Download N x C (N-2)

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PEROXISOMES AND OTHER MICROBODIES
Textbook: pp. 356-360 (also, look up “centrifugation”)
There is certainly no lack of small membrane-bound vesicles in the eukaryotic cell! But
these vesicles can be divided into basically two types: those that are fully derived from the
RER/golgi system and those that are not. The latter are the so-called microbodies, of variable
size but often smaller than mitochondria. The microbodies are now usually called peroxisomes.
The existence of microbodies as small, single-membrane vesicles that looked somewhat different
from the vesicles of similar size called lysosomes, was first discovered by a graduate student
studying cells with the quite newly available electron microscope. He just called them
“microbodies”. By the mid-1960s the cell biologist and biochemist Christian de Duve, using
density centrifugation in an ultracentrifuge, had obtained quite pure preparations of
microbodies. The one property which all microbodies had was that they contained large amounts
of catalase, much more than any other other organellee in the cell. Christiam de Duve had first
isolated lysosomes which, unlike peroxisomes, are derived from the RER/Golgi system.
Analysis of the lysosome-enriched fraction from the density centrifugation showed that
lysosomes have an acidic lumen (about pH 4.5) and that their enzymes are all acid hydrolases
with an acidic optimum pH for their activity. But, as your textbook (p. 356) states there was one
enzyme , called urate oxidase that had an optimal circumneutral pH for their activity. It was
very unlikely that this enzyme would be able to function in lysosomes with their acid lumenal
pH, so de Duve began the search for the contaminating organelles (the microbodies) containing
urate oxidase, and as was soon found out, catalase. Christian de Duve used the techniques of
density gradient ultracentrifugation, enzyme analysis, and electron microscopy to isolate
and characterize the “microbodies’. Because he found that all microbodies contain the enzyme
catalase, which catalyze the decomposition of hydrogen peroxide (H2O2), de Duve called the
microbodies peroxisomes. And that is the term that is now used for most microbodies. Even
when certain microbodies are not called peroxisomes, they are realized to “just” modified
peroxisomes.
Christian de Duve was a pioneer of the use of the ultracentrifugation. A centrifuge is
called an ultracentrifuge if its rotor is spun above about 50,000 x g to 200,000 x g and more.
There are a number of different ways of separating different organelles by centrifugation, but the
two most common are differential centrifugation and density gradient centrifugation. The
former depends upon the rate at which various organelles reach the bottom of the centrifuge tube
to form a pellet. Organelles that do not reach the bottom so rapidly remain in the supernatant. On
the next page a schematic of how differential centrifugation can be use to separate various
organelles is shown. A differential centrifugation is always used as a first step. For example, if
one wants peroxisomes one does need to keep the nuclei, so one spins them down as pellet in a
so-called “low-speed spin.” You should know the order in which various organelles are pelleted
in differential centrifugation and whether or not an ultracentrifuge would be required.
In order to obtain a finer distinction between organelles of similar size and density the
technique of density gradient centrifugation. A gradient of sucrose is set up in an
ultracentrifuge tube and then the supernatant from the differential centrifugation is layered on top
of the gradient. The sucrose gradient slows down the rate at which organelles of similar, but not
identical, density (and size) are spun towards the bottom of the tube. Instead of forming pellets at
the bottom of the tube, the organelles form bands at various levels in the tube, depending mainly
on their density.
DIFFERENTIAL CENTRIFUGATION
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ULTRACENTRIFUGATION
www.ncbi.nlm.nih.gov/bookshelf/picrender.fcg
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Textbook p. 648-649
FRACTION FROM DIFFERENTIAL
CENTRIFUGAL THAT CONTAINS
LYSOSOMES. MITOCHONDRIA, AND
PEROXISOMES
SUCROSE DENSITY
GRADIENT
DENSITY GRADIENT CENTRIFUGATION
The purified microbodies contained, apart from urate oxidase, the H2O2
–decomposing enzyme called catalase). This enzyme must bind two H2O2 molecules at the same
time. One of the H2O2 molecules in the active site acts as a reductant and the other acts as an
oxidant.
CATALASE
2 H2O2
2 H2O + O2
The lumen of peroxisomes, and other microbodies, is called the matrix. Peroxisomes
usually have a “crystalline core” which is composed of catalase (in plant peroxisomes) and
usually urate oxidase in animal peroxisomes. It is important to note, that all peroxisomes
contain large amounts of catalase (by definition) even if it does not make up the crystalline core.
In fact, catalase is the marker enzyme for peroxisomes just as succinate dehydrogenase and
cytochrome c oxidase are marker enzymes for mitochondria. The reason for the catalase in
peroxisomes is that enzyme reactions that have H2O2 as the product are located in these
organelles. Note that unlike the accidental production of H2O2 by the respiratory electron chain
of mitochondria, the enzymes in the peroxisomes form H2O2 as their proper product.
Peroxisomes contain about 40 different enzymes which are involved in various biochemical
transfromations.
Functions of peroxisomes
(1) Oxidation of very long-chain fatty acids (VLCFA). In animal cells, short and
medium length fatty acids (22 C, most commonly 18 C, or less) are oxidized
mainly in the mitochondria. The four enzymes of the β-oxidation cycle produce
an acetyl CoA molecule at each turn of the cycle. In the scheme I show below I
have included all molecular structures to emphasize, yet again, why a biochemical
oxidation often means “the removal of H-atoms”. You do not have to memorize
the structures or names that are of the compounds, other than acyl-CoA, acetylCoA, acyl-CoA dehydrogenase, FAD/FADH2, and NAD+/NADH + H+)
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THE β-OXIDATION CYCLE IN MITOCHONDRIA
This called the β-oxidation cycle because:
(1) For fatty acids the carboxyl carbon is called the α-carbon and the next carbon is
called the β-carbon. In the β-oxidation cycle, these first two carbons are “cut
off” as acetyl-CoA and leaving behind an (N-2) x C acyl CoA.
(2) It is called a “cycle” because the shorter acyl-CoA resulting from one cycle is then
subjected to the same process, and so on until the fatty acid has been chopped into
2 C of acetyl-CoA..
What you should know in addition is:
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(1) The first stage in the oxidation is the removal of two H-atoms by FAD. The
FADH2 is oxidized in the mitochondrial electron transport chain. The acyl-CoA
dehydrogenase is an integral membrane protein like succinate dehydrogenase.
(2) The second, and last oxidation, uses NAD+ as the oxidant. The resulting NADH is
oxidized in the electron transport chain.
(3) The operation of the β-oxidation cycle in mitochondria results in the maximum
amount of ATP that is possible from a fatty acid.
(4) The overall result of the β-oxidation cycle is that fatty acids with an even number
of C-atoms are converted to N/2 acetyl-CoA units (where N is the total number of
fatty acids in the fatty acids).
(5) In the process FADH2 and NADH are produced, which are then oxidized by the
mitochondrial electron transport chain with the production of ATP.
(6) The acetyl-CoA units are metabolized in the TCA cycle as usual.
FAD
Acyl-CoA
NxC
FADH2
NAD+ NADH + H+ CoASH
Acyl-CoA + Acetyl-CoA
(N-2) x C
2C
The diagram below is a simple little diagram that reminds us of the importance of the βoxidation cycle in mitochondria.
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However the diet of animals also contains fatty acids
that contain 24-26 C-atoms and these fatty acids (actually their acyl-CoA derivatives) cannot be
transported into mitochondria and cannot be oxidized by the mitochondrial acyl-CoA
dehydrogenase. These so-called very long chain fatty acids (VLFA) are transported into the
peroxisomes and are oxidized by a β-oxidation cycle that is similar but not identical to that
occurring in mitochondria.
CAT
H2O2
O2
FAD
FADH2
H2O + 0.5 O2
NAD+ NADH + H+
CoASH
Acyl-CoA
Acyl-CoA + Acetyl-CoA
NxC
(N-2) x C
2C
The major difference between the β-oxidation cycle in the mitochondria and
peroxisomes, apart from the fact that the enzymes are not exactly the same, is in the first step.
The reducing power of the FADH2 is wasted in peroxisomes, being “burnt off” by its oxidation
with molecular O2. The H2O2 that is formed is rapidly decompsoed by the catalase (CAT) before
it can leak from the peroxisomes. But the reducing power is not wasted, instead it is transferred
in a complex fashion to the mitochondria. The acetyl-CoA is also transferred to the
mitochondria. The peroxisomes do not oxidize the VLFA completely to acetyl-CoA. Instaed as
soon as medium length acyl-CoAs are also transferred to the mitochondria. I point this out
because it shows just how much transport must occur between all the organelles in cells.
MITOCHONDRION
PEROXISOME
O2
MLCFA
CO2
VLCFA
Acetyl-CoA
ATP
H+
NADH
VLCFA
ADP
H+
O2
H+
H+
3 H+
+
H+ H
H+
H+
H2 O
H+
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β-OXIDATION CYCLE
NADH
TCA CYCLE
TRANSPORT SYSTEM
H+ FLOW
ATP SYNTHASE
VLCFA = very long chain fatty acid
MLCFA = medium length chain fatty acid
(2) Metabolism of nitrogen-containing compounds. Animals require urate oxidase
to oxidase urate (Becker et al. 2009). Urate is formed during the breakdown of
nucleic acids (urate is a purine) and must itself be catabolized in the process of
eliminating excess N-atoms from the body. Oxidases, unlike dehydrogenase, form
H2O2 as a product. Hence the need to perform this reaction in the peroxisomes
where catalase is located.The The general reaction of oxidases can be given as:
OXIDASE
RH2 + O2
R + H2 O 2
Where R is oxidized with respect to RH2. Dehydrogenases, on the other hand,
catalyze the general reaction:
DEHYDROGENASE
RH2 + O2
R
NAD+
NADH + H+
OR
DEHYDROGENASE
RH2 + O2
R
FAD
FADH2
The NADH or the FADH2 is then oxidized in the TCA cycle or is used a
reducing agent in biosynthesis. In either case, no H2O2 is produced. (Well OK! The
complex, proton-pumping NADH dehyrogenase in the mitochondrial inner
membrane does produve a small amount of H2O2 as an accidental product.) Notice
that the oxidation of the FADH2 during the oxidation VLCFA is by an FADH2
oxidase (producing H2O2) and not a FADH2 dehydrogenase.
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(3) Detoxification of various foreign substances. The smooth endoplasmic
reticulum is the most verstaile organelle catalyzing the detoxification of foriegn
compounds (xenobiotics). But certain toxic compouds are oxidied in the
peroxisomes. Such toxic compounds include the D-amino acids using the enzyme
D-amino acid oxidase. Note that polypeptides are composed of only L-amino
acids and these are handled by other enzymes located outside the peroxisomes. As
with other oxidases, the D-amino acid oxidase produces H2O2 as a product.
The biogenesis of peroxisomes.
As stated previously, peroxisomes are not made by the RER/Golgi system, although the
RER itself is involved. So how are they made?
(1) The membrane (phospholipids) comes from the SER in the form of
phospholipids attached to phospholipid-binding proteins (to keep them soluble in
the cytosol) and also as already formed phospholipid bilayer vesicles. The latter
probably come from the RER because the vesicle contains a few integral
proteins, called peroxins, see later).
(2) The proteins are completely synthesized in the cytosol and are then imported
into the peroxisome membrane or matrix. In other words, protein import into
peroxisomes is post-translational, as it is in mitochondria. However, unlike
mitochondria, peroxisomes contain no DNA so they cannot make any of their
own proteins. Proteins imported into mitochondria are almost always fully
unfolded before their import. But peroxisomes can also import unfolded
proteins, even proteins with more than one polypeptide (or subunit)!
(3) The heme groups, required as a co-factor for catalase, must be imported into the
peroxisome by a heme transporter.
(4) Large peroxisomes can also form (by “division) a number of smaller
peroxisomes, which can themselves then grow bigger by incorporating more
phospholipids from the SER and importing more proteins.
The actual proteins required for formation of the peroxisomes (e.g. catalase) are not the
only proteins required. There is also a need, for example, of proteins to help the peroxisomal
proteins be imported into the peroxisomes. Proteins which are required for peroxisomal
biogenesis, but that are not actually invoved in the functioning of completed peroxisomes are
called peroxins (Pex). There are least 25 different peroxins.
For polypeptide import into an organelle the polypeptide must have a targetting sequence
consisting of a number of amino acids in the primary sequence. (In the co-translation import of
polypeptides into the RER, the targetting sequence is called the signal sequence. This sequence
is located at the N-terminal end of the polypeptide. The N-terminal end is the end of the
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polypeptide that is synthesized first. Why does this have to be the case for co-translational
import?) The peroxisomal targetting sequence (PTS) can be on the C-terminal or the Nterminal end, depending on the specfic polypeptide. C-terminal PTSs are recognized by different
peroxins than are the N-terminal PTSs.
RER
INSERTION OF
SOME PEROXINS
SER
EMPTY FATTY ACID-BINDING
PROTEIN
PHOSPHOLIPID
TRANSFER
PHOSPHOLIPID
TRANSFE
FATTY ACID-BINDING PROTEIN
PHOSPHOLIPID ATTACHED
UPTAKE OF
PEROXINS
A MATRIX
PROTEIN
EMPTY “FERRY”
PEROXIN
INSERTION OF PHOSPHOLIPID
INTO MEMBRANE. FATTY ACIDBINDING PROTEIN NOW
UNLOADED
UPTAKE OF MORE
PEROXINS
“FERRY” PEROXIN
LOADED WITH A
MATRIX PROTEIN
DIVISION OF
PEROXISOME
I have drawn the above diagram based upon an analysis of the recent literature dealing
with peroxisomal biogenesis.
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Mechanism of protein import (uptake) by peroxisomes
The study of the mechanism of protein import into peroxisomes has turned out to be
complex. The diagram I have drawn below gives an idea of the current thought on the topic.
Once again, I will say that your textbook over estimates the extent to which proteins are taken up
in their unfolded state.
MATRIX PROTEINS eg catalase
or urate oxidase
EMPTY “FERRY” PEROXIN
PICKS UP A MATRIX
PROTEIN
UNLOADED “FERRY” PEROXIN
LEAVES MEMBRANE: DRIVEN
BY ATP HYDROLYSIS
ADP + Pi
CYTOSOL
LOADED “FERRY” PEROXIN
ENTERS MEMBRANE AND
ENTERS MATRIX
ATP
MATRIX
ALL THE PROTEINS I HAVE
DRAWN IN THE MEMBRANE
ARE VARIOUS PEROXINS
MATRIX PROTEIN IS LEFT
BEHIND IN THE
PEROXISOMAL MATRIX