1021-4437/02/4902- $27.00 © 2002
Russian Journal of Plant Physiology, Vol. 49, No. 2, 2002, pp. 211–215. From Fiziologiya Rastenii, Vol. 49, No. 2, 2002, pp. 238–242.
Original English Text Copyright © 2002 by Salahas, Cormas, Zervoudakis.
Phosphoenolpyruvate carboxylase (PEPC; EC
22.214.171.124) and pyruvate orthophosphate dikinase
(PPDK; EC 126.96.36.199) located in the cytosol and chloro-
plasts of the C
mesophyll cells, respectively, are oligo-
meric enzymes of the C
-photosynthetic pathway [1, 2].
Both enzymes from C
plant leaf extracts have been
reported to be unstable under dilution and/or cold labil-
ity [3–5]. The cold lability of PEPC and PPDK has been
attributed to the dissociation of the tetrameric enzymic
form to dimers or monomers at low temperatures [3, 6].
It has also been found that pH, protein concentration,
and a number of compatible solutes modify the cold
lability of both enzymes [4, 7–9]. The PPDK cold labil-
ity varies widely between C
plant species  and cul-
tivars within species [11, 12]. Recently, a wide varia-
tion of PEPC cold inactivation among various C
has been observed . It was also shown that PEPC
from two C
differ in sensitivity to
cold inactivation . Moreover, cold treatment of
leaves from C
plants causes a loss of PPDK [7, 10] but
not of PEPC activity .
Because of the similarity between both enzymes
extracted from different plants in terms of molecular
mass, oligomericity and inactivation due to dilution
and/or cold treatment, we tried to investigate the cold
inactivation of PEPC and PPDK from the same C
and enzyme protection by some compatible solutes.
MATERIALS AND METHODS
L. is a C
growing naturally at the area of Patras. Mature leaves
were collected under full sunlight, placed into plastic
bags and taken immediately to the laboratory.
Extraction and desalting of extracts.
Leaves (1 g)
ﬂoating on water were illuminated (1 mmol/(m
room temperature for 45 min in order to fully activate
PEPC and PPDK and then ground in a mortar with puri-
ﬁed sea sand and 4 ml of extraction medium containing
100 mM Tris–HCl, pH 7.8, 10 mM MgCl
, 1 mM
EDTA, 5 mM 1,4-dithio-DL-threitol (DTT), 2 mM
and 2% (w/v) polyvinylpyrrolidone (PVP)
supplemented with a small amount (100 mg) of insolu-
ble PVP. The extract was centrifuged at 8000
5 min, and the clear supernatant was desalted using a
1 cm column of Sephadex G-25 (Sigma, United
States) which had been equilibrated with 50 mM Tris–
HCl, pH 7.5, containing 0.1 mM EDTA, 4 mM MgCl
and 5 mM DTT. The efﬂuent volume, used as desalted
extract, was double that layered on the column. All
steps were carried out at room temperature.
PEPC activity was assayed in 3-ml ﬁnal volume of
a buffer containing 100 mM Tris–HCl, pH 8.0, 5 mM
, 10 mM MgCl
, 4.5 units of malate dehydro-
Cold Inactivation of Phosphoenolpyruvate Carboxylase
and Pyruvate Orthophosphate Dikinase from the C
G. Salahas*, E. Cormas**, and G. Zervoudakis**
*Department of Greenhouses and Floriculture, Technological Institute, Mesologgi, Greece
**Department of Biology, Laboratory of Plant Physiology, University of Patras, Patras, Greece;
fax: 30(631)-58207; e-mail: firstname.lastname@example.org
Received March 15, 2001
—Phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) cold
inactivation was studied in leaf extracts from
Atriplex halimus L.
Both enzyme activities gradually reduced as
the temperature and the total soluble protein decreased. Mg
at a concentration of 10 mM stabilized PEPC and
PPDK activities against cold inactivation. At low Mg
concentration (4 mM), PEPC was strongly protected by
phosphoenolpyruvate, glucose-6-phosphate, and, partially, by
-malate, while PPDK was protected by PEP, but
not by its substrate, pyruvate. High concentrations of compatible solutes (glycerol, betaine, proline, sorbitol and
trehalose) proved to be good protectants for both enzyme activities against cold inactivation. When illuminated
leaves were exposed to low temperature, PPDK was partially inactivated, while the activity of PEPC was not
Key words: Atriplex halimus - cold inactivation - in vitro stabilization - phosphoenolpyruvate carboxylase -
pyruvate orthophosphate dikinase
* This article was submitted by the authors in English.