1022-7954/01/3705- $25.00 © 2001
Russian Journal of Genetics, Vol. 37, No. 5, 2001, pp. 581–583. Translated from Genetika, Vol. 37, No. 5, 2001, pp. 708–711.
Original Russian Text Copyright © 2001 by Zasypkin, Lapinskii, Primak.
Mannosephosphate isomerase (Mpi, EC 220.127.116.11) is
an enzyme catalyzing conversion of D-mannose-6-
phosphate into D-fructose-6-phosphate [1–3]. This
enzyme is polymorphic in many species. On electro-
phoregrams heterozygous variants are distinguished as
two bands. This is consistent with monomeric quater-
nary structure of the enzyme subunits . Since molec-
ular weight of the enzyme is not high, electrophoresis
in 6 to 7% polyacrylamide gels at 200 V should not last
more than 50 min or 1 h .
Glucosephosphate isomerase (Gpi, EC 18.104.22.168)
catalyzes conversion of D-fructose-6-phosphate into
D-glucose-6-phosphate [1–3]. Molecular weight of this
enzyme is far higher. For this reason, for best allozyme
separation, electrophoresis in similar to the Mpi condi-
tions should last for 2 to 2.5 h. Heterozygous variants
are detected on electrophoregrams as three bands,
which is typical to the markers with dimeric quaternary
structure . Although standard methods of detection
of both enzymes are rather simple and reliable [2, 3],
they have some shortcomings.
Mannose-6-phosphate and fructose-6-phosphate
(the substrates unique only for these enzymes) are typ-
ically used in the form of cheap barium salts (other salts
are much more expensive and less stable upon storage).
However, the Ba
cations can convert the dye anions
needed for the reaction mixture into insoluble salts. At
slight substrate overdosage, these salts can form non-
speciﬁc blue background in the block, thus affecting
staining quality of both enzymes.
We suggest to use enzymatic pathway for direct pro-
duction of mannose-6-phosphate from D-mannose, and
fructose-6-phosphate from D-fructose. This can make
Mpi a widely available genetic marker improve the
quality of staining at detection of both Mpi and Gpi due
to the absence of nonspeciﬁc background.
The method is based on the observation that in the
presence of adenosine triphosphate and magnesium
ions, hexokinase (EC 22.214.171.124.) catalyzes attachment of
the ATP phosphate group (which is thus converted into
ADP) to the sixth position of most D-hexoses. More-
over, the afﬁnity of hexokinase to D-mannose is the
highest with regard to other hexoses . The enzyme’s
afﬁnity to D-fructose is also sufﬁciently high.
The requirements for the reagents are as follows. We
usually utilize lyophilized hexokinase (Fluka, Switzer-
land), which can be stored in laboratory refrigerator for
about 20 years without loss of activity. The quality of
ATP and the manufacturer are inessential. D-mannose
and D-fructose may be of any quality.
The reaction mixture after solution of all compo-
nents and prior to staining is incubated at 37
30 min. Similarly to a standard technique, all other com-
ponents are separately dissolved in 5 ml of 0.5 M Tris–
HCl buffer (pH 7.1 to 7.5) (solution B).
Composition of the reaction mixture
(solution A for substrate production):
D-mannose 10 mg (for Mpi detection)
D-fructose 10 mg (for Gpi detection)
ATP 10 mg
Hexokinase 0.5 mg
0.5 Tris–HCl buffer, pH 7.1–7.5 5 ml
Composition of the reaction mixture (solution B):
MTT 3 mg
NADP 5 mg
Gpi (Glucosephosphate isomerase; WS) 10 µl
G-6-PDH (glucose-6-phosphate dehydrogenase; WS) 10 µl
PMS(phenazinemethane sulphate; WS) 1 ml
A Modified Method for Detection of Post-Phoretic Activities
in Mannose- (Mpi, EC 126.96.36.199)
and Glucose- (Gpi, EC 188.8.131.52) 6-Phosphate Isomerases
M. Yu. Zasypkin, A. G. Lapinskii, and A. A. Primak
Institute of Biological Problems of the North, Russian Academy of Sciences, Magadan, 685000 Russia;
fax: (41322)344-63; e-mail: email@example.com
Received May 17, 2000; in ﬁnal form, October 30, 2000
—A method for detecting activities of mannose- and glucose-6-phosphate isomerases based on
enzyme production of the substrates is described. The results obtained for several animal taxa are illustrated by