Practical Stuff Flashcards
Which organism has the most base pairs of DNA?
Frog - 2.8 x 10^13
Roughly how many base pairs are found in a human gene?
10^4 bases
How is animal DNA arranged?
DNA arranged in chromosomes in nucleus
What occurs when bacterial DNA is packaged for use in a lab?
Two different conformations of DNA arise, one supercoiled (helix) one coiled (circular)
How will a supercoiled and coiled DNA vary on an electrophoresis trace?
Supercoiled is more compact so will travel further than the equivalent coiled DNA
What is used to cut DNA into smaller pieces?
Restriction endonucleases, made naturally by bacteria
What are restriction endonucleases used for by bacteria?
Used to protect from pathogenic invasion (e.g. virus), breaks down DNA
How does a bacterial cell protect it’s own DNA from breakdown by restriction endonucleases?
Methylates the target sequence so the RE cannot bind
What is an important feature of all restriction sites?
They are palindromic
What does EcoR1 cut between?
G/AATTC and CTTAA/G
What does Taq1 cut between?
T/CGA and AGC/T
How would you work out the probability of a specific sequence n bases long occurring?
1/(4^n) e.g 6 bases = 1/(4^6)
Are shorter or longer sequences more likely to occur in DNA?
Shorter sequences, higher probability, you fucking dick
After DNA is cut with restriction enzymes, how are they separated?
Use agarose gel electrophoresis and compare to fragments of known size
Which part of the DNA chain has a negative charge?
The phosphate in the DNA phosphate backbone (at pH of above 1)
What type of molecule is agarose?
A polysaccharide, which forms open gels
Which way will DNA strands travel through electrophoresis plate?
Travel from anode to cathode
What is Ethidium Bromide used for in electrophoresis?
Used to visualise DNA, intercalates between bases and fluoresces at 300nm (UV light)
Why does an uncut plasmid and a plasmid cut at one point run at different positions on the gel?
Even though they have the same no. of bases, the uncut plasmid would be more compact so travel further along gel
When drawing a calibration curve what should you plot?
log10 number of base pairs (x) against distance migrated (y)
Do animals store amino acids?
No you fucking cretin, excess AA are deaminated, carbon skeletons used as fuel
What are amino acids interconverted between to remove nitrogen?
Interconverted between amino acids and ketoacids (oxo acids)
What enzyme moves amino groups between alpha-amino acids and 2-oxoacids?
Transaminases/Aminotransferases, transfer from an alpha-amino acid to an alpha-keto acid (2-oxoacid)
What catalyses the oxidative deamination of glutamate?
Glutamate Dehydrogenase
What is another word for transaminases?
Aminotransferases
What is the cofactor for transaminases?
Pyridoxal Phosphate (derived from Vitamin D)
What is the Keq for transaminases?
~1
Pyruvate (2-oxoacid) + Glutamate —> ?
Alanine + 2-Oxoglutarate
What coenzyme does glutamate dehydrogenase use?
NAD+ and NADP+
How was the heart extract prepared?
Heart was minced, homogenised in ice-cold buffer, centrifuged at 20,000g for 20mins, supernatant dialysed exhaustively against ice cold buffer
What colour are the 2,4 dinitrophenyl-hydrazones of oxoacids?
Yellow
How are amino acids detected on TLC?
Spraying with ninhydrin, appear purple
What is the average size of a eukaryotic cell?
1-2micrometers
How do you convert eyepiece units into regular units?
Use eyepiece graticule scale and calibration slide, match up one end of the eyepiece graticule/calibration slide and find how long it is
How is movement generated by cells?
Using motor proteins (kinesin, myosin) to convert chemical ATP energy into kinetic energy
What type of filament does myosin travel along?
Actin filaments
What type of filament does kinesin/dynein travel along?
microtubules
Which protozoa travels faster, paramecium or amoeba?
Paramecium travels faster
What contains a pseudopodia (false foot)?
Amoeba
How do you determine the optimum pH of an enzyme?
Vary pH and measure initial rate of reaction of enzyme
What is it important to do when investigating effect of enzyme concentration on reaction rate?
Important to use a shot incubation time to ensure [Substrate] is not affecting results e.g, initial rate
What do you plot on a line weaver burke?
1/v (Y) against 1/[S] (x) (v=reaction rate, [S]=substrate concentration)
What is the equation for V?
V = Vmax.[S]/(Km+[S])
What is the equation for Kcat?
Kcat = Vmax/[E0], turnover number = number of substrate molecules each enzyme converts per unit time
What points occur at the Y and X intercept?
X=0=1/Vmax Y=0=-1/Km
What is Km?
Michaelas constant, substrate concentration when reaction rate is half of Vmax, measure of how strongly something binds, small Km = strong binding
What is a competitive inhibitor?
Inhibitor binds reversibly to active site, enzyme can still process substrate (Vmax same), but cannot bind S (Km changes), so gradient increase, rotating around 1/Vmax point (X=0)
What is a non-competitive inhibitor?
Inhibitor binds to a remote site, affects activity not ability to bind, so Vmax changes, Km same, line rotates around Y=0 (-1/Km)
What is an irreversible inhibitor?
Binds to active site and shuts down activity, so [E] decreases
Where is the electron transport chain?
On the inner mitochondrial membrane
What is oxidative phosphorylation?
Process in mitochondria by which ADP is phosphorylated to ATP
Where do the electrons arise from to reduce water?
NADH or FADH2 provide the electron flow in the ETC
What happens if there is no H+?
No proton gradient, ATP cannot be synthesised by rotation of ATP synthase
What is said of phosphorylation of ADP and electron transport?
They are coupled
What does complex I do to what coenzyme?
Reoxidises NADH to NAD+
What complexes are also hydrogen pumps?
Complex I, III, IV (1, 3, 4)
What does complex II do to what coenzyme?
Reoxidises FADH2 to FAD+
Does only FADH2 have to be used by coenzyme II?
No, FAD containing enzymes can oxidise substrates such as succinate (bound FAD->FADH2)
What else can complex II oxidise?
Succinate
Which carrier carries electrons from complex I/II to III?
Coenzyme Q (Ubiquinone)
What is the mobile electron carrier that carries electrons between complex III and IV?
Cytochrome C
What is the final electron acceptor?
Oxygen
How many ATPs are generated by one NADH/FADH2
2.5 or 1.5
What ion is present at cytochrome c’s core?
Fe2+/Fe3+
What two forms does ubiquinone cycle between?
CoQ and CoQH2
Is Coenzyme Q membrane soluble?
Yes
Where is cytochrome c present on the membrane?
Membrane associated, not soluble so travels up and down inter membrane space membrane
Which complex is present only in the membrane and matrix side?
Complex II - it is not a proton pump
What is used to measure the rate of ATP production?
Oxygen electrode, isolated mitochondria given oxidisable substrate and known amount of ADP
What is the overall equation of the oxygen electrode?
O2 + 4Ag+ + 4Cl- + 4H+ —> 4AgCl + 2H2O
What are the conditions of the oxygen electrode?
Platinum cathode, silver-silver chloride anode, bathed in saturated KCl, separated by PTFE membrane (known as the Clarke electrode)
What happens in state 2 respiration?
When mitochondria have substrate but no ADP to phosphorylate
What happens in stage 3 respiration?
ADP and substrate present (Added to state 2), initial rate of oxygen use is proportional to amount of ADP added
What happens in state 4 respiration?
ADP exhausted, rate of oxygen consumption drops to 0 (if you add more ADP the return to state 3)
Define uncoupler and give examples
Compound which can dissolve in the membrane and carry hydrogen ions back across the membrane, dinitrophenol
What complex is malate oxidised through?
Complex I, NADH pathway, using NAD+ dependant enzyme malate dehydrogenase
What complex is succinate oxidised through?
Complex II, FADH2 pathway, using Flavoprotein succinate dehydrogenase
How do we calculate P:O ratio?
On % oxygen against time trace: rule straight lines across state 2 and 4, then rule down state 3 line, measure height of whole table, measure height between state2/3 and state 3/4 crossing over points, (height of state/height of whole table)x oxygen at 100%, then: moles of ADPadded/moles of oxygen used
Why is dinitrophenol proved to be an uncoupler?
O2 uptake increases considerably, P:O ratio decreased, this is because ADP cannot be phosphorylated as no proton gradient but ETC still carries on so O2 still produced
What are the effects of amytal?
oxygen uptake ceases using malate (w/ or w/out dinitrophenol), denatures complex I, but complex II, III and IV are fine (succinate)
What are the effects of antimycin A?
Affects complex III and IV - so no oxygen uptake w/ malate or succinate
What are the effects of oligomycin?
Affects ATP synthase, oxygen consumption when DNP present
What are the effects of potassium cyanide?
Affects complex III and IV - so no oxygen uptake with malate or succinate
What are the effects of rotenone?
Affects complex I, but not complex II
What is the role of 2,4 Dinitrophenylhydrazine solution in practical B?
Used to label oxyacids yellow and stop reaction
