1. supernatant 1 contains cytosol proteins
2. supernatant 2 contains PMP's
3. pellet 2 contains IMP's

• A- have an enzyme that connects a certain sugar
• B - have an enzyme that connects a diff. sugar
• AB - have both enzymes (universal recipient)
• O - have no sugars attached (universal donor)

ratio of lipids to proteins

proteins vary

lipid composition varies

• signaling pathway in immune response
• sorting proteins to different locations in cells (apical vs. basolateral)
• endocytosis
• virus budding

• use labeled antibodies that bind to antigen of protein interested.
• Can see fluorochrome label to deteremine location.

• abs from different animals have different constant regions

• better way of seeing protein
• primary antibody binds protein antigen
• secondary antibody binds to the constant region of the primary antibody (secondary's antigen)
• secondary has fluorochrome tag

• proteins completely segregated= one side red, the other green
• proteins were mixed completely together = orange color

• werent actively transported because fused cells in prescence of ATP synthesis inhibitor and proteins still mixed
• werent made becuase fused cells were treated with protein synthesis inhibitor and proteins still mixed

• length of hydrocarbon tail( longer tails=less fluid)
• degree of saturation in HC tail (unsaturated=more fluid)

• converts stearic acid(0 dbls) to oleic acid(1dbl)
• membrane needs to maintain proper stearic:oleic acid ratio to maintain membrane fluidity

• lateral shift (shorter)
• transverse diffusion (longer)

• grow by fusion of membrane
• vesicle buds from golgi and enter the plasma membrane as it transfers its contents to the ECF.
• EC vesicle heads become IC PM heads

• uses antibodies to stain specific proteins after gel electrophoresis
• use electrophoresis to transfer onto blot paper

• mutation in RBC membrane components. most commonly ankyrin.
• misshapes RBC's and they are destroyed by spleen.

Tight junction?

adherens Junction?

• septate junction-IMPs form a fence
• IMPs connect to cytoskeleton

• form barrier for diffusion between cells and diffusion of proteins within the membrane

• arrange claudins and occludins; act as scaffolds

• IMPs that mediate Ca2+ depedent cell adhesion
• form adherens junctions and desmosomes
• provide structural integrity to tissues

• easily grown and manipulated
• similar to higher orgs
• short life
• genome sequenced

• crumbs protein makes apical region of the cell
• discs lost proteins make sure junctions are made properly to maintain polarity of cell.

• 3-D microscope
• illuminates specimen with a pinpoint beam of light

• expose model organism to mutagen
• identify mutant offspring
• identify mutated gene; clone it
• corresponding gene product(protein) must be one of the causes for the mutation.

• transfer signals
• selectively permeable
• to be deformable and fluid
• serve as barrier

• Nonpolar
• water and some small polar molecules

3 passive

1. simple diffusion across bilayer (nonmediated)
2. simple diffusion thru channel (nonmediated)
3. facilitated diffusion (mediated)
4. Active transport (mediated)

channel proteins?

2 carrier proteins?

• do not need to bind solute for solute to pass through channel (simple diffusion thru channel)

1. passive transporters - need to bind the solute, but need no energy to get through membrane (facillitated diffusion)
2. active T - need to bind solute and need energy to force molecules against concentration gradient

How are ion channels unlike a pore? 3 kinds of one diff?

• selective for certain ions
• gated - charge, ligand, or stress

- selectivity loop made of 5 conserved AA's

- pore lined with caronyls that have partial negative charge to stabilize K and let it remove its hydration shell

-K atoms position themselves with a ring of 4 O's above and below them.

- opening only a certain size

waht subunit acts as the switch?

How?

• S4 helix moves up or down depending on charge inside the membrane
• open when cytoplasm is + becuase it repels S4's + residues and vice versa for closed

full mechanism of voltage gated K channel?

3 stages?

1. Resting - gate closed as cytoplasm is negative, which attracts the S4 helix keeping it down. this keeps the S6 helix(gate) from opening
2. Activated - gate opens as cytoplasm becomes + which repels S4 from cytplm and causes gate to open.
3. Inactivation - ball and chain floats in the pore within milliseconds blocking it. eventually the cell repolarizes and S4 is attracted back to the cytoplasm and the gate closes

1. need solute to bind to transporter protein
2. moves down its conc. gradient
3. saturable
4. can be regulated
5. bidirecitonal

1. find the mRNA of your protein
2. pick a small section that is unique to the protein
3. make double standard (dsRNA) for this section which = small interfering RNA (siRNA)
4. put siRNA in cells
5. siRNA complementary strands bind to mRNA for protein which is then chopped up by the cell.
6. NO mRNA for protein

1. cells move against conc. gradient
2. unidirectional
3. needs to bind solute, change in affinity required
4. ATP used

1. 3 Na bind on the high affinity cytoplasmic side of membrane
2. allows ATP to autophosphorylate itself
3. causes a conformational change so binding sites facing ECF. Na affinity is low so they release and K affinity is high so 2 K bind allowing dephosphorylation
4. causes a conformational change back to facing ICF where 2 K are released due to low affinity

It moves one solute down its conc. gradient, but make it take with it a solute up its conc. gradient

relies on change in affinity

1. Na binds from ECF causing an increased affinity for glucose.
2. glucose binds which opens the protein to the cell.
3. Na moves down its Conc. gradient to be kicked out by Na/K ATPase.
4. release of Na causes a decreased affinity for glucose so it is released as well into the cell.

• caused by the process of reactants always being supplied and products always being used/removed
• reaction always steadily going forward

what do enzymes do in a rxn?

how are they like chemical catalysts?

1. do not change thermodynamics, just lowers the activation energy in the rxn
2. small amount needed, not used up
3. accelerate reaction

1. operate at mild temps and neutral pH
2. bind to specific substrates

• once substrate binds, the enzyme further changes conformation to hid the substrate from cytoplasm

1. hold substrates correctly
2. manipulate charges on substrate
3. physical or chemical stress bond

Km

VMax?

• substrate conc. at 1/2 Vmax. measures the binding affinity of substrate
• the saturation point of the enzyme. Maximum velocity of the rxn

high km =

low km =

low affinity

high affinity

• inhibitors
• alter enzyme synthesis
• zymogens
• use cofactors
• phosphorylation

irreversible inhibitors

reversible inhibitors (3)?