EXAM 3 CELL PHYS

What are the amino chemically made up of?

• Amine group

• Carboxylic acid group

• Side chains (different side chain for each amino acid)

  • 20 standard amino acids

What are the 4 amino acid classes (based on charge and polarity)?

• Non polar

• Polar

• Positively charged

• Negative charged

Non- polar amino acids characteristics:

• Largest class of amino acids

• High solubility in octane, low solubility in water (hydrophobic)

• Nonpolar, hydrophobic side chains

Polar amino acid characteristics:

and

Which ones could be phosphorylated?

• Water-soluble

• Don’t carry a charge at normal physiologic PH

  • Serine, Theonine, and tyrosine can be phosphorylated

Which amino acids are positively charged, and what are their characteristics?

• Lysine, arginine, and histidine

  • They have a positively charged side chain

Which amino acids are negatively charged, and what are their characteristics?

• Aspartic acid and Glutamic acid

  • at physiologic PH they lose a proton

Primary Structure Amino acid

The primary structure of a protein is simply a chain of amino acids in the right order

• It is the direct translation of the mRNA

• The primary structure of a protein is easy to represent, as it is simply a linear list of amino acids (GIVCEQAS) (amino acid sequence)

• n terminal for a primary AA structure is always on the ____

• c terminal for a primary AA structure is always on the ____

• Ribosome starts off at the ___ terminus

• N terminal is always on the left (free amine)

• C termnial is always on the right (free COOH)

  • Ribosome starts off at the N terminus

What are the two shapes that are common to most amino acids?

these to shapes make a _______ structure of amino acids

Why are they so stable?

Shapes:

• Alpha helix and Beta sheet

Structure:

• Secondary structure

Stability

• A lot of hydrogen bonds are formed with these hexises- very stable

Why are non-polar molecules hydrophobic?

• non-polar molecules can’t form hydrogen bonds

• Water has a lot of hydrogen bonds

  • When a nonpolar amino acid is introduced to water, water can not form hydrogen bonds

  • Entropy goes up

  • That’s why tertiary structure puts hydrophobic AA in the inside away from water

Tertiary Structure formation is primarily driven by _______ interactions

hydrophobic interactions

• But also include hydrogen bonding and ionic interactions

In __________ structured amino acid Interactions between side chains cause the local shapes to collapse into a single __________ stucture

In tertiary AA’s, interactions between side chains cause the local shapes to collapse into a single 3-dimensional structure

• Also have hydrogen

How are tertiary structured proteins stabilized?

• By disulfide bonds between cysteine residues

Define Quaternary Structure

Give an example_______

Some proteins form stable associations between multiple polypeptides to form a single protein unit

• One peptide interacting with other peptides

  • Tertiary and quaternary structures interacting

• An excellent example is hemoglobin, which is made up of 2 α-chains and 2 β-chains

What are examples of Posttranslational Modifications?

Side chain interaction interaction drive a lot of proteins 3d shape, changing shape, could chain activity

• Phosphorylation (threonine, serine, or tyrosine- nonpolar, but with phosphate they are strongly negatively charged)

• Glycosylation

• Ubiquitination

• sumoylation

• Disulfide bonds (could stabilize 3-dimensional structures: 2 cysteines that react together to form a covalent bond- 3-dimensional structure is harder to undo)

• acetylation

• lipidation

• methylation

• hydroxylation

Know phosphorylation and disulfide bonds *****

Protein function is dependent on _____, ______ and _______

Size, shape, and chemistry

• Protein folding leads with posttranslational modification leads to a unique size, shape, and chemistry for each protein

• Clinically used: Identifying proteins= identify organisms making those protein= useful for diagnosis

What are antibodies?

Antibodies are defense proteins that are designed to recognize the unique 3 dimensional surface of other proteins

Antibodies can recognize proteins that should not be there (e.g. from an invasive organism/virus) and prompt an immune response

We can utilize the properties of antibodies to recognize specific proteins and thereby diagnose specific diseases

Each antibody is targeted to one specific _______

Each antibody is targeted to one specific protein

Direct Fluorescent Antibody Test (DFA)

(A type of Immunohistochemistry (IHC))

1. Check the brain stem

2. Create histology slides with brain stem sample]

3. Dry and fix in acetone

4. Add rabies antibody with fluorescent tag

5. Wash several times to remove unbound antibody (weakly bound proteins come off)

6. Mount on a coverslip

7. Check fluorescence

Using an antibody to look for an invasive protein!!!

(Using antibodies against rabies to test or rabies proteins)

Tube Agglutination Test

• Antigen (bacteria) too large to solubilize; stays in solution

• On their own, they suspend well; when they are cross-linked, they precipitate

• Bacteria stays up = not enough antibodies to efficiently cross-link

• Bacteria fall down/ precipitates= Crosslink occurs, which means there is a lot of antibodies there

1. Put an antigen (e.g. Brucella suis) into a tube

2. Add serum from a suspect animal

3. If the serum contains antibodies to the antigen

(e.g. Brucella suis), the samples should

agglutinate and precipitate

 If not, the sample will remain suspended

How can the amount of antibodies be estimated?

Amount of antibody present can be estimated by titrating (diluting) the serum

• How many times can I dilute the serum and still have it participate in the bacteria

• Dilute the serum a small number of times before it stops precipitating = not many antibodies

• Dilute the serum a large number of times and still precipitating = very positive/ a lot of antibodies

 Higher titers = more antibody (>1/160 indicative

of infection)

How is the elisa test conducted?

1. Coat the bottom of the well with an antigen (EIA)

2. Take the serum (filled with ANTIBODIES but if it was exposed to EIA It should have EIA specific antibodies; it should bind strongly to antigen) should be from the specimen and add it to the well

3. Wash the plate and see which plate has EIA antibodies

4. Wash antigen that is bound to an enzyme

5. Enzyme should stick if there is antibodies against EIA

6. IF there is no enzyme there indicator molecule will stay blue but if there is an enzyme, it will turn yellow

What is the advantage of ELISA?

Small amount of enzyme can make a lot of color

How can concentration on antigens be estimated in ELISA?

• How intense the end color is

  • Yellow= positive

  • blue= negative

• Or by titering the serum

What are we testing for using ELISA ?

• using EIV antigens ( or target antigen) to test for anti- EIV antibodies in serum

The Electron Transport System Overview

• Location: Mitochondrial inner membrane

• Split into 4 relatively independent complexes

• Complex I

  • could oxidize NAH

  • Complex II (succinate dehydrogenase)

• Complex III

• Complex IV

• Electron carriers connect the complexes

• Coenzyme Q

• pumping protons across the membrane

Cytochrome C

• cytochrome c moves enegery to compelex w and then across the membrane

Electron Transport System Summary

Since complexes I, III, and IV are pumping protons against their gradient,

energy is stored in the proton gradient

This energy would like to be discharged by pushing protons back across

the inner mitochondrial membrane: proton motive force

The proton motive force (pmf) is used to synthesize ATP

ATP Synthase

“motor”

• ATP synthase is a marvelously complex motor that uses the pmf

to produce ATP

• In the center of the complex there is a partially occluded proton

channel - for a proton to move through, the channel has to turn

• This channel rotation in turn changes the conformation of the

synthase portion of the enzyme

• Since protons release energy by going through the channel, they

can ‘push’ the channel around

• The synthase uses the energy of the protons pushing the

channel around to add a phosphate to ADP

• Each full rotation of the channel, causes the synthesis of 3 ATP,

and it takes 10 H+ to turn the channel one full rotation (3.33

H+/ATP)

Each NADH moves approximately ____ protons

across the membrane

10

Each FADH moves approximately __protons

across the membrane

6

When the proton gradient is developed, it takes approximately __protons to produce 1 ATP

4

NADH makes approximately __ ATP

2.5 ATP

FADH makes approximately ___ATP

1.5

LECTURE 18 Carbohydrate Metabolism III

What is Glycogen?

Glycogen is a highly branched polysaccharide made up of glucose with α-1,4 bonds (straight) and branching α-1,6 bonds (branched) (know that there is 2)

• A single glycogen polymer may have up to 60,000 glucose residues

• Each glycogen polymer is built around a protein – glycogenin, and glycogen particles

contain glycogen and enzymes involved in making and using glycogen

What is the primary storage method for carbohydrates in animal cells?

Glycogen

Glycogen is the primary storage method for carbohydrates in animal cells

(The liver stores sugar for the rest of the body )

(glucose is a osmolite; glycogen is not completely soluble)

(fat can not provide glucose, fat can not turn back into glucose- also an only be used aerobically)

• 5-8% weight of liver (in well fed state)

• 1-2% weight of muscle

• Much more glycogen is stored in muscle than liver (lower % weight, but much more muscle mass)

Why Glycogen? (not glucose or fat)

• Glucose intake during meals is infrequent – without storage and release, blood glucose would

fluctuate wildly between feeding and fasting

• Glucose is an osmolyte – adding glucose to a cell increase the osmotic pressure inside the cell

• Storing glucose in a cell will cause water to enter the cell to keep the osmolality balanced

• This would rapidly lead to membrane rupture if significant amounts of glucose were stored

• Glycogen, on the other hand, is not an osmolyte (it is insoluble, so it doesn’t contribute to

osmolality)

• Large amounts of glycogen can be stored in a cell without increasing osmotic pressure

• Cell can store glucose without rupturing

• Glycogen has much less energy density than fat (4 kCal/g vs 9 kCal/g)- based on energy density, fat

storage is much more efficient, and glycogen storage is limited, but:

1. Fatty acids cannot be converted back into glucose ( we’ll look at this in the lipid metabolism section), and glucose is

an absolute requirement

2. Glycogen can be much more rapidly mobilized than fat

3. Glucose from glycogen can be used anaerobically, fat cannot

What is the regulatory step for speeding up/ slowing down the production of glycogen?

Glycogen Synthase***

• but only straight bonds

Step 5: Glycogen Branching Enzyme

Glycogen Phosphorylase and Glycogen Debranching

Enzyme

Processing Glucose-6-Phosphate

Glucose-6-Phosphatase

Regulation: Insulin and Glucagon/Epinephrine