Cytoskeleton

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• Lecture 3
• Cytoskeleton
• Enzymes

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The Cytoskeleton
Components Microtubules Microfilaments Inter
mediate filament-like structures Functions of
Cytoskeleton Structure and support Intracellul
ar transport Contractility and
motility Spatial organization
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Microtubules and Microfilaments
Hollow tubular Components of many intracellular
structures Mitotic spindle Cilia
Flagella Longitudinal rows called protofilaments
(13 protofilaments in a microtubule) Protofilam
ents are a heterodimer
- Microtubles and microfilaments can assemble and
dissemble - Polymerization requires energy ATP
for microfilaments, GTP for microtubules -
microtubules and microfilaments are polarized
(plus and minus ends) - polymerization is more
rapid on positive ends
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Current model for the assembly of intermediate
filaments from protein monomers
Structural support of membranes (nuclear envelope
and PM)
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Microtubules Function in Mitosis and Cytokinesis
Mitosis process by which previously replicated
chromosomes are aligned, separated, and
distributed to daughter cells -
microtubules are an integral part of
mitosis Cytokinesis process by which a cell is
partitioned into two progeny cells
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Fluorescence microscopy can show changes in
microtubule arrangements in the cell
A
B
C
D
E
F
G
H
I
J
K
Changes in microtubule arrangements at different
stages in the cell cycle of wheat root meristem
cells. Microtubules are stained green and yellow.
DNA is blue. A-D Pre-prophase E-H Prophase G,
H Late prophase I-K Anaphase
For information on fluorescence microscopy,
please visit http//www.bioscience.org/lecture/mol
biol/micro/frame.htm
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Microtubules function in mitosis and cytokinesis
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Microtubules function in mitosis and cytokinesis
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Microtubules function in mitosis and cytokinesis
Electron micro-graph of a cell plate forming in a
maple seedling
Nuclear envelope
Vesicles
Microtubule
Nucleus
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Myosin-mediated transport of organelles along
actin microfilaments
Myosin a motor protein (other examples
kinesin, dynein) hydrolyses ATP
when activated by binding to actin -gt energy
moves from minus to plus end
http//www.ncbi.nlm.nih.gov/books/bookres.fcgi/mcb
/ch18anim3.mov
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Plasmodesmata
- Plasmodesmata are tubular extensions of the
plasma membrane - 40-50 nm in diameter -
Traverse the cell wall - Connect cytoplasm of
adjacent cells ? cytoplasm of cells form a
continuum, called the symplast (symplastic
transport) - Two types of plasmodesmata primary
and secondary - primary plasmodesmata form
during cytokinesis - secondary plasmodesmata
form between cells after their cell walls
have been deposited
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Plasmodesmata Structure
Longitudinal view
Cross section
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Plasmodesmata Structure
Schematic view
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Enzymes History
Introduction Greek in leaven Louis
Pasteurliving intact cells responsible Eduard
Buchner ..1897..juice of ground yeast ..could
ferment sugar Wilhelm Kuehne (1837-1900) coined
the term "enzyme in leaven Isolation of
pepsin, the first enzyme to be obtained in pure,
by John Northrop, James Sumner and Wendell
Stanley in 1926 ? Nobel Prize for Chemistry 1946
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Enzyme Properties

• Organic catalysts.cofactor and coenzyme
• Specific in reaction
• Active in small amounts
• Unaffected under stable conditions
• Will not affect the equilibrium of the reaction

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Triose-phosphate isomerase (TIM)
Catalyzes the reversible inter-convertion of
dihydroxyacetone phosphate and D-glyceraldehyde
3-phosphate
- A dimer of identical subunits consisting of
about 250 amino acid residues - Contains eight
a-helices (blue and red) on the outside and
eight parallel ?-strands on the inside (violet
and yellow) -gt aß-barrel - Active site in
center of barrel.
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How do enzymes work?
- Form Enzyme-substrate complex "Lock and key"
model (Emil Fischer, 1894)
- Lower activation energy ? faster reaction
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How do enzymes work?
Lower activation energy ? faster reaction
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Types of Enzymes

• Hydrolytic enzymes (a-amylase)
• Oxidation-reduction enzymes (dehydrogenases,
  oxidases)
• Phosphorylases (GDP-mannose pyrophosphorylase)
• Transferases (aspartate aminotransferase)
• Isomerases (triose phosphate isomerase)
• Carboxylases (Ribulose bisphosphate carboxylase)
• Kinases (Ribulose-5-phosphate kinase)

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Distribution of Enzymes

• Mitochondria
• Chloroplast
• Nucleus
• Cytoplasm
• Extracellular
• Membrane

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Enzyme kinetics
Fast
Slow
v Vmax S Km S
Km is a reflection of the affinity of the enzyme.
The smaller the value of Km, the tighter the
binding.
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Enzyme kinetics
Example Hexokinase in brain and muscle binds
glucose at Km 5 x 10-5 M Glucokinase in liver
also binds glucose at Km 2 x 10-2 M Both
enzymes convert glucose to glucose-6-phosphate
both enzymes also occur in plants. Which enzyme
has a higher affinity for glucose?
v Vmax S Km S
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Factors affecting enzyme activity

• Substrate concentration, Enzyme concentration
• Temperature, Hydrogen ion concentration (pH),
  Inhibitors

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Uninhibited enzyme
Lineweaver-Burk double-reciprocal plot
- plotting kinetic data as 1/Vi vs. 1/S -
easily find Km and Vmax - find inhibitors to
block enzyme activity
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Competitive and non-competitive enzyme inhibition
No Inhibition
Competitive Inhibition
Non-competitive Inhibition
or not possible at all
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Competitive and non-competitive enzyme inhibition
No Inhibition
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Types of Enzyme Inhibitors
Non-specific inhibitors denature the
enzyme Example acids, basis, temperature Irrever
sible inhibitor may form covalent
bonds Example Heavy metals such as Ag, Hg2,
Pb2 have strong affinities for SH
groups Reversible inhibitorsweak, non-covalent
bonds Example Advil, Tylenol Application
Design herbicides and insecticides Example
Round-up inhibits enolpyruvylshikimate-3-
phosphate synthase ? blocks synthesis of
aromatic amino acids produced by the
shikimic acid pathway Round-up is toxic
to plants but not animals
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Regulation of Enzyme Activity

• Control of enzyme concentration
• Compartmentalization
• Covalent modification
• Feedback inhibition

To further refresh your memory on Energy and
Enzymes, you may visit http//4e.plantphys.net/ch
apter.php?ch2