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Lehninger's Principles of Biochemistry.pdf
01 Foundations of Biochemistry
PART I: STRUCTURE AND CATALYSIS
02 Water
03 Amino Acids, Peptides, and Proteins
04 Three-Dimensional Structure of Proteins
05 Protein Function
06 Enzymes
07 Carbohydrates and Glycobiology
08 Nucleotides and Nucleic Acids
09 DNA-Based Information Technologies
10 Lipids
11 Biological Membranes and Transport
12 Biosignaling
PART II: BIOENERGETICS AND METABOLISM
13 Principles of Bioenergetics
14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway
15 Principles of Metabolic Regulation: Glucose and Glycogen
16 Citric Acid Cycle
17 Fatty Acid Catabolism
18 Amino Acid Oxidation and the Production of Urea
19 Oxidative Phosphorylation and Photophosphorylation
20 Carbohydrate Biosynthesis in Plants and Bacteria
21 Lipid Biosynthesis
22 Biosynthesis of Amino Acids, Nucleotides, and Related Molecules
23 Hormonal Regulation and Integration of Mammalian Metabolism
PART III: INFORMATION PATHWAYS
24 Genes and Chromosomes
25 DNA Metabolism
26 RNA Metabolism
27 Protein Metabolism
28 Regulation of Gene Expression
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1
chapter
THE FOUNDATIONS
OF BIOCHEMISTRY
1.1 Cellular Foundations 3
1.2 Chemical Foundations 12
1.3 Physical Foundations 21
1.4 Genetic Foundations 28
1.5 Evolutionary Foundations 31
With the cell, biology discovered its atom . . . To
characterize life, it was henceforth essential to study the
cell and analyze its structure: to single out the common
denominators, necessary for the life of every cell;
alternatively, to identify differences associated with the
performance of special functions.
—François Jacob, La logique du vivant: une histoire de l’hérédité
(The Logic of Life: A History of Heredity), 1970
We must, however, acknowledge, as it seems to me, that
man with all his noble qualities . . . still bears in his
bodily frame the indelible stamp of his lowly origin.
—Charles Darwin, The Descent of Man, 1871
F ifteen to twenty billion years ago, the universe arose
as a cataclysmic eruption of hot, energy-rich sub-
atomic particles. Within seconds, the simplest elements
(hydrogen and helium) were formed. As the universe
expanded and cooled, material condensed under the in-
fluence of gravity to form stars. Some stars became
enormous and then exploded as supernovae, releasing
the energy needed to fuse simpler atomic nuclei into the
more complex elements. Thus were produced, over bil-
lions of years, the Earth itself and the chemical elements
found on the Earth today. About four billion years ago,
life arose—simple microorganisms with the ability to ex-
tract energy from organic compounds or from sunlight,
which they used to make a vast array of more complex
biomolecules from the simple elements and compounds
on the Earth’s surface.
Biochemistry asks how the remarkable properties
of living organisms arise from the thousands of differ-
ent lifeless biomolecules. When these molecules are iso-
lated and examined individually, they conform to all the
physical and chemical laws that describe the behavior
of inanimate matter—as do all the processes occurring
in living organisms. The study of biochemistry shows
how the collections of inanimate molecules that consti-
tute living organisms interact to maintain and perpetu-
ate life animated solely by the physical and chemical
laws that govern the nonliving universe.
Yet organisms possess extraordinary attributes,
properties that distinguish them from other collections
of matter. What are these distinguishing features of liv-
ing organisms?
A high degree of chemical complexity and
microscopic organization. Thousands of differ-
ent molecules make up a cell’s intricate internal
structures (Fig. 1–1a). Each has its characteristic
sequence of subunits, its unique three-dimensional
structure, and its highly specific selection of
binding partners in the cell.
Systems for extracting, transforming, and
using energy from the environment (Fig.
1–1b), enabling organisms to build and maintain
their intricate structures and to do mechanical,
chemical, osmotic, and electrical work. Inanimate
matter tends, rather, to decay toward a more
disordered state, to come to equilibrium with its
surroundings.
1
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2
Chapter 1
The Foundations of Biochemistry
This is true not only of macroscopic structures,
such as leaves and stems or hearts and lungs, but
also of microscopic intracellular structures and indi-
vidual chemical compounds. The interplay among
the chemical components of a living organism is dy-
namic; changes in one component cause coordinat-
ing or compensating changes in another, with the
whole ensemble displaying a character beyond that
of its individual parts. The collection of molecules
carries out a program, the end result of which is
reproduction of the program and self-perpetuation
of that collection of molecules—in short, life.
A history of evolutionary change. Organisms
change their inherited life strategies to survive
in new circumstances. The result of eons of
evolution is an enormous diversity of life forms,
superficially very different (Fig. 1–2) but
fundamentally related through their shared ancestry.
Despite these common properties, and the funda-
mental unity of life they reveal, very few generalizations
about living organisms are absolutely correct for every
organism under every condition; there is enormous di-
versity. The range of habitats in which organisms live,
from hot springs to Arctic tundra, from animal intestines
to college dormitories, is matched by a correspondingly
wide range of specific biochemical adaptations, achieved
(a)
(b)
(c)
FIGURE 1–1 Some characteristics of living matter. (a) Microscopic
complexity and organization are apparent in this colorized thin sec-
tion of vertebrate muscle tissue, viewed with the electron microscope.
(b) A prairie falcon acquires nutrients by consuming a smaller bird.
(c) Biological reproduction occurs with near-perfect fidelity.
A capacity for precise self-replication and
self-assembly (Fig. 1–1c). A single bacterial cell
placed in a sterile nutrient medium can give rise
to a billion identical “daughter” cells in 24 hours.
Each cell contains thousands of different molecules,
some extremely complex; yet each bacterium is
a faithful copy of the original, its construction
directed entirely from information contained
within the genetic material of the original cell.
Mechanisms for sensing and responding to
alterations in their surroundings, constantly
adjusting to these changes by adapting their
internal chemistry.
Defined functions for each of their compo-
nents and regulated interactions among them.
FIGURE 1–2 Diverse living organisms share common chemical fea-
tures. Birds, beasts, plants, and soil microorganisms share with hu-
mans the same basic structural units (cells) and the same kinds of
macromolecules (DNA, RNA, proteins) made up of the same kinds of
monomeric subunits (nucleotides, amino acids). They utilize the same
pathways for synthesis of cellular components, share the same genetic
code, and derive from the same evolutionary ancestors. Shown here
is a detail from “The Garden of Eden,” by Jan van Kessel the Younger
(1626–1679).
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within a common chemical framework. For the sake of
clarity, in this book we sometimes risk certain general-
izations, which, though not perfect, remain useful; we
also frequently point out the exceptions that illuminate
scientific generalizations.
Biochemistry describes in molecular terms the struc-
tures, mechanisms, and chemical processes shared by
all organisms and provides organizing principles that
underlie life in all its diverse forms, principles we refer
to collectively as the molecular logic of life. Although
biochemistry provides important insights and practical
applications in medicine, agriculture, nutrition, and
industry, its ultimate concern is with the wonder of life
itself.
In this introductory chapter, then, we describe
(briefly!) the cellular, chemical, physical (thermody-
namic), and genetic backgrounds to biochemistry and
the overarching principle of evolution—the develop-
ment over generations of the properties of living cells.
As you read through the book, you may find it helpful
to refer back to this chapter at intervals to refresh your
memory of this background material.
1.1 Cellular Foundations
The unity and diversity of organisms become apparent
even at the cellular level. The smallest organisms consist
of single cells and are microscopic. Larger, multicellular
organisms contain many different types of cells, which
vary in size, shape, and specialized function. Despite
these obvious differences, all cells of the simplest and
most complex organisms share certain fundamental
properties, which can be seen at the biochemical level.
Cells Are the Structural and Functional Units of All
Living Organisms
Cells of all kinds share certain structural features (Fig.
1–3). The plasma membrane defines the periphery of
the cell, separating its contents from the surroundings.
It is composed of lipid and protein molecules that form
a thin, tough, pliable, hydrophobic barrier around the
cell. The membrane is a barrier to the free passage of
inorganic ions and most other charged or polar com-
pounds. Transport proteins in the plasma membrane al-
low the passage of certain ions and molecules; receptor
proteins transmit signals into the cell; and membrane
enzymes participate in some reaction pathways. Be-
cause the individual lipids and proteins of the plasma
membrane are not covalently linked, the entire struc-
ture is remarkably flexible, allowing changes in the
shape and size of the cell. As a cell grows, newly made
lipid and protein molecules are inserted into its plasma
membrane; cell division produces two cells, each with its
own membrane. This growth and cell division (fission)
occurs without loss of membrane integrity.
1.1
Cellular Foundations
3
Nucleus (eukaryotes)
or nucleoid (bacteria)
Contains genetic material–DNA and
associated proteins. Nucleus is
membrane-bounded.
Plasma membrane
Tough, flexible lipid bilayer.
Selectively permeable to
polar substances. Includes
membrane proteins that
function in transport,
in signal reception,
and as enzymes.
Cytoplasm
Aqueous cell contents and
suspended particles
and organelles.
centrifuge at 150,000 g
Supernatant: cytosol
Concentrated solution
of enzymes, RNA,
monomeric subunits,
metabolites,
inorganic ions.
Pellet: particles and organelles
Ribosomes, storage granules,
mitochondria, chloroplasts, lysosomes,
endoplasmic reticulum.
FIGURE 1–3 The universal features of living cells. All cells have a
nucleus or nucleoid, a plasma membrane, and cytoplasm. The cytosol
is defined as that portion of the cytoplasm that remains in the super-
natant after centrifugation of a cell extract at 150,000 g for 1 hour.
The internal volume bounded by the plasma mem-
brane, the cytoplasm (Fig. 1–3), is composed of an
aqueous solution, the cytosol, and a variety of sus-
pended particles with specific functions. The cytosol is
a highly concentrated solution containing enzymes and
the RNA molecules that encode them; the components
(amino acids and nucleotides) from which these macro-
molecules are assembled; hundreds of small organic
molecules called metabolites, intermediates in biosyn-
thetic and degradative pathways; coenzymes, com-
pounds essential to many enzyme-catalyzed reactions;
inorganic ions; and ribosomes, small particles (com-
posed of protein and RNA molecules) that are the sites
of protein synthesis.
All cells have, for at least some part of their life, ei-
ther a nucleus or a nucleoid, in which the genome—
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4
Chapter 1
The Foundations of Biochemistry
the complete set of genes, composed of DNA—is stored
and replicated. The nucleoid, in bacteria, is not sepa-
rated from the cytoplasm by a membrane; the nucleus,
in higher organisms, consists of nuclear material en-
closed within a double membrane, the nuclear envelope.
Cells with nuclear envelopes are called eukaryotes
(Greek eu, “true,” and karyon, “nucleus”); those with-
out nuclear envelopes—bacterial cells—are prokary-
otes (Greek pro, “before”).
Cellular Dimensions Are Limited by Oxygen Diffusion
Most cells are microscopic, invisible to the unaided eye.
Animal and plant cells are typically 5 to 100 m in di-
ameter, and many bacteria are only 1 to 2 m long (see
the inside back cover for information on units and their
abbreviations). What limits the dimensions of a cell? The
lower limit is probably set by the minimum number of
each type of biomolecule required by the cell. The
smallest cells, certain bacteria known as mycoplasmas,
are 300 nm in diameter and have a volume of about
10⫺14 mL. A single bacterial ribosome is about 20 nm in
its longest dimension, so a few ribosomes take up a sub-
stantial fraction of the volume in a mycoplasmal cell.
The upper limit of cell size is probably set by the
rate of diffusion of solute molecules in aqueous systems.
For example, a bacterial cell that depends upon oxygen-
consuming reactions for energy production must obtain
molecular oxygen by diffusion from the surrounding
medium through its plasma membrane. The cell is so
small, and the ratio of its surface area to its volume is
so large, that every part of its cytoplasm is easily reached
by O2 diffusing into the cell. As cell size increases, how-
ever, surface-to-volume ratio decreases, until metabo-
lism consumes O2 faster than diffusion can supply it.
Metabolism that requires O2 thus becomes impossible
as cell size increases beyond a certain point, placing a
theoretical upper limit on the size of the cell.
There Are Three Distinct Domains of Life
All living organisms fall into one of three large groups
(kingdoms, or domains) that define three branches of
evolution from a common progenitor (Fig. 1–4). Two
large groups of prokaryotes can be distinguished on bio-
chemical grounds: archaebacteria (Greek arche-, “ori-
gin”) and eubacteria (again, from Greek eu, “true”).
Eubacteria inhabit soils, surface waters, and the tissues
of other living or decaying organisms. Most of the well-
studied bacteria, including Escherichia coli, are eu-
bacteria. The archaebacteria, more recently discovered,
are less well characterized biochemically; most inhabit
extreme environments—salt lakes, hot springs, highly
acidic bogs, and the ocean depths. The available evi-
dence suggests that the archaebacteria and eubacteria
diverged early in evolution and constitute two separate
Eubacteria
Eukaryotes
Purple bacteria
Gram-
positive
bacteria
Green
nonsulfur
bacteria
Animals
Ciliates
Fungi
Plants
Cyanobacteria
Flavobacteria
Thermotoga
Flagellates
Microsporidia
Extreme
halophiles
Methanogens
Extreme thermophiles
Archaebacteria
FIGURE 1–4 Phylogeny of the three domains of life. Phylogenetic relationships are often illustrated by a “family tree”
of this type. The fewer the branch points between any two organisms, the closer is their evolutionary relationship.
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1.1
Cellular Foundations
5
All organisms
Phototrophs
(energy from
light)
Autotrophs
(carbon from
CO2)
Examples:
•Cyanobacteria
•Plants
Heterotrophs
(carbon from
organic
compounds)
Examples:
•Purple bacteria
•Green bacteria
Chemotrophs
(energy from chemical
compounds)
Heterotrophs
(carbon from organic
compounds)
FIGURE 1–5 Organisms can be classified according to their source
of energy (sunlight or oxidizable chemical compounds) and their
source of carbon for the synthesis of cellular material.
domains, sometimes called Archaea and Bacteria. All eu-
karyotic organisms, which make up the third domain,
Eukarya, evolved from the same branch that gave rise
to the Archaea; archaebacteria are therefore more
closely related to eukaryotes than to eubacteria.
Within the domains of Archaea and Bacteria are sub-
groups distinguished by the habitats in which they live.
In aerobic habitats with a plentiful supply of oxygen,
some resident organisms derive energy from the trans-
fer of electrons from fuel molecules to oxygen. Other
environments are anaerobic, virtually devoid of oxy-
gen, and microorganisms adapted to these environments
obtain energy by transferring electrons to nitrate (form-
ing N2), sulfate (forming H2S), or CO2 (forming CH4).
Many organisms that have evolved in anaerobic envi-
ronments are obligate anaerobes: they die when ex-
posed to oxygen.
We can classify organisms according to how they
obtain the energy and carbon they need for synthesiz-
ing cellular material (as summarized in Fig. 1–5). There
are two broad categories based on energy sources: pho-
totrophs (Greek trophe-, “nourishment”) trap and use
sunlight, and chemotrophs derive their energy from
oxidation of a fuel. All chemotrophs require a source of
organic nutrients; they cannot fix CO2 into organic com-
pounds. The phototrophs can be further divided into
those that can obtain all needed carbon from CO2 (au-
totrophs) and those that require organic nutrients
(heterotrophs). No chemotroph can get its carbon
Lithotrophs
(energy from
inorganic
compounds)
Examples:
•Sulfur bacteria
•Hydrogen bacteria
Organotrophs
(energy from
organic
compounds)
Examples:
•Most prokaryotes
•All nonphototrophic
eukaryotes
atoms exclusively from CO2 (that is, no chemotrophs
are autotrophs), but the chemotrophs may be further
classified according to a different criterion: whether the
fuels they oxidize are inorganic (lithotrophs) or or-
ganic (organotrophs).
Most known organisms fall within one of these four
broad categories—autotrophs or heterotrophs among the
photosynthesizers, lithotrophs or organotrophs among
the chemical oxidizers. The prokaryotes have several gen-
eral modes of obtaining carbon and energy. Escherichia
coli, for example, is a chemoorganoheterotroph; it re-
quires organic compounds from its environment as fuel
and as a source of carbon. Cyanobacteria are photo-
lithoautotrophs; they use sunlight as an energy source
and convert CO2 into biomolecules. We humans, like E.
coli, are chemoorganoheterotrophs.
Escherichia coli Is the Most-Studied Prokaryotic Cell
Bacterial cells share certain common structural fea-
tures, but also show group-specific specializations (Fig.
1–6). E. coli is a usually harmless inhabitant of the hu-
man intestinal tract. The E. coli cell is about 2 m long
and a little less than 1 m in diameter. It has a protec-
tive outer membrane and an inner plasma membrane
that encloses the cytoplasm and the nucleoid. Between
the inner and outer membranes is a thin but strong layer
of polymers called peptidoglycans, which gives the cell
its shape and rigidity. The plasma membrane and the
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6
Chapter 1
The Foundations of Biochemistry
Ribosomes Bacterial ribosomes are smaller than
eukaryotic ribosomes, but serve the same function—
protein synthesis from an RNA message.
Nucleoid Contains a single,
simple, long circular DNA
molecule.
Pili Provide
points of
adhesion to
surface of
other cells.
Flagella
Propel cell
through its
surroundings.
Cell envelope
Structure varies
with type of
bacteria.
Outer membrane
Peptidoglycan layer
Inner membrane
Inner membrane
Peptidoglycan layer
Inner membrane
Gram-negative bacteria
Outer membrane;
peptidoglycan layer
Gram-positive bacteria
No outer membrane;
thicker peptidoglycan layer
Cyanobacteria
Gram-negative; tougher
peptidoglycan layer;
extensive internal
membrane system with
photosynthetic pigments
Archaebacteria
No outer membrane;
peptidoglycan layer outside
plasma membrane
FIGURE 1–6 Common structural features of bacterial cells. Because
of differences in the cell envelope structure, some eubacteria (gram-
positive bacteria) retain Gram’s stain, and others (gram-negative
bacteria) do not. E. coli is gram-negative. Cyanobacteria are also
eubacteria but are distinguished by their extensive internal membrane
system, in which photosynthetic pigments are localized. Although the
cell envelopes of archaebacteria and gram-positive eubacteria look
similar under the electron microscope, the structures of the membrane
lipids and the polysaccharides of the cell envelope are distinctly dif-
ferent in these organisms.
layers outside it constitute the cell envelope. In the
Archaea, rigidity is conferred by a different type of poly-
mer (pseudopeptidoglycan). The plasma membranes of
eubacteria consist of a thin bilayer of lipid molecules
penetrated by proteins. Archaebacterial membranes
have a similar architecture, although their lipids differ
strikingly from those of the eubacteria.
The cytoplasm of E. coli contains about 15,000
ribosomes, thousands of copies each of about 1,000
different enzymes, numerous metabolites and cofac-
tors, and a variety of inorganic ions. The nucleoid
contains a single, circular molecule of DNA, and the
cytoplasm (like that of most bacteria) contains one or
more smaller, circular segments of DNA called plas-
mids. In nature, some plasmids confer resistance to
toxins and antibiotics in the environment. In the labo-
ratory, these DNA segments are especially amenable
to experimental manipulation and are extremely use-
ful to molecular geneticists.
Most bacteria (including E. coli) lead existences as
individual cells, but in some bacterial species cells tend
to associate in clusters or filaments, and a few (the
myxobacteria, for example) demonstrate simple social
behavior.
Eukaryotic Cells Have a Variety of Membranous
Organelles, Which Can Be Isolated for Study
Typical eukaryotic cells (Fig. 1–7) are much larger than
prokaryotic cells—commonly 5 to 100 m in diameter,
with cell volumes a thousand to a million times larger than
those of bacteria. The distinguishing characteristics of
eukaryotes are the nucleus and a variety of membrane-
bounded organelles with specific functions: mitochondria,
endoplasmic reticulum, Golgi complexes, and lysosomes.
Plant cells also contain vacuoles and chloroplasts (Fig.
1–7). Also present in the cytoplasm of many cells are
granules or droplets containing stored nutrients such as
starch and fat.
In a major advance in biochemistry, Albert Claude,
Christian de Duve, and George Palade developed meth-
ods for separating organelles from the cytosol and from
each other—an essential step in isolating biomolecules
and larger cell components and investigating their
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(a) Animal cell
1.1
Cellular Foundations
7
Ribosomes are protein-
synthesizing machines
Peroxisome destroys peroxides
Cytoskeleton supports cell, aids
in movement of organells
Lysosome degrades intracellular
debris
Transport vesicle shuttles lipids
and proteins between ER, Golgi,
and plasma membrane
Golgi complex processes,
packages, and targets proteins to
other organelles or for export
Smooth endoplasmic reticulum
(SER) is site of lipid synthesis
and drug metabolism
Nucleolus is site of ribosomal
RNA synthesis
Nucleus contains the
genes (chromatin)
Ribosomes
Cytoskeleton
Golgi
complex
Nuclear envelope segregates
chromatin (DNA ⫹ protein)
from cytoplasm
Plasma membrane separates cell
from environment, regulates
movement of materials into and
out of cell
Rough endoplasmic reticulum
(RER) is site of much protein
synthesis
Mitochondrion oxidizes fuels to
produce ATP
Chloroplast harvests sunlight,
produces ATP and carbohydrates
Starch granule temporarily stores
carbohydrate products of
photosynthesis
Thylakoids are site of light-
driven ATP synthesis
Cell wall provides shape and
rigidity; protects cell from
osmotic swelling
Vacuole degrades and recycles
macromolecules, stores
metabolites
Plasmodesma provides path
between two plant cells
Cell wall of adjacent cell
FIGURE 1–7 Eukaryotic cell structure. Schematic illustrations of the
two major types of eukaryotic cell: (a) a representative animal cell
and (b) a representative plant cell. Plant cells are usually 10 to
100 m in diameter—larger than animal cells, which typically
range from 5 to 30 m. Structures labeled in red are unique to
either animal or plant cells.
Glyoxysome contains enzymes of
the glyoxylate cycle
(b) Plant cell
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Chapter 1
The Foundations of Biochemistry
structures and functions. In a typical cell fractionation
(Fig. 1–8), cells or tissues in solution are disrupted by
gentle homogenization. This treatment ruptures the
plasma membrane but leaves most of the organelles in-
tact. The homogenate is then centrifuged; organelles
such as nuclei, mitochondria, and lysosomes differ in
size and therefore sediment at different rates. They also
differ in specific gravity, and they “float” at different
levels in a density gradient.
Differential centrifugation results in a rough fraction-
ation of the cytoplasmic contents, which may be further
purified by isopycnic (“same density”) centrifugation. In
this procedure, organelles of different buoyant densities
(the result of different ratios of lipid and protein in each
type of organelle) are separated on a density gradient. By
carefully removing material from each region of the gra-
dient and observing it with a microscope, the biochemist
can establish the sedimentation position of each organelle
(a)
Differential
centrifugation
Tissue
homogenization
❚
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Low-speed centrifugation
(1,000 g, 10 min)
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Tissue
homogenate
❚
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▲
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Supernatant subjected to
medium-speed centrifugation
(20,000 g, 20 min)
❚
❚❚
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▲ ▲
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Pellet
contains
whole cells,
nuclei,
cytoskeletons,
plasma
membranes
Supernatant subjected
to high-speed
centrifugation
(80,000 g, 1 h)
Supernatant
subjected to
very high-speed
centrifugation
(150,000 g, 3 h)
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Pellet
contains
mitochondria,
lysosomes,
peroxisomes
❚
❚
❚❚
❚ ❚❚
❚
❚❚
❚❚
❚
❚
❚
❚
❚
❚
❚
❚
❚
❚
Pellet
contains
microsomes
(fragments of ER),
small vesicles
❚
❚
❚
❚
❚
❚
❚
❚
❚
❚
❚
❚
❚
❚
❚
❚
❚
❚
❚
❚
❚
❚
Pellet contains
ribosomes, large
macromolecules
Supernatant
contains
soluble
proteins
FIGURE 1–8 Subcellular fractionation of tissue. A tissue such as liver
is first mechanically homogenized to break cells and disperse their
contents in an aqueous buffer. The sucrose medium has an osmotic
pressure similar to that in organelles, thus preventing diffusion of wa-
ter into the organelles, which would swell and burst. (a) The large and
small particles in the suspension can be separated by centrifugation
at different speeds, or (b) particles of different density can be sepa-
rated by isopycnic centrifugation. In isopycnic centrifugation, a cen-
trifuge tube is filled with a solution, the density of which increases
from top to bottom; a solute such as sucrose is dissolved at different
concentrations to produce the density gradient. When a mixture of
organelles is layered on top of the density gradient and the tube is
centrifuged at high speed, individual organelles sediment until their
buoyant density exactly matches that in the gradient. Each layer can
be collected separately.
(b)
Isopycnic
(sucrose-density)
centrifugation
Centrifugation
Sample
Sucrose
gradient
Less dense
component
More dense
component
Fractionation
8
7
6
5
4
3
2
1
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