OBSERVATIONS ON THE RELATIVE
ELECTROPHORETIC MOBILITY OF PISUM
SEED AMYLASES
J. Przybylska1, S.
Blixt2, Z. Zimniak-Przybylska1,
and J.
George1
1- Institute of Plant Genetics,
Polish Academy of Sciences,
Poznan, Poland
2- Weibullsholm Plant Breeding Institute, Landskrona,
Sweden
Two procedures, called here
Method A and Method B, were used
to detect variation in electrophoretic
patterns of pea seed amy-
lases (2,3). In Method A proteins were
separated in 9% polya-
crylamide gels, in a discontinuous buffer system
according to
Davis (1); following electrophoresis gels were incubated
for 5 hrs
in 1% solution of
soluble starch in 0.2 M acetate buffer, pH 5.3,
and then stained with I2-KI solution(2). In Method B the
electrode
buffer was 0.125 M Tris-H3B03, pH 8.9,
instead of Tris—
glycine, pH 8.5, and hydrolyzed starch was
incorporated into gels
instead of being put into the incubation mixture
(3). In both
procedures, electrophoresis was continued until after the
dye mar-
ker (bromophenol blue) had reached the anodic edge of a
gel.
Zymograms obtained with Method A
showed two anodic variant
zones of enzyme activity; the faster moving
bands, forming variant
zone Amy-1, were well defined while the slower
bands of Amy-2
variant zone were very faint, scarcely discernible (2).
In Method
B separation and detection of amylase-variants were
significantly
improved but Amy-1 variants were not revealed; marked
destaining
near the start of migration was at first not attributed to
the
Amy-1 variant zone (3).
In further investigations with
the use of Method B different
commercial products of hydrolyzed and
soluble starch were tried.
Results obtained with the hydrolyzed starch
and with the soluble
starch "Analar" from BDH were as reported earlier.
However, zymo-
grams obtained with the use of a soluble starch
purchased from
POCh, Gliwice, Poland, showed slow migrating bands
forming an ad-
ditional variant zone. The additional zone seemed to
correspond
to the Amy-1 zone on zymograms obtained with the use of
Method A
(Fig. 1). It was therefore assumed that under conditions
of
Method A and Method B variants forming Amy-1 and Amy-2 zones
mi-
grate in a reverse order. This was confirmed by eluting the
Amy-1
and Amy-2 variant bands from a gel obtained with Method A,
fol-
lowed by re-electrophoresis under conditions of Method B (Fig.
2).
It should be mentioned, however, that in the case of the
Amy-2
variant, besides the characteristic fast moving band two
addi-
tional slow moving bands could be seen. These additional
bands
are probably artifacts, possibly aggregations.
The order of electrophoretic
mobilities of the respective
Amy-1 variants in gels obtained with
Method A and Method B seems
to be similar (see Fig. 1). However, on
screening a wider plant
material differences may be
found.
