Plant Physiol. (1987) 85, 598-600
0032-0889/87/85/0598/03/$O 1.00/0
Changing Activity of Glucose-6-Phosphate Dehydrogenase from
Pea Chloroplasts during Photosynthetic Induction'
Received for publication February 17, 1987 and in revised form June 24, 1987
XIAo-HUA YUAN2 AND LOUISE E. ANDERSON*
Department ofBiological Sciences, University ofIllinois at Chicago, Box 4348, Chicago, Illinois 60680
ABSTRACT
Light inactivation of glucose 6-phosphate dehydrogenase is rapid and
occurs before photosynthetic 02 evolution is measureable in intact chloroplasts. Likewise, dark activation is rapid. The major light induced
change in the kinetic parameters of glucose 6-phosphate dehydrogenase
is in maximal velocity.
The first enzyme of the oxidative pentose phosphate pathway,
glucose 6-P dehydrogenase (EC 1.1.1.49), is inactivated when
pea chloroplasts are irradiated (3). This light inactivation probably prevents operation of a futile cycle involving glucose 6-P,
NADPH, and oxidative and reductive pentose phosphate pathway enzymes. We have now examined the kinetics of light
inactivation of glucose 6-P dehydrogenase in intact chloroplasts
during photosynthetic induction and the kinetic parameters of
the active (dark) and less active (light) forms of the enzyme.
Fickenscher and Scheibe (9) have partially purified cytosolic
glucose 6-P dehydrogenase from peas. Unlike the purified chloroplastic enzyme (20), the cytosolic enzyme was not DTT or
DTT/thioredoxin sensitive. On the basis ofthis lack of sensitivity
to DTT and the results of antibody experiments with extracts
from leaves which had been illuminated, Fickenscher and
Scheibe concluded that the cytosolic enzyme was not light inactivated. But in their antibody experiments the error in activity
determinations for the cytosolic enzyme from darkened and
illuminated leaves was 18 and 23%, respectively (Fig. 3 in Ref.
9). Since errors are additive, the error in the determination of
light inactivation was then the same as the expected change in
activity if the cytosolic enzyme were 40% inactivated by light.
Because previous work in this laboratory (4-6) indicated that the
cytosolic enzyme was light and DTT inactivated, we have reinvestigated the DTT-dependent inactivation of the enzyme in
whole leaf extracts.
MATERIALS AND METHODS
Photosynthetic 02 Evolution and Determination of Kinetic
Parameters. Chloroplasts were isolated from pea (Pisum sativum
L. var Little Marvel) seedlings and photosynthetic 02 evolution
assayed as described by Marques and Anderson (15) except that
the chloroplast concentration was increased. Where activity with
time of illumination was followed, the chloroplast content in the
ISupported by National Science Foundation grant DBM 84 17081
and Department of Energy grant 85-ER-60367.
2Permanent Address: Department of Biology, Peking University, Beijing, China.
02 evolution assay was equivalent to 250 to 280 Ag Chl ml-'.
Where the changes in kinetic parameters were investigated, the
chloroplast content during illumination was equivalent to 400 to
600ug Chl ml-'.
Glucose 6-P dehydrogenase activity was assayed spectrophotometrically. Change in A340 was followed on a Varian Cary 210
or 219 recording spectrophotometer.
When light inactivation and dark reactivation were measured
in the same experiment as photosynthetic 02 evolution, 10 ,l
aliquots (2.5-2.8 ,ug Chl) were removed from the 02 evolution
assay mixture directly into the assay cuvette, which contained
2.5 mm glucose 6-P (Na+), 0.2 mm NADP, 50 mM Hepes (K+),
10 mm KCI, 5 mM MgCI2, 1 mM EDTA (pH 7.8), and Triton X100 (0.03%) (final volume, 1 ml). The assay temperature was
24°C.
When the kinetic parameters were determined, an aliquot (1.5
ml) was removed from the O2 evolution assay mixture after 5
min incubation in the dark or 10 min incubation in the light,
diluted to 15 ml with 50 mM Hepes (K+), 10 mm KCI, 5 mM
MgCl2, 1 mM EDTA (pH 7.8), and centrifuged (27,000g, 15
min). Glucose 6-P dehydrogenase was partially reactivated during this procedure. After centrifugation aliquots (100 ,l, equivalent to 4-6 Ag Chl) of the supernate were used for the estimation
of kinetic parameters. The components of the assay were as
above except for the varied substrate and that Triton X- 100 was
omitted. Glucose 6-P was varied from 0.03 to 4 mm by even
reciprocal intervals for the Km (glucose 6-P) estimation. NADP
was varied from 0.2 to 2 AM by even reciprocal intervals for the
Km (NADP) estimation. Km and Vm. values were estimated from
steady state velocities with the aid of the IBM 3081 K64 computer
at the University of Illinois Chicago Computer Center and the
program of Hanson et al. (10). The reciprocal of the variance
was used as a weighting factor for estimation of mean Km and
SE.
For determination of the effect of pH and Mg2e on the kinetics
of the dark form of the enzyme, Tris (Cl-) (100 mM) replaced
Hepes (K+) in the assay and KCI, EDTA, and Triton X-100 were
omitted. Chloroplasts were lysed in the assay buffer, which
included MgCl2 when the pH was 7.8. The extract was centrifuged (27,000g, 20 min) to remove membranes and the supernatant solution was used as a source of enzyme.
Experiments with Whole Leaf Extracts. Pea plants (10-12 d
old) were placed in a dark cabinet overnight. In the morning 10
leaves were cut from the plants, rinsed in buffer, and homogenized with a glass tissue grinder in 4 ml of 100 mM Tris (Cl-)
(pH 7.8) containing Triton X-100 (0.1%) in darkness. The homogenate was centrifuged (27,000g, 5 min) and the supernatant
treated with DTT. In these experiments the Hepes (K+), KCI,
EDTA, MgCl2 buffer used in the assay in the experiments with
chloroplast extracts was replaced with 100 mm Tris (Cl-) (pH
7.8).
Protein was estimated by the method of Bradford (8).
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598 Biologists. All rights reserved.
Copyright © 1987 American Society of Plant
599
GLUCOSE-6-PHOSPHATE DEHYDROGENASE
RESULTS AND DISCUSSION
Inactivation of Glucose 6-P Dehydrogenase in Intact Chloroplasts. Light inactivation of glucose 6-P dehydrogenase in intact
pea chloroplasts is very rapid and occurs before photosynthetic
02 evolution is detected (Fig. 1). Activation of reductive pentose
phosphate cycle enzymes occurs at a comparable rate in this
system (16). Huber (13) has suggested that glucose 6-P dehydrogenase activity might contribute to the buildup of intermediates
during induction. However, in experiments which are comparable to the experiments reported here, we measured metabolite
levels and found that glycerate 3-P, triose-P, hexose 1,6-bisP,
sedoheptulose 1,7-bisP and ribulose 1,5-bisP levels remained low
and apparently constant during the first 2 min of illumination
(16). In the present experiments the dehydrogenase was almost
completely inactivated during the first 30 s of illumination. We
conclude that it is not likely that breakdown of glucose 6-P by
the dehydrogenase during light inactivation contributes substantially to the chloroplast metabolite pool.
Dark activation of glucose 6-P dehydrogenase is also very rapid
(Fig. 1). If levels of gluconate 6-P increase in proportion to
glucose 6-P dehydrogenase activity, then gluconate 6-P inhibition
of ribulose 5-P kinase (1) and ribulose bisP carboxylase (21)
probably helps to prevent operation of the reductive pentose
phosphate pathway in the dark.
Metabolically the most significant effect of light inactivation
of stromal glucose 6-P dehydrogenase is probably the decrease
in maximal velocity (Fig. 1; Table I). (This follows from a
consideration of the Michaelis-Menten equation. A change in
maximal velocity always results in a directly proportional change
in v, but a change in Km results in no change in v when S is high
and an inversely proportional change in v only when S is very
low.) The Km value for glucose 6-P decreases with the dark/light
transition, but the change is less than an order of magnitude and
may simply reflect the change in conformation of the enzyme
(20). The difference in the Km (NADP) values is small and only
slightly larger than the summed error in the determinations. It
seems unlikely that this change affects metabolism. Conversely,
the most significant effect of dark modulation is most likely the
increase in maximal velocity. The Km values reported here for
the enzyme is crude stromal extracts are comparable to those
reported for the purified enzyme (20). In earlier experiments,
under quite different conditions, we found no significant change
Table I. Km Values of Glucose 6-Phosphate Dehydrogenasefrom Pea
Chloroplasts Before and After Light Inactivation
Chloroplasts were illuminated for 10 min. The enzyme underwent
partial reactivation during centrifugation of the chloroplast extracts. Light
inactivation remaining at the time of these assays was between 40 and
65%. Dark maximal velocity values ranged from 2.9 to 10.5 ,umol
NADPH formed min-' mg-' Chl.
Varied
Substrate
Km
Dark Form
Light Form
JAM
1.3 ± 0.2 (2)
NADP
0.9 ± 0.1 (2)'
340 ± 20 (3)
140 ± 10 (3)C
Glucose-6-P"
b If
a Number in parentheses is number of separate determinations.
the substrate for the chloroplast enzyme is the d anomer as it is for other
glucose 6-P dehydrogenases (19), the Km (glucose 6-P) values are 220 uMm
for the dark form and 87 AM for the light form of the enzyme.
c Note
that this value is an average value in these experiments. The real value
at any time will be a function of the light treatment of the chloroplasts.
0.1'
0.017
f
Q
0.5
1
Glucose-6-P (nM)
FIG. 2. Effect of glucose 6-P levels on the activity of the dark form of
chloroplastic glucose-6-P dehydrogenase at pH 7.8 (0) and pH 7.2 (0).
The assay media was 100 mm Tris (Cl-), 0.2 mM NADP, and glucose
6-P at the indicated concentrations. MgCl2 (5 mM) was included in the
pH 7.8 assay. The protein concentration in the cuvette was 650 yg ml-'.
The n value was 1 when the pH was 7.8, and 2 when the pH was 7.2.
Similar results were obtained in a second experiment.
in the apparent affinity for glucose 6-P when the dehydrogenase
was light inactivated (4). In contrast light activation affects both
maximal velocity and Km in the case of the reductive pentose
phosphate cycle enzyme fructose bisphosphatase ( 15) and coopcm
E
erativity in the case of NADP-linked glyceraldehyde 3-P dehydrogenase
(14).
E
E
At pH 7.2, and in the absence of added Mg2", conditions which
S
E
may approximate the stroma in the dark chloroplast (1 1), the
kinetics become sigmoidal when glucose 6-P is the varied subz
strate (Fig. 2). Non-Michaelian kinetics have been reported for
E
Ec
the enzyme from several plant species (7, 12, 17, 18). Apparently
even in the dark the dehydrogenase will not be active unless
glucose 6-P concentrations exceed a threshold level. Because Pglucose isomerase has Michaelis-Menten kinetics (Km for glucose
6-P is about 0.2 mm, LE Anderson, MJ Hansen, unpublished
data),
glycolysis will be favored in the dark when hexose monoP
Time (min)
levels are low.
FIG. 1. Glucose 6-P dehydrogenase activity (data points) and rate of
DTT Inactivation of Glucose 6-P Dehydrogenase in Whole
C02-dependent 02 evolution (solid line without data points) from isolated Leaf Extracts. Clearly most of the enzyme in the whole leaf
chloroplasts after dark/light and light/dark transition at 25C. Light off extract, which is probably at least 50% cytosolic (6, 9), is DTT
and on as indicated by dark or light bar at top of figure. Similar results inactivated (Fig. 3). In experiments in which the leaves were
were found in two additional experiments.
chopped with razor blades and the debris, whole cells, and
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0
0
.-
0.,
a
z
Copyright © 1987 American Society of Plant Biologists. All rights reserved.
YUAN AND ANDERSON
600
Plant Physiol. Vol. 85, 1987
LITERATURE CITED
-~~~~~~~~~~~
40
Eo
-
Tkne (hr)
FiG. 3. Effect of DTT the activity of glucose 6-P dehydrogenase
in whole leaf extracts. Aliquots (10 1, 15 usg protein) were assayed for
activity. (0), control; (A), 50mm DTT; (A), 100 mM DTT. Similar results
were obtained in two additional experiments.
on
chloroplasts removed by filtration and centrifugation, DTT inactivated most of the dehydrogenase in the supematant solution
likewise (data not shown). Consistent with our previous results,
the present results indicate that native cytosolic glucose 6-P
dehydrogenase from pea leaves is DTT inactivated. Experiments
in which intact plants were irradiated were less definitive. There
about 25% inactivation of the enzyme in whole leaves and
in the supernatant after leaves were chopped with razor blades,
as above (data not shown). In earlier experiments (6) the chloroplastic and cytosolic forms of the enzyme separated electrophoretically were both apparently inactivated when seedlings
were irradiated. Notably, the dehydrogenase in crude whole leaf
extracts was quite stable (Fig. 3): Fickenscher and Scheibe (9)
reported a rapid loss of activity after breaking the leaf cells. It
seems likely that the cytosolic glucose 6-P dehydrogenase purified
by Fickenscher and Scheibe (9), like our first preparation of the
chloroplastic enzyme (2), lost sensitivity to DTT as a result of
partial denaturation during isolation.
was
Acknowledgments-We thank Lawrence Sykora and staff at the University of
Illinois at Chicago Greenhouse for cultivating the pea plants. Dominic Henry made
the figures with the aid of the IBM 3081 K64 computer at the University of Illinois
Chicago Computer Center.
1. ANDERSON LE 1973 Regulation ofpea leaf ribulose-5-phosphate kinase activity.
Biochim Biophys Acta 321: 484488
2. ANDERSON LE 1977 Isolation of pea leaf chloroplast glucose-6-phosphate
dehydrogenase by affinity gel chromatography. Plant Physiol 59: S-6
3. ANDERSON LE 1986 Light/dark modulation of enzyme activity in plants. In
JA Callow, ed, Advances in Botanical Research, Vol 12. Academic Press,
London, pp 1-46
4. ANDERSON LE, J DUGGAN 1976 Light modulation of glucose-6-phosphate
dehydrogenase. Plant Physiol 58: 135-139
5. ANDERSON LE, SC NEHRLICH 1977 Photosynthetic electron transport system
controls cytoplasmic glucose-6-phosphate dehydrogenase activity in pea
leaves. FEBS Lett 76: 64-66
6. ANDERSON LE, TCL NG, K-EY PARK 1974 Inactivation of pea leaf chloroplastic and cytoplasmic glucose 6-phosphate dehydrogenase by liht and
dithiothreitol. Plant Physiol 53: 835-839
7. ASIHARA H, A KOMAMINE 1976 Characterization and regulatory properties of
glucose-6-phosphate dehydrogenase from black gram. Physiot Plant 36: 5259
8. BRADFORD MM 1976 A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding. Analyt Biochem 72: 248-254
9. FICKENSCHER K, R SCHEIBE 1986 Purification and properties of the cytoplasmic
glucose-6-phosphate dehydrogenase from pea leaves. Arch Biochem Biophys
247: 393-402
10. HANSON KR, R LING, E HAVIR 1967 A computer program for fitting data to
the Michaelis-Menten equation. Biochem Biophys Res Commun 29: 194197
1 1. HELDT HW, CJ CHON, R MC LILLEY, AR PORTIS 1978 Role of factose and
sedoheptulosebisphosphatase in the control of CO2 fixation. Evidence fiom
the effects of Mge' concentration, pH and H202. In DO Hall, J Coombs,
TW Goodwin, eds, Proceedings of the 4th International Congress on Photosynthesis, Reading, England, pp 469-478
12. HOOVER JD, SH WENDER, EC SMITH 1977 Isoenzymes of glucose-6-phosphate
dehydrogenase from tobacco cells. Phytochemistry 16: 195-197
13. HuBER SC 1979 Orthophosphate control of glucose.6-phosphate dehydrogenase light modulation in relation to the induction phase of chloroplast
photosynthesis. Plant Physiol 64: 846-851
14. MACIOSZEK J, LE ANDERSON 1987 Changing kinetic properties of the two
enzyme phosphoglycerate kinase/NADP-4iinked glycerlehyde-3Vhophate
dehydrogenase couple from pea chioroplss during photosynthetic induction. Biochim Biophys Acta 892:185-190
15. MARQUES IA, LE ANDERSON 1985 Changing kinetic properties of fiuctose-1,6bisphosphatase from pea chloroplasts during photosynthetic induction. Plant
Physiol 77: 807-8 10
16. MARQUES IA, DM FORD, G MUSCHINEK, LE ANDERSON 1987 Photosynthetic
carbon metabolism in isolaed pea chl6roplasts: Metabolite levels and enzyme activities. Arch Biochem Biophys 252: 458-466
17. MUTO S, I URITANI 1972 Glucose 6-phosphate dehydrogenase from swet
potato: effect of various ions and their ionic strength on enzyme activity.
PlantCellPhysiol 13: 111-118
18. SCHAEFFER F, RY STANIER 1978 Glucose-6-phosphate dehydrogenase of Anabaena sp. Arch Microbiol 116: 9-19
19. SCHRAY KJ, SJ BENKOVIC 1978 Anomerization rates and enzyme specificity
for biologically important sugars and sugar phosphates. Accounts Chem Res
11: 136-141
20. SRIVASTAVA DK, LE ANDERSON 1983 Isolation and characterization of lightand dithiothreitol-modulatableglucose-6-phosphate dehydrogenase from pea
chloroplasts. Biochim Biophys Acta 724: 359-369
21. TABITA FR, BA McFADDEN 1972 Regulation of ribulose-1,5-diphosphate
carboxylase by 6-phospho-D-gluconate. Biochem Biophys Res Commun 48:
1153-1159
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