Passage
Oxidative stress occurs in cells when reactive oxygen species (ROS) such as H₂O₂, superoxide (O₂⁻) , and hydroxyl radicals (^•OH) interact destructively with biological molecules. Several studies have indicated that histidine and certain peptides that contain histidine can decrease oxidative stress.A common marker of oxidative stress in cells is leakage of the enzyme lactate dehydrogenase (LDH) into the extracellular space. Researchers incubated cultured rat cells (PC12 cells) with H₂O₂ in the presence of different tripeptides, each containing one alanine, one glycine, and one histidine residue. Every possible sequence was tested, but the researchers found that the sequence GAH was the most effective in limiting extracellular LDH activity. After obtaining these preliminary results, the following experiments were conducted.Experiment 1PC12 cells were incubated with or without 5 mM H₂O₂ and with varying concentrations of GAH for 4 hours. Cells were centrifuged, and the supernatant (ie, extracellular material) was extracted and assayed for LDH activity (Figure 1) .
Figure 1 Extracellular LDH activity from PC12 cells exposed to H₂O₂ and varying concentrations of GAH. p-values are relative to 5 mM H₂O₂ and 0 mg/mL GAH.Experiment 2LDH was purified and assayed for activity in the presence of 5 mM H₂O₂ and varying concentrations of GAH (Figure 2) .
Figure 2 Activity of purified LDH with varying concentrations of GAHExperiment 3PC12 cells were incubated with or without 5 mM H₂O₂ in the presence and absence of 1 mg/mL GAH. Cells were then stained with the fluorescent marker TUNEL, which binds strongly to apoptotic cells. The percentage of apoptotic cells in each condition is shown in Figure 3.
Figure 3 Percentage of apoptotic cells in the presence or absence of H₂O₂ and GAH
-The purpose of measuring purified LDH activity with various amounts of GAH was most likely:

Question

Passage
Oxidative stress occurs in cells when reactive oxygen species (ROS) such as H₂O₂, superoxide (O₂⁻) , and hydroxyl radicals (^•OH) interact destructively with biological molecules. Several studies have indicated that histidine and certain peptides that contain histidine can decrease oxidative stress.A common marker of oxidative stress in cells is leakage of the enzyme lactate dehydrogenase (LDH) into the extracellular space. Researchers incubated cultured rat cells (PC12 cells) with H₂O₂ in the presence of different tripeptides, each containing one alanine, one glycine, and one histidine residue. Every possible sequence was tested, but the researchers found that the sequence GAH was the most effective in limiting extracellular LDH activity. After obtaining these preliminary results, the following experiments were conducted.Experiment 1PC12 cells were incubated with or without 5 mM H₂O₂ and with varying concentrations of GAH for 4 hours. Cells were centrifuged, and the supernatant (ie, extracellular material) was extracted and assayed for LDH activity (Figure 1) .

Passage Oxidative stress occurs in cells when reactive oxygen species (ROS) such as H<sub>2</sub>O<sub>2</sub>, superoxide (O<sub>2</sub><sup>−</sup>) , and hydroxyl radicals (<sup>•</sup>OH) interact destructively with biological molecules. Several studies have indicated that histidine and certain peptides that contain histidine can decrease oxidative stress.A common marker of oxidative stress in cells is leakage of the enzyme lactate dehydrogenase (LDH) into the extracellular space. Researchers incubated cultured rat cells (PC12 cells) with H<sub>2</sub>O<sub>2</sub> in the presence of different tripeptides, each containing one alanine, one glycine, and one histidine residue. Every possible sequence was tested, but the researchers found that the sequence GAH was the most effective in limiting extracellular LDH activity. After obtaining these preliminary results, the following experiments were conducted.Experiment 1PC12 cells were incubated with or without 5 mM H<sub>2</sub>O<sub>2</sub> and with varying concentrations of GAH for 4 hours. Cells were centrifuged, and the supernatant (ie, extracellular material) was extracted and assayed for LDH activity (Figure 1) . <strong>Figure 1</strong> Extracellular LDH activity from PC12 cells exposed to H<sub>2</sub>O<sub>2</sub> and varying concentrations of GAH. p-values are relative to 5 mM H<sub>2</sub>O<sub>2</sub> and 0 mg/mL GAH.Experiment 2LDH was purified and assayed for activity in the presence of 5 mM H<sub>2</sub>O<sub>2</sub> and varying concentrations of GAH (Figure 2) . <strong>Figure 2</strong> Activity of purified LDH with varying concentrations of GAHExperiment 3PC12 cells were incubated with or without 5 mM H<sub>2</sub>O<sub>2</sub> in the presence and absence of 1 mg/mL GAH. Cells were then stained with the fluorescent marker TUNEL, which binds strongly to apoptotic cells. The percentage of apoptotic cells in each condition is shown in Figure 3. <strong>Figure 3</strong> Percentage of apoptotic cells in the presence or absence of H<sub>2</sub>O<sub>2</sub> and GAH -The purpose of measuring purified LDH activity with various amounts of GAH was most likely: A) to demonstrate that GAH does not directly inhibit LDH. B) to determine baseline extracellular LDH activity. C) to show that GAH is an effective LDH antagonist. D) to characterize the GAH-LDH binding site.

Figure 1 Extracellular LDH activity from PC12 cells exposed to H₂O₂ and varying concentrations of GAH. p-values are relative to 5 mM H₂O₂ and 0 mg/mL GAH.Experiment 2LDH was purified and assayed for activity in the presence of 5 mM H₂O₂ and varying concentrations of GAH (Figure 2) .

Figure 2 Activity of purified LDH with varying concentrations of GAHExperiment 3PC12 cells were incubated with or without 5 mM H₂O₂ in the presence and absence of 1 mg/mL GAH. Cells were then stained with the fluorescent marker TUNEL, which binds strongly to apoptotic cells. The percentage of apoptotic cells in each condition is shown in Figure 3.

Figure 3 Percentage of apoptotic cells in the presence or absence of H₂O₂ and GAH
-The purpose of measuring purified LDH activity with various amounts of GAH was most likely:

Quizplus · Accepted Answer

11ecba2d_0f19_db0b_a3bf_bd604f9ca4d9_MD0008_00 Peptides and proteins can interact with biological systems in a variety of ways, and the mechanisms by which they work must be determined experimentally. The results of an experiment often have more than one interpretation, and additional experiments must be performed to determine which interpretation is correct. The passage states that LDH leakage is a marker of oxidative stress, and researchers measured extracellular LDH activity in the presence and absence of GAH. Experiment 1 shows that in the presence of GAH, extracellular LDH activity is reduced, indicating that GAH may decrease oxidative stress, which would in turn decrease LDH leakage. However, it is also possible that GAH does not affect LDH leakage and that less LDH activity is observed because GAH directly inhibits LDH. To address this possibility, the researchers measured the activity of purified LDH (ie, no cells present) in the presence and absence of GAH in Experiment 2. They found that LDH activity was constant regardless of the amount of GAH used, and therefore they were able to demonstrate that GAH does not directly inhibit LDH activity but instead most likely prevents LDH leakage. (Choice B) Baseline extracellular LDH activity was determined in Experiment 1 when cells were incubated in the absence of H₂O₂ and GAH. No cells were used in Experiment 2, so this experiment does not show baseline extracellular LDH activity. (Choice C) Antagonists are molecules that bind to receptor proteins to inhibit a response. There is no evidence presented in the passage that GAH binds to any receptor proteins. (Choice D) Experiment 2 indicates that there are no direct interactions between GAH and LDH, so LDH most likely does not have a GAH binding site. Educational objective: The mechanisms by which biological molecules interact must be determined experimentally. The results of an experiment may have multiple interpretations, and additional experiments must be performed to determine which interpretation is correct.

Passage Oxidative Stress Occurs in Cells When Reactive Oxygen Species (ROS)