Cell Phys Exam 3

Glycine

Non polar

Alanine

Non polar

Valine

Non polar

Cysteine

Non polar

Proline

Non polar

Leucine

Non polar

Isoleucine

Non polar

Methionine

Non polar

Tryptophan

Non polar

Phenylalanine

Non polar

Lysine

Positive Charged

Arginine

Positive Charge

Histidine

Positive Charge

Serine

Polar

Can phosphorylate

Threonine

Polar

Can phosphorylate

Tyrosine

Polar

Can phosphorylate

Asparagine

Polar

Glutamine

Polar

Aspartic Acid

negative Charge

Glutamic Acid

Negative charge

Protein primary structure

amino acid chain

Protein secondary structure

Alpha helices and beta sheets formed locally by interactions along the amide backbone. Stabilize sections of protein

Protein tertiary structure

Hydrophobic interactions between the side chains cause local shapes to collapse into a single 3-D structure. Stabilized by disulfide bonds between cysteine residues

Protein quaternary strucutre

No present in all proteins!

Stable associations between multiple polypeptides to form a single protein unit.

Phosphorylation

Posttranslational modification

Adds a phosphate to serine, tyrosine, or threonine.

Disulfide bond

Posttranslational Modification

Covalently links the “S” atoms of two difference cysteine residues.

Acetylation

Posttranslational Modification

Adds an acetyl group to an N-Terminus of a protein or at lysine residues.

Methylation

Posttranslational Modification

Adds a methyl group, usually at lysine or arginine residues.

Antibodies

Dense proteins that recognize the unique 3-D surface of other proteins and prompt immune response when needed.

Direct Fluorescent Antibody Test (DFA)

Type of immunohistochemistry (IHC)

1. Slides made from tissue sample and fix with acetone.

2. Add rabies antibody with fluorescent tag.

3. Wash several times to remove unbound antibody.

4. Mount coverslip and check for fluorescence. If found, positive.

Tube Agglutination Test (Agglutination Assay)

• Put antigen into a tube

• Add serum from suspect animal

• If antibodies for the antigen are present, the sample should agglutinate and precipitate.

• Amount of antibody resent can be estimated by tittering the serum

Enzyme Linked Immunosorbent Assay (ELISA)

1. Coat plate with antigen

2. Add sample, some will bind to antigens

3. Add enzyme

4. If enzyme changes from blue to yellow, +

High probability of false positive

Sorting: Protein Targeting

Sorting of new proteins to different locations.

Ligand Binding: Changing 3-D structure

Change in protein shape caused by change in interactions, transferrable through the secondary structure network.

What causes confirmational changes to proteins?

• pH

• Hydroxylate

• Binding of a ligand

• Temperature

• phosphorylation

Degradation: Ubiquitin and the Proteosome

Ubiquitin is a protein that can attach to other proteins. Ubiquitin is then targeted by proteosomes that degrade the protein. This interaction reduces protein expression.

Proteolytic Activation: zymogens

Cleavage of a masking sequence activates a protein so it can do its job properly. Allows inactivation of proteins that can be dangerous if inappropriately active.

Allosteric inhibitors or activators

Compounds that bind to the regulatory subunit (not active site) and change the activity of an enzyme

Feedback inhibition

Later product inhibits an earlier enzyme to prevent overproduction of the final product

Feed-Forward activation

Earlier reactant activates a later enzyme to prevent unnecessary buildup of starting reactants.

GLUT 4

Insulin dependent glucose transporter

What tissues have an absolute glucose requirement?

Brain and RBC

What tissues are insulin dependent?

Heart, skeletal muscle, and adipose

What tissues absorb glucose?

Kidney and small intestine

What tissues regulate glucose?

Liver and pancreas

Glucose trapping

A phosphate is added to glucose to create glucose-6-phosphate and traps it in the cell. This allows for higher levels of glucose within the cell.

Hexokinase vs. Glucokinase

Hexokinase- low Km value=high glucose affinity

Active when glucose is low

In most tissues

constitutive

Feedback through G6P

Main metabolic enzyme

Glucokinase- high Km=low glucose affinity

active when glucose levels are high

In the liver and pancreatic bcells

inducible

not inhibited

Phosphofructokinase-1

Main regulatory enzyme in glycolysis

Converts fructose-6-phosphate to fructose-1,6-biphosphate

Pyruvate Kinase

Converts phosphoenol-pyruvate to pyruvate in glycolysis

2ADP→2ATP

Lactate Dehydrogenase

In low oxygen conditions, the TCA cycle cannot occur, so Pyruvate is converted to lactic acid via this enzyme

Cori Cycle

allows lactic acid to be processed into glucose for replenishment of blood glucose and reduction of metabolic acidosis. Uses ATP

Alcohol dehydrogenase

Converts pyruvate to ethanol in yeast to replenish NAD+

Where does the TCA cycle occur?

Interior of the mitochondria

TCA cycle

• Oxidizes pyruvate to three molecules of CO2

• Requires O2

• Products are oxidized in ETS

TCA during Ischemia

• Oxaloacetate is depleted and steps 1-3 stop

• Steps 4-5 run forward producing succinate

• Steps 6-8 run backward producing succinate

Reperfusion

Build up of succinate allows step 6 to proceed very rapidly, producing large amounts of FADH2. Upon entering ETS, FADH2 produces a large amount of free radical causing damage.

Where does the ETS occur?

Inner mitochondrial membrane

Succinate dehydrogenase

Complex II in ETS

Proton motive force

Created by Complexes I, III, and IV pumping protons against their gradient. Used to synthesize ATP via ATP Synthase.

Why is glycogen used for energy storage rather than glucose?

Glycogen is an osmolyte

Why is glycogen used for energy storage rather than fatty acids?

Fatty acids cannot be converted back into glucose (absolute requirement for glucose)

Much more rapidly mobilized

can be use anaerobically

Insulin

• Dephosphorylates phosphorylase kinase through Protein Phosphate 1 and reduction of glycogenolysis

• Dephosphorylation of glycogen synthase through PP1 and GSK3 inactivation and increase in glycogen synthesis

Glucagon

Phosphorylation of phosphorylase kinase through PKA and increase in glycogenesis

Phosphorylation of glycogen synthase through PKA and reduction of glycogen synthesis

Glucogenic Amino Acids

All except Leucine and Lysine

Both Glucogenic and Ketogenic Amino Acids

Phenylalanine, tyrosine, threonine, isoleucine, and tryptophan

Ketogenic Amino Acids

Leucine and Lysine

Branched Chain Amino Acids

Leucine, Isoleucine, and valine

Big component of skeletal muscle proteins and used by brain to synthesize neurotransmitters

Can only be metabolized in the muscle

High in blood

Aromatic Amino Acids

Preferentially metabolized by the liver

Ammonia

Strong base, highly toxic

Increases blood pH when in circulation

Interferes with the brain’s ability to regulate K+

Where does the urea cycle occur?

The liver

Urea Cycle Regulation

high glutamate concentrations activates N-acetylglutamate synthetase to produce N-acetylglutamate. This acts as an allosteric activator for carbamoyl phosphate synthetase to balance urea production with nitrogen waste in AA catabolism

Regulation of Fatty Acid Synthesis

Acetyl-CoA carboxylase is deactivated by phosphorylation by AMP-activated kinase, glucagon signaling, or epinephrine signaling

Activated by high citrate concentrations and dephosphorylation by insulin signaling (PP1)

Regulation of lipogenesis/lipolysis

Hormone Sensitive Lipase is activated by phosphorylation (lipolysis)

Acetyl-CoA Carboxylase (lipogenesis)

Citrate→ Lipogenesis

AMP→ Lipolysis

Insulin→ lipogenesis

Epinephrine → lipolysis

Glucagon→ lipolysis

Phosphofructokinase 1 (PFK1)

Main regulatory enzyme of glycolysis

Fructose 2,6 Bisphosphate

Allosteric activation of PFK1 (glycolysis)

Phosphofructokinase 2/Fructose Biphosphate 2 (PFK2/FBP2) (glycolysis)

Activates Fructose 2,6 Bisphosphate through dephosphorylation

Glycolysis Hormone pathway

Insulin→ protein, phosphatases

Fructose Bisphosphate 1 (FBP1)

Main regulatory enzyme of gluconeogenesis

Fructose 2,6 Bisphosphate

Allosteric activation of FBP1

Phosphofructokinase 2/Fructose Biphosphate 2 (PFK2/FBP2) (Gluconeogenesis)

Activates Fructose 2,6 Bisphosphate through phosphorylation

Gluconeogenesis hormone pathway

Glucagon/epinephrine → PKA

Glycogen Synthase

Main regulatory enzyme of Glycogenesis

Glycogen Synthase activation

Dephosphorylation

Glycogenesis Hormone pathway

Insulin → protein, phosphatases

Glycogn Phosphorylase

Main regulatory enzyme of glycoenolysis

Activation of Glycogen Phosphorylase

Phosphorylation

Glycogenolysis hormone pathway

Glucagon/epinephrine → PKA

Acetyl-CoA Carboxylase

Main Regulatory enzyme of lipogenesis

Acetyl-CoA Carboxylase activation

Dephosphorylation

Lipogenesis hormone pathway

Insulin → Protein, phosphates

Hormone sensitive lipase

Main regulatory enzyme of lipolysis

Hormone sensitive lipase activation

Phosphorylation

Lipolysis hormone pathway

Glucagon/epinephrine → PKA