Molecular Cell Biology Exam 1--

Fatty Acids are made from Acetyl CoA by what two cytosolic enzymes?

Acetyl-CoA Carboxylase and Fatty Acid Synthase

What does Desaturase enzymes do? Where are they located?

Introduce double bonds; present in the ER

Where are diacylglycerophospholipids synthesized?

ER

Where are sphingosine and N-acyl sphingosine synthesized?

ER

Where does the additino of a polar head group to ceramide take place?

Golgi

How do fatty acids move around?

Bound to fatty acid binding proteins; fatty acid binding proteins that have a beta-sheet pocket

What is the first step of phospholipid synthesis?

Acetyl-Coa is converted to a fatty acid by acetyl-CoA carboxylase and fatty acid synthase

What is the second step of phospholipid synthesis?

Fatty acid + CoA will create Fatty acyl CoA

What is the third step of phospholipid synthesis?

Fatty acyl CoA esterfied with glycerol phosphate to create Phosphatidic acid within the membrane. (2 CoA's are lost)

4th step of phospholipid syn., Phosphatidic acid is converted to what molecule by which enzyme?

Converted to Diacylglycerol by phosphotase.

What does phosphotase do? Where is it located?

Removes a phostphate, located in the ER membrane

Phosphatidic acid is basically?

a phospholpid without a head group

5th step of phospholipid syn., Diacylglycerol is converted to what by which enzyme?

Phosphatidyl choline by choline phosphotransferase

What does choline phosphotransferase do? Location?

transfers a choline and phosphate group to diacylglycerol. By product is CMP. Produces Phosphatidyl Choline

What do flippases do?

Catalyse movement of phospholipids from cytosolic leaflet to the exoplasmic leaflet. (phosphatidyl choline)

What enzyme converts Glycerol phostphate + fatty acyl CoA to phosphatidic acid?

Acyl transferases, by GPAT/LPAAT located in the ER membrane

Where is HMG-CoA synthesized?

Cytosol

What is the rate limiting step of cholesterol biosynthesis?

HMG-CoA reductase

Where is HMG-CoA reductase located? What is it's structure?

ER membrane; 8 transmembrane helices embedded in membrane, 5 of which form sterol-sensing domain

What happens when cholesterol levels are high?

Cholesterol binds to sterol-sensing domain of HMG-Reductase, causes it to bind to Insig-1 and Insig-2. Induce enzyme to be ubuiqinated and targeted for degradation

List the steps of Cholesterol Biosynthesis.

1. Acetyl-CoA + Acetoacetyl CoA --> HMG-CoA

2. HMG-CoA --> Mevalonate by HMG-CoA reductase

3. Mevalonate --> IPP

4. IPP --> Farnesyl pyrophospate

5. Farnesyl pyrophosphate --> Squalene

6. Squalene --> Cholesterol

What is Atherosclerosis?

Deposition of lipids, cholesterol, and extracellular material in inner wall of arteries. Causes distortion or changes in arterial wall structure, lead to clots.

Be familiar with the mechanisms for cholesterol and phospholipid transport between organelles.

By vesicle transport; by hypothetical proteins to allow transport through membranes; by binding proteins for cholesterol and hydrophobic groups

What do phospholipids form when dispersed in solution?

Micelles, liposomes, and phospholipid bylayers

Phosphoglycerides are derived from what?

glycerol 3-phosphate

Be able to draw head groups for phosphoglycerides.

Head group for PE, PC, PS, PI

Where are levels of plasmalogen most abundant?

Brain and heart tissue

Know the structure for Plasmalogen.

Draw with the different head groups of PE, PC, PS, and PI

What are sphingolipids derived from?

sphingosine, which is an amino alcohol with a long hydrocarbon chain

Know structure of sphingosine as well as the head groups of SM and GlcCer!!

Practice drawing

Cholesterol is a precursor for?

Bile acids, steroid hormones, and Vitamin D (skin and kidneys)

What are the 3 properties of membranes?

1. Hydrophobic core is impermeable barrier

2. Stability - Vander waals interaction, hydrophobic interaction stabilize fatty acid groups; ionic/hydrogen bonds stabilize polar head groups

3. Phosopholipid bilayers spontaneously form closed sealed compartments

What are flipases powered by?

ATP

Which leaflet are PE, PS, and PI located? What kind of bilayer do they form?

Located on the cytosolic leaflet and form more fluid bilayers

Which leaflet is phosphatidyl choline and sphigomyelin located? What do they form?

Located in the exoplasmic leaflet and form less fluid bilayers

How is cholesterol distributed in the leaflets?

Distributed evenly in both leaflets

What are lipid rafts?

more ordered, less fluid bilayers that contain cholesterol and sphingomyelin. Are Microdomains surrounded by more fluid phosphoglycerides

What does membrane fluidity depend on?

Lipid composition, Structure of hydrophobic tails, and temperature.

What do saturated fatty acids form?

aggregate forming a gel-like state of membranes

What do unsaturated fatt acids form?

form less stable interactions and more fluid membranes

What is the effect of cholesterol on membrane structure?

Decreases membrane fluidity - interaction of steroid ring with hydrophobic tails immobilize lipids. Increase membrane thickness in phosphoglycerid bilayers but not in sphingomyelin bilayers

How many times does a lipid molecule exchange places?

10^7 times per second

What direction do phospholipid bilayers usually perform

laterally and rotational movement

What is a FRAP experiment?

Detect lateral movements of proteins and lipids within the plasma membrane

Describe Integral membrane proteins

- Contain membrane spanning alpha-helices.

- 20-25 hydrophobic amino acids

- hydrophilic peptide bonds in interior

- hydrophobic chains interact with fatty acyl groups by hydrophobic/vanderwaals interactions

- ionic interactions between hydrophilic amino acids and polar head groups

What is Glycophorin A?

A single pass integral membrane protein that forms a dimer. Sugar residues are located on extracellular domain

Describe membrane spanning beta-strands?

- Form porins(channels)

- Outer membrane of mitochondria/chloroplasts

- trimers of identical subunits

- each subunit has 16 beta strands

- barrel - hydrophilic interior and hydrophobic exterior

What is an Acylation lipid anchor?

Fatty acyl groups(myristate or palmitate) attach to glycine residue in N-terminus. Occur in cytosol

What is an Penylation lipid anchor?

hydrocarbon chains attached to cysteine residue near C-terminus

What is a GPI anchor?

Exoplasmic anchor. Phosphatidyl inositol binds to fatty acyl groups. Phosphoethanolamine binds to sugar residues and C-terminus

What extra group is added on for an A antigen blood group?

GalNAc = N-acetylgalactosamine

What extra group is added for an B antigen blood group?

Gal = galactose

How does ionic detergents work?

denature the proteins. Have a charged group.

How is nonionic detergents different from ionic detergents?

Lack charged group. Useful for extracting membrane proteins before purified. Will not denature proteins.

What forms when CMC concentrations are high we non-ionic detergents?

Micelles, become solubilized

What happens with CMC concentrations are low with non-ionic detergents?

Dissolves the protein but does not form micelles

How are peripheral membrane proteins removed?

High salt solutions that break ionic bonds. Soluble in aqueous solutions, so no need to be solubilized by non-ionic detergents

Are peripheral membrane proteins attached to membrane?

No.

What is the bilayer permeable to?

Small hydrophobic and small uncharged, polar molecules. Slightly permeable to water and urea.

What does diffusion depend on?

- concentration gradient across bilayer

- hydrophobicity

- size

- if charged, also depends on electric potential across membrane

What do cell cultures require?

- Solid surfaces

- growth media that is rich in nutrients

What are primary cell cultures?

Differentiate into cell culture

What are cell strains?

Have a finite life span from one initial primary culture

What is a cell line?

culture of cells with an indefinite lifespan and is immortal. Taken from a cell strain.

What are cell sorters(flow cytometry) uses for?

- Purify different types of white blood cells

- T cells separated from other cells using fluorescent tagged antibodies against specific proteins on their cell surfaces

What are magnetic beads?

- used to sort cells

- coat beads with antibodies specific to proteins on surface of cells

- only cells with surface protein can be separated using magnets

What is the resolution limit of light microscopes?

0.2 micrometers

How is resolution improved using light microscopes?

using shorter wavelengths or increasing refractive index and angular aperture

What is phase contrast microscopy?

- see thin layers of cells, but not thick tissues

- examine location and movement of large organelles

- generate image where degree of brightness depends on refractive index

What is Differential interference contrast?

- suited for small details and thick objects

- thin optical section through object

- contrast generated by differences in index of refraction of object and medium

What is the major function of fluorescence microscopy?

- localize specific cellular molecules, like proteins

- express fluorescent proteins with recombinant DNA technology

What are some advantages of fluorescent microscopy?

- sensitivity

- specificity: immunofluorescence

- cells can be fixed or living

What is used for fluorescent microscopy?

fluorescent dyes/proteins called flurochromes. These may be indirectly or directly associated with the cellular molecule

Fluorescent microscopy absorb light... .

Absorb light at one wavelength and emit at a specific and longer wavelength

What is a technique to tag proteins with GFP for visualization in living cells?

- Gene of interest is fused with GFP gene

- this recombinant DNA is transfected into living cells

- wait for protein expression

- chimeric fusion protein can be visualized in living cells

- useful for studying protein localization

What must be removed from gene of interest X? why?

the stop codon for it to be added into frame for the GFP to fluoresce

What is immunofluorescent micropscopy?

- fixed cells

- fluorescently tagged primary antibody

- tagged secondary antibody

- fluorescently labelled antibody to tagged proteins like myc or FLAG

What are the limitations of fluorescence microscopy?

- blurred images

- thick specimens

What is confocal microscopy?

- laser as energy source

- laser scans specimens across and down

- use pinhole in front so light from other focal planes are blocked

What does FRAP stand for?

Fluorescence recovery after photobleaching

What does FRET measure?

distance between proteins using chromophores

What thickness of specimens are used for transmission EM?

50-100nm

What does transmission EM visualize?

- 2D sectional image that reveals surface details

- subcellular organelles

Samples need to be prepared how for transmission EM?

Be fixed and stained. Can be stained with heavy metals

What is scanning EM?

view unsectioned, metal coated specimens which will create a 3D surface image

What is the resolution for scanning EM?

10nm

What can be used to detect antibodies bound to a protein? (metal)

gold particles coated with protein A. Protein A binds to Fc segment of all antibody molecules

What is the resolution for cryoelectron microscopy?

5nm

What samples are used for cryoelectron microscopy?

- hydrated, unfixed and unstained

- observed in its native hydrated state

whats the method used for cryoelectron microscopy?

aqueous suspension of sample is applied on a grid and held by a special mount, froze in liquid nitrogen

What is a variation of cryoelectrion microscopy?

cryoelectron tomography, allows determination of 3D structure

Which organelles have double membranes?

mitochondria, nuclear envelopes, and chloroplasts

How are plasma membranes of cells broke open?

- suspended in isotonic sucrose

- sonication

- homogenization

- placed in hypotonic solution which ruptures cell membranes

How are organelles separated by?

- differential centrifugation

- density gradient centrifugation

How long and at what G is required to get nuclei from homogenate in differential centrifugation?

600g for 10 minutes

How long and at what G is required to obtain mitochondria, chloroplasts, lydsosomes, and peroxisomes by differential centrifugation?

15,000g for 5 minutes

How long and at what G is required to obtain plasma membrane, microsomal franctions, etc by differential centrifugation?

100,00g for 60 minutes

How long and at what G is required to obtain ribosomal subunits and small polyribosomes by differential centrifugation?

300,000g for 2 hours

What is density gradient centrifugation?

separates organelles bases on their density

Where is catalase located?

proxisomes