mcb review exam ii ji woong park. logistics this review covers lectures by dr. mercer, nichols, and...
TRANSCRIPT
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MCB Review Exam II
Ji Woong Park
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Logistics
• This review covers lectures by Dr. Mercer, Nichols, and first and third lecture by Dr. Bose.
• It accounts for 60/100 points in the second exam.• Be sure to not just study but be able to answer
2013 Exam II (at least for my section) – you will thank me on Tuesday.
• In addition, understand everything on this review slide as well.
• There will be NO math problem on this exam.
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Primary Active Transport: Na,K-ATPase
• 3 Na outward / 2 K inward / 1 ATP• Km values: Nain ≈ 20 mM Kout ≈ 2 mM• Inhibited by digitalis and ouabain• Palytoxin “opens” ion channel• 2 subunits, beta and alpha (the pump)• Two major conformations E1 & E2• Turnover = 300 Na+ / sec / pump site @ 37 °C
3 Na
2 K
ATP
ADP + Pi
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Unidirectional Transport Assays
Cells growingin multi-well plates
1. Cells washed in isotonic buffered solution
2. Required transport inhibitor(s) added
3. Flux medium containing radioactive isotope added
4. At required times flux medium rapidly removed and cells washed (3-4 x) in ice-cold isotonic saline
5. Final wash removed, cells lysed and radioactivity and protein content of samples determined
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LIMITING JUNCTION/TIGHT JUNCTION
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CLAUDINS
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“GATING” OF CONNEXONS
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Aquaporins- ADH Stimulated Water Permeability
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SORTING SIGNALS
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10
Selectivity filter diversity
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11
Ion gradients and membrane potential
Na 117K 3Cl 120Anions 0Total 240
Na 30K 90Cl 4Anions 116Total 240
[+ charge] = [- charge]
0 mV
[+ charge] = [- charge]
-89 mV
How does this membrane potential come about?
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12
At Electrochemical Equilibrium:
The concentration gradient for the ion is exactly balanced by the electrical gradient
There is no net flux of the ion
There is no requirement for any energy-driven pump to maintain the concentration gradient
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13The Goldman Hodgkin Katz Equation
outClinNainK
inCloutNaoutKm [Cl]P[Na]P[K]P
[Cl]P[Na]P[K]Plog60mVV
Resting Vm depends on the concentration gradients and on the relative permeabilities to Na, K and Cl. The Nernst Potential for an ion does not depend on membrane permeability to that ion.
The GHK equation describes a steady-state condition, not electrochemical equilibrium.
There is net flux of individual ions, but no net charge movement.
The cell must supply energy to maintain its ionic gradients.
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14Currents During an Action Potential
Time Course of Currents
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15
Sodium Channel Gating States
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Trypsin – a protease that cleaves after basic residues (R or K).
Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides
Slide courtesy of Andrew Link
Protein of Interest:
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Products from Trypsin digest.
Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides
Slide courtesy of Andrew Link
Average length of tryptic peptides = 10 aa residues
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Select an Individual Peptide in the Mass Spectrometer
Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides
Slide courtesy of Andrew Link
Performed by adjusting the electrical fields in the mass spectrometer.
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Impart energy to the peptide by colliding it with an inert gas (Argon or Helium).
Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides
Slide courtesy of Andrew Link
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Measure the masses of the fragment ions.
Identifying a Protein by Mass Spectrometry on Its Tryptic Peptides
Slide courtesy of Andrew Link
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Control
Mix Lysates
Identify and Quantify Proteins by Mass Spec
Fractionate Proteins on SDS-PAGEDigest Bands with Trypsin
Treatment 1 Treatment 2
Protein Quantitation with Mass Spectrometry
Bose et al., PNAS 103: 9773-8, 2006
Introduce Stable Isotope by Metabolic Labeling
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Studying EGFR Signal Transduction with Quantitative Proteomics
Introduce Stable Isotope by Chemical Labeling
Zhang et al., MCP 4: 1240-50, 2005
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Her2/neu and Breast Cancer
• 1987 – Southern blots of genomic DNA from breast cancer patients shows Her2 gene amplification. – Sample 3 & 4: normal level– Sample 1 & 2: 2-5 x normal– Sample 6 & 26: >5 x normal– Sample 18: > 20 x normal
• Correlation between Her2 gene copy number and patient survival
Slamon, et al., Science 1987
100%
Time (months)
Patie
nt S
urvi
val
80%
60%
40%
20%
0%
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Her2/neu and Breast Cancer
• Transgenic mice bearing the MMTV-Her2/neu construct develop breast cancer in all 5 pairs of mouse mammary glands.
• Tumor formation with Her2 in this tg model is more rapid than with the Myc oncogene.
Muller et al., Cell 1988
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Drugs to Target Receptor Tyrosine Kinases
HE
R2
EG
FR
HE
R2
HE
R2
Homodimer Heterodimer
Extracellulardomain
Tyrosine-kinasedomains
Monoclonal Antibodies
ATP-mimetic Tyrosine Kinase Inhibitors
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The RARα Nuclear Hormone Receptor in Acute Promyelocytic Leukemia (APL)
• APL has a characteristic translocation 15;17 that forms the PML-RARα fusion protein.
• Retinoic Acid (RA) binding converts PML-RARα from a transcriptional repressor to a transcriptional activator.
• All-trans retinoic acid (ATRA) has made APL the most treatable and best prognosis form of adult acute leukemia.
PML Retinoic Acid
Binding
DNA Binding
Retinoic Acid Receptor α
ATRA
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GPCR signaling Controls Blood Pressure via the Renin-Angiotensin System
Angiotensinogen
Angiotensin I
Angiotensin II
Renin (kidney)
ACE (lung)(Angiotensin Converting Enzyme)
Angiotensin II Receptor (GPCR)
TACE inhibitors
TAngiotensin Receptor Blockers
Common Blood Pressure Medicines
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Erythropoietin (EPO) binds to a Cytokine Receptor
Nucleus
STAT5
STAT5
DNA
JAK2 Tyr Kinase
EPO receptor
Munugalavadla and Kapur, Reviews in Onc-Hem, 2005
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EPO Deficiency causes Anemia of Chronic Kidney Disease
Chronic kidney disease causes a fall in EPO secretion and this results in decreased red blood cell production (i.e.- anemia). Therefore patients with chronic
kidney disease are given recombinant EPO to prevent anemia.
Kidney
BoneMarrow
Increased Red Blood Cell Production
EPO
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Good Luck!