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# Why are integral proteins embedded into membranes?
Because they have alot of hydrophobice amino acid side chains. Membranes are hydrophobic by nature.
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# How can you get an intergral membrane protein out of the membrane?
By adding a ionic detergent, it will "wash" the protein out of the membrane so it can be viewed independently.
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# Which detergent will get a membrane protein out of the membrane? a nonionic or a ionic?
Both
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# What detergent are we going to use?
ionic detergent = SDS
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# What are we assuming the SDS-PAGES's charge is in this class?
negative
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# What is the point of ripping a membrane protein out of a membrane?
To get in solubilized in the water
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# What is the starting material for membrane proteins
RBCs
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# What are the only membrane that mature RBCs have?
Plasma membranes
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# Why were the RBCs looked at first?
Because they only consists of a plasma membrane and nothign else (no ers, golgi, etc.)
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# How do you get to study the plasma membrane of an RBC and nothign else?
By "popping" it and getting the "ghost"
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# How do you get the RBC "ghosts?"
Hemolysis (pop and reseal after osmotic shock)
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# The hemolysis of a RBC is hypotonic or hypertonic?
Hypotonic
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# How do you physically just get the ghosts of the RBC?
Preform hemolysis on the RBCs, centrifuge it, then throw out the supernatant (goo inside), to leave the "ghosts"- plasma membrane
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# RBC ghosts are _____ plasma membranes
Pure
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# If you want to know what pure plasma membrane are you need to study the ...
RBC ghosts
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# If you add SDS-PAGE to the RBC ghosts what will it do to them?
rip the membrane proteins out of the membrane, and cover them in detergent
-
# "top of the gel" means
Where we started the experiment
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# When SDS-PAGE is added to a membrane, what proteins come off? Peripheral or integral?
Both of them, peripheals are easily washed away and integrals are ripped out by the detergent.
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# what is your first assumption about membrane proteins?
That they are all negatively charged and all soluble.
-
# How do you start the electrophoresis?
By turning on the electricity and having the proteins pull towards the positive pole (because proteins *are negative* opposites attract)
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# How do we know when to turn off the electricity for the electrophoresis?
When the proteins hit the colored dye at the "bottom"
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# How would you determine which proteins are peripheal and which are integral in gel electrophoresis?
Cetrifuge the RBC ghosts in a high salt solution. then re-do the gel electrophoresis. Peripheal proteins will have come off due to the high salt, and all that is left is the intergral proteins.
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# How would you know what the molecular weight of certian proteins are?
By indicating bands with known molecular weights.
-
# You can identify a protein by its...
molecular weight
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# With SDS coating proteins, they move by ___________ towards the _________ pole.
- * electrophoresis
- * positive
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# The smaller proteins move ________ in electrophoresis
faster
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# What is electrophoresis used for?
Which proteins are integral and which are peripheral.
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# What is Actin?
- * A long fibrous protein
- * peripheal protein
- * CytoSkeleton like
- * Ties together integral protiens
- * (cytoskeletal filaments)
-
# Sugars are on the...
outside of the cell
-
# You will only see what three things facing the outside of the cell?
- * Sugars
- * Glycolipids
- * Glycoproteins
-
# All cytofilaments are ....
polymers of proteins
-
# Cytofilaments are assembled from a pool of protein subunits and ___________.
polymerized
-
# Each protein is a polymer of...
amino acid
-
# Polymerize proteins to make a....
filament
-
# Filament means...
a polymer of proteins
-
# Protein subunits start as monomers and are polymerized to....
filaments
-
# protein filaments are depolymerizied into
monomeric subunits
-
# If a cell wants to divide what must they do to the cytofilaments?
depolymerize them
-
# Some filaments are _________, always changing by assembly and disassembly
dynamic
-
# If filaments grow more than they shrink they get ....
longer
-
# If filaments shrink more than they grow they get...
shorter.
-
# What are the three main kinds of cytoskeletal filaments?
- * microtubules
- * intermediate filaments
- * microfilaments
-
# What is the typical diameter size of a microtubules?
25 nm
-
# What is the typical diameter size of a intermediate filaments?
10 nm
-
# What is the typical diameter size of a microfilaments?
5-7 nm
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# How can you theorectially tell which cytoskeltal filament is which?
By cutting them and measuring the diameter of it.
-
# What are microtubules?
- * A "tube of tubulin"
- * Tubulin subunits are dimers (heterodimers)
- * heterodimers made of Alpha tubulin and Beta tubulin
-
# In a microtubules, aplha and beta tubulins _________ to make the dimers.
polymerize
-
# Do you need atp for alpha and beta tubulins to spontaneously form micrtobules in vitro?
No
-
# what is the main idea behind intermediate filaments?
that you can put many of them together and just end up with one big intermediate filaments
-
# Microfilaments are made of..
actin
-
# What does Micofilaments, actin filaments, f-actin and filamentous actin have in common?
they are all the same thing
-
# Actin monomers are...
just actin
-
# if you put actin monomers together by polymerization you get...
Filament (polymer)
-
# How do filaments become stronger?
By turning into a double helix of filaments
-
# When we say microfilaments is really means the...
double helix of filaments
-
# Globular actin is not just a glob because
it has structure to it
-
# ATP is required to make what kind of filaments?
Actin
-
# GTP is required to make what kind of filaments?
microtubules
-
# "various globular proteins" are needed to make what kind of filament?
intermediate filaments
-
# What End are microtubules built on?
The "+" end
-
# Where are intermediate filaments built on?
internal (all over the place)
-
# What end do microfilaments grow on?
the "+" end
-
# Do microtubules have polarity?
yes
-
# Do intermeidate filaments have polarity?
no
-
# do microfilaments have polarity?
yes
-
# What does a GTPase do?
breaks down GTP
-
# What does a ATPase do?
breaks down ATP
-
# What is the enzymatic activity of microtubules?
GTPase
-
# what is the enzymatic activity of intermediate filaments?
none
-
# what is the enzymatic activity for microfilaments?
ATPase
-
# What are microtubules motor proteins?
Kinesins, dyneins
-
# what are microtubules motor proteins?
Myosins
-
# what are intermeidate filaments motor proteins?
they have none
-
# What are the microtubules major group of associated proteins?
MAPs
-
# What are the major group of associated proteins for microfilaments?
Actin-binding proteins
-
# What is the structure of microtubules?
Stiff, hollow tube
-
# What is the structure of intermediate filaments?
tough, ropelike fibers
-
# What is the structure of microfilaments?
flexible, helical filament
-
# Who has microtubules?
all eukaryotes
-
# Who has intermediate fibers?
only animals
-
# who has microfilaments?
all eukaryotes
-
# What is the primary function of microtubules?
- * MAINLY intracellular transport
- * Support
- * Cell Organization
-
# What is the primary function of intermediate filaments?
structural support
-
# What is the primary fuction of microfilaments?
Motility, contractility
-
# The molecular weights of intermediate fibers are usually...
all over the place
-
# What would pure microtubules look like on SDS-PAGE w/high salt?
two seperate bands (alpha and beta tubulin)
-
# what is the genral molecular weight of the tubulins in pure microtubulins on SDS-PAGE?
about 66 kDa
-
# What would microfilaments look like on SDS-PAGE w/high salt?
One band of actin
-
# What is the general molecular weight of the actin in microfilaments on SDS-PAGE?
about 43 kDa
-
# SDS breaks down everything into it's __________ form.
monumeric
-
# How do you use SDS-PAGE to identify a protein?
It's size againt the molecular standards
-
# How can you stop cell division?
Stop microtubules from growing
-
# Microtubules are repeating units of ....
heterodimers (alpha and beta)
-
# what is the tubulin dimer?
The subunit for the protofilament
-
# What is a protofilament?
One line of tubulin
-
# Protofilaments form...
Microtubulin
-
# Tubulin dimers, microtubules are _________, no need to add energy.
spontaneous, they form on their own.
-
# In microtubules, alpha is on the _______ end.
minus
-
# in microtubules, beta is on the ________ end.
plus
-
# Microtubles always grow at the ______ end.
beta
-
# Does GTP hydrolysis is microtubles provide energy?
No
-
# The complete formation of a microtubule is?
- * The alpha and beta forms a tubulin dimer
- * The tubulin dimer forms a protofilament
- * 13 protofilaments form a microtubule
-
# Microtubule is ...
A clynider made of 13 protofilaments side by side (cytoplasmic)
-
# If you preform a cross section on a microtubule how many protofilaments would you see?
13
-
# Is GTP hydrolysis necessary for the Microtubule to grow?
No.
-
# Colchicine inhibits _________.
Microtubule growth
-
# what is the role of GTP in microtubule assembly?
To allow protofilaments to grow
-
# What are the three binding site on the beta of a tubulin dimer?
- * Ca ++ binding site
- *Colchine Binding Site
- * Exchangeable GTP Binding Site
-
# You cannot add to a protofilament unless the beta in the last tubulin dimer has..
GTP
-
# Charged tubulin is?
Tubublin that is GTP bound.
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