membranes is glutathione transferase (GT). Membrane vesicles from intact mitochon-
dria can be generated by consecutive swelling, shrinking and sonication of the
suspended organelles. The vesicular mixture is then separated by sucrose density
centrifugation into the three main types of mitochondrial membranes (Fig. 3.7b).
The distribution of marker enzyme activities in the various fractions demonstrates
that the outer membrane has a lower density compared to the inner membrane. The
glutathione transferase-containing contact zones are positioned in a band between the
30–70% sucrose density gradient
20 h at 150 000 g
Outer membranes
(a)
(b)
(c)
Contact sites
Inner membranes
Intact
mitochondria Swelling
shrinking
sonication
Vesicular
mixture
Outer
membrane
Inner
membrane
Contact site
Mitochondrial matrix
GT Cytosol
SDH
MAO
% sucrose Enzyme activity(units per fraction)
20
70
30
40
50
60
1.0
0.2
0
0.4
0.6
0.8
Gradient fraction
MAO GT SDH
Pore
246 8 101214161820
Fig. 3.7Scheme of the fractionation of membranes derived from liver mitochondria. Shown is the distribution
of marker enzymes in the micro compartments of liver mitochondria (MAO, monoamino oxidase; SDH,
succinate dehydrogenase; GT, glutathione transferase) (a), the separation method to isolate fractions highly
enriched in the inner cristae membrane, contact zones and the outer mitochondrial membrane (b), as well
as the distribution of mitochondrial membranes after density gradient centrifugation (c).
94 Centrifugation