Biology Reference
In-Depth Information
Fig. 4
Sucrose step gradient after centrifugation. The organelles from a microsomal fraction obtained from
pollen tubes grown for 240 min (MF
240
) were separated. (
a
) The interphases which correspond to the enriched
organelle fractions as indicated are clearly visible. (
b
) An example of an ATP hydrolysis assay analysis using the
P-, V-, and F-type ATPases as marker enzymes for the tonoplast, the inner mitochondrial membrane, and the
plasma membrane, respectively
3.5 Marker Enzymes
and ATP Hydrolysis
Assay
The marker enzyme concept is based on the fact that certain
enzyme activities co-localize with just one organelle membrane.
However, due to membrane and protein traffi cking some proteins
or enzyme activities can be detected in many organelles but the
main signal is always specifi c for one organelle. An excellent descrip-
tion of the marker enzyme concept, its critical evaluation, and
detailed protocols for measuring enzyme activities are given [
10
-
12
].
Here, we present an ATP hydrolysis assay to distinguish between
the P-type H
+
ATPase of the plasma membrane, the V-type H
+
ATPase of the tonoplast, and the F-type H
+
ATPase of the inner
mitochondrial membrane. The ATP hydrolysis assay is based on the
detection of the released inorganic phosphate and was fi rst described
by Fiske and Subbarow in 1925 [
13
] with modifi cations [
14
,
15
].
Due to the fact that the different ATPase types can be inhibited by
specifi c inhibitors, the ATP hydrolysis assay can be performed in
the absence and the presence of a specifi c inhibitor to determine
the amounts of vanadate-sensitive (P-type ATPase), bafi lomycin-
sensitive (V-type), and azide-sensitive (F-ATPase) ATP hydrolysis.
1. Label plastic half-micro cuvettes. Perform the assay in triplicates
and add cuvettes for the following blank values: buffer blank
(no substrate (ATP), no protein), substrate blank (with substrate,
no protein), and vesicle blank (no substrate, protein).
2. Add an appropriate volume of reaction buffer to the cuvettes.
The total volume of the reaction is 300
μ
l. Consider the volumes
