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Plant Physiol. (1998) 116: 605-615
Magnesium-Chelatase from Developing Pea Leaves1
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Mg-chelatase
catalyzes the ATP-dependent insertion of Mg2+ into
protoporphyrin-IX to form Mg-protoporphyrin-IX. This is the first step
unique to chlorophyll synthesis, and it lies at the branch point for
porphyrin utilization; the other branch leads to heme. Using the
stromal fraction of pea (Pisum sativum L. cv Spring)
chloroplasts, we have prepared Mg-chelatase in a highly active (1000 pmol 30 min
1 mg1) and stable form. The
reaction had a lag in the time course, which was overcome by
preincubation with ATP. The concentration curves for ATP and
Mg2+ were sigmoidal, with apparent
Km values for Mg2+ and ATP of
14.3 and 0.35 mm, respectively. The
Km for deuteroporphyrin was 8 nm. This Km is 300 times lower
than the published porphyrin Km for
ferrochelatase. The soluble extract was separated into three fractions
by chromatography on blue agarose, followed by size-selective
centrifugal ultrafiltration of the column flow-through. All three
fractions were required for activity, clearly demonstrating that the
plant Mg-chelatase requires at least three protein components. Additionally, only two of the components were required for activation; both were contained in the flow-through from the blue-agarose column.
The biosynthesis of heme and chlorophyll are integral parts of
chloroplast development and the acquisition of photosynthetic capacity.
Both end products are required in specific but vastly different amounts
and are synthesized within the chloroplast via a common tetrapyrrole
biosynthetic pathway (Beale and Weinstein, 1990 Previous work has shown that Mg-chelatase activity requires ATP (Pardo
et al., 1980 More recently, there have been several major developments in the study
of Mg-chelatase. The most important development has come from work on
the photosynthetic bacterium Rhodobacter sphaeroides, in
which it was shown that Mg-chelatase activity requires three proteins
(Gibson et al., 1995 An important characteristic of the prokaryotic Mg-chelatase systems is
that they are completely soluble, and the derived amino acid sequences
show no hydrophobic stretches capable of spanning a membrane (Gibson et
al., 1995 Chemicals and Biochemicals
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
; Porra, 1997). The
branch point for the formation of chlorophyll and heme is the use of
Proto by the two enzymes that catalyze metal insertion. Ferrochelatase
catalyzes the insertion of Fe2+ into Proto,
whereas Mg-chelatase catalyzes the insertion of
Mg2+ into Proto. Because the flux of common
precursors to the respective end products is likely to be controlled by
modulation of the branch-point enzymes, we were interested in the
properties of these enzymes. This report describes the properties of a
totally soluble enzyme system from pea (Pisum sativum L. cv
Spring) chloroplasts that catalyzes the Mg-chelatase reaction.
; Walker and Weinstein, 1991b). Although the reaction is
formally similar to the ferrochelatase reaction, insertion of a
divalent cation into Proto, there is no ATP requirement for the
Fe-insertion reaction. Our previous work has shown that the
Mg-chelatase reaction requires at least two different protein components (Walker and Weinstein, 1991b; Walker et al., 1992). We also
showed that the reaction proceeds by a two-step mechanism, involving
activation followed by Mg2+ insertion; both steps
require ATP (Walker and Weinstein, 1994). This hypothesis was based on
the following observations. There is a lag phase in the kinetics that
can be overcome by preincubation of the crude enzyme fraction with ATP
before the porphyrin substrate is added. ATP
S can substitute for ATP
in the preincubation, but not for the Mg2+
insertion step. The final reaction rates are enhanced if the preincubations have a higher protein concentration, suggesting protein-protein interaction in the activation step.
). This was shown by cloning and expressing the
products of the bchD, bchH, and bchI
genes in Escherichia coli, and combining the E. coli extracts to reconstitute Mg-chelatase activity in vitro.
Similarly, in the cyanobacterium Synechocystis PCC6803,
three genes were identified (chlD, chlH, and
chlI), cloned, and expressed in E. coli;
reconstitution of Mg-chelatase activity required all three gene
products (Jensen et al., 1996a). The requirement for
three subunits catalyzing an ATP-dependent metal ion insertion is
analogous to the situation for Co insertion in vitamin B12 biosynthesis
in Pseudomonas denitrificans (Debussche et al., 1992). Homologs for two of the bacterial Mg-chelatase genes have been identified in eukaryotic plants, olive and ch42
for bchH and bchI, respectively (Koncz et al.,
1990; Hudson et al., 1993; Nakayama et al., 1995; Gibson et al., 1996;
Jensen et al., 1996b). The sequence homologies between the two
bacterial and plant genes and the requirement for ATP suggest that
higher plants also require three subunits for Mg-chelatase activity.
Evidence for this supposition comes from the finding that there are
three nonallelic mutations that affect Mg-chelatase activity in
isolated barley (Hordeum vulgare L.) chloroplasts (Jensen et
al., 1996b), and that activity can be reconstituted by mixing
chloroplast extracts from any two nonallelic mutants (Kannangara et
al., 1997). Although two of the plant genes have been cloned and
expressed, there is currently no system available to test whether the
expressed subunits are active (Nakayama et al., 1995; Gibson et al.,
1996).
; Jensen et al., 1996a; Petersen et al., 1996). The same is
true for the two plant genes that have been identified (Hudson et al.,
1993; Nakayama et al., 1995; Gibson et al., 1996; Jensen et al.,
1996b). Although most of our work on the in vitro characterization of
the Mg-chelatase reaction has used membrane-containing chloroplast
fractions, we have observed that Mg-chelatase activity can be
solubilized by chloroplast lysis in buffers that contain low
concentrations of MgCl2 (Walker and Weinstein,
1995). Because the higher plant system is also soluble, it is important
to determine whether the observations that led to our proposal of a
two-step mechanism were a function of having membranes in the system.
Thus, we have continued our studies on the enzyme extracted from pea
chloroplasts. The soluble enzyme system has been characterized with
respect to substrate requirements, potential inhibitors, and the lag
phase in the kinetics. This system has also been separated into three
fractions, and the role of these fractions in activation and
Mg2+ insertion has been investigated.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
S
was purchased from Boehringer Mannheim. All other biochemicals were
purchased from Sigma, and all organic solvents and salts were of
analytical grade or better. Centrifugal ultrafiltration devices (for
protein concentration and fractionation by size) were purchased from
Amicon (Beverly, MA).
Plant Material and Chloroplast Isolation
Pea (Pisum sativum L. cv Spring) seeds were purchased from Asgrow (Doraville, GA). Seeds were washed with tap water to remove excess Captan fungicide and were then allowed to imbibe in water for 1.5 to 3 h. The plants were grown at about 26 to 32°C for 7 to 8 d under a 12-h light/12-h dark cycle in trays of moist vermiculite. Seedlings were harvested for chloroplast isolation between 3 and 6 h after the final light cycle started.). Remaining Percoll and BSA were removed by washing the intact
plastids with grinding buffer lacking BSA.
Preparation of SPs
The intact chloroplast pellet was resuspended in a small volume of hypotonic lysis buffer (10 mm Tricine, 2 mm EDTA, 2 mm DTT, 10% [w/v] glycerol, and 0.0025% [w/v] PMSF, pH 7.85) and stored at
75°C before homogenizing. The frozen
suspension was thawed and diluted to a protein concentration of about
20 mg/mL with the same buffer, then homogenized in a 10-mL
Potter-Elvehjem (Wheaton, Millville, NJ) glass homogenizer to get a
mixture of broken chloroplasts. The mixture was centrifuged at
280,000g for 35 min at 4°C. The pellet was discarded and
the supernatant was concentrated to a protein concentration between 10 and 20 mg/mL using Centriprep 10 concentrators (Amicon). This soluble
system is designated SP. A typical eight-tray preparation of pea
seedlings (starting with 3.5 L of seeds) yields approximately 300 mg of SP.
Fractionation of SP on Blue-Agarose
A Cibacron Blue 3GA-agarose (4% cross-linked) column (1.5 × 7 cm, about 12-mL gel) was equilibrated with hypotonic lysis buffer. Eight milliliters of SP containing about 90 mg of protein was loaded onto the column at a flow rate of 0.2 mL/min, and the first 5 mL of effluent was discarded. The column was then washed with hypotonic lysis buffer (70 mL at 2 mL/min), and the next 15 mL was collected and designated FT. The remaining wash buffer was discarded. Then, the column was washed with 30 mL of hypotonic lysis buffer supplemented with 2 m NaCl at a flow rate of 2 mL/min. The eluate was collected and concentrated to 3 mL using Centriprep 10 concentrators. This fraction was desalted by gel filtration using a spin column and ACA 202 spectra gel (Spectrum, Los Angeles, CA). The desalted fraction was diluted to 10 mL; this fraction was designated as BB. Mg-chelatase activity of fractions FT and BB was checked using either a stopped or a continuous assay. Preparation of chloroplast fractions containing LM/S and separation of the LM and S by ultracentrifugation was as described previously (Walker et al., 1992).
Further Fractionation by Size
In some cases the FT was further fractionated by size using centrifugal ultrafiltration with membranes having different pore sizes. Low-molecular-mass components (<100 kD) were obtained in the filtrate after centrifugation (3,000g for 15 min at 4°C) of FT through a Microcon 100 concentrator (Amicon). This fraction is designated FT-lo. The higher-molecular-mass components (>100 kD) were concentrated in the retentate, but the retentate also contained contaminating low-molecular-mass components at their original concentration. To remove these low- molecular-mass components, the retentate was diluted 15-fold with hypotonic lysis buffer (without additional PMSF) and the centrifugal ultrafiltration was repeated. This process was repeated two more times, and the final retentate was resuspended in one-half the original volume (giving another 6-fold dilution). Theoretically, the overall dilution of low-molecular-mass components in the high-molecular-mass fraction was 20,000-fold, assuming no interactions. The washed high-molecular-mass fraction of FT is designated FT-hi. using BSA as a
standard.
Mg-Chelatase Activity Measurements
Stopped Assay
Mg-chelatase activity was measured by an adaptation of the method previously described (Walker and Weinstein, 1991b). However, because
the enzyme samples no longer contained membranes, they could be
conveniently incubated in 1.5-mL microcentrifuge tubes that were placed
in a heating block set to 30°C and covered with two layers of
aluminum foil. Samples were incubated in a total volume of 100 µL of
Tricine-EDTA (10-2 mm) buffer containing 9 µm Deutero, 30 mm
MgCl2, 2 mm ATP with an
ATP-regenerating system (10 mm phosphocreatine/creatine
kinase [2 units/mL]), 5 to 8% (w/v) glycerol, and 1 to 1.75 mm DTT, final pH, 7.85. Reactions were started by addition
of samples and were allowed to proceed for 30 min with constant shaking
(100 rpm on an orbital shaker) before termination of the reaction by
addition of 750 µL of cold acetone. To this mixture 150 µL of cold
0.12 n NH4OH was added. The samples
were vortexed and centrifuged (13,000g) for 2 min to
sediment precipitated proteins. The supernatant was reserved, and the
fluorescence of the product was read directly in this 75% (v/v)
acetone extract. No second acetone extraction or hexane extraction was
necessary.
). Activities are expressed as units (the number of
picomoles of Mg-Deutero produced in a 30-min assay).
Continuous Assay
The continuous assay was carried out as previously described (Walker et al., 1992). In a typical assay an aliquot of the sample was
thawed and added directly to a fluorescence cuvette containing the
assay buffer equilibrated to 30°C. The assay buffer was exactly as
described above, except that the MgCl2
concentration was reduced to 20 mm to prevent formation of
turbidity. The total final incubation volume was 600 µL. In some
experiments with the continuous assay, some of the plastid-derived
enzyme components were preincubated for 10 min at 30°C in the
presence or absence of other components required for the reaction. In
these experiments Deutero was absent during the preincubation period.
At the end of the preincubation period, Deutero plus the remaining
components were added to the cuvette to initiate the reaction. The
preincubation volumes were always less than one-half of the final
volume. Unless otherwise indicated the preincubation contained 2 mm ATP (plus regenerating system, see above) and 20 mm MgCl2. For the
porphyrin-concentration curves, the shutter was opened for 3 s
every 60 s instead of every 30 s. Reaction rates were
determined from the slope of the linear portion of the continuous assay
curves. The fluorescence yield of the reaction products in aqueous
buffers was sensitive to the presence or absence of protein in the
assay buffer. Thus, the standard curves were constructed in buffer
containing 1.5 mg/mL SP. The average deviation for duplicate
measurements was 8%.
Immunoblotting
Proteins in various chloroplast fractions were separated by electrophoresis on SDS polyacrylamide (10% [w/v]) gels, according to the method of Fling and Gregerson (1986). Ten micrograms of protein
from each fraction was loaded onto the gel. The separated proteins were
transferred to an Immobilon-P filter (Millipore) using a semi-dry
electroblotter (JKA-Biotech, Brønhøj, Denmark) according to the
manufacturer's directions. One filter was probed with rabbit
antibodies to a recombinant protein expressed in Escherichia coli, which was encoded by a truncated form of the ch42
gene from Arabidopsis thaliana. The other filter was probed
with mouse antibodies to a recombinant protein expressed in E. coli, which was encoded by a truncated form of the
olive gene from Antirrhinum majus. Both
antibodies were generously provided by C. Gamini Kannangara (Carlsberg
Research Laboratory, Copenhagen, Denmark). Incubation of the primary
antibodies with the filters was performed according to Sambrook et al.
(1989). The primary antibody was detected using secondary antibodies
conjugated to alkaline phosphatase (Sigma) according to the supplier's
directions.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Preparation and Characterization of Soluble Mg-Chelatase
We have shown that Mg-chelatase can be prepared from pea plastids in a completely soluble form by using a buffer with a low Mg concentration to wash the active components off the membranes (Walker and Weinstein, 1995). Unfortunately, this preparation required the
presence of ATP and Mg2+ during enzyme
preparation and storage to maintain the highest activity. Even in the
presence of these components, the enzyme preparation was not very
stable; 38% of the activity was lost when stored on ice for 3 h
(data not shown). Ten percent (w/v) glycerol and increased DTT
concentration (2 mm) vastly improved the stability of the
enzyme preparation when these components were included in the
chloroplast-lysis buffer and in all subsequent steps of fractionation.
Compared with fresh enzyme there was only a 7% loss of activity when
stored for 2 months at 75°C followed by 0°C for 5 h (data
not shown). There was no loss of activity if the 75°C material was
thawed and used immediately. The activities of these preparations were
high; the specific activity was 1050 pmol 30 min1 mg1 protein when
measured at a protein concentration of 7 mg/mL. As with LM/S (Walker et
al., 1992), the activity versus protein-concentration curve is not
linear until a threshold protein concentration between 2 and 3 mg/mL is
reached in the assay (data not shown).
Inhibition
). This early
work used the stopped assay, and there were difficulties with achieving
initial velocity conditions for Michaelis-Menten kinetics. To overcome
these problems, the continuous assay was used with the following
modifications: SP was preincubated with ATP and
Mg2+ for 10 min at 30°C to overcome the lag
period (see below). After the preincubation period the sample was
diluted to the final volume with buffer containing ATP and
Mg2+, and varying amounts of porphyrin were
added. Slopes for the fluorescence increase with time were recorded
over a 5- to 10-min period after the addition of porphyrin (the shorter
time periods were required for the lower porphyrin concentrations). In
Figure 1 the results of a typical
experiment with Proto are shown (average Km = 13.5 nm). For most experiments the more soluble and
stable porphyrin, Deutero, was used. The Km
for this artificial substrate was also in the nanomolar range
(Km = 7.9 nm). The
Km values were determined by fitting the
data to the Lineweaver-Burk double-reciprocal plot; the correlation
coefficients for linearity were always greater than 0.96. Km values in the nanomolar range were
confirmed by following the progress curve of substrate disappearance at
several different initial concentrations of Deutero. The
Km values, determined from the integrated
rate equation (Segel, 1975), fell between 1 and 10 nm.
These samples were also preincubated with ATP and Mg2+ before dilution and addition of porphyrin.
At concentrations of Deutero greater than 500 nm, some
substrate inhibition was observed. At 4 µm Deutero the
velocity was 80% of the maximal velocity measured.
View this table:
Table I.
Substrate requirements for soluble Mg-chelatase
The Km values for Deutero and Proto were
obtained from double-reciprocal linear replots of initial velocities
obtained from the continuous assay after activation (see text). The
apparent Km values for ATP and Mg2+
were obtained using the linearized version of the Hill equation (Segel,
1975 ), with initial velocities determined using the stopped assay.
View larger version (18K):
[in a new window]
Figure 1.
Mg-chelatase follows Michaelis-Menten kinetics
with respect to porphyrin concentration. Initial velocities were
determined from the slopes of the continuous assay after activation and
subsequent addition of Proto. The assays contained 0.28 mg of SP. The
preincubation volume was 40 µL. Inset, Lineweaver-Burk plot of the
same data (r2 = 0.974). In this experiment
the Km for Proto was 18.9 nm,
and the Vmax corresponded to 794 units/mg.
Fractionation of SP by Pseudo-Affinity Chromatography
We have previously reported that pea plastid Mg-chelatase can be separated into a LM and a S by centrifugation (Walker et al., 1992).
Both fractions were required for activity. SP can also be separated
into two fractions, FT and BB, by pseudo-affinity chromatography on
blue-agarose. Both fractions were required to reconstitute activity
(Table II). To avoid having to
concentrate the FT, the SP was applied to the column at a fairly high
protein concentration, 11 mg/mL. If there was substantial activity
(>10% of the recombined activity) in the FT alone, this fraction
could simply be reapplied to a smaller fresh blue-agarose column. For recovery of the active component(s) in BB, elution of the column with
buffer containing ATP (up to 10 mm) could not replace
elution with 2 m NaCl. FT contained 30% of the applied
protein, and BB contained 50% of the applied protein.
|
Components Required for Activation
Biochemical Rationale for Three Components in
Higher-Plant Mg-Chelatase
75°C. The four fractions LM, S, FT, and BB were
tested for the presence of two Mg-chelatase subunits by immunoblotting
(Fig. 2). Antibodies to a fragment of the
ch42-encoded protein (I homolog) could be used to detect the
presence of the I component of Mg-chelatase in FT and S. This component
was clearly absent in BB and LM. Antibodies to the
olive-encoded protein (H homolog) showed the presence of the
H component in BB and in LM. No H component could be seen in FT, but
there appeared to be some of the H component present in the S. Antibodies to a putative eukaryotic D component were not available at
this time.
View larger version (46K):
[in a new window]
Figure 2.
Immunoblots of chloroplast fractions showing the
presence of Mg-chelatase subunits I and H. A, Localization of the I
component; B, localization of the H component. SPs were prepared from
intact chloroplasts and fractionated on a column of blue-agarose to
obtain a FT and a BB. Alternatively, intact chloroplasts were lysed and the thylakoid membranes removed by centrifugation to leave an LM/S.
After addition of 10 mm MgCl2, LM/S was
separated into LM and S by ultracentrifugation.
View larger version (18K):
[in a new window]
Figure 3.
Continuous assay and preincubation of the
blue-agarose fractions FT and BB. The fractions were preincubated in
buffer containing ATP and MgCl2. After the preincubation
the remaining components were added for the continuous assay. The
preincubations contained: 1, FT; 2, FT plus BB; 3, no preincubation;
and 4, BB. In all samples the final assay contained 338 µg of FT and
578 µg of BB. The activity for 1 is 84.5 units.
S can substitute for ATP in the activation
stage, but not in the Mg2+-insertion stage.
; Jensen et al., 1996a). Two
of these proteins, products of the bchI and bchD
genes, are required for activation (Willows et al., 1996). Our results
with activation in the two different preparations from pea chloroplasts
also predict at least a three-component system for higher plants. The
fact that both fractions are required for activation in the LM/S system
suggests that at least two components are required for the activation
step. Because the FT is sufficient for activation, at least two
components are present in this fraction. The fact that BB also is
required for activity implies the existence of a third component. In
combination with our immunoblot results, the fractionation and
activation data allowed us to predict which fractions contained the
"missing" third component. The S of LM/S contains I, as indicated
by probing with the antibody to the ch42 gene product.
Because the S alone does not support activation, the D component is
probably in the LM along with the H component (as indicated by probing
with the antibody to the H component). As indicated by the immunoblots, FT contains I. Because only FT is required for activation, D is probably also present in this fraction, which means that D is not
retained by the blue-agarose column.
Separation of the FT Components (I and D) by Size
Subunit Requirements for Activation and Mg2+ Insertion
We have prepared a totally soluble fraction of chloroplasts that
is highly active in the Mg-chelatase reaction. The stability conferred
on this preparation by glycerol and extra DTT has allowed for the
characterization of the soluble enzymatic activity with respect to
substrate requirements, potential inhibitors, preincubation effects,
and behavior during subfractionation. The new preparation with glycerol
and extra DTT is as active as our previous soluble preparation, but it
has the advantage of being more stable and does not require the
presence of substrates to maintain the stability (Walker and Weinstein,
1995 Received July 29, 1997;
accepted October 27, 1997.
Abbreviations:
ATP We thank Dr. C. Gamini Kannangara (Carlsberg Research
Laboratory, Copenhagen, Denmark) for providing the antibodies to the ch42- and olive-encoded proteins. We also thank
Drs. Caroline J. Walker and Megen Bruce-Carver (Clemson University,
Clemson, SC) for critically reading the manuscript.
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eds, Molecular Cloning: A Laboratory Manual, Vol 3.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 18.1-18.88
Segel IH
(1975)
Enzyme Kinetics.
John Wiley & Sons, New York
Walker CJ,
Hupp LR,
Weinstein JD
(1992)
Activation and stabilization of Mg-chelatase activity by ATP as revealed by a novel in vitro continuous assay.
Plant Physiol Biochem
30:
263-269
Walker CJ,
Weinstein JD
(1991a)
Further characterization of the magnesium chelatase in isolated developing cucumber chloroplasts. Substrate specificity, regulation, intactness and ATP requirements.
Plant Physiol
95:
1189-1196
Walker CJ,
Weinstein JD
(1991b)
In vitro assay of the chlorophyll biosynthetic enzyme Mg-chelatase: resolution of the activity into soluble and membrane-bound fractions.
Proc Natl Acad Sci USA
88:
5789-5793
Walker CJ,
Weinstein JD
(1994)
The magnesium insertion step of chlorophyll biosynthesis is a two-stage reaction.
Biochem J
299:
277-284
Walker CJ,
Weinstein JD
(1995)
Re-examination of the localization of Mg-chelatase within the chloroplast.
Physiol Plant
94:
419-424
[CrossRef]
Walker CJ,
Willows RD
(1997)
Mechanism and regulation of Mg-chelatase.
Biochem J
327:
321-333
Walker CJ,
Yu G,
Weinstein JD
(1997)
Comparative study of heme and Mg-protoporphyrin (monomethyl ester) biosynthesis in isolated pea chloroplasts: effects of ATP and metal ions.
Plant Physiol Biochem
35:
213-221
Willows RD,
Gibson LCD,
Kannangara CG,
Hunter CN,
von Wettstein D
(1996)
Three separate proteins constitute the magnesium chelatase of Rhodobacter sphaeroides.
Eur J Biochem
235:
438-443
[ISI][Medline]
View this table:
Table III.
Reconstitution of activity with three chloroplast
fractions obtained by combining LM/S and blue-agarose fractionation
The BB fraction from the blue-agarose column contains the H component
(see Fig. 2). The S fraction of LM/S contains the I component (see Fig.
2). The third component, D, was obtained from the blue-agarose FT of
solubilized LM, LM-FT (see text). The following amounts of protein were
used in a 200-µL assay: S, 200 µg; BB, 350 µg; and LM-FT, 27 µg.
; Nakayama et al., 1995). In R. sphaeroides this component is thought to function as a dimer
(Willows et al., 1996). Although there is no information on the
putative D component in higher plants, the expressed protein from
R. sphaeroides purifies as a 550-kD multimer (Willows et
al., 1996). Thus, these two components contained in the FT were
separated by centrifugal ultrafiltration through a 100-kD cut-off
filter. The filtrate (FT-lo) contains the low-molecular-mass
components, and the retentate contains the high-molecular-mass
components and some low-molecular-mass components. The
contaminating low-molecular-mass components were removed from the
retentate by repeated dilution and centrifugal ultrafiltration of the
retentate FT-hi. The FT-hi and FT-lo were tested for activity in the
various combinations with the BB (Table IV). Again, it is clear that three
different fractions are required for activity. The best activity in the
combination of any two fractions gives only 6% of the activity when
all three are combined.
View this table:
Table IV.
Reconstitution of activity with three fractions
obtained from blue-agarose and molecular size fractionation
The H (BB) component was retained on the column and eluted with NaCl.
The I and putative D components were in the FT, and were separated from
each other by centrifugation through a 100-kD cut-off membrane. The
FT-hi was retained by the membrane, and the FT-lo passed through the
membrane. The FT-hi fraction was repeatedly washed to remove
low-molecular-mass contaminants. The following amounts of protein were
used in a 200-µL assay: FT-lo (I), 25 µg; BB (H), 193 µg; and
FT-hi (D), 145 µg.
View this table:
Table V.
Protease sensitivity of the three fractions required
for Mg-chelatase activity
The individual fractions were treated with trypsin (120 units) or
trypsin plus trypsin inhibitor (1200 units) for 15 min on ice. At the
end of this treatment, trypsin inhibitor was added to the samples that
did not already have it, then the other two fractions and the
substrates were added for a standard stopped assay. The following
amounts of protein were used for the protease treatments and assays:
FT-hi (D), 145 µg; BB (H), 193 µg; and FT-lo (I), 16 µg.
View larger version (18K):
[in a new window]
Figure 4.
Activation requires preincubation of the FT-hi (D)
and FT-lo (I). Fractions were preincubated in various combinations,
then the remaining components were added for the continuous assay. The
preincubations contained: 1, FT-hi plus FT-lo (D + I); 2, FT-hi plus
FT-lo plus BB (D + I + H); 3, no preincubation; 4, FT-hi plus BB (D + H); and 5, FT-lo plus BB (I + H). In all samples the final assay
contained the following amounts of protein from each fraction: FT-hi,
362 µg; FT-lo, 62 µg; and BB, 481 µg. The activity for 1 is 62 units.
), we assessed the
effect of preloading the H-containing fraction with Deutero.
Preincubation of BB with Deutero for 5 min at 30°C, followed by
addition of activated FT (preincubated with ATP as above) and the
remaining buffer components for a standard continuous assay, resulted
in a 32% decrease in the rate of reaction compared with when Deutero and the BB were added to the activated FT at the same time.
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
). This preparation also has about the same level of activity as
that recently reported for a soluble preparation from barley
(Hordeum vulgare L.) etioplasts (Kannangara et al., 1997).
However, it should be noted that despite the good activity in these
preparations, the activity is almost 10-fold lower than can be measured
in intact pea chloroplasts (Walker et al., 1997).
) and to our initial
estimates with SP (Guo, 1996). It is clear that our initial estimates
were too high, because too much of the substrate was used up during the
reaction. Several modifications to the assay procedure made it possible to get a more accurate value. First and foremost, the continuous assay
made it possible to get an accurate initial rate. Elimination of the
lag period by preincubation of SP with ATP ensured that the linear
phase of the reaction occurred soon after substrate addition. This was
most important for the lower porphyrin concentrations. During the
actual fluorescence measurements it was important to control the
excitation shutter carefully. Reaction rates were significantly higher
when the shutter was opened every minute instead of every 30 s.
Most likely the excitation light was causing photodestruction of the
substrate, product, or both. This phenomenon may have been the cause of
the substrate inhibition observed at higher substrate concentrations.
report a Km for Proto of
40 nm in intact chloroplasts from greening wheat. Although
this value is in reasonable agreement with our value for the soluble
system, it is difficult to predict by how much the membrane barrier
should increase the Km. The other report is
from the in vitro reconstitution of the expressed R. sphaeroides proteins (Willows et al., 1996). In this report the
ratio of subunits to each other was optimized and the initial
velocities were determined from the linear portion of the continuous
assay. Despite the similarity to our assay procedure, the reported
Km values for Deutero and Proto were
approximately 30-fold greater than that in the pea chloroplast SP. This
disparity may simply reflect the different physiological needs of the
different organisms or differences attributable to slightly different
assay conditions.
). The Km for Deutero
was 2.4 µm, 300 times greater than that of Mg-chelatase.
The Vmax for ferrochelatase was 5.2 nmol
min1 mg1 chlorophyll.
For Mg-chelatase in intact pea chloroplasts the value is similar
(Walker et al., 1997). Because Vmax is
defined for saturating substrate values, the presence or absence of a permeability barrier is not an issue for this parameter. We recently measured a 5- to 10-fold higher flux of porphyrins through the Mg2+, compared with the Fe2+, branch
of the pathway in pea chloroplasts (Walker et al., 1997). If porphyrin
substrate is limiting, the difference in Km
values is more than sufficient to account for the higher flux into the Mg2+ branch of the pathway. Regulation of the
branch point by relative affinity for the substrate could work in
addition to a mechanism of day versus night regulation, based on the
demonstrated circadian rhythmicity of subunit H expression (Gibson et
al., 1996; Jensen et al., 1996b).
), and is twice as high
as the Km for ATP with the reconstituted R. sphaeroides enzyme (Willows et al., 1996). With LM/S the
shape of the curve was also sigmoidal, although this was not the case with the reconstituted R. sphaeroides enzyme. It is too
early to ascribe physiological significance to the sigmoidal character of the curve. However, one of the subunits, I, has a clearly defined ATP-binding sequence (Koonin, 1993; Nakayama et al., 1995), and with
the expressed R. sphaeroides protein this subunit purifies as a dimer (Willows et al., 1996). Thus, two binding sites for ATP
could result in the cooperativity in ATP binding, which is indicated by
the sigmoidal ATP concentration dependence. It is important to note
that we have found no significant differences between the behavior of
SP and LM/S with respect to ATP: ATP is required in the preincubation
to overcome the lag phase, and inactivation of the reaction by addition
of an ATP trap demonstrates that ATP is still required in the second
stage of the reaction. As with LM/S, ATPS will work in the first
step but not in the second (Walker and Weinstein, 1994).
). In contrast, the
reconstituted expressed R. sphaeroides enzyme has a
Km of 1.7 mm (Willows et al.,
1996). The values from the two higher-plant systems are in reasonable agreement. We expect that Mg2+ is involved in at
least three discrete roles: (a) as a substrate for the chelatase, (b)
as a co-substrate with ATP, and (c) for maintenance of protein-protein
interactions. This third role can be inferred from data showing that
MgCl2 concentrations well above those required
for binding with ATP (at least 20 mm) are required to
optimize the preincubation stage of the reaction (Guo, 1996). During
this stage there is no porphyrin present, so the high Mg concentration
during this step cannot be required for the chelation reaction per se.
). To ensure that there was
not a problem attributable to accessibility caused by the membranes, we
retested the effects on activity of the following compounds: Mg-Proto,
Mg-Proto monomethylester, and protochlorophyllide. As with intact
plastids, the potential inhibitors had no significant effect on
activity. Thus, at the level of modulating Mg-chelatase enzyme
activity, feedback inhibition does not play a direct role in regulation
of chlorophyll synthesis. It was recently shown that pretreatment of
seedlings with 
-aminolevulinic acid causes an increase in the
endogenous level of Mg-Proto and Mg-Proto monomethylester, and
plastids from these plants had decreased Mg-chelatase levels (Averina
et al., 1996). The effective plastid porphyrin concentrations were not
given, and we have observed nonspecific inhibition by exogenous
porphyrins (Walker and Weinstein, 1991a). Therefore, it is difficult to
know if the effect is physiologically important. However, if the effect
is physiological, Walker and Willows (1997) have suggested that the
results can be reconciled if the buildup of intermediates inhibits the
import of one or more of the Mg-chelatase subunits into the
chloroplasts. Such a mechanism has been proposed for the regulation of
protochlorophyllide reductase import (Reinbothe et al., 1995).
; Walker et
al., 1992). We also showed that a minimum of two components were
required for the activation step by using the fractionated LM/S system (Walker and Weinstein, 1994). We have now shown that the soluble proteins from pea chloroplasts can be resolved into three separate protein fractions that are required for activity. This resolution could
be accomplished by two slightly different procedures, although both
procedures rely on fractionating SP on a blue-agarose column. When
inferences from the bacterial systems are combined with the results
from the immunoblots, we can make tentative assignments regarding the
subunit composition of each of our fractions. For the blue-agarose
fractions, BB contains H, FT contains D and I, FT-lo contains I, and
FT-hi contains D. For the LM/S fractions, LM contains H and D, LM-FT
contains D, and S contains I. The assignments for D must be tentative,
because there is no antibody available for the higher-plant D
component.
). This
activity was not stimulated by the addition of the soluble fraction. In
this case the plastids were ruptured in an enriched medium containing
high concentrations of all of the substrates, including
Mg2+, ATP, and Proto (Lee et al., 1992). It is
possible that this combination held the putative complex together,
although it is also possible that the high Mg2+
concentration caused a nonspecific association of the individual components with the membranes. Of course, most of our fractionations have been carried out in the absence of Mg2+, and
it is possible that this ion will hold the complex together. However,
in the experiment in which the FT (containing I and D components) was
activated in the presence of 2 mm ATP and 30 mm MgCl2, the I and D components could still be
separated by the most gentle of the techniques, centrifugal
ultrafiltration. Thus, if a complex is formed, something more than
Mg2+ and ATP will be required to stabilize it for
detection as a complex.
), and demonstrates another of the similarities between the two
systems.
), and its tight association with Proto during purification of the expressed R. sphaeroides protein from E. coli extracts. This role for the H subunit is consistent with the
hypothesis that a product of the activation process will act on the
porphyrin bound to the H subunit. Two mechanistic questions can be
asked. Is the reaction facilitated if the H subunit is preloaded with porphyrin? And are both components from the activation reaction required for catalysis? A positive answer to the first question would
support the hypothesis that H is the porphyrin-binding subunit. No
enhancement in rate was observed when the BB fraction was preincubated with porphyrin before addition to the activated FT. This result neither
supports nor negates the suggestion that the H subunit is responsible
for porphyrin binding. Our approach to the second question involved
separating the FT-lo (I) and FT-hi (D) fractions from each other after
activation, and adding each activated component to the BB fraction (H)
separately. Although we cannot rule out the possibility that an
activated component could have been inactivated during the process of
separation, the results would suggest that all three components are
required for the Mg2+-insertion step. This result
is not consistent with activation of a single catalytic subunit by the
other subunit.
1
This work was supported by the U.S. Department
of Energy (grant no. DE-FG05-95ER20170).
FOOTNOTES
2
Present address: Lombardi Cancer Center, E512,
The Research Building, Georgetown University Medical Center, 3970 Reservoir Road NW, Washington, DC 20007.
*
Corresponding author; e-mail wjon{at}clemson.edu; fax
1-864-656-0435.
ABBREVIATIONS
S, adenosine
5
-O-(3-thiotriphosphate).
BB, blue-bound protein
fraction that is bound to a blue-agarose column.
Deutero, deuteroporphyrin.
FT, flow-through protein fraction that does not bind
to a blue-agarose column.
FT-hi, high-molecular-mass (>100 kD)
fraction of FT.
FT-lo, low-molecular-mass (<100 kD) fraction of
FT.
LM, membrane fraction after ultracentrifugation of LM/S.
LM-FT, protein fraction obtained from a low-salt wash of LM, which then
flows through a blue-agarose column .
LM/S, chloroplast fraction that
contains light membranes and soluble proteins.
Proto, protoporphyrin-IX.
S, supernatant fraction after ultracentrifugation of
LM/S.
SP, soluble proteins from the chloroplast.
ACKNOWLEDGMENTS
LITERATURE CITED
Top
Abstract
Introduction
Methods
Results
Discussion
References
Copyright Clearance Center: 0032-0889/98/116/0605/11
© 1998 American Society of Plant Physiologists
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