Passage
NDPK is an enzyme in budding yeast that uses ATP to phosphorylate nucleotide diphosphates (NDPs and dNDPs) , yielding the triphosphate form of each (NTPs and dNTPs) . dNTPs are necessary for DNA synthesis, yet yeast cells that lack NDPK grow and divide at the same rate as wild-type yeast. Therefore, triphosphate generation is most likely carried out by one or more other enzymes.The enzyme TMPK converts deoxythymidine monophosphate (dTMP) to the diphosphate form (dTDP) by transferring the γ-phosphate of ATP to dTMP. Yeast cells that lack TMPK activity cannot synthesize DNA and become arrested during replication. It was proposed that TMPK may partially compensate for NDPK loss by phosphorylating dTDP as well as dTMP.To better understand the role of TMPK in nucleotide phosphorylation, wild-type TMPK and the inactive E75K variant were purified by affinity chromatography, and the following experiments were performed.Experiment 1After purification, a sample containing 3 μg of protein was added to a reaction mixture containing radiolabeled nucleotide monophosphates (NMPs or dNMPs) and unlabeled ATP. The reaction proceeded for 30 minutes, and products were separated by thin-layer chromatography (TLC) on a PEI-cellulose plate. PEI-cellulose separates nucleotides by charge (Figure 1) , and migration distance decreases with increasing negative charge. Spots were visualized by autoradiography, and it was determined that 0.30 nmol of dTMP and 0.15 nmol of uridine monophosphate (UMP) became phosphorylated, respectively.
Figure 1 TLC analysis of nucleotide monophosphates after exposure to TMPK and ATP. (Note: Different nucleotides with the same phosphorylation level migrate different distances.) Experiment 2The experiment was repeated using radiolabeled dTDP as the starting material. The reaction proceeded in buffer only, in the presence of E75K TMPK, or in the presence of wild-type TMPK. Products were separated by TLC and visualized (Figure 2) .
Figure 2 TLC analysis of dTDP phosphorylation with buffer only (Lane 1) , E75K TMPK (Lane 2) , or wild-type TMPK (Lane 3)
Adapted from Chien CY, Chen BR, Chou CK, Sclafani RA, Su JY. The yeast Cdc8 exhibits both deoxythymidine monophosphate and diphosphate kinase activities. FEBS Lett. 2009;583(13) :2281-6.
-Experiment 2 was performed to determine whether phosphorylation of dTDP was due to residual NDPK activity in the purified enzyme solution. Which of the following shows the expected outcome of Experiment 2 if NDPK were still present after purification?

Question

Passage
NDPK is an enzyme in budding yeast that uses ATP to phosphorylate nucleotide diphosphates (NDPs and dNDPs) , yielding the triphosphate form of each (NTPs and dNTPs) . dNTPs are necessary for DNA synthesis, yet yeast cells that lack NDPK grow and divide at the same rate as wild-type yeast. Therefore, triphosphate generation is most likely carried out by one or more other enzymes.The enzyme TMPK converts deoxythymidine monophosphate (dTMP) to the diphosphate form (dTDP) by transferring the γ-phosphate of ATP to dTMP. Yeast cells that lack TMPK activity cannot synthesize DNA and become arrested during replication. It was proposed that TMPK may partially compensate for NDPK loss by phosphorylating dTDP as well as dTMP.To better understand the role of TMPK in nucleotide phosphorylation, wild-type TMPK and the inactive E75K variant were purified by affinity chromatography, and the following experiments were performed.Experiment 1After purification, a sample containing 3 μg of protein was added to a reaction mixture containing radiolabeled nucleotide monophosphates (NMPs or dNMPs) and unlabeled ATP. The reaction proceeded for 30 minutes, and products were separated by thin-layer chromatography (TLC) on a PEI-cellulose plate. PEI-cellulose separates nucleotides by charge (Figure 1) , and migration distance decreases with increasing negative charge. Spots were visualized by autoradiography, and it was determined that 0.30 nmol of dTMP and 0.15 nmol of uridine monophosphate (UMP) became phosphorylated, respectively.

Figure 1 TLC analysis of nucleotide monophosphates after exposure to TMPK and ATP. (Note: Different nucleotides with the same phosphorylation level migrate different distances.) Experiment 2The experiment was repeated using radiolabeled dTDP as the starting material. The reaction proceeded in buffer only, in the presence of E75K TMPK, or in the presence of wild-type TMPK. Products were separated by TLC and visualized (Figure 2) .

Figure 2 TLC analysis of dTDP phosphorylation with buffer only (Lane 1) , E75K TMPK (Lane 2) , or wild-type TMPK (Lane 3)
Adapted from Chien CY, Chen BR, Chou CK, Sclafani RA, Su JY. The yeast Cdc8 exhibits both deoxythymidine monophosphate and diphosphate kinase activities. FEBS Lett. 2009;583(13) :2281-6.
-Experiment 2 was performed to determine whether phosphorylation of dTDP was due to residual NDPK activity in the purified enzyme solution. Which of the following shows the expected outcome of Experiment 2 if NDPK were still present after purification?

Quizplus · Accepted Answer

11ecba2d_0ee7_5901_a3bf_25d3555b07d1_MD0008_00 Protein purification separates a protein of interest from all other proteins in a cell culture, allowing the activity of that protein to be assayed. However, purification techniques are imperfect, and trace amounts of proteins other than the protein of interest may remain in purified solutions. To confirm that an observed activity is due to the protein of interest, an inactive form of the protein can be expressed, purified by the same method as the active form, and assessed for activity. Identical protocols are used to obtain both the wild-type and inactive protein solutions, so they are expected to have the same impurities. Therefore, contributions to the observed activity from impurities such as residual NDPK will be apparent in both solutions. If phosphorylation of dTDP were due to residual NDPK activity, that activity would also be expected in the purified E75K solution. Therefore, if residual NDPK is the cause of dTTP production, both Lane 2 (E75K) and Lane 3 (wild-type) should have two spots: one at the top that corresponds to unmodified dTDP, and one near the origin that corresponds to newly formed dTTP. The fact that the dTTP spot did not appear in Lane 2 of the actual experiment (Figure 2) indicates that there was no residual NDPK, so the purification technique successfully removed NDPK. (Choices B and D) The reaction in Lane 1 was carried out in buffer only as a negative control (ie, no purified protein was added to the buffer), so no enzymes of any type were present and dTTP will not be observed in this lane. A dTTP spot in Lane 1 would indicate external contamination, and the experiment would have to be repeated. (Choice C) Lanes 2 and 3 contain identical reaction mixtures, with the exception that the E75K variant of TMPK in Lane 2 is inactive. Therefore, if there is residual NDPK activity, it should be present in both lanes, not just in Lane 2. Educational objective: Protein purification is imperfect, and trace amounts of proteins other than the protein of interest may be present in a purified solution. Any observed activity can be attributed to the protein of interest (rather than impurities) if a purified solution of an inactive variant does not yield the same activity.

Passage NDPK Is an Enzyme in Budding Yeast That Uses ATP