Biology Reference
In-Depth Information
the 1980s [
11
,
12
] and is the most widely adopted method to
prepare highly enriched plasma membranes from plants [
13
,
14
].
The method is based on surface properties of the membrane [
15
]
that alter during plant development and by biotic or abiotic stress
factors due to changes in lipid and protein composition. The over-
all composition of the plasma membrane can differ between plant
species and tissues as well as from one variety to another. Those
differences and alterations were clearly demonstrated by pro-
teomic and lipodomic approaches [
16
-
19
]. Changes in the mem-
brane surface properties may affect yield and purity of plasma
membrane preparations. This has to be kept in mind, if aqueous
polymer two-phase partitioning is adopted. An example of this
method is described, which has been shown to produce highly
enriched plasma membrane fractions for maize (
Zea mays
L.) and
wheat (
Triticum aestivum
L.) roots [
20
-
22
]. Aqueous polymer
two-phase systems for
Arabidopsis thaliana
(L.) Heynh. leaves
and
Nicotiana tabaccum
L. BY2 cells or for other species and tis-
sues were described elsewhere [
15
,
23
-
25
].
Sucrose density gradient centrifugation is widely used for
tonoplast preparation [
26
-
28
]. Tonoplast and plasma membranes
are similar in buoyant density but different in surface properties.
To avoid contamination by plasma membranes, we use the fi rst
lower phase after aqueous polymer two-phase partitioning
(depleted in plasma membranes) to separate tonoplast from intra-
cellular membranes (ICM). The latter fraction contains endoplas-
mic reticulum, Golgi, microbodies, etc. and may be also used for
further analysis.
Marker analysis is a crucial point after cell fractionation, because
marker abundance may also be changed by stress or other factors
[
18
]. Purity of a membrane preparation can be investigated either
by specifi c staining using transmission electron microscopy, marker
enzyme activity or protein-immunoblot analysis [
15
,
20
-
22
].
To avoid contamination by soluble proteins that are entrapped
inside membrane vesicles or attached to the membrane during
preparation samples have to be washed [
29
]. Interaction of attached
proteins with the membrane or integral proteins can be destroyed
by high salt concentrations. In our studies we use physiological
salt conditions for the removal of proteins, which avoids the loss of
peripheral membrane proteins. Class III peroxidases appear to
interact with membranes by a transmembrane spanning domain [
9
,
30
]. Thus proteins have to be solubilized before polyacrylamide
gel electrophoresis (PAGE). Non-ionic detergents or a combina-
tion of the zwittergent CHAPS and aminocaproic acid (ACA) have
been used for solubilization of plasma membrane-bound peroxi-
dases in the past [
4
,
18
,
31
].
After sample preparation, proteins can be separated by native
or in-native PAGE to get a higher resolution of the numerous per-
oxidases. Native isoelectric focussing (IEF) is a standard method to
characterize peroxidase isoenzymes [
32
]. Besides estimation of the
