ISSN 0003-6838, Applied Biochemistry and Microbiology, 2018, Vol. 54, No. 3, pp. 320–323. © Pleiades Publishing, Inc., 2018.
Original Russian Text © A.T. Eprintsev, M.O. Gataullina, 2018, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2018, Vol. 54, No. 3, pp. 299–303.
Isolation, Purification, and Properties
of Peroxisomal Malate Dehydrogenase from Maize Mesophyll
A. T. Eprintsev
* and M. O. Gataullina
Voronezh State University, Voronezh, 394006 Russia
Received July 4, 2017
Abstract⎯Peroxisomal malate dehydrogenase (EC 126.96.36.199) with a specific activity of 533 U/mg (144-fold
purification) and a yield of 5% was obtained in a homogeneous state by a purification scheme including
sucrose gradient centrifugation from maize mesophyll. The Michaelis constants for the forward and reverse
reactions were determined to be 11.6 mM and 256 μM, and the pH optimum was 9.5 and 9.0, respectively.
Analysis of the molecular weight of the native enzyme and its subunits showed that the peroxisomal malate
dehydrogenase was a homodimer. It was established that the isolated and purified isoform of the enzyme had
a higher affinity for malate and NAD
in comparison with the mitochondrial and cytoplasmic isoforms.
Keywords: malate dehydrogenase, Zea mays, mesophyll, purification, Michaelis constant, peroxisomes
Malate dehydrogenase (MDH, NAD-dependent
oxidoreductase, EC 188.8.131.52) is widely distributed in
nature due to its polyfunctionality . MDH has a wide
spectrum of isoforms, which are usually characterized
by specific localizations in different compartments of
the plant cell. This allows the enzyme to participate in
Krebs cycle functioning, the glyoxylate pathway,
osmolyte synthesis, and nitrogen metabolism . Pre-
vious studies [3–6] described effective methods of puri-
fication of MDH from plants, bacteria, and animals,
which opened prospects to study their catalytic, kinetic,
and other characteristics of the enzyme.
Several MDH isoforms are present in the plant cell.
The physicochemical and regulatory properties of
mitochondrial and cytoplasmic isoenzymes charac-
terized by high polymorphism from the mesophyll of
maize leaves were studied [3, 7].
The study of the functioning of the MDH system in
peroxisomes, in which oxidation is not associated with
energy generation, is of particular interest. The pro-
cesses in peroxisomes are an essential link uniting
metabolite flows in the plant cell to the integrated sys-
tem [4, 8, 9].
It is known that glyoxylate cycle enzymes appear at
certain stages of ontogeny in peroxisomes; they are
involved in the reutilization of membrane lipids and
proteins of dying cells . NAD
-MDH, which can
participate in malate/oxaloacetate synthesis, amino
acid synthesis, metabolite transport between organelles,
and adaptive response to stress effects, also belongs to
such enzymes [11–14]. The malate dehydrogenase
enzyme complex is a convenient object for the study of
the mechanisms of the functioning of active centers of
oxidoreductases, regulation of enzymatic activity, and
NADH regeneration in the study of other enzymatic
systems (cascade enzyme), as well as for the solution of
various problems of enzymology and molecular biology
. In the food industry, MDH is used to determine the
quality of food products (measurement of the malate
content) and the synthesis of expensive oxaloacetate via
the transformation of a relatively cheap malate. MDH is
used as an enzyme marker in the immunoassay analysis
of many antigens, as well as in the diagnosis of various
diseases . Therefore, the search for sources for the
production of MDH that differing both in molecular
structure and substrate affinity remains important. In
this study, the mesophyll of maize leaves characterized
by a high metabolism level was used to isolate the per-
oxisomal form of the enzyme.
The goal of this study was to isolate and purify
malate dehydrogenase isoforms to homogeneity from
the mesophyll of maize leaves and to study their phys-
ico-chemical and catalytic properties of the homoge-
MATERIALS AND METHODS
Object of study. The objects of the study were
10-day-old maize seedlings (Zea mays, cultivar Voro-
nezhskaya 76) grown hydroponically at 25°C.
To isolate mesophyll tissue, maize leaves (2 g) were
homogenized in 10 mL of 100 mM Tris-HCl buffer,
pH 8.0, containing 10 mM MgCl
, 4 mM DTT, and
4 mM EDTA. The homogenate was filtered through