Biology Reference
In-Depth Information
GB Gel buffer
Guaiacol 2-Methoxyphenol
HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid
hrCNE High resolution clear native electrophoresis
ICM Intracellular membranes
IEF Isoelectric focussing electrophoresis
LB Loading buffer
MALDI Matrix-assisted laser desorption/ionization
modSDS-PAGE Modifi ed SDS-PAGE
PAGE
Polyacrylamide gel electrophoresis
PEG
Poly-ethylene glycol
PM
Plasma membrane
PMSF
Phenylmethylsulfonyl fl uoride
PVPP
Polyvinylpolypyrrolidone
TEMED
N , N , N , N
-Tetramethyl-ethylenediamine
TIFF
Tag Image File Format
TMB
Tetramethylbenzidine
Tris
Tris(hydroxymethyl)aminomethane
2-amino-2-hydroxymethyl-
propane-1,3-diol
1
Introduction
The superfamily of plant heme -containing peroxidases (E.C.
1.11.1.7) is one of the most intensively investigated protein fami-
lies in plant research. Besides intracellular ascorbate peroxidases
(class I peroxidases), peroxidases of the secretory pathway (class
III) belong to this protein superfamily [ 1 ]. The number of class III
peroxidases is extremely high and they are involved in several pro-
cesses like plant development, cell metabolism, cell elongation,
pathogen defense, symbiosis, fruit ripening, senescence and stress
responses etc. [ 2 ]. The steadily increasing number of identifi ed and
predicted peroxidase sequences is documented in the PeroxiBase at
http://peroxibase.toulouse.inra.fr/ [ 3 ]. A recent in silico analysis
suggests that a signifi cant high number of isoenzymes may be
membrane-bound [ 4 ]. So far, the majority of investigations stud-
ied soluble peroxidases [ 5 - 7 ], whereas much less is known of
membrane-bound isoenzymes [ 8 - 10 ].
Here we present a protocol that allows investigation of soluble
and membrane-bound class III peroxidases simultaneously in the
same sample. After harvesting of plant material, the sample is frac-
tionated to get a higher resolution for the numerous peroxidase
isoenzymes. Soluble isoenzymes are prepared by differential cen-
trifugation, concentrated by ammonium sulfate precipitation and
used without further fractionation for analysis. In contrast to the
soluble isoenzymes, the membrane fraction is further separated.
The state of the art for plasma membrane preparation is aqueous
polymer two-phase partitioning. This technique was introduced in
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