PRIMERA TAREA

1. Classic Experiment 5.1
Separating Organelles
n the 1950s and 1960s, scientists used two techniques to study cell
I 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 equilibrium-
density centrifugation to uncover yet another organelle.
Background First, he identified the light mitochondrial fraction, which is
made up of hydrolytic enzymes that are now known to com-
Eukaryotic cells are highly organized and composed of cell pose the lysosome. Then, in a series of experiments described
structures known as organelles that perform specific func- here, he identified another discrete subcellular fraction,
tions. While microscopy has allowed biologists to describe which he called the perioxisome, within the mitochondrial-
the location and appearance of various organelles, it is of rich fraction.
limited use in uncovering the organelle’s function. To do this,
cell biologists have relied on a technique known as cell frac-
tionation. Here, cells are broken open, and the cellular com-
The Experiment
ponents are separated on the basis of size, mass, and den- de Duve studied the distribution of enzymes in rat liver cells.
sity using a variety of centrifugation techniques. Scientists Highly active in energy metabolism, the liver contains a
could then isolate and analyze cell components of different number of useful enzymes to study. To look for the presence
densities, called fractions. Using this method, biologists had of various enzymes during the fractionation, he relied on
divided the cell into four fractions: nuclei, mitochondrial- known tests, called enzyme assays, for enzyme activity. To
rich fraction, microcosms, and cell sap. retain maximum enzyme activity, he had to take precautions,
de Duve was a biochemist interested in the subcellular which included performing all fractionation steps at 0°C be-
locations of metabolic enzymes. He had already completed cause heat denatures protein that would compromise en-
a large body of work on the fractionation of liver cells, in zyme activity.
which he had determined the subcellular location of nu- de Duve used rate-zonal centrifugation to separate cel-
merous enzymes. By locating these enzymes in specific cell lular components by successive centrifugation steps. He re-
fractions, he could begin to elucidate the function of the or- moved the rat’s liver and broke it apart by homogenization.
ganelle. He has noted that his work was guided by two hy- The crude preparation of homogenized cells was then sub-
potheses: the “postulate of biochemical homogeneity” and jected to relatively low-speed centrifugation. This initial step
“the postulate of single location.” In short, these hypothe- separated the cell nucleus, which collects as sediment at the
ses propose that the entire composition of a subcellular pop- bottom of the tube, from the cytoplasmic extract that re-
ulation will contain the same enzymes, and that each en- mains in the supernatant. Next, de Duve further subdivided
zyme is located at a discrete site within the cell. Armed with the cytoplasmic extract into heavy mitochondrial fraction,
these hypotheses and the powerful tool of centrifugation, de light mitochondrial fraction, and microsomal fraction. He
Duve further subdivided the mitochondrial-rich fraction. accomplished separating the cytoplasm by employing suc-

2. cessive centrifugation steps of increasing force. At each step, compartment, it would separate from the lysosomal enzymes
he collected and stored the fractions for subsequent enzyme in each gradient tested. de Duve performed the fractiona-
analysis. tions in this series of gradients, then performed enzyme as-
Once the fractionation was complete, de Duve performed says as before. In each case, he found uricase in a separate
enzyme assays to determine the subcellular distribution of population than the lysosomal enzyme acid phosphatase and
each enzyme. He then graphically plotted the distribution of the mitochondrial enzyme cytochrome oxidase (see Figure
the enzyme throughout the cell. As had been shown previ- 5.2). By repeatedly observing uricase activity in a distinct
ously, the activity of cytochrome oxidase, an important en- fraction from the activity of the lysosomal and mitochon-
zyme in the electron transfer system, was found primarily drial enzymes, de Duve concluded that uricase was part of
in the heavy mitochondrial fractions. The microsomal frac- a separate organelle. The experiment also showed that two
tion was shown to contain another previously characterized other enzymes, catalase and D-amino acid oxidase, segre-
enzyme glucose-6-phosphatase. The light mitochondrial gated into the same fractions as uricase. Because each of
fraction, which is made up of the lysosome, showed the char- these enzymes either produced or used hydrogen peroxide,
acteristic acid phosphatase activity. Unexpectedly, de Duve de Duve proposed that this fraction represented an organelle
observed a fourth pattern when he assayed the uricase ac- responsible for the peroxide metabolism and dubbed it the
tivity. Rather than following the pattern of the reference en- perioxisome.
zymes, uricase activity was sharply concentrated within the
light mitochondrial fraction. This sharp concentration, in 5
contrast to the broad distribution, suggested to de Duve that
the uricase might be secluded in another subcellular popu- 4
lation separate from the lysosomal enzymes. 3
To test this theory, de Duve employed a technique known Cytochrome oxidase
as equilibrium density-gradient centrifugation, which sepa- 2
rates macromolecules on the basis of density. Equilibrium 1
density-gradient centrifugation can be performed using a
number of different gradients including sucrose and glyco- 20 40 60 80
gen. In addition, the gradient can be made up in either wa-
5
ter or “heavy water” that contains the hydrogen isotope deu-
Relative concentration
terium in place of hydrogen. In his experiment, de Duve 4
separated the mitochondrial-rich fraction prepared by rate-
3
zonal centrifugation in each of these different gradients (see Uricase
Figure 5.1). If uricase were part of a separate subcellular 2
1
20 40 60 80
5
4
Organelle
fraction 3
Increasing density of
1.09 Acid phosphatase
Lysosomes
2
(1.12 g/cm3)
sucrose (g/cm3)
1.11
1.15 1
Mitochondria
(1.18 g/cm3)
1.19
20 40 60 80
1.22 Percent height in tube
Peroxisomes
1.25 (1.23 g/cm3)
v FIGURE 5.2 Graphical representation of the enzyme analy-
sis of products from a sucrose gradient. The mitochon-drial-rich
Before After
centrifugation centrifugation fraction was separated as depicted in Figure 5.1, and then en-
zyme assays were performed. The relative concentration of active
v FIGURE 5.1 Schematic depiction of the separation of the enzyme is plotted on the y-axis; the height in the tube is plotted
lysosomes, mitochondria, and perioxisomes by equilibrium on the x-axis. The peak activities of cytochrome oxidase (top) and
density centrifugation. The mitochondrial-rich fraction from rate- acid phosphatase (bottom) are observed near the top of tube. The
zonal centrifugation was separated in a sucrose gradient, and the peak activity of uricase (middle) migrates to the bottom of the
organelles are separated on the basis of density. [From Lodish tube. [Adapted from Beaufay et al., 1964, Biochem J. 92:191.]
et al., 3rd edition, page 166.]

3. Discussion
de Duve’s 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 en-
zymes in a given cell fraction gave him clues to its function.
His careful work resulted in the uncovering of two or-
ganelles: the lysosome and the perioxisome. His work also
provided important clues to the organelles’ function. The
lysosome, where de Duve found so many potentially de-
structive 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 perox-
ide. In 1974, de Duve received the Nobel Prize for Physiol-
ogy and Medicine in recognition of his pioneering work.