Principles of Science Flashcards
How are histological samples are prepared?
- Fixation – sample placed in fixative for length of time (formalin, 24hr, 70% alc)
- Process – paraffin wax & solvent (histolene)
- Section – cut with rotary micro turn
- Stained – haematoxylin (blue nuclei) & eosin (pink protein)
What skills are involved in examining histological sections
Directions of sectioning
- Longitudinal – horizontal (along)
- Transverse – vertical (across)
Recognise examples of the main tissue types
1. Simple Epithelial tissue ○ Form boundaries/barriers (dark purple staining) Squamous – blood vessels (passive) Cuboidal – glands (semi-acive) Columnar – gut (active)
2. Connective tissue
○ Lie in between epithelia & muscle
○ Contains proteins & provides active tissue support
○ ECM – support network of proteins
○ Dense Irregular CT (tendon) – multidirectional collagen bundles
3. Muscle
○ Skeletal/striated – longitudinal = striations
○ Cardiac
○ Smooth
4. Nerves
○ Nerve bundles from CNS
○ Bundles found in muscle tissue
Explain what is meant by a “protein structural hierarchy”
Primary – linear chain of sequential amino acids
Secondary – AA chains folded into alpha-helix or beta-plated sheets
Tertiary – interact with protein regions & forms final protein structure
Quaternary – multiple protein complex which define function
Describe the structure of a peptide bond, its properties and influence on protein structure.
Structure = A-carbon w/ 4 bonds – R group, H, amino group & carboxyl group
Properties = links carboxyl & amino groups together
Influence = planar – double bond, O pulls electrons, fold rotating bonds around alpha carbon
Describe the types of chemical bonding responsible for determining protein conformation.
Chemical Bonding Protein Conformation
Covalent Primary (peptide bond)
Hydrogen Secondary
Ionic Tertiary & Quaternary (electrostatic)
Hydrophobic Tertiary & Quaternary (Van der Waals)
Explain the following terms: α-helix; coiled-coil; parallel and antiparallel β-pleated sheets; domain
A-helix – hydrogen bonding 4 residues apart, clockwise, R group outward
Parallel B – H bonds interact w/ chains, planar (sheet), tetrahedral (bent), amino top & carboxyl down orientation (1 direction)
Antiparallel B – 2 peptide strands running in opposite directions held together by hydrogen bonding between the strands
Tertiary Domain – region of polypeptide chain which forms a stable independent structural tertiary unit
Use haemoglobin as an example of a protein possessing quaternary structure, the function of which depends on conformation.
Haemoglobin
□ a carrier protein, transporting 02 or C02 around the body
□ made up from 2 pairs of different polypeptide chains (4 subunits)
- Starts in T (tense) state
- one oxygen attaches to haemoglobin
- conforms quaternary structure into R (relaxed) state
- higher affinity for oxygen & o2 molecules attach quickly = sigmoid curve
Outline the structure of collagen, explaining how its triple gamma-helix differs from that of an alpha-helix
Collagen
□ 3 helical chains that wind around central axis
□ Gly-X-Y
□ X = amino acid (proline/lysine or hydroxylysine)
□ Y = hydroxyl group (proline or hydroxyproline)
□ Gly = glycine in centre of each helix
Differs = collagen R group inwards (alpha outwards), 1 vs 3 chains
Explain the detrimental functional consequences that may ensure when conformational variants are formed, using prion proteins as examples and to explain their role in BSE and Scrapie
Prions
□ Transmissible via contaminated feed (eating other infected animals)
□ Protein is an infectious agent (conformational change)
BSE = bovine spongiform encephalopathy
□ Toxic build up of plaque in brain neural tissue
Scrapie = sheep
□ PrPc transforms into PrPSc causing fibrils to aggregate into neural tissue as
plaque
Define the following terms: substrate; product; reaction rate; equilibrium constant; rate constant; activation energy; induced fit
Substrate – beginning component
Product – end component
Reaction rate – how fast substrate turns into product or vice versa
Equilibrium constant – ratio between both rate constants (forward & back)
Rate constant – measuring combined rate of reaction forward or backward
Activation energy – how much free energy is needed for transition state
Induced fit – active site moulds itself around a substrate (flexible)
Outline the physical basis of how enzymes achieve massive reaction rate increases as compared with uncatalysed reactions.
- Enzymes lower activation energy needed for a transition state
- Enzymes only speeds up rate to reach equilibrium
- No difference in free energy or equilibrium constant
Explain in qualitative terms using a graphical representation of enzyme activity plotted against substrate concentrations. (Michaelis-Menten Kinetics)
- Hyperbolic profile
- Inc. substrate conc. = inc. rate of reaction
- Plateaus at Vmax (all binding sites saturated)
- Enzymes determine speed of forward & back rate constants
Outline the properties of an allosteric enzyme and explain qualitatively how its action differs from enzymes exhibiting Michaelis-Menten kinetics.
Allosteric enzyme properties
- Bind to allosteric sites on enzymes at start/end/branching parts of metabolic
pathway
Feedback regulation – conforms active sites of enzymes
- Inhibitors stabilise T states - Activators stabilise R states
Differs – use half Vmax, activator shift curve left (less sub.), inhibitor shift right
(more sub.)
Outline the control of enzymatic activity by: phosphorylation/dephosphorylation, protein and/or nucleotide binding, Ca2+ binding in EF-hand, proteolysis.
Phosphorylation
□ Control covalent bonding
□ Kinase enzymes involved (serine/threonine & tyrosine)
□ Specificity – to adjacent residues, determine which AA get phosphorylated
□ Requires ATP –> ADP + P (added to protein)
Dephosphorylation
□ Phosphatases enzymes involved
□ Phosphate gets removed from protein
Regulatory Proteins/Nucleotides
□ Inactive – cAMP-dependant protein kinase (regulatory & catalytic subunits)
□ Cyclic AMP activates enzyme kinase activity
□ Active – cAMP in regulatory subunits & catalytic subunits separate
Ion Binding (Calcium EF-hand)
□ High volume of Ca enter cell (usually kept out)
□ Calcium binds to tight loop connected to 2 a-helixs E & F
Proteolysis
□ Sequential activation of protease activity
□ ensures rapid signal amplification.
□ Protease factors –> prothrombin –> thrombin –> fibrogen –> fibrin –> clot
