Journal of Experimental Botany, Vol. 42, No. 237, pp.

537-540, April 1991

Equilibration of Adenylates by Maize Leaf

Adenylate Kinase: Effects of Magnesium on
Apparent and True Equilibria
LESZEK A. KLECZKOWSKI 1 and DOUGLAS D. RANDALL
Department of Biochemistry, 117 Schweitzer Hall, University of Missouri, Columbia, MO 65211, USA
Received 16 July 1990

ABSTRACT
In this study, the effect of magnesium on equilibration of adenylates by purified maize leaf adenylate kinase (AK) was
investigated. The equilibration was expressed in terms of either apparent equilibrium constant, defined as K,pp =
(ATP toul )(AMP u ,)/(ADP tot .,) 2 , or true equilibrium constant, defined as tftnie = (Mg-ATP)(AMP fr )/(Mg-ADP)(ADP fre ,). At a
fixed concentration of free magnesium (1-8-1-9 mM), the K,pp and Ktrac were constant at 0-76 + 0-10 and 6-O20-75, respectively.
On the other hand, at the free magnesium range of 00014 to 8-3 mM, the Kmpp varied from 0-30 to 1-27, while A^true remained
constant at 5-93 0-31. The data indicate that, contrary to previous speculations, leaf AK does not maintain an equilibrium of
total adenylates. Rather, the enzyme governs an equilibrium of Mg-ADP, free ADP, Mg-ATP, and free AMP, which are the true
substrates/products of the AK. reaction. Some implications of this finding for studies on energy metabolism in plant tissues are
discussed.
Key words: Adenylate kinase, adenylate energy charge, adenylates, C 4 -photosynthesis, magnesium.

INTRODUCTION
Adenylate kinase (AK) catalyses a freely reversible reaction of 2 ADP<->ATP + AMP. The enzyme requires magnesium for catalysis, which reflects the fact that AK
utilizes one molecule each of the magnesium-complexed
and free adenylates as substrates in either direction of the
reaction, i.e. Mg-ADP and free ADP as well as Mg-ATP
and free AMP, respectively. This property has been
demonstrated for AK from yeast (Khoo and Russell,
1970), animal tissues (Bowen and Kerwin, 1956; Rose,
1968; Rhoads and Lowenstein, 1968; Blair, 1970; Punch
and Fromm, 1972) and, recently, for AK from maize
leaves (Kleczkowski and Randall, 1986; Kleczkowski,
Randall, and Zahler, 1990). The leaf enzyme shows higher
rates in the direction of AMP utilization (reverse reaction)
(Hatch, 1982; Kleczkowski and Randall, 1986; Manetas,
Stamatakis, and Samaras, 1986) and its Km values for
substrates are in the micromolar range (Kleczkowski et
al., 1990). These characteristics seem important since in
C4 plants AK is believed to be metabolically linked to
pyruvate, orthophosphate dikinase activity, which produces AMP and phosphoenolpyruvate, the latter being
1

the primary carbon acceptor during C4 photosynthesis

(Hatch, 1982; Nakamoto and Edwards, 1987; Usuda,
1988).
The elucidation of the nature of true substrates of
maize AK (Kleczkowski et al., 1990) poses a question
regarding the magnitude of the true equilibrium constant
(Klruc) of leaf AK. Previously, it has been assumed that
leaf AK maintains an equilibrium of total adenylates
(expressed as A^app) rather than that of the magnesiumcomplexed and free adenylates (Kobayashi, Inoue, Furuya, Shibata, and Heber, 1979; Pradet and Raymond,
1982). There has been no consensus concerning the exact
magnitude of the A^app (values ranging from 0-4 to 1-2),
although it has been recognized that the constant may
vary depending on magnesium concentration (Bomsel and
Pradet, 1968; Pradet and Raymond, 1982).
In the present study, apparent and true equilibrium
constants of maize AK were determined, and the role of
magnesium as a 'regulator' of the AK mass action ratio
was assessed. The data indicate that, similarly to AK
from yeast and non-plant tissues, the maize enzyme

To whom all correspondence should be addressed at: Plant Molecular Biology Laboratory, NLVF, P.O. Box 51, 1432 As-NLH, Norway.

Oxford University Press 1991

538

Kleczkowski and RandallEquilibration of Adenylates by Adenylate Kinase

maintains an equilibrium of Mg-ATP, free AMP, MgADP, and free ADP, rather than that of total adenylates.
MATERIALS AND METHODS
Reagents
Maize leaf AK was purified as previously described (Kleczkowski and Randall, 1986). The enzyme was homogeneous as
determined by SDS-electrophoresis (Kleczkowski and Randall,
1986) and by western blotting using rabbit antibodies prepared
against this protein (Kleczkowski and Randall, 1987, 1988).
AMP and ATP were from P-L Biochemicals, while ADP was
from Sigma. Pyruvate kinase, lactate dehydrogenase (both from
rabbit muscle), hexokinase (yeast), and glucose-6-phosphate
dehydrogenase (Leuconostoc mesenteroides) were from Sigma.
Assays of adenylates
Prior to experiments, total concentrations of AMP, ADP,
and ATP in stock solutions were determined enzymatically, as
follows: (a) assay of total AMP100 mM Tricine (pH 7-8),
0-5 mM ATP, 50 mM MgCl2, 10 mM phosphoenolpyruvate,
60 mM KC1, 0-2 mM NADH, and one unit each of pyruvate
kinase, lactate dehydrogenase and maize AK; (b) assay of total
ADP100 mM Tricine (pH 7-8), 5-0 mM MgCl2, 10 mM phosphoenolpyruvate, 60 mM KC1, 0-2 mM NADH, and one unit
each of pyruvate kinase and lactate dehydrogenase; (c) assay of
total ATP100 mM Tricine (pH 7-8), 4-0 mM MgCl2, 50 mM
D-glucose, 0-5 mM NAD and one unit each of hexokinase and
glucose-6-phosphate dehydrogenase. Coupling enzymes were
desalted on a small Sephadex G-25 column prior to assays. All
assays were carried out at 25 C by monitoring NAD(H) oxidation/reduction at 340 nm. Control assays (minus adenylates)
were always carried out to correct for non-specific NAD(H)
oxidation/reduction. Assay volumes were 1-0 cm3, for each
nucleotide.
Contents of incubation mixtures for determination of the
K,pp and K{Tac of AK and details of the incubation, are described
in legends to Fig. 1 and Table 1. Equilibrium concentrations of
total adenylates were based on assays of total ATP, as described
above. As found in preliminary experiments, both the amount
of AK and the duration of the incubation in studies described
in Table 1 and Fig. 1 were more than sufficient for a full
equilibration of adenylates. After determination and/or calculation of total adenylates in the equilibration mixtures, at a given
total magnesium, the data were fed to a computer program
described by O'Sullivan and Smithers (1979) which allowed
calculations of the magnesium-complexed and free adenylates
and of free magnesium. The following stability constants for
complexation of magnesium with adenylates were used:
*M..AMP = 69-4 M - \ tfM,.ADp = 390OM-\ and *M,-ATP = 69 700

M" 1 (O'Sullivan and Smithers, 1979).

The true equilibrium constant of AK was defined as Kirat =
(Mg-ATP)(AMPfree)/(Mg-ADP)(ADPfrec). The apparent equilibrium constant was K.Pp = (ATP1M,,)(AMP10Ul)/(ADPloUl)2One unit of AK activity was defined as the amount of the
enzyme required to oxidise one /tmol NADH under assay
conditions previously described (Kleczkowski and Randall,
1988).

RESULTS
Two approaches were undertaken to determine the Klnic
and A^.ppof maize AK. Firstly, conditions of the equilibration were chosen where a total pool of adenylates (a sum
of total AMP, ADP, and ATP) varied at a relatively

constant free magnesium (Table 1). Specifically, the ratio

of total magnesium to total adenylates varied from 6 to
16 (for the 126 to 337 ^M range of adenylates at 20 mM
MgCl2), but free magnesium varied only from about 1 -77
to 1-90 mM. Under these conditions, both KlTXlt and A",,,,,
values were constant at 602 0 7 5 and 076 0 1 0 , respectively. The small variation of free magnesium in experiments described in Table 1 was unlikely to have any
considerable effect on the AK-mediated equilibria of
adenylates (see also Fig. 1).
The second approach concerned the effect of free
magnesium on the KBpp and KtTUC of the enzyme (Fig. 1).
In this case, the equilibration was carried out at a fixed
concentration of total adenylates (215 mM) and varying
free magnesium. A range of free magnesium from 00014
to 8-3 mM caused a 4-fold change of the magnitude of
the K,pp of AK. At the two extremes of free magnesium
concentration, the Kipp had a common value of about
0-30-0-33. An increase of free magnesium from 0-0014 to
015 mM raised the Kapp up to the value of 1 -27. Further
increase of free magnesium resulted in lowering of the
Free magnesium had no effect on the Klnie of maize
AK (Fig. 1). The A"lrue was 5-930-31, which compares
to the value of 602 + 0-75 determined under conditions
of a relatively constant free magnesium and a changing
total adenylate concentration (Table 1).
DISCUSSION
The evidence with respect to the KtTUC and A^pp of maize
AK (Table 1; Fig. 1) essentially confirms the results of
previous studies on the enzyme from yeast and non-plant
tissues (Bowen and Kerwin, 1956; Rose, 1968; Blair, 1970;
Purich and Fromm, 1972). These earlier investigations
provided both an experimental and/or a theoretical basis
TA BLE 1. Determination of an equilibrium constant of maize leaf
adenylate kinase
Incubation mixtures (I 0 cm 3 each) contained 100 mM Tricine (pH 8-0),
40-5 pM total AMP, 73 ^M total ATP, indicated concentration of total
ADP, and 20 mM MgCl 2 . Reactions were initiated by addition of 0-1
unit of purified maize AK. Incubation was carried out at 25 C for 150
min and then mixtures were heated at 100 C for 10 min, centrifuged
at 10000xg for 5 min to remove denatured protein and frozen
overnight. Equilibrium concentrations of adenylates (total, free and
magnesium-complexed) were determined and calculated as described in
the Materials and Methods section. Errors quoted here are 9 5 %
confidence limits.
Initial [ADP 1OU] ]

12-8

25-6
70-4
800
140-8
179-2
2240

5-24
5-97
7-27
4-92
6-71
6-26
5-74
x = 6O20-75

0-63
0-72
0-88
0-63
0-87
0-81
0-75
x = 0-760-10

Kleczkowski and RandallEquilibration of Adenvlates by Adenylate Kinase

1.4

- 10
1.2
'

(
- 8

1.0

o
/

aa.

oo

0.6

0.4

0.8

(J
(

- 6

- 4

- 2

0.2

-6

-5

-4

-3

-2

Log CMg f r e e ], M
FIG. 1. Effects of free magnesium on the apparent and true equilibria
of adenylates governed by maize leaf adenylate kinase. Incubation
mixtures contained 100 mM Tncine (pH 80), 215 mM total ADP,
varying [MgCl2] (0-0014-8-3 mM), and 0-1 unit of purified maize leaf
AK. Incubation was carried out for 5 h (25 C) and then the enzyme
was precipitated by heating at 100 C for 8 min, followed by freezing
at minus 20 C. Equilibrium concentrations of adenylates (total, free
and magnesium-complexed) and those of free magnesium were determined and calculated as described in the Material and Methods section.
The K,nc calculated from these data was 5-93 0 3 1 (95% confidence
limit).

for the current understanding of the action of AK.

Namely, they have established the nature of true substrates of AK and demonstrated that the mass action
ratio of total adenylates (A^pp) governed by AK is profoundly affected by changes of free magnesium. Rose
(1968) has predicted, using stability constants for the
complexation of adenylates with magnesium which were
slightly different from those used in the present study,
that the KBpp of AK (at pH 7-5, 35 C) would be the
lowest at infinitively low and infinitively high free magnesium concentration, approaching values of 0-37 and 017,
respectively, while the highest Ktpp of 111 would be
reached at about 0 2 mM free magnesium. A more elaborate model, developed by Blair (1970), predicts values of
the Kspp which are essentially similar to those found
experimentally in the present study: about 0-3 for the
extremes of magnesium concentration, and the value of
1-3 for the highest A",pp (at about 0-2 mM free magnesium).
Also, in the Blair model, the Klrve of AK would not be
affected by free magnesium, being fixed at 5-31, which is
similar to the values of 602 and 593 calculated from our
results (Table 1; Fig. 1). As emphasized by Blair (1970),
and further discussed by Punch and Fromm (1972), the
dependence of the A^.pp of AK on free magnesium is to

539

be expected and can be mathematically predicted, based

on the nature of true substrates of AK.
The knowledge about true substrates and equilibrium
constants of AK is of importance for studies on many
aspects of energy metabolism, including the so called
'adenylate energy charge' (AEC) concept, developed by
Atkinson (1968). The predicted response of enzymes or
processes to AEC assumes that the ^ a p p of AK is fixed
at about 0-8 (Atkinson, 1968; Bomsel and Pradet, 1968).
The AEC concept does not take into account the strong
dependence of the A^app of AK on magnesium, nor the
fact that magnesium does fluctuate under physiological
conditions. The most notable example of this for plant
tissues might be a change in magnesium concentration
during light/dark transition in the chloroplast stroma
(Krause, 1977; Portis, 1981). The inherent property of
AK from yeast and non-plant tissues to utilize one
molecule each of the magnesium-complexed and free
adenylates as substrates, and the variation of its K,pp
depending on levels of free magnesium have frequently
been cited as the key argument against validity of the
AEC theory (Rose, 1968; Blair, 1970; Purich and Fromm,
1972, 1973); it appears now that this argument holds for
higher plant tissues as well.
Despite its dependence on free magnesium, the A^pp of
AK might still be a useful indicator of energy metabolism
in plant tissues. Any measurement of levels of total
adenylates in vivo (e.g. in chloroplasts or in the cytosol),
resulting in A^app values lower than 0 3 or higher than 1-3,
could indicate that the adenylates are not equilibrated by
AK (see also Kobayashi et al., 1979; Pradet and Raymond, 1982). Rose (1968) has demonstrated, using human
blood red cells, that knowledge about the Kapp might also
help in an indirect estimation of free magnesium in a
given tissue, especially at low concentrations of this ion.
As shown in Fig. 1, the experimentally determined A^pp
might be related to a given free magnesium level(s)
(assuming, of course, that adenylates are fully equilibrated
by AK). It remains to be seen whether this approach,
used successfully with erythrocytes, finds any application
with respect to plant tissues.
ACKNOWLEDGEMENTS
We thank Dr Warren L. Zahler for his expert advice
during the development of this research. This research
was supported in part by the National Science Foundation
grant #DMB-8506473. This is journal report No. 11 296
from Missouri State Agricultural Experiment Station.
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ATKINSON,

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