May 13, 2016
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Pre-Medical
There are a couple of questions on the C/P section bank for which I don't understand the explanation. I'd appreciate if you could explain to me why what I picked is incorrect and why the correct answer is in fact correct. Here are the questions I am having trouble with:

61) Dnmt3a was purified using which type of column chromatography?
A. Affinity
C. Cation-exchange - I picked this because Histidine (+ charge) was being purified with Ni-nitrilotriacetic acid-agarose chromatography.

63) Which statement about the interaction between Dnmt3a and the DNA substrate is supported by the data in Figure 1? - I don't understand why C is the correct answer.

78) Which solution component will have the lowest concentration at the end of the kinetic assay described in the passage?
B. ADP
C. ATP
I thought both of these can be correct since as ATP is being made it is being used up. And the same applies to ADP. Why is B the correct answer?

89) Which change in the protonation state of Glu-487 is most likely responsible for the change in MP activity at either low or high pH? - I am stuck between A and D. Shouldn't both of these be correct?

94) The observed activity in the H232R variant is most likely the result of Arg: - I am stuck between A and B.
 

theonlytycrane

5+ Year Member
Mar 23, 2014
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Medical Student
There are a couple of questions on the C/P section bank for which I don't understand the explanation. I'd appreciate if you could explain to me why what I picked is incorrect and why the correct answer is in fact correct. Here are the questions I am having trouble with:

61) Dnmt3a was purified using which type of column chromatography?
A. Affinity
C. Cation-exchange - I picked this because Histidine (+ charge) was being purified with Ni-nitrilotriacetic acid-agarose chromatography.
Valid thinking for choice (C). Ni-nitrilotriacetic acid-agarose is the giveaway. If it was cation exchange, the resin described would be negatively charged so that cations stick to it. Ni is a metal that can be be coordinated to, but isn't anionic. The His is likely deprotonated and uses a lone-pair to coordinate to the metal to stick to it.

63) Which statement about the interaction between Dnmt3a and the DNA substrate is supported by the data in Figure 1? - I don't understand why C is the correct answer.
This question is kind of subtle and I don't really like it either. So we know that Dnmt3a is associated with DNA and methylates it. At first, the experimental procedure isn't super clear whether biotinylated DNA or non-biotinylated DNA (or both) will stick to the enzyme. The only hint is that the samples are loaded onto plates with avidin, which apparently strongly binds biotin. So the Dnmt3a + biotinylated DNA must stick to the avidin and the non-biotinylated DNA washes away. With this said, the y-axis on the graph shows methylation rates being measured so biotinylation isn't preventing Dnmt3a from methylating the DNA (choice A is incorrect). If the stability of the 30mer and 590mer were the same, they would probably be methylated at the same rate, but the graph shows 509mer being methylated high in condition 1 (choice B is incorrect). We washed away all the non-biotinylated DNA so we don't know the methylation rate of it (choice D is incorrect). By POE, it must be (C). In condition 1 we see higher methylation rates for 590mer. The reason the rates are kind of the same for conditions 2 and 3 is that for those the biotinylated DNA and non-biotinylated DNA were added either at the same time or in the opposite order as condition 1, so the Dnmt3a didn't get to bind as much of the biotinylated DNA to methylate it as in condition 1.

78) Which solution component will have the lowest concentration at the end of the kinetic assay described in the passage?
B. ADP
C. ATP
I thought both of these can be correct since as ATP is being made it is being used up. And the same applies to ADP. Why is B the correct answer?
They couple the phosphorylation of galactose with some other stuff. So after galactose gets phosphorylated, ADP is generated. ADP feeds in as a substrate to the PEP -> pyruvate reaction (last step of glycolysis) to convert ADP to ATP. Pyruvate then feeds as a substrate to pyruvate -> lactate via lactate dehydrogenase which uses NADH and oxidizes it to NAD+. They do all this craziness to assay the entire conversion by decreasing NADH levels. As they see NADH levels decline, they infer that this chain of events is taking place.

Overall, any ATP that is used get regenerated instead of staying in ADP form.

89) Which change in the protonation state of Glu-487 is most likely responsible for the change in MP activity at either low or high pH? - I am stuck between A and D. Shouldn't both of these be correct?
In the passage it says that the active site has Glu that acts like a general acid during catalysis. That means that it should be protonated, so that it can give up the proton during the reaction. At low pH this is okay. At a high pH, Glu will lose the proton and then no longer be able to function in the way it needs to for catalysis. The graph at the bottom shows how the relative activity drops off after pH ~ 6.

94) The observed activity in the H232R variant is most likely the result of Arg: - I am stuck between A and B.
I didn't really like this one either. Both answers sound okay to me. They reject (A) by saying that the phosphorylated-His is different than Arg. But I assumed that Arg would similarly be phosphorylated like His was. The answer says that Arg likely does some kind of allosteric activation which is a bit of a stretch to me. I see the increased activity level, associate His and Arg both being basic a.a. and think that both likely interact in the same way. (B) makes sense because it says GK-P undergoes significant motion to do it's activity. I think this question needs clarification, but you're thinking about it great if you narrowed it down to A and B.
 

aldol16

2+ Year Member
Nov 1, 2015
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61) Dnmt3a was purified using which type of column chromatography?
A. Affinity
C. Cation-exchange - I picked this because Histidine (+ charge) was being purified with Ni-nitrilotriacetic acid-agarose chromatography.
You should commit to memory Ni and His tags. Putting His tags on proteins is very common in anticipation of a pulldown. A column filled with Ni will result in His residues coordinating to the Ni.

Can't answer the rest because I'm too lazy to look them up.
 
OP
K
May 13, 2016
77
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Status
Pre-Medical
Thank you very much to you both for replying! Your knowledge always amazes me!