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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 theRER/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 fortheir 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) containingurate 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 variousorganelles 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.
Textbook p. 648-649
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 dehydrogenaseandcytochrome 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, acetyl- CoA, acyl-CoA dehydrogenase, FAD/FADH2, and NAD+/NADH + H+)
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 Cacyl 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:
(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-CoAdehydrogenase 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.
Acyl-CoA Acyl-CoA + Acetyl-CoA
N x C (N-2) x C 2 C
The diagram below is a simple little diagram that reminds us of the importance of the β-oxidation cycle in mitochondria.
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.
Acyl-CoAAcyl-CoA + Acetyl-CoA
N x C (N-2) x C 2 C
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.
VLCFA = very long chain fatty acid MLCFA = medium length chain fatty acid
(2)Metabolism of nitrogen-containing compounds. Animalsrequire 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 + H2O2
Where R is oxidized with respect to RH2. Dehydrogenases, on the other hand,
catalyze the general reaction:
DEHYDROGENASE
RH2 + O2 R
DEHYDROGENASE
RH2 + O2R
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.
(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 smallerperoxisomes, 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
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 N-terminal end, depending on the specfic polypeptide. C-terminal PTSs are recognized by different peroxins than are the N-terminal PTSs.
I have drawn the above diagram based upon an analysis of the recent literature dealing with peroxisomal biogenesis.
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.