UCL Transamination Practical
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- Created by: cgeorge
- Created on: 14-04-14 20:07
What do transaminases do?
move amino groups between amino acids and oxoacids
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What does glutamate dehydrogenase do?
catalyses the oxidative deamination of glutamate
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(transaminases) Which amino acid has an amino group removed and where is it transferred to?
the alpha amino acid has an amino group removed and transferred to a 2-oxoacid
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What does this produce?
A new amino acid and a new 2-oxo acid
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What is the aim of interconverting amino acids and oxo acids?
to channel the amino nitrogen into urea for excretion
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What are the two routes for this aim of the interconverting?
one is glutamate and one is aspartate
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What does the first option of these two routes do?
it is oxidatively deaminated, which releases an ammonium ion
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What does the second option of these two routes do?
it can contribute its amino group directly for synthesis of urea
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What do transaminases have as a cofactor?
tightly bound pyridoxal phosphate which is derived from vitamin B6
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Glutamate dehydrogenase is unusual in that it..
uses either NAD+ or NADP+ as a coenzyme
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What happens to the toxic ammonium ion released from the oxidative deamination of glutamate?
it is transported (as glutamine) to the liver, where it is converted to carbamoyl phosphate, which enters the urea cycle
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After the heart extract is minced, homogenised in an ice cold buffer, what happens?
it is centrifuged at 20000g x 20 minutes. The supernatant is then exhaustively dialysed (this means many changes of dialyses buffer) against ice cold buffer (0.05mol L-1 phosphate, pH 7.4)
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What is the purpose of TLC (thin layer chromatography)?
it is a quick and convenient way of separating many types of compound
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How is this [answer to previous question] brought about?
by allowing a solvent mixture to flow up (by capillary action) a thin layer of particles, which may be silica gel, aluminium oxide or cellulose (among other media). Molecules partition between the mobile phase and the stationary phase according to th
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How are the 2, 4 dinitrophenyl-hydrazones of the oxoacids visualised?
they are yellow and can be seen directly
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How are the amino acids visualised?
they are purple and can be seen when sprayed with ninhydrin (and heated)
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After the extracts have been added (which are added after the reactants), ethyl acetate is added to tubes 5- 10 and the tubes are shaken well. What does this do?
extracts the 2,4 dinitrophenyl-hydrazones
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After the ethyl acetate is added to tubes 5-10, all ten tubes were centrifuged. What was the purpose of this?
to remove precipitated protein and to separate the ethyl acetate (5-10)
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Tube 5 gives you a standard of what?
the 2,4 dinitrophenyl-hydrazone of pyruvate
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Tube 9 gives a standard of what?
the 2,4, DNPH of 2 oxo glutarate
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Tube 10 gives a standard of what?
the DNPH alone
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What is solvent system I?
ethanol: aqueous ammonia (conc) 70:30 (amino acids)
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What is solvent system II?
n-butanol: ethanol: aqueous ammonia (conc) 70: 10: 20 (oxoacids)
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The dinitrophenylhydrazine...
runs irregularly near the top so ignore the top spots and concentrate on the pyruvate DNP and 2-oxoglutarate DNP spots
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Pyruvate DNP…
often produces two spots close together (isomers). More rarely, 2 spots are seen with 2-oxoglutarate DNP as well
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(Use the drawings of your plates for the rest of the questions.) What's in
Nothing (no amino acids produced)
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What’s in position 2?
Glutamate
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what’s in position 4?
glutamate (technically shouldn’t be there, it’s because of limiting factors)
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What’s in position 9?
Oxoglutarate
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What’s in position 5?
Pyruvate
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What’s in position 6?
DNPH (buffer compounds)
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What can we derive is in position 3?
alanine (and some left over glutamate)
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What can we derive is in position 7?
Oxoglutarate
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What can we derive is in position 8?
oxoglutarate
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What can we derive is in position 10?
Nothing
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Tubes 1 and 5, the reactants are pyruvate and the products are pyruvate. What was the reaction?
no reaction
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Tubes 2 and 6, the reactants are glutamate and the products are glutamate. What was the reaction?
no reaction
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Tube 9, the reactants were oxoglutarate and the products are oxoglutarate. What was the reaction?
no reaction
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Tubes 4 and 8, the reactants are NAD+ and glutamate and the products are oxoglutarate. What was the reaction?
deamination
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Tube 10, the reactants were none and none. What was the reaction?
no reaction
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Tubes 3 and 7, the reactants are pyruvate and glutamate and the products are oxoglutarate and alanine. What was the reaction?
transamination
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Other cards in this set
Card 2
Front
What does glutamate dehydrogenase do?
Back
catalyses the oxidative deamination of glutamate
Card 3
Front
(transaminases) Which amino acid has an amino group removed and where is it transferred to?
Back
Card 4
Front
What does this produce?
Back
Card 5
Front
What is the aim of interconverting amino acids and oxo acids?
Back
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