Biochem Exam 5

Recombinant DNA Techniques:

Restriction Endonucleases
1. Restriction enzymes split DNA into specific fragments
2. They are bacterial enzymes that cleave DNA at a highly specific sites
  1. Recognize cleavage sites of 4-8 nucleotides in length as they are palindromic (repeating Letters) → then will cleave each strand of DNA
3. They are very common in bacteria → can be used to eliminate infectious viral DNA
4. Some enzymes will make staggered cuts resulting in sticky ends while others will make straight cuts causing blunt ends
Nucleic Acid Fragment Separation
1. At phys pH, nucleic acids are negatively charged bc of the negatively charged phosphate group in the backbone
2. Agarose gels can be made in order to separate DNA fragments (typically 0.7-2.0%)
  1. Can visualize gel results bc of dye like Ethidium bromide which has fluoresce under UV light
3. Smallest weight fragments will run the farthest and the fastest
Separation of DNA by electrophoresis
1. Negatively charged DNA migrates toward the anode in the presence of the electri field
2. Mobility depends on size and shape
  1. Smaller, more compact molecules run faster
DNA Cloning
1. Helps make identical copies of a piece of DNA like a gene
2. Target DNA is inserted into a vector plasmid and it replicated → plasmids are then introduced to bacteria and through transformation the bacteria picks up the DNA
3. Plasmids
  1. Circular DNA that are separate from the DNA genome
  2. They can replicate autonomously (origins of replication)
  3. Have antibiotic resistance genes
4. Method
  1. Use same restriction enzyme on genetic sequence and on plasmid → put specific cut sequence into plasmid and use ligation → vector has been formed → transformation into bacterial cells → propagation and replication
Eukaryotic gene expression in bacteria
1. Eukaryotic gene from genome will not express properly in bacteria, promoters are different
  1. Eukaryotes have exons and introns → leaving introns in eukaryotic genes cause issues and bacteria can’t splice out introns bc they dont have the correct mechanisms
    1. However, mRNA is intron free as they have already been spliced out → allowing a work around
2. mRNA can be taken out of eukaryotic cells → add reverse transcriptase catalysis → cDNA is created (and can be correctly expressed in bacteria)
Amplifying DNA by PCR
1. Ingredients for PCR:
  1. Target DNA
  2. Pair of DNA primers
  3. DNA polymerase (heat-resistant) to help make DNA molecules
  4. dNTPs
2. Thermal DNA Denaturation (melting)
  1. Two dna strands separate when temp increases a lot
  2. They will re-anneal when temp is lowered again (ie reversible)
  3. DNA denaturation typically occurs at about 260 nm on UV spectrophotometry
    1. Nucleic acid absorbance increases when melting occurs
3. Steps of one PCR cycle
  1. Stand separations (95 degrees)
  2. Annealing of primers (55 degrees)
  3. Extension of primers by DNA synthesis (72 degrees)
4. Q-PCR → can be used to show quantitative differences in gene levels

Techniques in Biochemistry

Proteome → set of proteins expressed and modified (is varying characteristic of cell → unlike genome)
Subcellular Fractionation of tissue
1. Homogenization
  1. Cells are disrupted to form a homogenate (is a mixture of all the cellular components of a cell → but no intact)
2. Differential centrifugation
  1. The homogenate is centrifuges slowly and undergoes centrifugation a couple more times to get pellet (filled with cellular materials) and a supernatant which has soluble cytosolic proteins
Protein Purification
1. Requires an assay that determines if the protein of interest is present
  1. Assay specifically tells us how much enzyme activity is present, not how much protein is present
2. Protein mixtures can be separated based on
  - 1. Solubility
    2. Charge
    3. Size
    4. And specific binding activity (affinity for a ligand)
  1. However, many methods used for separation involve some form of chromatography → allows protein to remain fully folded too
3. Salting out
  1. Proteins will precipitate out of a solution at high salt concentrations
  2. Salt conc at which this occurs differs for different proteins
  3. Many proteins lose their activity in the presence of high salt
  4. At high salt conc → solubility of protein will decor as water molecules become bound to salt ions in the aqueous solution instead → centrifuge is used to precipitate protein
    1. After initial centrifugation, resuspend the pellet so that purification can continue again
4. Dialysis
  1. Removal of small molecules from a solution of a macromolecule by their diffusion through a semipermeable membrane into a buffer solution
  2. Salt can be removed from protein solution by dialysis
  3. Proteins are retained in the dialysis bag while the small other molecules diffuse into the surrounding medium.
Chromatography
1. Column Chromatography
  1. Separation of mixture of proteins over a solid phase (porous matrix) using a liquid phase to move and mobilize the proteins
  2. Proteins w/ lower affinity for sold will wash out first and proteins with high affinity for solid will remain on longer and wash off later
2. Ion Exchange Chromatography
  1. Separation by charge
  2. Proteins move through based of net charge relevant to which pH is being used
    1. Cation exchangers:
      1. Fixed negative charges in the solid matrix
      2. more neg proteins will move faster through and elute earlier as positive proteins will interact more with it
    2. Anion exchanger
      1. Has fixed positive charges in the solid matrix
      2. More positive proteins will elute through as negative proteins will get caught
3. Affinity Chromatography
  1. Separation by binding affinity
  2. Unwanted proteins will wash through the column and proteins of interest will be eluted by a ligand solution
4. Size Exclusion Chromatography
  1. Protein mixture is added to the column containing cross-linked polymers
  2. Proteins are separated by size →
    1. Larger molecules elute more freely → appearing in earlier fractions
    2. Small molecules → enter the pores → longer paths → takes longer to elute
Electrophoresis for Protein Analysis
1. Separation on analytical scale
2. Electric field pulls proteins according to their charge
  1. Gel matrix hinders mobility if proteins according to their size and shape
  2. Common gel is polyacrylamide → (called PAGE)
3. SDS PAGE
  1. SDS - sodium dodecyl sulfate - is a detergent
  2. SDS micelles bind to proteins → facilitate unfolding → apply a uniform negative charge to proteins and original shape is no longer present
    1. This in turn, causes only the size of the protein to be analyzed
  3. Reducing SDS PAGE
    1. DTT or BME – further denature proteins by reducing disulfide linkages → messing up tertiary folding and breaking up quaternary structure
4. Visualization of proteins after gel
  1. Coomasie blue staining binds to proteins not the gel
  2. Each band on gel represented a diff protein
  3. Smaller proteins move through faster and are found closer to end of the gel
  4. First lane is molecular weight markers
  5. SDS PAGE can be used to calculate the molecular weight of a protein
    1. SDS gets rid of charge implications → only relevant to weight
    2. Uses some proteins as comparison that already have a known standardized weight
5. Isoelectric focusing
  1. Separation in gel of proteins on the basis of their relative acidic and basic amino acids
  2. Mixture of gel placed into a pH gradient → proteins will migrate to where their isoelectric points are (the pH where they have no charge)
6. 2D Electrophoresis
  1. 2D gel electrophoresis where proteins are separated in one direction by isoelectric focusing
    1. Gel then is attached to SDS PAGE and electrophoresis is performed at 90 degrees angle to the direction of the isoelectric.
  2. Horizontal separation reflection difference in pI
  3. Vertical separation reflects differences in molecular weight
  4. Thousands of proteins can be resolved using this technique
  5. Individual protein spots can be cut out of the gel and identified using mass spectrometry.

Signal Transduction Pathways

Which amino acids can be phosphorylated
1. Tyrosine, serine, threonine
Biological Role of Signal Transduction
1. Cells receive signa;s from the environment beyond the plasma membrane
  1. Types of signals can include: hormones, neurotransmitters, light, touch, and pheromones
2. These signals can change the cell’s composition and function
3. Signal transduction is the process by which an extracellular signal is amplified and converted to a cellular response
Principles of Signal Transduction
1. Release of the primary messenger as a result of a stimulus
  1. Hormones are examples of primary messengers
2. Reception of the primary messenger
  1. A signal molecule binds to the membrane receptor (normally a transmembrane receptor) - except for steroid receptors
3. Transduction of the signal
  1. Information that the signal has arrived is converted into other chemical forms
  2. The signal is often amplified and the cell will then increase production of a secondary messenger
    1. Some examples include cAMP, Ca++ and PIP3
4. Response
  1. Activation and sometimes inhibition of the effectors
5. Feedback pathways
  1. Negative feedback loops regulate the entire signaling pathway
6. Termination of signal
  1. Cells should be able to stop signaling processes to stay responsive to new signals
Basic Types of Receptors
1. A receptor is a membrane-bound or soluble protein or protein complex that exerts a physiological effects after brining its natural ligand
2. G protein coupled receptor (GPCRs)
  1. are alpha helical integral membrane spanning proteins (7TM, heptahelical of serpentine receptors
3. G proteins
  1. Are heterotrimeric membrane-associated guanosine-binding proteins that bind GTP (active form) or GDP (inactive form)
4. An effector enzyme in the plasma membrane
  1. Is regulated by the activated G protein
Epinephrine interacts with cells via GPCRs
1. Epi is a hormone produced by the adrenal glands
  1. Works to mediate stress response
  2. Binds to receptors in muscle or liver cells to induce the breakdown of glycogen
  3. Binds to receptors in adipose cells to induce lipid hydrolysis
  4. Binds to receptors on heart cells to increase heart rate
7TM receptors are a protein complex of 7 transmembrane proteins with a ligand binding site on the extracellular matrix side
Adenylate cyclase
1. Activation of the clyclases leads to the synthesis of the 2nd messenger, cyclic adenosine monophosphate (cAMP)
2. cAMP is a secondary messenger
  1. Allosterically activates a variety of enzymes including cAMP dependent protein kinase A (PKA)
    1. PKA leads to activation of enzymes that release glucose from glycogen
3. Activation of PKA by cAMP
  1. When cAMP is low → 2 catalytic subunits are bound to 2 regulatory units (complex is inactive)
  2. When cAMP is high → R subunits bind to cAMP → reorganization → release catalytic subunits in their active form
4. Signal Amplification
  1. Activation of a few GPCRs leads to the activation of a few adenylyl cyclase enzymes
    1. Every active adenylyl cyclase enzyme makes several cAMP molecules → activate several PKA enzymes
    2. Thousands of glycogen-degrading enzymes are activated in the liver tissue
5. Intracellular levels of cAMP are regulated between two enzymes
  1. Adenylyl cyclase and cyclic nucleotide phosphodiesterase
6. G proteins can spontaneously resent themselves through GTP hydrolysis
7. Signaling molecule can dissociate from the receptor terminating the signal
The Epinephrine-initiated pathway can be shut down numerous ways
1. cAMP phosphodiesterase converts cAMP to AMP → AMP does not activate PKA
2. G alpha has inherent GTPase activity that cleaves the bound GTP to GDP → GDP can reassociate and terminate the activity of the G protein
3. Epinephrine’s receptor interaction is reversible, so once the concentration of epinephrine falls → the receptor will not longer be active
Receptor Tyrosine Kinases
1. Consists of
  1. An extracellular ligand binding domain
  2. Intracellular catalytic domain
  3. Extracellular and intracellular parts are connected by a single transmembrane segment
2. Catalytic domains have tyrosine kinase activity
  1. It adds a phosphate group to itself (autophosphorylation) which leads to a conformational change that allows the specific binding and catalytic phosphorylation of binding proteins
  2. It adds a phosphate group to a tyrosine in specific target proteins
3. The receptors for insulin and epidermal growth factor are prototypes of the family
Gated Ion Channels
1. Regulate transport of ions across cell membrane
2. They respond to changes in membrane potential and ligand specific binding to specific receptor sites
3. Membrane are electrically polarized
  1. The inside of the cell is typically negative (-50–70 mV)
    1. This is largely due to the asymmetric transport of cations by the sodium potassium pump
  2. Flow of ionic specifies across the membrane depends on its concentration gradient and overall electrical potential
Structure of insulin
1. Is a peptide hormone that consists of 2 chains linked by 2 interchain disulfide bonds
2. Alpha subunit is facing outside and beta subunit is on the inside
Insulin Pathway
1. Insulin present → binds to insulin receptor → activated receptor through cross-phosphorylation → protein-protein interaction → enzymatic reaction → protein-lipid interaction → activated PIP3-dependent protein kinase → enzyme reaction → activated Akt protein kinases → increase glucose transporter on cell surface
Insulin signaling is terminated by the action of phosphatases
1. Protein phosphatases remove phosphates from the activated proteins in the insulin signal-transduction pathway, terminating the insulin signal
2. Lipid phosphatases contribute to signal termination by converting PIP3 into PIP2
Steroid Hormones
1. Steroids are oxidized derivatives of sterols
2. Steroids have the sterol nucleus but lack the alkyl chain found in cholesterol.
3. They are more polar than cholesterol.
4. Steroid hormones are synthesized from cholesterol in gonads and adrenal glands.
5. They are carried through the body in the bloodstream, usually attached to carrier proteins.
6. Many of the steroid hormones are male and female sex hormones