Plant & Cell Physiol. 2 2 ( 1 ) : 115-126 (1981)
Regulation of C4 Photosynthesis: Inactivation of Pyruvate,Pi
Dikinase in Leaf and Chloroplast Extracts in Relation
to Dark/Light Regulation in Vivo
T. Sugiyama1-3 and M. D. Hatch 2
1
2
Department of Agricultural Chemistry, School of Agriculture, Shizuoka University,
836 Ohya, Shizuoka 422, Japan
Division of Plant Industry, CSIRO, P.O. Box 1600, Canberra City, A.C.T. 2601, Australia
Key words: Ci photosynthesis, regulation — Dark/light mediated regulation —
Pyruvate,Pi dikinase, regulation — Zea mays, Ct photosynthesis.
Pyruvate,Pi dikinase catalyses the conversion of pyruvate to PEP via the
following reaction: pyruvate+ATP + P i ^ P E P + A M P + PPi. This enzyme is
exclusively located in the mesophyll chloroplast of C4 plants and undergoes rapid
light-mediated activation and dark-mediated inactivation in leaves (Hatch 1978,
Hatch and Osmond 1976). Studies on the action spectrum for activation, and the
effects of DCMU, suggest that this regulation is closely linked with the photosynthetic
electron transport system (Yamamoto et al. 1974).
Earlier studies with leaf extracts from maize (Hatch and Slack 1969) demonstrated that the dark inactivated enzyme was activated by Pi and that the active
enzyme isolated from illuminated leaves was inactivated by a process requiring ADP
or ATP. There is evidence that activation requires a high molecular weight heat
labile protein (Hatch and Slack 1969, Sugiyama 1974).
Abbreviations: PEP, phosphoenolpyruvate;'DTT, dithiothreitol; AP5A, P1,P5-di(adenosine-5'-)
pentaphosphate.
This
research was performed in the Division of Plant Industry, CSIRO, Canberra, Australia.
3
Present address: Department of Agricultural Chemistry, Faculty of Agriculture, Nagoya University,
Chikusa, Nagoya 464, Japan.
115
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Active pyruvate, Pi dikinase in leaf or chloroplast extracts isolated from illuminated
leaves was inactivated by incubating with ADP. With chloroplast extracts neither ATP
nor AMP alone was effective. Half the maximum rate of inactivation was observed
with about 55 fiu ADP. The following evidence supported the view that ADP-mediated
inactivation had a co-requirement for low concentrations of ATP [Buchanan (1980)
Ann. Rev. Plant Physiol. 31: 341], adding hexokinase and glucose prevented inactivation by ADP [Feldhaus et al. (1975) Eur. J. Biochem. 57: 197], when GDP
and UDP were added in place of ADP they mediated rapid inactivation only when
ATP was also provided; GTP was not effective. ATP was apparently optimally
effective at about 1 /M or less. The rate of inactivation was approximately proportional
to the square of extract concentration suggesting dependancy on a factor in the extracts
in addition to active enzyme. The involvement of one or more heat labile protein
factors was confirmed by trypsin treatment of extracts. Pyruvate,Pi dikinase inactivated
by treatment with ADP was reactivated by incubating with Pi, a property common to
the inactive enzyme extracted from darkened leaves. Thiol/disulphide interconversion
was apparently not critical in the regulation of pyruvate,Pj dikinase.
116
T. Sugiyama and M. D. Hatch
In the present study we investigated in more detail the nucleotide-mediated
inactivation of pyruvate,Pj dikinase in leaf and chloroplast extracts. Evidence is
provided that ADP-mediated inactivation has a co-requirement for a very low
concentration of ATP, and that one or more protein factors are also involved.
Materials and Methods
Preparation of leaf and chloroplast extracts—Detached maize leaves were illuminated
in the laboratory for about 45 min (600 Win""2, Phillips HPL lamp) to activate
pyruvate,Pi dikinase. Deribbed lamina (3 g) were vigorously ground in a chilled
mortar with sand and 5 ml of 100 mil Tris-HCl buffer, pH 8.0, containing 10 mM
MgCl2, 1 mM EDTA, 10 mM DTT and 0.2 g of Polyclar AT. The extract obtained
by filtering through Miracloth was centrifuged at 15,000 Xg for 5 min at 13°C.
The supernatant was processed on a 20 ml column of Sephadex G-25 (equilibrated
with 50 mM Tris-HCl, pH 8.0, containing 10 mM MgCl2, 0.1 mM EDTA and either
10 mM DTT or 2-mercaptoethanol) at room temperature and about 4 ml of the
excluded protein band was collected. Samples of this extract were added immediately to reactions to follow the inactivation of pyruvate,P, dikinase.
For preparing chloroplast extracts the midrib was removed from preilluminated
leaves (see above) and 10-12 g were sliced into 1-2 mm sections in the light. This
tissue was immediately blended at 0°C in a Sorvall Omnimixer in 120 ml of 20 mM
Tris-HCl, pH 8.3, containing 0.4 M sorbitol, 5 mM MgSC>4, 2 mM DTT, 2 mM isoascorbate, 0.2 mM EDTA and 0.2% (w/v) bovine serum albumin. After blending
for 10 sec at 40% of line voltage the homogenate was filtered through Miracloth and
then centrifuged at 1,000 Xg for 3 min. The pellet was resuspended in 30ml of
the above buffer medium and centrifuged at the same speed. The washed pellet
(containing intact mesophyll chloroplasts, see Hatch 1977) was suspended in 3 ml
of 20 mM Tris-HCl, pH 8.3, containing 5mM DTT, 10 mM MgCl2, 0.2 mM EDTA
and 2 mg/ml bovine serum albumin. This suspension was frozen (—80°C, solid
COg) and then thawed to break the cholroplasts and the soluble extract, essentially
free of chlorophyll, was obtained by centrifuging at 0°C for 10 min at 18,000 X^.
The supernatant was treated on a 15 ml column of Sephadex G-25 equilibrated with
the resuspension buffer described above but without bovine serum albumin, and
3.5 ml of the excluded protein band was collected. This extract was incubated for
10 min at 25°C to recover any pyruvate,Pi dikinase converted to its cold inactive
form (see Hatch 1979) and then used to follow nucleotide mediated inactivation.
Inactivation of pyruvate,P\ dikinase—Details of the composition of reactions are
provided in the appropriate tables and figures. Generally 0.3 ml of extract was
added to a reaction of 0.4 ml total volume. The mixtures were incubated at 25°C
and at intervals samples of 25 fi\ were removed and placed in cuvettes containing
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Plant material and reagents—Zea mays (variety, Dekalb 805A) and other species
were grown in soil in a greenhouse maintained between 20 and 30°C. Expanded
3rd or 4th leaves from young plants were used. Biochemicals and reagent enzymes
were obtained from either Calbiochem (Sydney, Australia) or Sigma Chemical Co.
(St. Luis, Mo). Pyruvate,Pi dikinase (used in some inactivation assays) and PEP
carboxylase (used as a coupling enzyme for the assay of pyruvate,Pi dikinase) were
purified as previously described (Hatch 1979).
Regulation of pyruvate,Pi dikinase
117
Results
Inactivation in leaf and chloroplast extracts
We confirmed the earlier observation (Hatch and Slack 1969) that pyruvate, Pi
dikinase is rapidly inactivated in maize leaf extracts incubated with ADP or ATP.
Fig. 1 reports the results of various treatments of leaf extracts in the presence of the
adenylate kinase inhibitor AP5A (Feldhaus et al. 1975). Maize leaves contain high
levels of adenylate kinase (located in mesophyll chloroplasts, see Hatch and Osmond
1976) and 0.2 mM AP5A inhibits this enzyme by about 99.8%. Interconversion of
ADP with ATP and AMP was thereby minimized. Activity in the control with no
AP5A and no other additions remained stable or commonly increased slightly (Fig. 1).
Pyruvate,Pi dikinase activity declined in reactions provided with ADP or ATP
both in the absence (results not shown) and the presence of AP5A; no inactivation
was observed with AMP (results not shown). It should be noted that this experiment was conducted at 20°C; at 30°C the rate of inactivation would have been about
2.6 times greater (data not shown). With ATP the maximum rate of inactivation
was preceded by a lag. A complication in the plus AP5A treatments was that
inactivation also occurred in the control after a lag of about 10-15 min. One
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a complete system for assaying pyruvate, Pi dikinase. These were immediately
stirred and activity was measured by the absorbance change at 340 nm as previously
described (Hatch 1979).
Reactivation ofADP-inactivated enzyme—Enzyme in chloroplast extracts was inactivated by incubating with 1.3 ITIM ADP and 50 /J.M ATP as described above (also see
the legend of Fig. 2). This reaction mixture (0.7 ml) was then treated on a 4 ml
column of Sephadex G-25 previously equilibrated with 25 HIM HEPES-KOH
buffer, pH 7.5, containing 10 mM MgCl2, 5 mM pyruvate and 0.5 mM EDTA.
About 0.6 ml of the excluded protein band was collected. With this medium there
is little or no loss of pyruvate,Pi dikinase activity in the absence of DTT (K.S.R.
Chapman and M. D. Hatch, unpublished). Dilution resulting from this treatment
was estimated by measuring NADP malate dehydrogenase. Samples of this extract
were then incubated in reactions provided with either 6 mM DTT or 3 mM Pj or Pi
plus DTT.
Removal of ATP contaminating ADP—Our ADP stock contained about 3% ATP
(measured spectrophotometrically by the hexokinase/glucose-6-P dehydrogenase
coupled system) and AMP (detected chromatographically, see below). A sample of
stock ADP solution was chromatographed as a band on Whatman 3 MM paper for
15 hr with propyl acetate : formic acid : water (11 : 5 : 3 v/v) as the developing solvent.
The ADP band eluted from the paper with water was free of AMP and contained
only about 0.3% ATP. This preparation was used for the studies with chloroplast
extracts.
Assay of adenylate kinase and ATP—Adenylate kinase was assayed in the direction
of ATP formation by measuring NADP reduction at 340 nm in an assay system
containing 25 min HEPES-KOH, pH 8.0, 2.5 mM MgCh, 5 mM glucose, 1.25 mM
ADP, 0.25 mM NADP, 4 units hexokinase [freed of (NH 4 ) 2 SO 4 ] and 2 units of glucose6-P dehydrogenase in a total volume of 1 ml. ATP was determined in the same
system but with ADP omitted.
118
T. Sugiyama and M. D. Hatch
1.75
*
^
^
—
'°
Control (no AP 5 A)
-
ADP + HK + Glu
ri
£
1.25
A T P + f^PK + PP
^ \
\ ^
! 10°
>-
\
| 0.75
o
<
1
Control (+ AP5AI >
\
N.ATP
\
\ .
ADP + AMP
0.5010.25 - o NoAP5A
• PlusAP5A
ADP
ADP + ATP-J
1
15
i
30
TIME (min)
45
60
Fig. 1 Adenine nucleotide requirements for inactivation of pyruvate.Pi dikinase in maize leaf extracts. Samples of Sephadex G-25 treated extracts from illuminated leaves (see Materials and
Methods) were incubated at 20°C in a basic medium containing 40 mM Tris-HCl buffer, pH 8.0,
8 mM MgCU, 8 mM DTT and 0.1 mM EDTA. Additions as shown were 1 mM AP5A, 1 mM ADP,
1 mM ATP, 1 mM AMP, 2 mM glucose (Glu), 5 units hexokinase (HK), 2 mM creatine-P (CP) and
2 units creatine-P kinase (CPK). See Materials and Methods for other details.
interpretation is that hydrolytic enzymes in these crude extracts degrade AP5A, and
probably also ATP, to ADP thereby causing inactivation.
Other treatments suggested that both ADP and ATP were essential for inactivation of pyruvate,Pj dikinase. No inactivation was observed for at least 30 min
when ATP was provided with an ADP consuming system consisting of creatine-P
and creatine-P kinase (Fig. 1). Furthermore, there was no inactivation when hexokinase plus glucose was provided with ADP to consume ATP either contaminating
the ADP or subsequently formed from ADP in the reaction.
The studies below were conducted with chloroplast extracts which proved to
contain much lower levels of enzymes that degrade nucleotides including AP5A.
However, they were not depleted of adenylate kinase which is largely located in
chloroplasts. For these studies an ADP sample was prepared by chromatography
(see Materials and Methods) which contained less than 0.4% ATP compared
with about 3% in the original preparation.
With extracts derived from washed mesophyll chloroplasts ATP alone did not
induce inactivation of pyruvate,Pj dikinase (Fig. 2). ADP induced inactivation
both with and without AP5A and this was not increased by adding ATP (Fig. 2a, b).
However, when hexokinase plus glucose were added to deplete any ATP present
there was no inactivation with added ADP. Inactivation mediated by ADP was
unaffected when either hexokinase or glucose was added separately, or when boiled
hexokinase was added with glucose (Table 1). Hexokinase was only partially
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%
LU
3
•
•*•*>».
Regulation of pyruvate,Pi dikinase
119
0.24
0.20
30
45
60
Fig. 2 Adenine nucleotide mediated inactivation of pyruvate.Pi dikinase in mesophyll chloroplast
extracts. Samples of Sephadex G-25 treated chloroplast extracts (see Materials and Methods)
were incubated at 25°C in the following basic medium: 20 mu Tris-HCl, pH 8.3, 5 mM DTT, 8 miu
MgCU and 2 mg ml"1 bovine serum albumin. Additions as shown were 0.2 mM AP5A, 1.2 mM ADP
and ATP, 2.5 mM glucose (Glu) and 5 units of hexokinase (HK). See Materials and Methods for
other details.
Table 1 Evidence for co-requirement for ADP and ATP for dikinase inactivation in chloroplast
extracts
Rate of dikinase inactivation
[units (ml reaction)"1 hr"1] *
Expt.
Additions to reaction "
Percentage of ADP control in brackets
ADP
ADP+HK (5 units)
ADP+HK (boiled)+Glu
ADP+HK + Glu
0. 42 (100)
0.42 (100)
0.43 (102)
0.03 (7)
ADP
ADP+HK (0.4 units)+Glu
ADP+HK (1 unit)+Glu
ADP+HK (5 units) +Glu
0.44 (100)
0.31 (70)
0.15 (34)
0.025 (6)
ADP
GDP
GDP+ATP
ATP
0.62 (100)
0.085 (13)
0.23 (37)
0.03 (5)
" Reactions including AP5A were as described in the legend of Fig. 2. Additions were 1 mM ADP,
hexokinase (HK), units as shown, 3 mM glucose (Glu), 2 mM GDP, 50 fiM ATP. Reaction components
were mixed and incubated for 5 min at 25°C prior to adding the chloroplast extract.
4
Units (/<mol min"1) of enzyme activity lost per ml of reaction per hr. Estimated from the initial
slopes of the inactivation curves or the time at which the curves cut the 25% inactivation value for
treatments showing rapid inactivation.
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60 0
15
TIME (min)
T. Sugiyama and M. D. Hatch
100
1
L
%
2
O
1-
4
ontr
o
• Minus ATP
* 45)JM ATP
'
s
60
0.5
1.0
ADP (mMI
1.5
All
-45^1 M ADP + ATP
—
<
f
2
0
u
v.
\
\ \
DiKir
40
\
20
45uM ADP
^^-
1
\ . 450M M ADP ± ATP^^«
1
1.35mM ADP± ATP
1
20
40
60
t
80
TIME (min)
Fig. 3 Effect, of varying ADP concentration with or without ATP. ADP was added to chloroplast
extracts in reactions of the same basic composition described in the legend of Fig. 2 (including 0.2 mM
AP5A in all treatments). The ATP concentration was 45 /IM. Inset is a plot of the initial rates of
inactivation of pyruvate,Pi dikinase with varying ADP.
effective when added at lower levels. Notably, the low rate of inactivation resulting
when ADP was replaced by GDP was stimulated about 3-fold by the inclusion of
50 IXM ATP.
These results support the view that a low level of ATP is a co-requirement, with
ADP, for pyruvate,P| dikinase inactivation. As shown later about half the maximum rate of inactivation is observed with about 55 /*M ADP. At this concentration
of ADP, the contaminating ATP would be initially less than 0.2 /MM, and yet normal
inactivation kinetics were observed (no lag) even when AP5A was added to inhibit
conversion of ADP to ATP (see Fig. 3). Trials with simulated reactions (in the
presence of 0.2 mM AP5A which inhibits adenylate kinase by about 99.8%) demonstrated that the ATP level would increase by less than 2 fiM in 30 min when 55 /XM
ADP added initially. These observations suggest that the requirement for ATP
must be satisfied by concentrations of less than 1 /J.M.
Nucleotide requirements for inactivation
Half maximum rates of inactivation were observed with about 55 ^M ADP both
in the presence and absence of 50 /J,M ATP (Fig. 3). As discussed above the lack of
any response to adding ATP is explained by its low concentration requirement.
Initial rates of inactivation were very low with either UDP or GDP in place of
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UJ
</)
8
6
LATI
INAC
\
80
S
^. »
10
s
i/ATI
120
Regulation of pyruvate,Pi dikinase
121
100
* ATP
80
N«—UDP
60 -
>
>
40 -
^ U D P + ATP
GDP + A
20 -
\v£-ADP± ATP
=*
i
20
i
40
TIME (min)
I
1
60
80
Fig. 4 Nucleotide specificity for inactivation of pyruvate,Pi dikinase.
The basic reaction mixtures
(with chloroplast extracts, see Fig. 2) were provided, as indicated, with 1.1 mM ADP, GDP or UDP
plus or minus 50 /IM ATP. AP5A (0.2 mM) was included in all treatments.
ADP although the rate of inactivation accelerated with time (Fig. 4). However,
when UDP or GDP were provided with ATP the initial rate of inactivation was
increased several fold but was still only about 35% of that observed with ADP.
The accelerating rates of inactivation observed in reactions with GDP or UDP
alone apparently were not due to the generation of their nucleoside triphosphate
counterparts in the reaction since GDP-mediated inactivation was accelerated by
ATP but not GTP (Table 2). Instead, it seems more likely that AP5A, added to
inhibit adenylate kinase, may be slowly degraded to provide effective levels of ATP.
Table 2 Nucleotide requirement for pyruvate,Pi dikinase inactivation in chloroplast extracts
Rate of dikinase inactivation
Additions"
[units (ml reaction)"1 hr"1] b
Percentage of ADP control in brackets
ADP
GDP
GDP+GTP
GDP+ATP
GTP
ATP
0.58 (100)
0.07 (12)
0.03 (5)
0.22 (38)
0
0.03
(0)
(5)
" Concentrations were 1 mM ADP, 2 mia GDP, 50 /*M GTP, 50 /IM ATP. Other conditions were as
described in the legend to Fig. 2.
* See footnote in Table I.
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V
122
T. Sugiyama and M. D. Hatch
Cyclic AMP had no effect on inactivation either alone or in combination with ADP
or ADP plus ATP (data not shown).
Effect of extract concentration
The percentage inactivation of pyruvate,P( dikinase varied more less directly
with extract concentration (Fig. 5). Hence, in terms of units of enzyme activity
lost the rate of inactivation was closely related to the square of extract concentration.
An interpretation of these data is that, in addition to active enzymes, the extracts
contain a second factor influencing inactivation. The following section cites
100
*
^
•
T
'.Trypsin treated
90
o
Q
80
\
CJ
^ \ T r y p s i n + inhibitor
o
s 70
>
—
>
H
(J
w
\w
\\ \
\\ \X.
60
LU
<
z
50
\
X
Control (+ADP\
Q
40
-
\
30
80
r
*
i
i
i
20
40
60
80
i
100
TIME (min)
Fig. 6
Fig. 5 Effect of varying extract concentration on inactivation. Varying amounts of chloroplast
extract was added to reactions of the composition described in the legend to Fig. 2. The total volume
was 0.365 ml and APsA (0.2 min) was included in all treatments. For each dilution treatments were
run without nucleotides and with 1.1 mM ADP plus 50 fiM ATP. For the reaction containing 0.3 ml
of chloroplast extract the initial pyruvate,Pi dikinase activity was 0.58 unit ml-1. Values in brackets
are the initial rates of enzyme inactivation for each treatment. The inset figure shows the relationship
between the rate of inactivation [units (ml reaction)"1 hr-1] and the square of extract concentration.
Fig. 6 Effect of trypsin and boiling pretreatments on inactivation: evidence for involvement of a
protein factor. Trypsin (Worthington, TPCK treated) and soybean trypsin inhibitor (Sigma, Type
1-S) were dissolved to make 8 mg ml"1 and 10 mg ml"1 solutions, respectively. A sample of trypsin
was treated with two volumes of the inhibitor solution for 10 min at 25°C. Chloroplast extract (350 y\)
was then incubated with either 5 /i\ of trypsin or 15 /JI of the trypsin plus inhibitor mixture for 5 min at
25°C. The trypsin treated extract was then provided with 10 /il of inhibitor. Another sample of the
chloroplast extract was heated at 100°C for 5 min. Samples of these chloroplast extracts were then
assayed for ADP-dependent inactivation in the standard assay system (see Materials and Methods
and Fig. 2) except that the mixtures were supplemented with purified pyruvate,Pj dikinase giving a
final activity in the reaction of 0.21 unit ml"1.
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100
T
'I1
~"
Boiled extract
Regulation of pyruvate.Pj dikinase
123
evidence that this factor is a high molecular weight protein present in leaf and
chloroplast extracts.
Evidence for a protein factor for inactivation
Inactivation in leaf extracts from other species
With whole leaf extracts from the C4 species Sorghum vulgare and Panicum
100
se 6 0 -
20
40
60
80 100
TIME (min)
120
140
Fig. 7 Reactivation of ADP-inactivated pyruvate,Pi dikinase in chloroplast extracts.
Enzyme in a
chloroplast extract was inactivated by incubation at 30°C with 1.3 miw ADP and 50 fiM ATP (see
Fig. 2 for other details). When the enzyme was about 90% inactivated the reaction was processed on
a Sephadex G-25 column to remove nucleotides and change the buffer to HEPES-KOH, pH 7.5,
(see Materials and Methods). Samples were incubated with either 6 mM DTT, 3 mM Pi or Pj
plus DTT.
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The following evidence supported the above inference that a protein factor or
factors, separate from active pyruvate,Pi dikinase, was required for inactivation:
(i) Purified pyruvate,Pj dikinase was not inactivated by ADP or ATP (results not
shown); (ii) various treatments of Sephadex G-25 filtered leaf extracts resulted in
loss of ADP dependent inactivation activity without affecting pyruvate,Pi dikinase
per se including exposure to a high concentration of ( N H ^ S C ^ (1M), incubation
at 25°C for 2-3 hr, and incubation at 0°C without D T T ; (hi) all ADP-dependent
inactivation potential was lost by incubating extracts without Mg 2+ and DTT
(results not shown), by boiling the extracts, or by treating the extracts with trypsin
(see Fig. 6). The latter treatments also destroyed pyruvate,Pi dikinase present in
the extracts so that assays for inactivation were conducted by adding purified pyruvate,Pi dikinase. As shown in Fig. 6, when trypsin was pretreated with soybean
trypsin inhibitor prior to incubation with the extract there was little or no loss of
ADP-dependent inactivating capacity.
124
T. Sugiyama and M. D. Hatch
maximum there was an initially rapid ADP dependent inactivation of pyruvate,Pi
dikinase after which the enzyme was reactivated. Such an effect has also been seen
with maize and is attributed to rapid destruction of nucleotides by phosphatases and
other hydrolytic enzymes. Chloroplast extracts from Atriplex spongiosa, Sorghum and
Panicum maximum gave rapid rates of inactivation with ADP and low or negligible
rates with ATP.
Reactivation of ADP-inactivated enzyme
Discussion
Earlier studies demonstrated that inactive pyruvate,Pi dikinase extracted
from darkened leaves is activated by a Pi-dependent reaction involving a high
molecular weight protein (Hatch and Slack 1969, Sugiyama 1974); the active enzyme
isolated from illuminated leaves is irreversibly inactivated in air without DTT and
reversibly inactivated by incubating with ADP or ATP (Hatch and Slack 1968,
1969). The present studies further define the nucleotide mediated inactivation
process. They also support a view based on recent studies on Pi-dependent activation (K. S. R. Chapman and M. D. Hatch, unpublished) that dark/light regulation of pyruvate,Pi dikinase does not involve interconversion of dithiol and
disulphide forms of the enzyme. This is contrary to an earlier conclusion (Hatch
and Slack 1969) and indicates a basic difference between pyruvate,Pi dikinase and
several other dark/light regulated photosynthetic enzymes (Buchanan 1980, Walker
1976). Not only did nucleotide-mediated inactivation proceed in the presence of
high concentrations of DTT but the inactive enzyme, like the enzyme isolated from
darkened leaves, was reactivated by Pi without DTT. Clearly, dark treatment of
ADP, ATP, protein factor
Fig. 8 Summary of factors affecting activation
and inactivation of pyruvate.Pi dikinase (PPD)
in vivo and in vitro.
j and protein factor,
inhibited by AMP
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Enzyme inactivated by incubating chloroplast extracts with ADP was processed
on Sephadex G-25 to remove ADP and then incubated with P4 plus or minus DTT
(Fig. 7). Like the inactive enzyme from darkened leaves (unpublished results of
K. S. R. Chapman and M. D. Hatch and also H. Nakamoto and T. Sugiyama)
the activity of the ADP-inactivated enzyme was largely restored by incubating with
Pi alone. Inclusion of DTT with Pi gave very little additional activity and there was
little activation with DTT alone. In different preparations of crude leaf extracts
(Chapman and Hatch, unpublished) activation with Pi alone varied between 65
and 100% of that recorded with P, plus DTT.
Regulation of pyruvate,Pi dikinase
125
We gratefully acknowledge the enthusiasm and expert technical assistance of Tony Agostino.
References
Buchanan, B. B. (1980) Role of light in the regulation of chloroplast enzymes. Ann. Rev. Plant
Physiol. 31: 341-374.
Feldhaus, P., T. Frolich, T. Goody, M. Tsakov and R. H. Schirmer (1975) Synthetic inhibitors of
adenylate kinase in the assay for ATPases. Eur. J. Biochem. 57: 197-205.
Hatch, M. D. (1977) Light-dark mediated activation and inactivation of NADP malate dehydrogenase in isolated chloroplasts from Zea mays. In Photosynthetic Organelles, Special Issue of Plant
& Cell Physiol. Edited by S. Miyacbi et al. p. 311-314. Japanese Society of Plant Physiolosists
and Center of Academic Publications Japan.
Hatch, M. D. (1978) Regulation of enzymes in C4 photosynthesis. In Current Topics in Cellular
Regulation 14. Edited by B. L. Horecker and E. R. Stadtman. p. 1-27. Academic Press, New
York.
Hatch, M. D. (1979) Regulation of C4 photosynthesis: Factors affecting the cold-mediated inactivation of pyruvate.Pi dikinase. Aust. J. Plant Physiol. 6: 607-619.
Hatch, M. D. and C. B. Osmond (1976) Compartmentation and transport in C4 photosynthesis.
In Encyclopedia Plant Physiology, New Series 3. Edited by C. R. Stocking and U. Heber. p. 144—
184. Springer-Verlag, Berlin.
Downloaded from http://pcp.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 17, 2016
leaves and incubation of extracted enzyme with ADP give comparable inactive
forms neither of which require reduction of a disulphide for activation.
The nucleotide-mediated inactivation process is extremely complex. Not only
ADP (replaceable by GDP or UDP), but also low concentrations of ATP, are
essential together with at least one protein factor. These findings are summarized
in Fig. 8.
This system has some resemblance to the process operating to regulate bacterial
glutamine synthetase (Stadtman and Chock 1978). In this case an adenylylated
species of the enzyme is formed by an enzyme catalysed reaction with ATP, and P,
removes the adenyl group to form free enzyme and ADP. A second protein factor
which regulates adenylylation of glutamine synthetase is, in turn, modified by
uridylylation by UTP. Clearly studies with more purified leaf systems will be
essential to answer the question of whether the pyruvate,Pi dikinase regulation
process is similar in principle.
The dark/light regulation of pyruvate,Pi dikinase appears to be linked to the
photosynthetic electron transport system (Yamamoto et al. 1974) presumably
through the effects of light on adenine nucleotides and Pi. Since AMP inhibits Pidependent activation (Hatch and Slack 1969) the prevailing activity of the enzyme
would be influenced by the levels of ATP, ADP and AMP as well as P,. However,
the concentration of ATP required for optimal effects on inactivation is very low
(apparently less than 1 /J,M) SO that changes in ATP levels may not have a critical
influence on regulation of pyruvate,Pi dikinase. At least with isolated chloroplasts
there are not large changes of ATP levels with changing illumination or the addition
of substrates (Kobayashi et al. 1979). In general, one could expect rises in ADP
and AMP levels in the dark, thus favouring inactivation. Anticipating changes
in the stromal levels of Pj during changes in light intensity or metabolite levels is
more difficult. The possibility that other metabolites may interact in this regulation
of pyruvate,Pi dikinase by Pj, ADP and ATP cannot be dismissed.
126
T. Sugiyama and M. D. Hatch
(Received September 22, 1980; Accepted December 6, 1980)
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