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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