cell phys unit 3

positively charged amino acids

lysine, arginine, histidine

negatively charged amino acids

aspartic acid, glutamic acid

polar amino acids

serine, threonine, tyrosine, asparagine, glutamine

non-polar amino acids

glycine, alanine, valine, cysteine, proline, leucine, isoleucine, methionine, tryptophan, phenylalanine

primary structure of protein

chain of amino acids, direct translation of mRNA, N-ter to left, C-ter to right

secondary structure of protein

interactions along amide backbone (binds to itself) cause polypeptide to form locally stable shapes, will stabilize sections of protein and less flexible overall

two most common secondary structure shapes

alpha helix and beta sheet

tertiary structure of protein

interactions of side chains causes local shapes to collapse into single 3d structure, driven by hydrophobic interactions where hydrophobic molecules at center, includes hydrogen bonding and ionic interactions, but often stabilized by disulfide bonds between cysteine residues

quaternary structure of protein

stable associations between multiple polypeptides to form single protein unit, shape and surface chemistry affect what it will bind to, hemoglobin is a great example

posttranslational modifications

phosphorylation, disulfide bond, acetylation, lipidation, methylation, hydroxylation, glycosylation, ubiquitination, SUMOylation

can change side chain to change function

phosphorylation

adds phosphate to serine, threonine or tyrosine, will make them negatively charged

disulfide bond

covalently link the S atoms of two different cysteine residues

what about protein is useful for diagnosis?

size, shape, and chemistry

antibodies

defense proteins that recognize unique 3 dimensional surface of other proteins, can recognize proteins that should not be there and prompt immune response

each antibody is targeted to

one specific protein

how can antibodies be used for diagnoses?

use their properties to recognize specific proteins and thereby diagnose specific diseases

immunohistochemistry

uses antibodies to detect specific proteins or antigens in tissue

direct fluorescent antibody test steps

1. chop up brain to make histology slides

2. dry and fix in acetone

3. add rabies antibody with fluorescent tag

4. wash several times to remove unbound antibody, this will get rid of weak bonds that are not to rabies protein

5. mount coverslip

6. if it is fluorescent there is rabies!

tube agglutination test

1. put antigen (Brucella suis) in tube and add serum from animal

2. if serum has antibodies, they will bind to antigen and precipitate, if not the antigens will stay suspended in the solution

3. amount of antibody can be told by tittering/diluting the serum

the titer is the lowest concentration bacteria stopped precipitating at, higher titers= more antibody

enzyme linked immunosorbent assay (ELISA)

1. coat well with antigen

2. add serum

3. wash

4. add enzyme to speed up reaction

5. wash

6. add blue molecule

if antibody is there, enzyme will bind, and then blue molecule will bind to enzyme and turn yellow

how can equine infectious anemia be diagnosed?

by ELISA, which is quick due to enzyme but can have false positive and is more expensive, or coggin’s agar immunodiffusion test that is a type of agglutination assay

how can proteins be regulated?

sorting, ligand binding, degradation, proteolytic activation

sorting: protein targeting

immediately after protein is made, sometimes after as well, determines where protein will go, default is cytoplasm, others controlled by target peptides and signal peptides

signal peptides

AA at N-terminus, signal recognition particles carry the protein to the RER, then goes through golgi, then secretory vesicles/lysosomes out of cell

target peptides

target protein to specific organelle in the cell

ligand binding

if a small molecule (ligand) binds to a protein, it will create new interactions that will change its shape, and change the function

glucocorticoid ligand binding

when cortisol binds glucocorticoid receptor it will change shape, which can cause big change in shape elsewhere

what can change protein shape/function?

pH, ligand binding, phosphorylation

agonist form

binds to receptor and activates function

antagonist form

binds to receptor and blocks function

degradation

molecule will be degraded into amino acids, will reduce protein expression

ubiquitin

common protein, will attach to protein and its proteasome will degrade the protein, can be partially targeted based on N-sequence (called N-end rule)

proteolytic activation

proteins can be made in an inactive form called zymogens or proproteins, that can have a masking sequence that will be cleaved off to activate it

why is proteolytic activation important in the pancreas?

pancreas makes lots of proteases that could break it up

glucocorticoid receptor regulation

without any glucocorticoid, the receptor is bound to HSP complex and inactive, when cortisol enters it will bind and disassociate from binding complex to make GR dimer with cortisol, where it will travel to nucleus and enhance immunosuppression gene expression

ubiquitin with glucocorticoid dimer

always being degraded by ubiquitin, but the amount of GR dimer will determine how much the genes are turned on

delta G = 0

equilibrium, equally likely to go either way

Gibbs energy

amount of chemical energy stored in its bonds and arrangements, which way a reaction will go

delta G

energy of products - energy of reactants

- delta G

more likely to happen, reactions that release energy and products have less energy than reactants

Gibbs free energy

released energy from a favorable reaction that can be used for something else

irreversible reactions

reactions with a very large negative G that almost always proceed forward

Ea (activation energy)

energy needed to reach the intermediate state, how fast a reaction will go

with a catalyst _ is reduced

Ea

the higher the Ea

the slower the reaction will go

enzymes

biological (usually protein) catalysts that speed up biological reactions

non-protein enzyme

ribosomes, an RNA based enzyme

where do enzymes go?

folded into the 3d structure that forms an active site in the binding pockets, the enzyme has specific AAs that will bind to reactants and catalyze their transition to products

Km, Michaelis constant

reactant concentration at ½ max velocity, to describe how much reactant concentration affects reaction speed

low Km means

high enzyme affinity for substrate, speed of reaction won’t vary much on reactant concentration

altering reactant concentration near Km will

vary the rate of reaction significantly

anabolism

build up

catabolism

break down

rate limiting steps

slowest step in a pathway that regulates how quickly a pathway can run

committed steps

a reaction that commits a reactant to a certain pathway

enzymatic control of metabolism

regulate by activating or inhibiting enzymes

allosteric activator

binds and increase the speed of the reaction

allosteric inhibitor

bind and decrease speed of reaction

allosteric compound

bind NOT at active site but change the activity of an enzyme

feedback inhibition

later product will inhibit an earlier enzyme that prevents over-production of the final product

feed-forward activation

earlier reactant will activate a later enzyme to prevent unnecessary build up of starting reactants

equine type 1 polysaccharide storage myopathy

dominant mutation that causes glycogen synthase to be active in absence of glucose-6-phosphate, causing progressive weakness, loss of muscle mass, and colic-like symptoms

glycogen synthase

is fed forward by glucose-6-phosphate

how do you manage EPSM1?

low carbohydrate diet, exercise therapy, no breeding

GLUTs

use facilitated diffusion to move glucose down a concentration gradient

SGLT/SLC5A

secondary active transport that uses sodium gradient to move glucose up its concentration gradient

ideal blood sugar

100 mg/dL

absolute glucose tissues

GLUT1 tissues, brain and erythrocytes, need sugar because fat cannot get into brain and RBCs don’t have mitochondria so can only burn sugar anaerobically

hexokinase/glucokinase

add a phosphate to glucose to make glucose-6-phosphate so that more “glucose” can enter the cell

hexokinase

low Km, feedback inhibition by G6P, active at low glucose, main metabolic enzyme

glucokinase

high Km, activate at high glucose, triggers insulin secretion

phosphofructokinase-1

main regulatory enzyme for glycolysis

fructose-1,6-bisphosphate

split into two molecules

NAD+ to NADH

will take high energy electrons out of carbohydrates, gives neutral energy to use

pyruvate kinase

turns phosphoenol-pyruvate into pyruvate, get an ATP where most energy comes from in glycolysis

what two enzymes need to be controlled for glycolysis regulation

phospho-fructokinase and pyruvate kinase

regulate the enzymes with substrates with _ delta G, which are

big negative, glucose, fructose-6-phosphate, phosphoenol-pyruvate

how is NADH used for glycolysis regulation

produces NAD+ and H+, that replenishes NAD+ supply in step 6 of glycolysis, and the proton gradient is used to produce ATP

what is done to pyruvate in low oxygen conditions?

it is reduced

lactate dehydrogenase

turns pyruvate into lactic acid, only yields two ATP

benefits of lactic acid metabolism

replenishes NAD+ when O2 levels are low and allows bursts of energy without O2 uptake

drawbacks of lactic acid metabolism

reduces the energy production of glycolysis and eliminates energy production of TCA, lactic acid is a waste product that in high concentrations leads to acidosis

cori cycle

allows lactic acid to be processed as glucose, but will take 6 ATP from liver and give 2 to muscle, draining liver energy supplies

what happens to lactic acid in oxidative muscle?

it is converted to pyruvate and used to complete TCA cycle, can be used as 70% of heart energy with intense exercise

what does yeast do with pyruvate?

it will convert it to acetaldehyde which will convert to ethanol, replenishes NAD+

fibrobacteres

will turn pyruvate into acetate and succinate and make ATP, NAD+, and HS-CoA

megasphaera

will turn pyruvate into acetate and proprionate and make ATP, acetyl coA, and HS-CoA

ruminants

have megasphaera and fibrobacteres that use fermentation to turn pyruvate into volatile fatty acids which are their main energy source

grain poisoning

increases fermentation that will decrease rumen pH, increases lactobacillis that makes lactic acid to further decrease rumen pH, leads to ruminal acidosis

treatment for rumen acidosis

feeding bicard or replace ruminal fluid

citric acid cycle products

two cycles per glucose, one cycle: 1 GTP, 4 NADH, 1 FADH2, 3 CO2

citric acid cycle

pyruvate is oxidized into acyl-coA, which is added oxaloacetate to continue in the cycle, requires lots of O2 to take high energy electrons

where does the citric acid cycle happen?

mitrochondria

oxygen dependent pathways

first three steps of citric acid cycle

irreversible pathways

end at succinate

reversible pathways

“end” at oxaloacetate, will turn around and go to succinate

step 8 of citric acid cycle

makes oxaloacetate, and would really like to go backwards, without NAD+, NADH will build up and it will run backwards

what happens when too much succinate is made?

it will create lots of FADH2 as a byproduct that will enter electron transport system and cause significant oxidative damage

the reducer will _, and the oxidizer will _

lose electrons and be oxidized, gain electrons and be reduced

when something is oxidized it can look like

increased positive charge, decrease negative charge, gaining a bond to oxygen, losing a proton and an electron