Biology Reference
In-Depth Information
60 s
C
M + NM
Iono
Digit
SDS
0-Ca/BAPTA
Fig. 16
Adi
V
erential permeabilization experiment for estimating subcellular fractions of Rhod-2
indicator. A rabbit vagal sensory neuron was incubated with 1
m
M Rhod-2 AM for 1 h at 23
C and
then bathed in nominally Ca
2
þ
-free physiological saline to which 2 mM Na
4
BAPTA was added (0-Ca/
BAPTA). The total Rhod-2 fluorescence from the cell was monitored. Ionomycin (Iono, 2
m
M) was
applied to dissipate Ca
2
þ
gradients between subcellular compartments. Digitonin (Digit, 20
m
M)
permeabilized the plasma membrane selectively to release cytosolic Rhod-2 (intensity decrement
marked ''C''). Sodium dodecyl sulfate (SDS; 0.25%, w/v) permeabilized all cellular membranes to
release Rhod-2 from organellar compartments (decrement marked ''M
þ
NM''). The durations of
reagent applications are indicated by the bars at the bottom.
Rhod-2 to escape from mitochondria as well as nonmitochondrial organelles; this
causes a further decrement in fluorescence (labeled ''M
NM'' in
Fig. 16
). For
four neurons tested, the ratio of the noncytosolic fraction, M
þ
þ
NM, to the cyto-
solic fraction, C, was
þ
M
NM
C
¼
5
:
36
This provides one required algebraic condition; a second condition is
M
þ
NM
þ
C
¼
1
that is, intracellular Rhod-2 must be in the mitochondria, in nonmitochondrial
organelles, or in the cytosol. Since there are three variables, a third algebraic
condition is required, and this can be obtained by performing the permeabilization
experiment on cells whose incubation with Rhod-2 AM had been done in the
presence of a protonophore (e.g., CCCP, FCCP, 2,4-dinitrophenol),
28
which
28
It is advisable to use oligomycin (e.g., 10
m
M), a blocker of the mitochondrial F
1
F
0
-ATP synthase,
in conjunction with the protonophore. In discharging the mitochondrial membrane potential, the
protonophore eliminates the H
þ
electrochemical gradient that is used by the ATP synthase to generate
ATP. This causes the ATP synthase to run in reverse—as an ATPase—and rapidly deplete cellular ATP.
Oligomycin, by blocking ATPase action, helps to preserve the cellular ATP pool.
