Cells And Proteins Flashcards
Describe a hazard and the types of hazards
Hazards in the lab include toxic or corrosive substances, heat or flammable substances, pathogenic organisms and mechanical equipment
Describe what a risk is
Risk is the likelihood of harm arising from exposure to a hazard
Describe risk assessment
Risk assessment involves identifying control measures to minimise risk
Name these control measures
Control measures include using appropriate handling techniques, protective clothing and equipment, and aseptic technique
Describe linear and log dilutions
Dilution in a linear dilution series differ by an equal interval e.g 0.1, 0.2, 0.3 and so on.
Dilutions in a log dilution series differ by constant proportion e.g 10^-1, 10^-2, 10^-3 and so on.
Production of a standard curve to determine an unknown, and what is a standard curve
A standard curve is used to determine the concentration of a solution.plotting measured values for known concentrations to produce a line or curve allows concentration of an unk own to be determined from the standard curve.
Describe the use of buffers to control Ph
PH is a measure of the acidity or basicity of a solution acids<7 neutral=0 bases>7.A buffer is a solution whose pH changes very little when a small amount of acid or base is added to it.Addition of acid or alkali has a very small effect on the pH of a buffer, allowing the pH of a reaction mixture to be kept constant.
Describe the methods and uses of a colorimeter to quantify concentration and turbidity
A colorimeter is used to measure the concentration of a coloured solution or the turbidity(cloudiness).Calibration with appropriate blank(solvent), use of absorbance to determine concentration of a coloured solution using suitable wavelength filters, use of percentage transmission to determine turbidity, such as cells in suspension.A colorimeter works by passing a light beam at a specific wavelength, though a cuvette containing a sample solution.some of the light is absorbed by the sample therefore light of a lower intensity hits the detector.for turbidity a denser sample will show a lower degree of transmission.
Describe the Use of centrifuge to separate substances of differing density
Centrifugation allows substances to be separated according to their destiny. More dense components settle in the pellet; less dense components remain in the supernatant.
Describe paper and thin-layer chromatography and affinity chromatography
Paper and thin layer chromatography can be used for separating different substances such as amino acids and sugars. The speed that each solute travels along the chromatogram depends on its differing solubility in the solvent used.
Principle of affinity chromatography and its use in separating proteins
A solid matrix or gel column is created with specific molecules bound to the matrix or gel. Soluble, target proteins in a mixture, with a high affinity for these molecules, become attached to them as the mixture passes down the column. Other non-target molecules with a weaker affinity are washed out.
Principle of gel electrophoresis and its use in separating proteins and nucleic acids
Charged macromolecules move through an electric field applied to a gel matrix
Describe how native gels separate proteins
Native gels separate proteins by their shape, size and charge
Decribe wether native gels denature and how they separate proteins
Native gels do not denature the molecule so that separation is by shape, size and charge
Describe how SDS-PAGE separates proteins and what they do
SDS–PAGE separates proteins by size alone. SDS–PAGE gives all the molecules an equally negative charge and denatures them, separating proteins by size alone.
Describe how proteins can be seperated
Proteins can be separated from a mixture using their isoelectric points (IEPs)
Describe what isoelectric point is
IEP is the pH at which a soluble protein has no net charge and will precipitate out of solution.
Describe how proteins will precipitate if a solution is buffered to a specific pH
If the solution is buffered to a specific pH, only the protein(s) that have an IEP of that pH will precipitate
Describe how an IEP can be used in electrophoresis
Proteins can also be separated using their IEPs in electrophoresis
.
Soluble proteins can be separated using an electric field and a pH gradient. A protein stops migrating through the gel at its IEP in the pH gradient because it has no net charge.
What techniques are used to identify proteins
Immunoassay techniques are used to detect and identify specific proteins
What do immunoassay techniques use
These techniques use stocks of antibodies with the same specificity, known as monoclonal antibodies
How are antibody specific to a protein antigen linked
An antibody specific to the protein antigen is linked to a chemical ‘label’
Describe the label and other reporter enzymes
The ‘label’ is often a reporter enzyme producing a colour change, but chemiluminescence, fluorescence and other reporters can be used.
What else can the assay use to detect antibodies
In some cases the assay uses a specific antigen to detect the presence of antibodies.
What is western blotting and when is it used
Western blotting is a technique, used after SDS–PAGE electrophoresis
The separated proteins from the gel are transferred (blotted) onto a solid medium.used for identifying specific proteins that have been separated using SDS-PAGE gel electrophoresis.
How can proteins be identified
The proteins can be identified using specific antibodies that have reporter enzymes attached
Describe what bright-field microscopy is
Bright-field microscopy is commonly used to observe whole organisms, parts of organisms, thin sections of dissected tissue or individual cells
Describe what fluorescence microscopy is
Fluorescence microscopy uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues
Describe what aseptic technique is
Aseptic technique eliminates unwanted microbial contaminants when culturing micro- organisms or cells
What does aseptic technique involve
Aseptic technique involves the sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of microbial contaminants.
How can a microbial culture be started
A microbial culture can be started using an inoculum of microbial cells on an agar medium, or in a broth with suitable nutrients
What is does culture media promote
Many culture media exist that promote the growth of specific types of cells and microbes.
How are animal cells grown
Animal cells are grown in medium containing growth factors from serum
What are growth factors and their importance
Growth factors are proteins that promote cell growth and proliferation. Growth factors are essential for the culture of most animal cells
Describe in terms of culture primary cell lines and tumour cell lines and division
In culture, primary cell lines can divide a limited number of times, whereas tumour cells lines can perform unlimited divisions
Explain the importance of plating out of a liquid microbial culture
Plating out of a liquid microbial culture on solid media allows the number of colony- forming units to be counted and the density of cells in the culture estimated
Why is serial dilation needed for colony count
Serial dilution is often needed to achieve a suitable colony count
What is haemocytometry used for
Method and use of haemocytometer to estimate cell numbers in a liquid culture
What is vitale staining used for
Vital staining is required to identify and count viable cells
What is the proteome
The proteome is the entire set of proteins expressed by a genome
Why is the proteome larger then the number of genes, eukaryotes and why is this caused
The proteome is larger than the number of genes, particularly in eukaryotes, because more than one protein can be produced from a single gene as a result of alternative RNA splicing
What are non coding proteins called and what are they transcribed to produce
Genes that do not code for proteins are called non-coding RNA genes and include those that are transcribed to produce tRNA, rRNA, and RNA molecules that control the expression of other genes
How can the set of expressed genes by a given cell type change and under what circumstances
The set of proteins expressed by a given cell type can vary over time and under different condition
Describe factors which affect the set of proteins expressed by a given cell type
Some factors affecting the set of proteins expressed by a given cell type are the metabolic activity of the cell, cellular stress, the response to signalling molecules, and diseased versus healthy cells
Describe the system of eukaryotic cells and the affect of this on the membrane
Eukaryotic cells have a system of internal membranes, which increases the total area of membrane
Describe how the size of eukaryotic cells have an affect on the ratio of volume to surface area and towards the plasma membrane of the cell.
Because of their size, eukaryotes have a relatively small surface area to volume ratio. The plasma membrane of eukaryotic cells is therefore too small an area to carry out all the vital functions carried out by membranes
What does the ER OR endoplasmic reticulum form
The endoplasmic reticulum (ER) forms a network of membrane tubules continuous with the nuclear membrane
What is the Golgi apparatus
The Golgi apparatus is a series of flattened out membrane discs
What are lysosomes wand what to they digest
Lysosomes are membrane-bound organelles containing a variety of hydrolases that digest proteins, lipids, nucleic acids and carbohydrates
What transports materials membrane compartments
Vesicles transport materials between membrane compartments
where are lipids and proteins synthesised
Lipids and proteins are synthesised in the ER
Compare the rough ER and smooth ER in their number of ribosomes and location
Rough ER (RER) has ribosomes on its cytosolic face while smooth ER (SER) lacks ribosomes.
Where are lipids synthesised and inserted into
Lipids are synthesised in the smooth endoplasmic reticulum (SER) and inserted into its membrane
Where does the synthesis of all proteins begin in
The synthesis of all proteins begins in cytosolic ribosomes
Where is the synthesis of proteins in cytosolic ribosomes completed and where do they remain
The synthesis of cytosolic proteins is completed there, and these proteins remain in the cytosol
How is the RER formed
Transmembrane proteins carry a signal sequence, which halts translation and directs the ribosome synthesising the protein to dock with the ER, forming RER
What is a signal sequence and what does this determine
A signal sequence is a short stretch of amino acids at one end of the polypeptide that determines the eventual location of a protein in a cell
What continues after socking and where is the protein inserted.
Translation continues after docking, and the protein is inserted into the membrane of the ER
Describe what happens once protein are in the ER
Once the proteins are in the ER, they are transported by vesicles that bud off from the ER and fuse with the Golgi apparatus
What do proteins undergo after they move through the Golgi apparatus
As proteins move through the Golgi apparatus they undergo post-translational modification
Describe what happens when molecules move through the Golgi apparatus and the action of enzymes during this process.
Molecules move through the Golgi discs in vesicles that bud off from one disc and fuse to the next one in the stack. Enzymes catalyse the addition of various sugars in multiple steps to form the carbohydrates
What is the major modification
The addition of carbohydrate groups is the major modification
What happens to vesicles that leave the Golgi apparatus
Vesicles that leave the Golgi apparatus take proteins to the plasma membrane and lysosomes
Describe how vesicles move along microtubules to other…
Vesicles move along microtubules to other membranes and fuse with them within the cell
What are translated in ribosomes on the RER and what do they enter
Secreted proteins are translated in ribosomes on the RER and enter its lumen
What are examples of secreted enzymes
Peptide hormones and digestive enzymes are examples of secreted proteins
What are packaged into secretory vesicles and what do they move through
The proteins move through the Golgi apparatus and are then packaged into secretory vesicles
These vesicles move to and fuse…
These vesicles move to and fuse with the plasma membrane, releasing the proteins out of the cell
Describe how secreted proteins are synthesise and what they require to produce active proteins
Many secreted proteins are synthesised as inactive precursors and require proteolytic cleavage to produce active proteins
Describe what is meant by proteolytic cleavage
Proteolytic cleavage is another type of post- translational modification. Digestive enzymes are one example of secreted proteins that require proteolytic cleavage to become active.
Proteins are polymers…
Proteins are polymers of amino acid monomers
What do amino acids form and what are they linked by
Amino acids are linked by peptide bonds to form polypeptides
Amino acids are classified to their
Amino acids are classified according to their R groups: basic (positively charged); acidic (negatively charged); polar; hydrophobic
Describe how the wide range of functions are carried out by proteins
The wide range of functions carried out by proteins results from the diversity of R groups
What is the primary structure
The primary structure is the sequence in which the amino acids are synthesised into the polypeptide
Describe how a secondary structure is formed and the types of structures
Hydrogen bonding along the backbone of the protein strand results in regions of secondary structure — alpha helices, parallel or anti- parallel beta-pleated sheets, or turns
How is a tertiary structure formed
The polypeptide folds into a tertiary structure
How is the conformation of a tertiary structure stabilised
This conformation is stabilised by interactions between R groups: hydrophobic interactions; ionic bonds; London dispersion forces; hydrogen bonds; disulfide bridges
What are disulphide bridges
Disulfide bridges are covalent bonds between R groups containing sulfur.
How does a quaternary structure exist
Quaternary structure exists in proteins with two or more connected polypeptide subunits
What is a prosthetic group
A prosthetic group is a non-protein unit tightly bound to a protein and necessary for its function
Give an example of a prosthetic group
The ability of haemoglobin to bind oxygen is dependent upon the non-protein haem group.
Describe the affect of increasing or decreasing pH or temperature affect R group interactions
Increasing temperature disrupts the interactions that hold the protein in shape; the protein begins to unfold, eventually becoming denatured. The charges on acidic and basic R groups are affected by pH. As pH increases or decreases from the optimum, the normal ionic interactions between charged groups are lost, which gradually changes the conformation of the protein until it becomes denatured.
What is a ligand
A ligand is a substance that can bind to a protein
How is binding to ligands enabled
R groups not involved in protein folding can allow binding to ligands
How does the ligand relate to the binding site
Binding sites will have complementary shape and chemistry to the ligand
As the ligand binds to the protein describe how this permanently affects the protein
As a ligand binds to a protein-binding site the conformation of the protein changes. This change in conformation causes a functional change in the protein
Where do Allosteric interactions occur
Allosteric interactions occur between spatially distinct sites
Describe how an allosteric enzyme affects the other active sites and substrate molecules, and the biological importance of this
The binding of a substrate molecule to one active site of an allosteric enzyme increases the affinity of the other active sites for binding of subsequent substrate molecules. This is of biological importance because the activity of allosteric enzymes can vary greatly with small changes in substrate concentration.
What do allosteric proteins consist of
Many allosteric proteins consist of multiple subunits (have quaternary structure)
Describe how Allosteric proteins show co-operativity
Allosteric proteins with multiple subunits show co-operativity in binding, in which changes in binding at one subunit alter the affinity of the remaining subunits
What is the other site of an Allosteric enzyme
Allosteric enzymes contain a second type of site, called an allosteric site
What do modulators do
Modulators regulate the activity of the enzyme when they bind to the allosteric site
Describe the binding of a modulators affect on an enzyme
Following binding of a modulator, the conformation of the enzyme changes and this alters the affinity of the active site for the substrate
Describe what positive and negative modulators do
Positive modulators increase the enzyme’s affinity for the substrate, whereas negative modulators reduce the enzyme’s affinity
Describe how The binding and release of oxygen in haemoglobin shows co-operativity
Changes in binding of oxygen at one subunit alter the affinity of the remaining subunits for oxygen.
Describe The influence and physiological importance of temperature and pH on the binding of oxygen
A decrease in pH or an increase in temperature lowers the affinity of haemoglobin for oxygen, so the binding of oxygen is reduced. Reduced pH and increased temperature in actively respiring tissue will reduce the binding of oxygen to haemoglobin promoting increased oxygen delivery to tissue
Describe the affect of the addition or removal of a phosphate
The addition or removal of phosphate can cause reversible conformational change in proteins. This is a common form of post-translational modification
What do protein kinases do
Protein kinases catalyse the transfer of a phosphate group to other proteins
Where is the terminal phosphate-of ATP transferred to
The terminal phosphate of ATP is transferred to specific R groups
Protein phosphatases…
Protein phosphatases catalyse the reverse reaction
What does phosphorylation bring and what does it affect
Phosphorylation brings about conformational changes, which can affect a protein’s activity. The activity of many cellular proteins, such as enzymes and receptors, is regulated in this way Some proteins are activated by phosphorylation while others are inhibited.
Describe what .adding a phosphate group does
Adding a phosphate group adds negative charges. Ionic interactions in the unphosphorylated protein can be disrupted and new ones created.
