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Separating Organelles: Classic Experiment 5.1
Separating Organelles: Classic Experiment 5.1
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.
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-
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
40
60
80
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.