Classic Experiment 5.

Separating Organelles
n the 1950s and 1960s, scientists used two techniques to study cell
organelles: microscopy and fractionation. Christian de Duve was at
the forefront of cell fractionation. In the early 1950s, he used
centrifugation to distinguish a new organelle, the lysosome, from
previously characterized fractions: the nucleus, the mitochondrial-rich
fraction, and the microsomes. Soon thereafter, he used equilibriumdensity centrifugation to uncover yet another organelle.

Background
Eukaryotic cells are highly organized and composed of cell
structures known as organelles that perform specific functions. While microscopy has allowed biologists to describe
the location and appearance of various organelles, it is of
limited use in uncovering the organelles function. To do this,
cell biologists have relied on a technique known as cell fractionation. Here, cells are broken open, and the cellular components are separated on the basis of size, mass, and density using a variety of centrifugation techniques. Scientists
could then isolate and analyze cell components of different
densities, called fractions. Using this method, biologists had
divided the cell into four fractions: nuclei, mitochondrialrich fraction, microcosms, and cell sap.
de Duve was a biochemist interested in the subcellular
locations of metabolic enzymes. He had already completed
a large body of work on the fractionation of liver cells, in
which he had determined the subcellular location of numerous enzymes. By locating these enzymes in specific cell
fractions, he could begin to elucidate the function of the organelle. He has noted that his work was guided by two hypotheses: the postulate of biochemical homogeneity and
the postulate of single location. In short, these hypotheses propose that the entire composition of a subcellular population will contain the same enzymes, and that each enzyme is located at a discrete site within the cell. Armed with
these hypotheses and the powerful tool of centrifugation, de
Duve further subdivided the mitochondrial-rich fraction.

First, he identified the light mitochondrial fraction, which is

made up of hydrolytic enzymes that are now known to compose the lysosome. Then, in a series of experiments described
here, he identified another discrete subcellular fraction,
which he called the perioxisome, within the mitochondrialrich fraction.

The Experiment
de Duve studied the distribution of enzymes in rat liver cells.
Highly active in energy metabolism, the liver contains a
number of useful enzymes to study. To look for the presence
of various enzymes during the fractionation, he relied on
known tests, called enzyme assays, for enzyme activity. To
retain maximum enzyme activity, he had to take precautions,
which included performing all fractionation steps at 0C because heat denatures protein that would compromise enzyme activity.
de Duve used rate-zonal centrifugation to separate cellular components by successive centrifugation steps. He removed the rats liver and broke it apart by homogenization.
The crude preparation of homogenized cells was then subjected to relatively low-speed centrifugation. This initial step
separated the cell nucleus, which collects as sediment at the
bottom of the tube, from the cytoplasmic extract that remains in the supernatant. Next, de Duve further subdivided
the cytoplasmic extract into heavy mitochondrial fraction,
light mitochondrial fraction, and microsomal fraction. He
accomplished separating the cytoplasm by employing suc-

compartment, it would separate from the lysosomal enzymes

in each gradient tested. de Duve performed the fractionations in this series of gradients, then performed enzyme assays as before. In each case, he found uricase in a separate
population than the lysosomal enzyme acid phosphatase and
the mitochondrial enzyme cytochrome oxidase (see Figure
5.2). By repeatedly observing uricase activity in a distinct
fraction from the activity of the lysosomal and mitochondrial enzymes, de Duve concluded that uricase was part of
a separate organelle. The experiment also showed that two
other enzymes, catalase and D-amino acid oxidase, segregated into the same fractions as uricase. Because each of
these enzymes either produced or used hydrogen peroxide,
de Duve proposed that this fraction represented an organelle
responsible for the peroxide metabolism and dubbed it the
perioxisome.
5
4
3
Cytochrome oxidase
2
1
20
Relative concentration

cessive centrifugation steps of increasing force. At each step,

he collected and stored the fractions for subsequent enzyme
analysis.
Once the fractionation was complete, de Duve performed
enzyme assays to determine the subcellular distribution of
each enzyme. He then graphically plotted the distribution of
the enzyme throughout the cell. As had been shown previously, the activity of cytochrome oxidase, an important enzyme in the electron transfer system, was found primarily
in the heavy mitochondrial fractions. The microsomal fraction was shown to contain another previously characterized
enzyme glucose-6-phosphatase. The light mitochondrial
fraction, which is made up of the lysosome, showed the characteristic acid phosphatase activity. Unexpectedly, de Duve
observed a fourth pattern when he assayed the uricase activity. Rather than following the pattern of the reference enzymes, uricase activity was sharply concentrated within the
light mitochondrial fraction. This sharp concentration, in
contrast to the broad distribution, suggested to de Duve that
the uricase might be secluded in another subcellular population separate from the lysosomal enzymes.
To test this theory, de Duve employed a technique known
as equilibrium density-gradient centrifugation, which separates macromolecules on the basis of density. Equilibrium
density-gradient centrifugation can be performed using a
number of different gradients including sucrose and glycogen. In addition, the gradient can be made up in either water or heavy water that contains the hydrogen isotope deuterium in place of hydrogen. In his experiment, de Duve
separated the mitochondrial-rich fraction prepared by ratezonal centrifugation in each of these different gradients (see
Figure 5.1). If uricase were part of a separate subcellular

40

60

80

5
4
3
Uricase
2
1
20

40

60

80

Increasing density of
sucrose (g/cm3)

5
4

Organelle
fraction
1.09
1.11
1.15
1.19
1.22
1.25
Before
centrifugation

3
Acid phosphatase

Lysosomes
(1.12 g/cm3)

2
1

Mitochondria
(1.18 g/cm3)

20
Peroxisomes
(1.23 g/cm3)
After
centrifugation

FIGURE 5.1 Schematic depiction of the separation of the

lysosomes, mitochondria, and perioxisomes by equilibrium
density centrifugation. The mitochondrial-rich fraction from ratezonal centrifugation was separated in a sucrose gradient, and the
organelles are separated on the basis of density. [From Lodish
et al., 3rd edition, page 166.]

40

60

80

Percent height in tube

FIGURE 5.2 Graphical representation of the enzyme analysis of products from a sucrose gradient. The mitochon-drial-rich
fraction was separated as depicted in Figure 5.1, and then enzyme assays were performed. The relative concentration of active
enzyme is plotted on the y-axis; the height in the tube is plotted
on the x-axis. The peak activities of cytochrome oxidase (top) and
acid phosphatase (bottom) are observed near the top of tube. The
peak activity of uricase (middle) migrates to the bottom of the
tube. [Adapted from Beaufay et al., 1964, Biochem J. 92:191.]

Discussion
de Duves work on cellular fractionation provided an insight
into the function of cell structures, as he sought to map the
location of known enzymes. Examining the inventory of enzymes in a given cell fraction gave him clues to its function.
His careful work resulted in the uncovering of two organelles: the lysosome and the perioxisome. His work also
provided important clues to the organelles function. The
lysosome, where de Duve found so many potentially destructive enzymes, is now known to be an important site for
degradation of biomolecules. The perioxisome has been
shown to be the site of fatty acid and amino acid oxidation,
reactions that produce a large amount of hydrogen peroxide. In 1974, de Duve received the Nobel Prize for Physiology and Medicine in recognition of his pioneering work.