BICD 110 midterm 1
resting potential in animal cell
dependt on potassion ions pass through non gated channels keep potential negative at -70mV
electrical gradient
difference in electrical charges between the inside and outside of the cell
diffusion
diffuse across membrane with no assistance, includes small gasses and small uncharged molecules, travel high to low []
periphenial membrane proteins
do not directly contact the hydrophpbic core of the bilayer, bound to the membrane indirectly by interactign with lipid heads doesnt cross membrane but can be on both sides
F class pump
does not require the phosphylation of proteins to function, opposide to V class, release H down [] grad and is used to power ATP synthesis from ADP and Pi, commonly called ATP synthases
cryo-electron miscroscope allows vizualization of samples without fixing
electron beam to image molecule, uses rapid cooked sample, hold structure
light microscopy
examine cell and tissue, not colloed, have to be fixed and stained, hematoxylin purple stain nuc, Eosin pink for cell
ABC protein functins
export drugs and toxins from cells , can export hdrophobic and hydrophilic molecules, first found in tumor cells
plasma mbm
half proteins half lipids, protiens expressed in plasma membrane dictate function, defines cell shape and encloses cell, controls movement of molecules in and out
transmission EM
heavy metal must be used to prepare the sample in order to add contrast and scatter electron beam, sample absorbed to the EM coated inplastic and carbon, next hevay metal are added megatuve staining occurs, and excludes regions where the sample is adheredto the grid
electron micropscopy
high image resolution, teo types transmission EM and scanning EM
confocal microscopy images
images each focal plane seperatly and excludes light from other out of focus planes
how to keep cell culture alive
imitate in vivo conditions, pH, temp, ionic strength,
cholesterol in phosphatidylcholine
increases thickness of the membrane by making it more ordered Cholesterol increases the thickness of phosphoglyceride bilayers
cytoslic
innerleaflet mbm
basolateral
inside bloodstream, facing side
glucose transport from intestinal lumen to the blood
intesinal lumen(apical) 2na/glucose sympoter (high in gietary glucose, sodium, and cl) moves into cytosol, low na high K, glucose goes through GLUT2 into the blood which has high na low k, blood na/k pump bring already high na into blood and cycles K in and out of cytosol
differential centrifugation
isolate organelles, spin at different speeds and increase speed while decanting supernantent each time to get smaller molecules, can also separate with magnet, increasing dentist top to bottom of separation
stigasterol
main sterol in plant mbm
PE = Phosphatidylethanolamine,
makes curved membrane (small head group)
Peripheral membrane proteins
may noncovalently interact with phospholipid heads becasue they dont make direct contact with the hydrophobic core of the bilayer
most integral proteins have at least one _?
membrane spanning alpha helix
PC = Phosphatidylcholine, most abundant, makes flat sheet-like membrane(big head group)
most abundant, makes flat sheet-like membrane(big head group)
-Gangliosides:
most complex sphingolipids that are abundant in ganglion cells ofnervous tissue
flippase enzyme
move lipids across leaflet, but require ATP, unfavorable
antiporters
move two molecules in opposite directions, three na in push one ca out, cell likes low [] of ca and pushes it out, medications that prevent this stop ca from leaving the heart in pateints with heart faliure since more ca in the cell strengthes heart contractions
signal anchor sequence SA
no cleavble ER signal, positive charge residue near NH3 to make it go in the cytosol which has a negative charg, like foling thread the pushing through end to make c terminus in lumen
cholesterol in sphingomyelin bilayer
no effect on already ordered sphingomyelin bilayer
lysosomes
oftern budded from golgi, contain degradative enzymes that break down molecules into smaller components, acidic lumen pH 4.5 break down proteins
cystic fibrosis transmembrane conductance regulator (CFTR) is an ABC transporter
one function to move cl- down grad certain regulatory region not present in other ABC transporters must be phosphorylated
PS = Phosphatidylserine
only in inner leaflet (cytosolic), marker for apoptosiswhen detected outside cell
N- terminal and internal stop transfer anchor sequence
orient N terminus in lumen and c terminus in cytosol, when STA enter translocon, translocon open like clam and and makes a transmembrane anchor, continuing translation and c terminus remains in cytosol
limitaions of fluorescence microscopy
out of focus fluorescence, thich sections must be imaged separately and then reconstructed
exoplasmic
outter leaflet mbm
Na+/K+ pump
p class pump, moves 3 sodium out and 2 potassium in per ATP hydrolized against concentration, generated low Na high K in cytoplasm , moves sod and pot aginast grad, lead to neg charge in cell,
function of memebrane
pass ions, protect inside, diffusion, cell structure, permeability barrier, control movement of molecules, proteins expressed in mbm dictate cells function, define cell shape
biomembranes three claases of lipids
phosphoglycerides, sphingolipids, sterols
Golgi complex
process and sort secreted lysosomal and membrane proteins on the way to the surface of cell, a stack of flattened membranes called cisternae
scanning electron microscopy
produce 3D image of surface of an unsanctioned sample
Eukaryotes includes
protists, fungi, plants, animals
V class ion pumps
proton pump, does not require the phosphylation of proteins to function. ytansport only protons to generate low pH in plave vacules and animal lysosomes to make acidic , pump against grad into cell
FACS: fluorescence activated cell sorter
purify whole cell, fluorescent tqg and antibody to a cell surface molecule, purify different cells based on proteins expressed
p-pump
require phosphorylation of protiens subunit, at least one of two idential alpha catalytic subunits becomes phosphorylated on a highly conserved aspartae residue ex. H+/K+ pump in stomach H out K in(keep charge in membrane unchanged) Na+/K+ pump
sympoters
same direction ex. nodium move into cell favorable to continue unfavprable pushing glucose inside when it is already highly concetrated inside, also does this with amino acids (two na to 1 aa, glucose, or leucine)
more fluid
shorter chains, unsaturated, high temp,
SRP
signal recognition peptide, bind to ER signal sequence and large ribsomoal subunit and takes this complex to the ER and binds to its receptor on the membrane, SRP receptor, GTP hydrolysis accompanies the disassembly of SRP and its receptor takes unfinished protein chain and ribosome to site on ER membrane where translocation can take place
intestinal cells apical side
specialized in absorbtion of sugars, aa, and other molecules produced by fppd breakdown
topology of a membrane protein
specifies the number of transmembrane segments and their orientation across the membrane
Peroxisomes
spehrical organelle the contain oxidase to detoxify various molecules and break down fatty acids to produce acetyl groups fro biosynthesis
ER
synthesis 33% of protein, largest internal membrane system Rough: surface covered with ribosomes, site of synthesis and secreted proteins, smooth: site of fatty acid and phospholipid synthesis
membrane potential
the electric potential (voltage) across the membrane
why cant v class pumps acidify the lumen by itself
they are lelectrogenic =, the mbm potental will stop more protons from going acoss the membrane by adding negatuve anion cl with each H
immunofluorescence (IF) micoscopy
to visualize specific protiens within cellm cells or tissue chemically fixed, antubiotics applied and dyed,
apical
top surface facing side
porins
transmem proteins that lack and alpha helix, consist of trimers of identical subunits, form barrel with hydrophilic pore, found in membrane of gram-negatuve bacteria such as e coli to protect against harmful agents
channels
transport water, specific ions, or hydrophilic small molecules across the membrane down their gradients, facilitated diffusion/passive transport NO ATP -fast, no confirmational change
Chloroplast
two membrane organelle, thylakoid memebranes conaing enzymes of the photosynthetic pathway, convert light to ATP
Mitochondria
two membranes and generate ATP, 25% of cytoplasm volume, (f): complete the degredation of glucose by oxidation to generate ATP and power cell, folds called cristae, central space called matrix, own set of maternal DNA
transcellular transport
two stage process of the impirt of molecules through the plasma mbm on the apical side and thier export through the basolateral side
transporters consist of
uniporters and cotrasnporters
cotrasnporters
use energy stored in elecrochemical gradient to power transport of molecules, always coupled together, present in all orgs
PI = Phosphatidylinositol,
used in GPI lipid anchored membrane proteins
transporters
variety of ions and molecules, slower than channels, NO ATP
double labeled fluroescence
visualize distribution of two proteins
Fluorescence microscopy
visualize molecules within cell, absorb light at one wavelength and emit at another
inactivation of CFTR
water flows out with na which also follows cl to maintain electrical neutrality, in many CF patitents Phe508 is deleted so build up of mucus outside of cell
During transcellular transport of glucose in an intestinal epithelial cell, which of the following is/are moved against its gradient? Please choose all that apply. A) Import of glucose through Na+/glucose symporter B) Export of glucose through GLUT2 C) Import of sodium through Na+/glucose symporter D) Export of sodium through Na+/K+ pump E) Export of potassium through K+ channel
-Import of glucose through Na+/glucose symporter -Export of sodium through Na+/K+ pump
cell theory
-all living things are composed of one or more cells -every cell comes from a pre-existing cell, -cell is the basic unit of structure, function, and organization in all organisms -cell contains hereditary info which passes cell to cell during division -all cells have the same basic chemical compositions ex. similar set of proteins -all energy flow (metabolic and biochem) of our life occur w/in cell
how to study a cell
-cell culture: grow up mammalian cells, -primary cell cultres: directly from animal tissues or embryos -cell strain: lineage of cells originaling fromone primary culture -cell line: cells undergo oncogenic mutation and continue dividing HeLa(human), COS-7(monkey kidney), CHO (chinese hamster ovary)
what does an alpha helix consist of
20-25 hydrophobic uncharged aa only long enough to span membrane
Where are cholesterols found?
50-90% in plasma mbm and associated vesicles, too hydrophobic to form bilayer so they intercalate between phospholipd molecules to be incorpotaed in mbm, provide structurak support
concentration gradient
A difference in the concentration of a substance on diff sides of the membrane
ABC superfamily
ATP Binding Cassette -diverse function, each protein is specific for a single substrate, four core domains: two transmembrane domains and two cytosolic ATP domains
Although many types of vesicles are similar in size and density, if they are coated with different proteins, it is possible to isolate specific types of vesicles through the use of:
Antibodies attached to bacterial carriers and low speed centrifugation, antibodies will tag the specific vesicles
Type I membrane proteins have all of the following properties EXCEPT
B) Internal signal-anchor sequence
Protein insertion into the mammalian ER membrane typically occurs
B. as translation is happening (cotranslational)
prokayotes include
Bacteria and Archaea
All the following statements describe biomembranes EXCEPT: A. Different biomembranes may contain different proportions of the same phospholipids B. The two leaflets of the same membrane may contain different phospholipids C. Some biomembranes have free edges D. Some phospholipids and cholesterol may cluster to form lipid rafts
C. Some biomembranes have free edges, they do not they are all enclosed
what forms lipid rafts
Cholesterol and sphingolipids
Sterols
Cholesterol is the major animal sterol and is abundant in theplasma membrane of mammalian cells, but is absent from plantand most prokaryotic cells, not phospholipids but amphipathic
Which of the following classes of lipids is (are) present in biomembranes? A. Phosphoglycerides B. Sphingolipids C. Sterols D. All of the above
D. All of the above
As a budding cell biologist, you would like to study your favorite protein, titin, in muscle cells. (Titin is the single largest known protein in a cell, and stretches across a muscle cell sarcomere like a bungee cord during muscle contraction). If you would like to see and be able to distinguish (tell them apart) both titin and the actin filaments that structure the muscle cell, what kind of technique would be best to use?
Double label fluorescence microscopy because you can see the over lap of the two proteins in relation to eachother
region of trans mbm protein
Extracellular (hydrophilic) Membrane-spanning (hydrophobic) Cytosolic (hydrophilic)
Which of the four classes of ATP-powered pumps share overall similarity, including several subunits, the same general organization, and a similar function in being H+ transporters?
F-class pumps and V-class pumps, -F class move H+ down [] grad -v class move H= against [] grad
How are lipid-anchored exoplasmic proteins tethered to the membrane?
GPI anchor
what accompanies SRP disassembly
GTP hydrolysis accompanies the disassembly of SRP and its receptor takes unfinished protein chain and ribosome to site on ER membrane where translocation can take place
How do the following molecules normally travel across a membrane? Glucose up its gradient Glucose down its gradient O2 or CO2 down their gradients K+ down its gradient
Glucose up its gradient- symporter with Na+ Glucose down its gradient -uniporter O2 or CO2 down their gradients-Diffusion K+ down its gradient-channels
Phospholipids with short or unsaturated fatty acyl chains
Increase membrane fluidity, unsaturated=bdl bonds which creakes kinks and more disorder, shorter chains=less LDF so less ordered
The _____________________________ are transmembrane proteins.
Integral membrane proteins
The resting membrane potential in animal cells depends largely on nongated ____ channels.
K+, b/c resting mbm potential is -70mV
ABC superfamily proteins are thought to act as ATP-dependent flippases in transporting:
Lipophilic drugs out of mammalian cells
endosymbiotic theory
Mitochondria and chloroplasts evolved from symbiotic bacteria engulfed though endocytosis by the euk cell evidence: both have two membranes, inner likely from the bacterial membrane, the outer is a vestige of plasma mbm from engulfment, broth have own GNA genome, synthesis of proteins in these more closely related to bacteria
ATP powered pumps
Move specific ions or molecules across the membrane; require ATP molecules to function, against a gradient, active trasnport, slowest transfer b/c of confirmational change, all have binding site for ATP in cytosolic region of protein
If a cellular homogenate were subjected to differential centrifugation, which of the following would be expected to pellet first?
Nuclei, because 1/2 cell size, very large
Types of ATP powered pumps
P-class, V-class, F-class, ABC superfamily,
phosphoglycerides
PE = Phosphatidylethanolamine, makes curved membrane (small head group) PC = Phosphatidylcholine, most abundant, makes flat sheet-like membrane(big head group) PS = Phosphatidylserine, only in inner leaflet (cytosolic), marker for apoptosiswhen detected outside cell PI = Phosphatidylinositol, used in GPI lipid anchored membrane proteins
Prokaryote vs. Eukaryote cells
Prokaryotes:.5-1 micron, no nucleus, free-flowing single c circular chromosome eukaryotes: 10 micron, nucleus, mbm bound organelles, Both: ribosomes, transcription/translation, , proteins and ATP, ETC, plasma membrane, cell walls, flagella
cotranslational translocation
Simultaneous transport of a secretory protein into the endoplasmic reticulum as the nascent protein is still bound to the ribosome and being elongated.
Integral membrane proteins (Transmembrane
Span a phospholipid bilayer and consist of three segments- Have a helix or b sheet domain, some are receptors, not all transport material
Sphingolipids
Sphingomyelin is themost abundant -Sphingomyelin: Sphingomyelins are present abundantly in the cell membranes of animal cells especially in the myelin sheath of neurons -Glycosphingolipids: abundant in the brain and spinal cord (Glucosylceramide,(GlcCer) shown above) -Gangliosides: most complex sphingolipids that are abundant in ganglion cells ofnervous tissue
-Sphingomyelin
Sphingomyelins are present abundantly in the cell membranes of animal cells especially in the myelin sheath of neurons
electrochemical gradient
The diffusion gradient of an ion, which is affected by both the concentration difference of an ion across a membrane (a chemical force) and the ion's tendency to move relative to the membrane potential (an electrical force).
Ergosterol
The main sterol in fungal membranes
translocon
a complex of proteins forms a channel through ER membrane, continues the elongated protein chain into the central pore of the translocon
-Glycosphingolipids
abundant in the brain and spinal cord (Glucosylceramide,(GlcCer) shown above)
cytosolic side acylation (Lipid-anchored membrane proteins)
acylation, anchored fatty acyl group cov attacheed to N terminal glycine reside image: cytosolic leaflet with gly attached to potein chain with COO- in cytosol
why do integral proteins-spanning membrane have alpha helixes
alpha helixes are stably embedded becasue of energetically favorable interactions, single alpha helix suffiencent to wmbed protein,
charged residues in alpha helix guide assembly of multimeric proteins
also contain charged residues, guide assembly and stabilization of multimeric proteins
Lipid-anchored membrane proteins: GPI anchor
always have phosphatidylinositol, with sugar residues attached on top, with phosphoethanolamine attached above sugar holding protein
Phosholipids
amphopathic lipid with a phophate base head group and a two pronged hydrophobic tail
prenylation (Lipid-anchored membrane proteins)
anchored to membrane by hydrocabon chain attached to cysterine residue at or near the C-terminus, NH3 in cytosol
cholesterol
animal
method to obtain proteins of intrest
anitbodies, monoclonal antibodies generated in a single cell type, polyclonal antibodies generated multiple cell types, inject proteins of interest into animal and let antibodies against protein epitope(part of antigen the antibody attach to) be raised, can tag with magnet or fluorescent
A characteristic of all biomembranes is
asymmetry, Cholesterol, however, seems to be evenly distributed• When cells die, the asymmetry is no longermaintained and lipids normally enriched in thecytoplasmic leaflet can be detected in the exoplasmicone
N terminal ER signal sequence
at N terminus of protein, stretch of hydrophobic amino acids and removed by signal peptidase., not present in mature form of protein, 16-3o hydrophobic residues, all signal scontain one or more postively charges aa
transcriptional fusion
attach promoter and gfp, promoter express gfp and flurences shows what cells this promoter is activated
translational fusion
attach promoter with gene and gfp so protein is trascribed with gfp and follows protein
multipass proteins
both SA and STA alternating, two or more membrane spanning segments=helices
Lipid-anchored membrane proteins
bound covalently to lipid molecule, hydrophobic segment of the lipid embed into mbm and anchors protein
unitransporters
carrier single molecule or ion unidirectionally, down gradient, ex. glucose into cell, trasnporter called GLUT1
less fluid
cholesterol, long chains, low temp, sphingolipids
chemical gradient
concentration gradient for an ion across the plasma membrane
Nucleous
contains DNA of genome, half size of cell, site of transcription, mRNA leaves through nuclear pores, two membranes the outer membrane is continuous with rough ER