Unit 1 Flashcards
Phosphorylation Cascades
Involve a series of events with on kinase activating the next and so on.
Phosphorylation cascades can result in the phosphorylation of many proteins.
G-Proteins
Relay signals from activated receptors to target proteins such as enzymes and ion-channels
Process of a peptide/hydrophilic hormone
Reception- Transmembrane receptors change conformation when the ligand binds to the extracellular face; the signal is transduced across the plasma membrane.
Transduction- Transmembrane receptors act as signal transducers by converting the extracellular signals which alters the behaviour of the cell.
Hydrophilic Signalling
Hydrophilic signals molecules bind to transmembrane receptors so do not enter the cytosol.
Process of A Steroid Hormone
Steroid hormone passes across the plasma membrane.
The hormone binds to the receptor protein activates it.
the hormone receptor complex binds to the hormone response elements.
Binding at the HRE influences the rate of transcriptions.
What are the receptors for hydrophobic signalling molecules
Transcription Factors
Transcription Factors
Proteins that when bound to DNA can either stimulate/inhibit initiation of transcription.
Hydrophobic signalling
Hydrophobic signalling molecules can diffuse directly through the phospholipid bi-layer of the membrane, so bind to intracellular receptors.
How are cells ‘Switched on’
Binding changes the conformation of the receptor which initiates a response within the cell.
Receptor Molecules
Proteins with a binding site for specific signal molecules.
Examples of extracellular signalling molecules
Steroid hormones- Vitamin D
Peptide Hormones- Insulin
What drives the active transport of glucose in the small intestine.
The sodium gradient created by the sodium potassium pump drives the active transport of glucose
Sodium Potassium Pump
1- The transporter protein has its ion binding sites exposed to the cytoplasm. The protein has a high affinity for Na+ ions and 3 Na+ ions bind to the sites.
2- When the sodium ions are attached, the transporter protein is able to hydrolyse ATP. The phosphate attaches to the protein to phosphorylate it and this causes a conformational change.
3- This new conformation has its ion binding sites exposed to the outside of the cell, because it has a lower affinity for Na+ ions, they are released outside the cell.
4- This new conformation has a high affinity for K+ ions and 2 K+ ions bind to the proteins outside the cell, this triggers dephosphorylation.
5- Dephosphorylation causes the protein to revert to its original conformation with its binding sites exposed to the cytoplasm.
Step 6- This conformation has a low affinity for K+ ions so the are released into the cell.
How does the sodium potassium pump gain energy and what is its use for it.
Using energy from the hydrolysis of ATP to maintain ion gradients.
Membrane Potential
An electrical potential difference.
What enzymes hydrolyse ATP
ATPases
How does active transport go against the gradient
Using pump proteins that transfer substances across the membrane.
Pump proteins are transporter proteins that are coupled to an energy source.
Transporter Proteins
Bind to specific substance to be transported and undergo a conformational change to transfer the solute across the membrane.
Voltage-Gated Channes
Controlled by changes in ion concentration.
Ligand Gated Channels
Controlled by the binding of signal molecules.
Channels
Multi sub unit proteins with the sub units arranged to form water filled pores that extend across the membrane
Facilitated Diffusion
The passive transport of substances across the membrane through specific transmembrane proteins.
How are peripheral proteins held.
They have hydrophilic R groups on their surface and are bound to the surface membranes, mainly by ionic and hydrogen bond interactions.
What holds integral proteins in the bi-layer.
regions of hydrophobic R groups that allow strong hydrophobic intercations.
What term is used to describe the cell membrane.
Fluid Mosaic Model
What charge does a phosphate group add.
Negative Charge
Phosphotases
Catalyses the removal of a phosphate group.
Kinases
Catalyses the transfer of a phosphate group to other proteins.
What does the addition/removal of a phosphate cause in proteins.
Causes reversible conformational change.
How does pH and temp affect co-operativity of haemoglobin.
Decrease in pH/ increase in temp lowers affinity of haemoglobin for oxygen so binding of 02 is increased.
This means oxygen delivery to tissues is increased.
How is co-operativity shown in haemoglobin
The binding and release of oxygen
Process of modulators
Following binding of a modulator the conformation of the enzyme changes and this alters the affinity of the active site.
What do positive modulators do
Increase enzymes affinity for the substrate
What do negative modulators do
Decrease enzymes affinity for the substrate.
Function of modulators
To regulate the activity of an enzyme when it binds to the allosteric site
What structure do allosteric proteins have
Quaternary structure and consist of multiple sub units.
Denaturation
Increased temp disrupts interactions that hold proteins in shape, protein begins to unfold eventually becoming denatured.
As pH increases or decreases from optimum the ionic interactions between charged groups are lost which changes the conformation of the protein, it becomes denatured.
What happens when a substrate binds to the allosteric site.
It increases the affinity of the other active sites for binding of subsequent substrate molecules.
Ligand binding process
As a ligand binds to the protein binding site the conformation of the protein changes. This change in conformation causes a functional change of the protein.
What do binding sites have to ligands
Complementary shape and chemistry
Ligand
A substance that can bind to a protein.
Quaternary protein structure
Exists in proteins with 2 or more connected polypeptide sub units.
The spatial arrangements of said sub units is the quaternary structure.
Di-Sulfide Bridges
Covalent bonds between R groups containing Sulfur.
Tertiary Protein Structure
The polypeptide folds, this conformation is stabilised by R group interactions:
Hydrophobic interactions, ionic bonds, LDF’s, hydrogen bonds and disulfide bridges.
Secondary Protein Structure
Hydrogen bonding along the backbone of the protein strand results in regions of secondary structure;
Alpha Helices, Parallel or anti-parallel beta plated sheets, turns.
Primary Protein Structure
Sequence in which the amino acids are synthesised onto the polypeptide.
What are the three different types of R groups.
Basic (positive charge)
Acidic (negative charge)
Polar (hydrophobic)
How do R groups differ
Shape, charge, hydrogen, bonding capacity and chemical reactivity.
Examples of secreted proteins
Digestive Enzymes
Peptide Hormones
How do proteins become active after secretion
Through proteolytic cleavage, breaking peptide bonds between the amino acids in the protein.
Secretory Pathway
1- proteins that have to be secreted are translated in the ribosomes on the RER. They enter the lumen.
2- The proteins move through the Golgi Apparatus as normal and are packaged into vesicles.
3- The vesicles move to the membrane and fuse with it, releasing their contents into the cell
What are the major modifications of the proteins
The addition of complex carbohydrates, cutting/combining of strands, addition of phosphate groups.
Movement of proteins between membranes
Once the proteins are in the ER, they are transported by vesicles that bud off from the endoplasmic reticulum and fuse with the Golgi apparatus. As proteins move through the G.A they undergo post translational modifications. Vesicles move along microtubules to other membranes and fuse with them within a cell.
Synthesis of transmembrane proteins
The synthesis of all transmembrane proteins begins at the cytosolic ribosomes;
- A polypeptide that will become part of a transmembrane protein starts with a short strand of about 20 amino acids called a signal sequence. When the signal sequence emerges from the ribosome a cytosolic particle binds to it and halts translation.
2.The particle also directs the ribosome to dock with a protein pore on the endoplasmic reticulum thus forming Rough ER.
- After docking, the protein pore removes the cytosolic particle and the signal sequence so translation can continue.
- As it is translated the polypeptide chain is inserted directly into the membrane of the ER. The ribosome is released back into the cytosol once translation finishes.
Vesicles
Vesicles are fluid filled sacs which transport materials between different membrane components.
Lysosomes
Membrane bound organelles containing a variety of hydrolases that digest proteins, lipids, nucleic acid and carbohydrates.
Golgi Appartus
A series of flattened membrane discs.
Its job is to process proteins to be used inside and outside the cell.
Endoplasmic Reticulum
The ER forms a network of membrane tubules that branch off from the nuclear membrane.
It folds proteins and transports completed proteins to the Golgi apparatus.
Why can some vital functions not be carried out by them membrane in eukaryotes.
Because the plasma membrane is too small.
What do eukaryotes have due to their size.
Eukaryotes have a small surface area to volume ratio.
What do eukaryotic cells have to increase total area.
System of internal membranes.
Factors affecting proteins expressed.
Cellular Stress
Metabolic activity of the cell.
Response to signalling molecules.
Diseased vs Healthy cells.
Non Coding RNA Genes
Genes that do not code for proteins, that are transcribed to produce tRNA, rRNA and RNA.
Proteome
The entire set of proteins expressed by a genome.
How to calculate concentration of cells using a haecytometer
Multiply LxBxDepth x10,000 to get cm3
Cells along top/right boundary are counted
What is a hemocytometer used for
To count cell density
What proteins are added to cell culture
Growth factors are proteins that promote cell growth and proliferation. Growth factors are essential for culture of animal cells.
Examples of aceptic techniques
Sterilization of equipment and culture media by heat or chemical means.
How is a microbial culture setup
Innoculum of microbial cells on an agar medium or in a broth with suitable nutrients.
What is the purpose of aseptic techniques.
Eliminates unwanted microbial contaminants when culturing microorganisms or cells.
Fluorescence microscopy
Uses specific fluorescent labels to bind to and visualize certain molecules or structures within cells and tissues.
Bright field microscopy
Commonly used to observe whole organisms, thin sections of tissues or individual cells.
Western blotting
A technique used after SDS page. The separated proteins are transferred to a solid medium.
Immunoassay techniques
Used to detect and identify specific proteins. Where an antibody specific to the protein antigen is linked to a chemical label.
Centrifugation
Rapid revolution of a liquid in which more dense components settle in the pellet whereas less dense remain in the supernatant.
What is a risk assessment
The identification of control measures to minimize the risk.
What are examples of control measures
Appropriate handling techniques, protective clothing, protective equipment, aseptic technique.
What are some hazards in labs
Toxic chemicals
Corrosive chemicals
Flammable substances
Pathogenic organisms
Gel Electrophoresis
Uses a current flowing through a buffer to separate molecules by size and charge and shape.
Molecules are in their (native) folded state.
SDS page
Uses a current flowing through a buffer to separate proteins based on size alone. Molecules are in their non-native (unfolded) state.
What is a ligand
Molecules that bind specifically to another molecule.
What is a proteins iso-electric point
The pH at which a protein has no charge so is insoluble.
How to use iso electric point to separate proteins
Proteins at their iso-electric point are insoluble. This means that they will form a solid. They will be able to be separated from soluble proteins in a liquid at that point.
Affinity chromatography
1- A solid matrix or gel column is created with specific molecules bound to the matrix or the gel.
2- Soluble, target proteins in a mixture with a high affinity for these molecules becomes attached to them.
3- Other non target proteins with a weaker affinity are washed out.
4- The column is then washed with a buffer which lowers the affinity of target molecules allowing them to be collected.
How is a buffer used with iso-electric point.
If the solution is buffered to a specific pH only the proteins that have an IEP at that pH will precipitate.
Paper/Thin layer chromatography
Used for separating substances such as amino acids and sugars.
The speed at which each solute travels along the chromatogram depends on its solubility in the solvent used.
Colorimeter
The colorimeter is used to quantify the concentration of a pigmented compound.
Passes a light beam at a specific wavelength through a cuvette causing a simple solution.
What is a buffer
A solution that can resist pH change upon the addition of an acidic or base components.
Standard curves
Graphs or lights absorbance against concentration which can be used to figure out the solute concentration in unknown samples.
Linear dilutions
Dilutions that differ by an equal interval for example
0.1, 0.2, 0.3 and so on
Log dilution series
Differ by a constant proportion for example
0.1, 0.01, 0.001 and so on
Risk
The likely hood of harm arising from exposure to a hazard
How is GLUT-4 recruited.
Binding of insulin to its receptor causes a conformational change that triggers phosphorylation of the receptor. This starts a phosphorylation cascade inside the cell, which eventually leads to GLUT4-containing vesicles being transported to the cell membrane.
How is diabetes type 1 caused
Diabetes mellitus type 1 can be caused by failure to produce insulin
Diabetes mellitus type 2 is caused by loss of receptor function
How can the uptake of glucose be increased
By exercise triggering the recruitment of GLUT-4.
Resting membrane potential.
a state where there is no net flow of ions across the membrane
What does a nerve impulse require to take place
Changes in the membrane potential of the neurons plasma membrane.
Action potential
is a wave of electrical excitation along a neuron’s plasma membrane
Process of a nerve impulse.
Binding of a neurotransmitter to its receptor triggers the opening of ligand gated ion channels at at synapse.
Positive ions enter the cell and there is depolarization of the plasma membrane.
Adjacent voltage gated sodium channels are opened.
When the action potential reaches the end of the neuron it causes vesicles containing neurotransmitters to fuse with the membrane, this releases neurotransmitter, which stimulates are response in the connecting cell.
If sufficient ion movement occurs, and the membrane is depolarized beyond a solution threshold value, the opening of voltage gated sodium channels is triggered and sodium ions enter the cell down their electrochemical gradient.
This leads to a rapid and large change in the membrane potential.
A short time after opening the sodium channels become inactivated.
Voltage gated potassium channels then open to allow potassium ions to move out of the cell to restore the resting membrane potentials.
Restoration of resting membrane potential.
allows the inactive voltage-gated sodium channels to return to a conformation that allows them to open again in response to depolarisation of the membrane.
How are ion concentration gradients re established after nerve impulses
Ion concentration gradients are re- established by the sodium-potassium pump, which actively transports excess ions in and out of the cell.
Retina
Area within the eye that detects light.
Two types of photoreceptor cells
Cones and Rods
Rods
Rods function in dim light but don’t allow colour perception.
Cones
Are responsible for colour vision and only function in bright light .
How are photoreceptors in the eye formed.
Light sensitive molecules Retinal is combined with membrane protein opsin.
How is photo excited rhodopsin formed
Retinal absorbs a photon of light and rhodopsin changes concentration.
What type of molecule is activated by photo-excited rhodopsin.
Activates hundreds of molecules of a G-Protein called transducin.
What does transducin activate.
One molecule of the enzyme photodiesterase.
Function of photodiesterase
Catalyses the hydrolysis of a molecule called Cyclic GMP at a rate of a thousand molecules per second.
What does the reduction of cGMP affect
This results in the closure of ion channels in the membrane of the rod cells, which triggers nerve impulses in neurons in the retina.
How can rod cells respond to low intensities of light.
It has a very high degree of amplification.
What colour/wavelength do cone cells have a sensitivity to.
Red, blue, green or UV.
Function of the cytoskeleton
The cytoskeleton gives mechanical support and shape to cells and transports molecules along the cytosol.
What are microtubules made of
Alpha tubulin and Beta tubulin
Where do microtubules originate from
Microtubule organizing centre(MTOC)/ centrosome
What is the function of microtubules
control of the movement of membrane-bound organelles and chromosomes
How do microtubules form and breakdown
Polymerization and depolymerization of tubulin.
What role do microtubules play during cell division
They form the spindle fibers.
What is the first phase of the cell cycle
Interphase
What are the three stages of interphase
G1, S, G2
What occurs during G1
Growth phase
What occurs during S phase
DNA replication
What happens during G2 phase
Further growth
What are the 6 stages of mitosis
Mitosis consists of prophase, metaphase, anaphase and telophase and cytokinesis.
Prophase
— DNA condenses into chromosomes each consisting of two sister chromatids. Nuclear membrane breaks down; spindle microtubules extend from the MTOC by polymerisation and attach to chromosomes via their kinetochores in the centromere region.
Metaphase
Chromosomes are aligned at the metaphase plate (equator)
Anaphase
as spindle microtubules shorten by depolymerisation, sister chromatids are separated, and the chromosomes are pulled to opposite poles.
Telophase
the chromosomes decondense and nuclear membranes are formed around them.
Cytokinesis
the cytoplasm is separated into two daughter cells.
What is a checkpoint in cells.
Checkpoints are mechanisms within the cell that assess the condition of the cell during the cell cycle and halt progression to the next phase until certain requirements are met.
What are cyclins function
Cyclins combine with and activate cyclin- dependent kinases (CDKs). Active cyclin- CDK complexes phosphorylate proteins that regulate progression through the cycle. If sufficient phosphorylation is reached, progression occurs.
What occurs at the G1 checkpoint
retinoblastoma protein (Rb) acts as a tumour suppressor by inhibiting the transcription of genes that code for proteins needed for DNA replication
Phosphorylation by G1 cyclin-CDK inhibits the retinoblastoma protein (Rb)
This allows transcription of the genes that code for proteins needed for DNA replication.
What happens at the G2 checkpoint
the success of DNA replication and any damage to DNA is assessed
During the G2 checkpoint what happens if DNA damage is detected
DNA damage triggers the activation of several proteins including p53 that can stimulate DNA repair, arrest the cell cycle or cause cell death.
What happens at the metaphase checkpoint
At the metaphase checkpoint, progression is halted until the chromosomes are aligned correctly on the metaphase plate and attached to the spindle microtubules.
What does an uncontrolled reduction in cell cycle rate result on
A degenerative condition such as MS
What does an uncontrolled increase in the cell cycle result in
Cancers and Tumor formation
Porto-oncogene
is a normal gene, usually involved in the control of cell growth or division, which can mutate to form a tumour- promoting oncogene.
How is apoptosis triggered
Apoptosis is triggered by cell death signals that can be external or internal.
Example of internal death signal
DNA damage
Example of an external death signal
Signal molecules from lymphocytes
How are external death signals caused
External death signal molecules bind to a surface receptor protein and trigger a protein cascade within the cytoplasm
Caspases
Type of protease enzymes that cause destruction of the cell.
What happens in the absence of growth factors
Apoptosis
p53 protein
A protein that acts as a tumor suppressor and regulates cell division.
Electrochemical Gradient
Electrochemical gradient) is a combination of the concentration gradient.
AND
Potential/charge difference (across the plasma membrane)/membrane potential.
Monoclonal
An antibody derived from a single cell line
