Download SEPARATING ORGANELLES

8945d_013-016
6/18/03
11:05 AM
Page 13 mac85 Mac 85:1st shift: 1268_tm:8945d:
Classic Experiment
5.1
SEPARATING ORGANELLES
n the 1950s and 1960s, scientists used two techniques to study cell organelles:
I
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
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 organelle’s 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,
mitochondrial-rich 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 mitochondrialrich 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 mitochondrial-rich 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 0°C 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 rat’s liver and broke it apart by homogenization. The crude preparation of homogenized cells was
6/18/03
11:05 AM
Page 14 mac85 Mac 85:1st shift: 1268_tm:8945d:
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 successive 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 rate-zonal centrifugation in each of these
different gradients (see Figure 5.1). If uricase were part of
a separate subcellular 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
5
4
3
Cytochrome oxidase
2
1
20
Relative concentration
8945d_013-016
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 mitochondrial-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.]
8945d_013-016
6/18/03
11:05 AM
Page 15 mac85 Mac 85:1st shift: 1268_tm:8945d:
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.
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 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.