Intracellular Compartments: The endoplasmic reticulum, Golgi complex, endosomes, lysosomes, and pero

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Chapter 12

• Intracellular Compartments The endoplasmic
  reticulum, Golgi complex, endosomes, lysosomes,
  and peroxisomes

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Endo-membrane System

• RER and SER, Golgi complex, endosome (early and
  late), lysosome, nuclear envelope (continuous
  with ER)
• Transport vesicles move between these organelles
• Peroxisomes - not part of endomembrane system but
  a vesicle

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Endoplasmic Reticulum

• Flattened sacs or cisternae, tubules and vesicles
• Function to make proteins for ER, Golgi, etc and
  proteins for secretion RER
• Transitional elements (vesicles) shuttles
  lipids and proteins from ER to Golgi, resembles
  SER
• Function to make lipids for membranes
  triglycerides, cholesterol, related compounds
  SER
• Continuous with the RER no vesicles

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RER and SER
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Sub-cellular Fractionation

• Using differential centrifugation we can separate
  organelles and macromolecules based on size and
  density
• Sedimentation rate rate that things move
  through a solution
• Larger move faster
• Smaller move slower

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Centrifugation

• Three types of centrifugation
• Differential
• Equilibrium gradient
• Density gradient
• Sample prep bust up the cells in a cold
  isotonic solution to end up with a homogenate
  all the cellular components in a mixture

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Differential Centrifugation

• Separation is based on size and/or density
• Sedimentation Coefficient measurement of how
  rapidly the particle sediments when centrifuged

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Using Differential Centrifugation
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Density Gradient Centrifugation

• Also called rate-zonal centrifugation
• Place sample on a gradient such as sucrose (small
  concentration range)
• After centrifugation (low speeds), bands form
  where the organelle has the same density as the
  sucrose solution around it
• Larger fragments move farther than the smaller
  ones

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Equilibrium Density Centrifugation

• Also called buoyant density centrifugation
• Place sample on a gradient such as sucrose (large
  concentration range)
• After centrifugation (high speeds), bands form
  where the organelle has the same density as the
  sucrose solution around it
• Denser fragments move farther than the others
• Usually done after one of the other types of
  centrifugation steps

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RER Functions

• Synthesis and processing of proteins
• proteins that will be membrane bound or soluble
  outside of the cell
• Protein enters the ER 1 of 2 ways
• co-translationally right after leaving the
  ribosome
• Membrane bound anchored in membrane or to a
  lipid in the membrane
• Soluble released into the lumen of the ER
• post-translationally after they have been made
• for peroxisomes, mitochondria and chloroplasts

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Additional RER Functions

• Site where the addition of the carbohydrates
  starts, proper folding of polypeptides and
  assembly of multimeric proteins
• Enzymes present to help with these processes
• Formation of di-sulfide bonds by protein
  disulfide isomerase, make and break them
• Target for degradation if misfolded
• ERAD ER associated degradation
• moves protein to the cytoplasm for destruction by
  proteosome

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SER Functions

• Site of drug detoxification, carbohydrate
  metabolism, Ca2 storage, steroid biosynthesis
  (male/female hormones)
• Drugs detoxified by addition of OH groups using
  cytochrome P450 in liver cells
• In liver cells must break down glycogen to enter
  glycolysis
• Ca2 is moved by a ATP-dependent pump
  specifically in sarcoplasmic reticulum of muscle
  cells

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Drug Detoxification

• Liver, lung and intestines
• Hydroxylation requires an e- donor and O2
• NADPH when drug detox and steroid synthesis
• NADH when fatty acid synthesis
• Enzyme responsible are mixed-function oxidases or
  mono-oxygenases
• Can see an increase in SER and enzyme production
  following some drug overdoses such as
  barbiturates
• habitual use can lead to decreased effectiveness
  of other drugs because of the increase in SER
• antibiotics, anticoagulants and steroids

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Drug Detox continued

• Transfer e- from NADPH or NADH to cytochrome P450
  and then on to O2
• 1 O used for hydroxylation and the other is
  reduced to H2O
• Hydroxylation increase water solubility of
  hydrophobic drugs that can then be removed
• Aryl hydrocarbon hydroxylase another P450
  enzyme can metabolize polycyclic hydrocarbons and
  make them more dangerous
• cigarette smoke can activate this enzyme pathway

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Carbohydrate Metabolism

• Breakdown glycogen using to Glc-1-PO4 by glycogen
  phosphorylase
• Convert Glc-1-PO4 to Glc-6-PO4
• Then glucose-6-phosphatase creates glucose that
  can leave
• enzyme unique to ER
• liver, kidney and intestine
• Muscles and brain use as glucose 6-PO4

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Ca2 Storage

• Specialized SER to store Ca2 to increase the
  Ca2
• sarcoplasmic reticulum
• Use ATP-dependent Ca2 ATPase
• Ca2 released in response to a stimulus and can
  cause muscle contraction

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Biosynthesis of Membranes

• SER is primary site for membrane lipid synthesis
  phospholipids and cholesterol
• peroxisomes also may contribute cholesterol and
  dolichol
• Lipids on the cytosolic surface require special
  proteins to move it to the appropriate leaflet
  specific for each lipid type
• Flippase or phospholipid translocators moves
  lipids in the plasma membrane, specific for each
  lipid
• Phospholipid exchange proteins move lipids from
  ER to mitochondria and chloroplast, also specific

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Golgi Apparatus

• Very dynamic organelle
• Number per cell varies based on cell structure
  and function
• Linked physically and functionally to ER
• Chemically modifies, sorts and packages proteins
  from the ER
• Coated transport vesicles will move the ER
  material between the ER and Golgi, between sacs
  of Golgi and to other areas of the cell

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Golgi Apparatus

• 2 faces (sides) of the Golgi
• Forming or cis surface near the transitional
  elements of the ER
• Coated vesicles bring lipids and proteins to the
  cis-Golgi network (CGN)
• Maturing or trans surface furthest from the ER
• Coated vesicles bud with processed lipids and
  proteins from the trans-Golgi network (TGN) to
  other areas
• Medial cisternae between CGN and TGN
• majority of the lipid and protein processing here

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CGN, Medial Cisternae and TGN

• Chemically distinct each has their own
  collection of enzymes to carry out their
  functions biochemical polarity
• Different coated vesicles carry materials between
  each portion
• ER to CGN COPII
• from CGN to medial cisternae COPI only
• from TGN clathrin or COPl

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GlcNAc Transferase I Staining
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Lipid and Protein Movement

• 2 models
• Stationary cisternae model each is a stable
  structure
• Traffic moves by shuttle vesicles between
  cisternae cis ? trans
• Proteins for TGN not necessarily selected or
  concentrated, those for the CGN or ER are
  actively retained or retrieved
• Cisternal maturation model each is a transient
  structure
• Cisternae are transient transformation of one
  into the next and so forth, ER to cis to medial
  to trans, return enzymes to the cis by transport
  vesicles
• More than likely a combination of both

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Anterograde and Retrograde Transport

• Anterograde moves things from ER ? plasma
  membrane
• Transport vesicle membrane gets added to the
  plasma membrane
• Retrograde moves from plasma membrane ? cells
  interior
• Prevents depletion of membrane for other vesicles
  by bringing in the membrane

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Protein Glycosylation

• Protein processing in the ER an Golgi deal with
  glycosylation adding of carbohydrate residues
  to R group on amino acid - glycoprotein
• 2 types of glycosylation
• N-linked glycosylation sugar added to a NH2
  group on Asn residue
• Asn-X-Ser/Thr, X? Pro
• O-linked glycosylation sugar added to the OH
  group on Ser or Thr
• Glycosylation is a stepwise process dependent
  on previous steps
• Defective enzymes will halt the process

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N-linked Glycosylation
Enzymes spread throughout various compartments
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N-Linked
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Early Steps

• Glycosylation starts outside the ER with initial
  sugars added to dolichol, a membrane lipid
• Special flippase turns the glycolipid into the
  lumen of the ER where remaining sugars are added
• Core oligosaccharide is 14 sugar residues on a
  lipid called dolichol phosphate
• 2 GlcNAc, 9 mannose and 3 glucose

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N-linked Glycosylation Stage 1

• Core glycosylation Figure 12-7
• In the lumen of ER
• N-acetylglucosamine is attached to Asn
• Moved to the Asn by oligosaccharyl transferase
  that is anchored in the membrane
• May be done before protein is finished
• Aids in proper folding of the protein

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N-linked Glycosylation Stage 2

• Modification of sugar side chains
• Initial steps in the ER, again may be before
  protein is finished
• Glucosidase eventually removes 3 glucose residues
• Mannosidase removes one mannose residue

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Protein Folding

• Sugar tree helps in protein folding
• Calnexin (CNX) and calreticulin (CRT) bind to a
  monoglycosylated protein and interact with ERp57
  (thiol reductase) to make S-S bonds
• Glucosidase II will then remove the 3rd glucose
• UGGT UDP-glucoseglycoprotein glucotransferase
  acts as a sensor for proper folding
• If wrong, it adds back a glucose and CNX and CRT
  come back in and try again
• If right, can leave and move to Golgi

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Golgi Processing

• Protein then is moved to the Golgi for further
  processing
• CGN ? medial cisternae ? TGN terminal
  glycosylation
• Remove some core sugars
• Could be no additional modification
• Can add complex oligosaccharides GlcNAc,
  galactose, sialic acid or fucose

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Terminal Glycosylation

• Proteins leaving the Golgi with only high
  mannose, only complex oligosaccharide or a
  combination of both
• Sugars are on proteins only on the lumen side so
  that it will end up outside the cell when vesicle
  fuses with plasma membrane

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Important Glycosylation Enzymes

• Glucan synthetase makes the oligosaccharides
  from monomers
• Glycosyl transferases adds the sugars to
  protein
• Approximately 200 types highly complex
• Potential complexity in the oligo-side chains
• Proteins dispersed according to where the sugars
  are added on the branch

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Trafficking in Endomembrane System
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Protein Sorting

• Proteins have a tag to help direct them to the
  appropriate place
• Starts in the ER/Golgi and finishes in the TGN
• Tags can be
• Specific amino acid sequence
• Oligosaccharide side chain
• Hydrophobic domains
• Other structural features
• Tags may also exclude proteins from certain areas

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Lipid Sorting

• Tags may also help lipids get to the right
  orientation
• PO4 location on the lipids
• Degree of saturation of tails may also aid in
  proper localization

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ER-Specific Proteins

• Proteins destined to return to the ER have a
  retrieval tag KDEL (Lys-Asp-Glu-Leu)
• HDEL in yeast
• Movement of the protein is from the CGN to the ER
• Tag binds receptor in CGN and receptor undergoes
  conformation change and sends protein in a
  transport vesicle back to the ER
• Chimeric proteins
• can use the KDEL signal to return secreted
  proteins back to the ER

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Golgi Protein Sorting 3 Methods

• Retrieval tag keeps these proteins in the Golgi
• Formation of large complexes keep the proteins
  out of transport vesicles
• Dependent on the length of the membrane spanning
  domain hydrophobic domain
• Golgi membrane thickens as you move from the CGN
  to the TGN
• Protein stops moving when the thickness of the
  membrane exceeds the hydrophobic spanning region

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Targeting to Endosomes/Lysosomes

• Lysosomal enzymes move thru Golgi
• N-glycosylation and then lose glucose and mannose
  in the Golgi
• Lysosomal tag is a phosphorylated mannose residue
• Directs it to lysosome rather than for secretion
  because of the Man-6-PO4 receptor in the TGN

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Lysosomal Protein Processing

• Two enzymes prepare the protein for the lysosome
• Phosphotransferase in early compartment adds
  GlcNAc-1-PO4 to mannose on C6
• A mid-Golgi enzyme removes the GlcNAc, leaving a
  mannose-6-PO4 which binds to the mannose-6-PO4
  receptor (MPR) and is moved from TGN to the late
  endosome by clathrin coated vesicles
• Early endosome ages to become a late endosome and
  pH drops to 5.5
• MPR and protein separate because of pH receptor
  is recycled
• Late endosome can become a new lysosome or fuse
  with an existing lysosome

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Secretory Pathways

• Movement of proteins from ER to Golgi to
  secretory vesicles and secretory granules for
  release to the exterior of the cell
• 2 modes of secretion
• Constitutive secretion
• Regulated secretion

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Proof of Secretory Pathway
Follow radioactive proteins in salivary glands
after injecting radioactive amino acids
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Constitutive Secretion

• Continuous discharge, independent of external
  signal
• Mucous producing cells in gut or glycoproteins in
  the extracellular matrix
• Thought to be a default pathway as there are no
  retention signals
• Can remove the KDEL sequence and watch the
  protein be secreted
• No concentration step

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Regulated Secretion

• Controlled, rapid release usually because of a
  specific extracellular signal
• Secretory vesicles move to the plasma membrane
  and wait for their signal
• Proteins leave in an immature secretory vesicle,
  lose clathrin coat, undergo maturation
• Condensation, proteolytic processing and
  concentration of proteins
• Called zymogen granules or secretory granules

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Granules

• Release of neurotransmitters, insulin and
  digestive enzymes

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Exocytosis

• Proteins in vesicles to exterior of the cell
• Vesicle moves to the plasma membrane
• Membrane fuse
• Membrane ruptures discharging contents
• Vesicle membrane becomes part of the plasma
  membrane
• Glycoproteins and glycolipids remain attached to
  the membrane and face the extracellular space

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Exocytosis

• Vesicles move along microtubules to the plasma
  membrane
• Can inhibit movement with colchicine a drug
  that prevent tubulin polymerization
• Release of regulated secretion is caused by an
  increase in Ca2 concentration in the cell
• Due to an external signal that activates the
  second messenger InsP3
• Polarized secretion exocytosis restricted to
  specific surface of the cell and to different
  materials

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Endocytosis

• Ingest essential nutrients and as defense system
• Mechanism
• Plasma membrane folds inward
• Pinches off to form endocytic vesicle containing
  ingested materials
• Brings outside to inside

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Membrane Flow

• Exocytosis and endocytosis have opposite effects
  on the membrane
• Recycling of the proteins and lipids needed for
  exocytosis
• Endocytosis and retrograde transport bring in
  nutrients

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2 Types Endocytosis

• Phagocytosis
• Pinocytosis
• Receptor-mediated endocytosis (clathrin
  dependent)
• Clathrin independent endocytosis

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Phagocytosis

• Ingestion of large particles and even whole
  organisms
• Mechanism triggered by binding target
• Pseudopods form around debris and eventually
  engulfs particle
• Phagocytic vesicle (phagosome) that fuse with
  late endosome or matures to lysosome
• Phagocytes get rid of foreign material
• Macrophages get rid of cellular debris like
  RBCs
• Neutrophils microorganisms
• Occasional other specialized cell types

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Receptor-Mediated Endocytosis
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Receptor-Mediated Endocytosis

• Also called clathrin-dependent endocytosis
• Use specific receptors to bring in material
• Concentrates the things brought into the cell
• Mechanism
• Receptor moves to plasma membrane
• Receptor-ligand complex encounters specialized
  area - coated pits and accumulates
• Coated pits contain adaptin protein, clathrin and
  dynamin and causes invagination
• May get internalized without ligand
• Coated vesicle is pinched off by dynamin
• Release the clathrin coat which is recycled

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Fate of Receptor-Ligand Complex

• Vesicle containing the receptor-ligand complex
  binds to early endosome which causes release of
  ligand due to acidified environment
• Ligands can
• Move on to late endosome and ultimately digestion
• Move cargo to other side of cell to release cargo
  transcytosis
• Antibody transport from mom to baby
• Move to the TGN and enter a variety of paths in
  the endomembrane system
• Receptors may be recycled to the plasma membrane

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Clathrin Coated Pit
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Clathrin Coated Pits

• Coat is made of 2 multimeric proteins called
  clathrin and adaptor protein
• Adaptor protein allows for the regulation of the
  assembly and disassembly of the coat
• Also requires dynamin a cytosolic GTPase that
  constricts and closes the vesicle off from the
  membrane

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Clathrin Independent Endocytosis

• Fluid-phase endocytosis pinocytosis
• Non-specific internalization of fluid
• No concentration
• May sweep in receptors along with liquids
• Helps control membrane put there by exocytosis
  cell volume and cell surface area
• Occurs at a relatively constant rate

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Vesicle Targeting

• SNAREs help to target vesicle to the appropriate
  membrane
• Soluble NSF Attachment Protein (SNAP) REceptor
• 2 families
• v-SNARE on the transport vesicle
• t-SNARE on the target membrane
• Also requires Rab GTPase and tethering proteins
  that are coiled-coil and multi-subunit proteins
• Vesicle fusion caused by N-ethylmaleimide-Sensitiv
  e Factor and group of SNAPs

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Cellular Digestion

• Lysosomes contain digestive enzymes
• Degrades all major classes of macromolecules
• Building blocks move out to cytosol to be re-used
• Vary in size and shape
• Single membrane
• Acidic environment allows for digestion
• Enzymes are acid hydrolases
• Membrane is protected by heavy glycosylation on
  membrane components

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Lysosomes from Endosomes

• Enzymes made in the ER travel to the Golgi and
  then to early endosomes which mature to late
  endosomes
• Early endosome has all the enzymes but doesnt
  partake in digestion
• Final step is to activate the acid hydrolases
• pH has to become more acidic by ATPases pumping
  in H

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Functions of Lysosomes
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Digestive Process

• Heterophagic lysosomes digestion of materials
  of extracellular nature
• Autophagic lysosome digestion of materials of
  intracellular nature, autophoagosome
• Damaged or materials no longer needed
• Macrophagy remove organelles
• Microphagy remove small parts of cytoplasm
• Extracellular digestion sperm digests the
  surface of the egg to get into it for
  fertilization

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Lysosomal Storage Diseases

• Caused by harmful accumulation of specific
  substances because of missing hydrolases
• Enzyme missing for glycogen, glycosaminoglycans
  and glycolipids
• See skeletal defects, muscle weakness and mental
  retardation
• Usually becomes fatal

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Peroxisomes

• Single membrane but not derived from the ER
• Prominent in kidney and intestinal cells, algae,
  photosynthetic cells of plants and germinating
  seeds
• Catalase is most prominent enzyme that degrades
  H2O2 that comes from oxidative reactions with
  oxidases

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5 General Categories of Function

• H2O2 metabolism
• H2O2 made by moving an e- around
• Catalase can work in the catalatic mode that
  creates 2 H2O and 1 O2 or in the peroxidatic mode
  that uses e- from organic donor to make 2 H2O
• Detoxification of harmful compounds
• Gets rid of organic made in the peroxidatic mode
• Oxidation of fatty acids
• ?-oxidation of long-chain (16-20C), very
  long-chain (24-26C) or branched fatty acids
• Make into AcetylCoA that goes to the mitochondria

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5 General Categories Continued

• Metabolism of N-containing compounds
• Urate that come from the catabolism of nucleic
  acids and some proteins
• Urate oxidase also creates H2O2 from urate
• Catabolism of unusual substances
• Things that are rare such as D-amino acids that
  cannot be digested or used

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Peroxisome Formation

• Biogenesis
• Divide from pre-existing peroxisomes
• Catalase is tetrameric and requires heme