the spinach chloroplast aldolase was
found to be statistically much more similar to the NH2terminal sequences of animal and plant cytosolic aldolases
than it was to the NH2-terminal sequence of the procaryotic
1 399
ANTI- CYTOSOLIC
0
50
!\o
\@\
* CYT
o CHL
0~~~
50
PRE-INC
100
150
[CYT] (ug/mi)
Figure 5. Competitive inhibition of antibody binding to the two spinach aldolases by preincubation of antiserum raised against the cytosolic aldolase with pure cytosolic aldolase. Aliquots of antiserum,
diluted 1:100 in antibody binding buffer (1.2 mL each), were preincubated with increasing amounts of highly purified cytosolic aldolase
for 5 d at 40C with occasional mixing. Final concentrations of aldolase
in the preincubation mixtures ranged from 0 to 150 Ag/mL. These
preincubated mixtures were then substituted for antiserum in the
standard ELISA procedure. Data are plotted as a percentage of the
reactivity observed when no competing antigen (cytosolic aldolase)
was present in the preincubation mixtures (ordinate) versus the
concentration of cytosolic aldolase present in the preincubation mixture (abscissa).
M. smegmatis class I enzyme, particularily in that region
which corresponds to the first beta sheet in the secondary
structure of rabbit muscle aldolase; (c) Results of systematic
amino acid composition comparisons of diverse eucaryotic
and procaryotic fructose bisphosphate aldolases indicated that
the chloroplast enzyme is more closely related to the eucaryotic class I aldolases than it is to class I aldolases of procaryotic origin. Furthermore, the chloroplast aldolase, like other
eucaryotic aldolases, possesses a carboxy-terminal structure
which is essential for maximum catalytic activity (14) and is
composed of four subunits (12), characteristics which are not
exhibited by most procaryotic class I aldolases. Collectively,
these observations support the assignment of the chloroplast
aldolase to the eucaryotic "family" of class I fructose bisphosphate aldolases.
We have recently demonstrated that a subpopulation of
antibodies raised against the chloroplast aldolase also recognizes a homologous domain(s) in cytosolic aldolases derived
from animals as well as plants (our unpublished observation)
and that these homologous antibody sites consist predominantly of "linear" stretches of amino acids, rather than being
constructed of three-dimensional "conformational" motifs
which are characteristic of only the native form(s) of these
proteins. That is, the immunological cross-reactivity observed
between chloroplast and other eucaryotic aldolases is still
evident after the enzymes are subjected to harsh denaturing
conditions or after their primary structures are fragmented by
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Copyright © 1989 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 91, 1989
MARSH ET AL.
1 400
z
Protein
Chloroplast
Maize
Trypanosome
Drosophila
Rat A
Rabbit A
Human A
Human B
Rat B
M. smegmatis
Sequence Alignment
SSYADELVKTAKTVAS PGRGILAMDESk
MSAYCGK*K***I*N*AYIGT **K** *A** *tGTIFKRL
MSKRVEVLLTQLPAYNRLKTP*EA**IE***KMTA **K*L**A***JGSCSKRF
TTYFNYPSKELQ***REI*QKIVA4
*K****A***PPTHGKRL
MPHPYPALTPEQKK**ADI*HRIV4
*K* ***A** *GSIAKRL
MPHSHPALTPEQKK**SDI*HRIVA **K****A*** rGSIAKRL
PYQYPALTPEQKK**SDI*HRIVA **K****A*** rGSIAKRL
MAHRFPALTQEQKK**SEI*QSIVA *K****A*** VGTMGNRL
MAHRFPALT*EQKK**SEI*QRIV
*K****A*** VGTMGNRL
VNQQQADKMT *Q*FI*AALC ADGGELHL
Residues
Valuea
29
40
54
43
44
44
43
44
44
29
15.2
15.6
7.9
7.3
7.6
7.9
5.1
5.4
0.7
az = (optimized similarity score
mean of optimized scores from randomized sequences)/standard deviation of optimized scores
obtained from randomized sequences (15). In each case, 20 comparisons (ktup = 1) were made with shuffled sequences.
Figure 6. Amino acid sequence alignments and corresponding Z values for class I fructose bisphosphate aldolases. Amino acids are represented
by their one letter code. The sequence of the cytosolic maize aldolase was taken from ref. 10 and the sequences of the Trypanosoma, Drosophila,
rat A, rabbit A, human A, human B, and rat B aldolases were obtained from National Biomedical Research Foundation protein sequence
database. A "*" indicates the same amino acid appears in the corresponding position of the chloroplast aldolase. The highly conserved region
common to all eucaryotic, class I aldolase is enclosed in the large open box. Underlined residues in the rabbit A sequence correspond to the first
a-helix (left) and the first beta sheet (right) structures in the crystallographic structure of rabbit muscle aldolase (24).
enzymatic (trypsin) or chemical (cyanogen bromide) means.
We should be able to use an immunological approach to
identify and characterize one or more domains present in
class I eucaryotic fructose bisphosphate aldolases which have
been highly conserved during establishment of the large repertoire of class I eucaryotic aldolases currently found in
nature.
Finally, results of the present work have important implications concerning the probable evolutionary origin of the
chloroplast aldolase molecule. Two distinct models have been
proposed to explain the origin of proteins which reside in the
chloroplast and mitochondrial organelles of eucaryotic cells.
The "autogenic" model (reviewed in ref. 18) speculates that
these organellar proteins are encoded for by structural genes
which were created in situ during evolution by duplication
and modification of genetic information already present in
the eucaryotic genome. In contrast, the popular "endosymbiotic" model (reviewed in ref. 26) proposes that these structural genes were originally derived from the genomes of
procaryotic endosymbionts which long ago took up residence
within primative eucaryotic cells. The case for an endosymbiotic origin of the chloroplast organelle is considered to be a
strong one and in particular, results of comparative biochemical studies have revealed a potentially close relationship
between chloroplasts and the procaryotic blue-green algae
(cyanobacteria) (26).
A common method for investigating the probable evolutionary origin of the chloroplast form of a particular chloroplast/cytosolic enzyme pair is to perform "three-way" structural comparisons between the two members of the plant
enzyme pair and the corresponding enzyme derived from a
suitable procaryotic organism. The rationale is as follows: if
the chloroplast form ofthe enzyme pair arose by an endosym-
biotic mechanism, it should be structurally more similar to
the procaryotic enzyme while, if it arose by an autogenic
mechanism, the chloroplast form should be structurally more
similar to its cytosolic counterpart. Structural and/or functional comparisons of this sort have supported an endosymbiotic origin for a number of nuclear-encoded chloroplast
enzymes including glyceraldehyde-3-phosphate dehydrogenase (23), phosphoglucose isomerase, fructose 1,6-bisphosphatase and malate dehydrogenase (reviewed in ref. 27). However,
using the same approach, the structural similarities we have
found between the chloroplast and cytosolic forms of spinach
leafaldolase are difficult to accomodate within the framework
of the endosymbiotic model. In addition, it is important to
note that the chloroplast contains a class I (Schiffs base)
aldolase in contrast to the mechanistically, structurally and
presumably evolutionarily distinct class II (metallo) aldolase
( 19) found in all photosynthetic procaryotes studied thus far,
including the blue-green algae (1 9, 29).
Our findings, plus the observed distribution of class I and
class II aldolases in nature, seem to be more consistant with
the notion that the chloroplast form of fructose bisphosphate
aldolase was derived from an autogenic evolutionary origin.
According to this model, it is proposed that the nuclearlocalized structural gene which codes for the chloroplast aldolase arose by the duplication and subsequent modification
of an ancestral gene already present in the eucaryotic nucleus,
an established mechanism for the creation of isoenzymes in a
number of other systems (8). Such a mechanism would not
require the transfer of genetic information from the genome
of a procaryotic endosymboint to the nuclear genome of the
eucaryotic "host," a necessary complication of the endosymbiotic alternative. Comparative studies on the chloroplast and
cytosolic forms of plant triose phosphate isomerase (13) and
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Copyright © 1989 American Society of Plant Biologists. All rights reserved.
SIMILARITIES BETWEEN CHLOROPLAST AND CYTOSOLIC ALDOLASE
superoxide dismutase (reviewed in ref. 27) plus the mitochondrial and cytoplasmic forms of phenylalanyl-tRNA synthase
(7) suggest an autogenic origin for the organellar forms of
these enzymes as well. These findings, in conjunction with
the results of the present work, raise the intriguing possibility
that both endosymbiotic and autogenic mechanisms may
have contributed to the creation of nuclear-localized structural genes which code for the organellar form of a particular
protein pair.
ACKNOWLEDGMENTS
We thank Ms. Marie Ayers for excellent secretarial assistance and
Sylvia Yuen for sequence analysis.
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Copyright © 1989 American Society of Plant Biologists. All rights reserved.