Explain why the results of Experiment 2 point to some blockage of the glycolytic pathway by beta drummin.

Introduction to Biochemistry

4LMS0001

Interpretative assignment 2017-18

Your answers should be written in a Word document. This should only contain the text of the answers (and not the details of the experiments or the text of the questions). The maximum word count for the assignment is 1000 words. The work should be submitted electronically via StudyNet.

Objectives

· To introduce you to data interpretation and develop your interpretive skills.

· To show how metabolism can be studied.

· To show how studies of metabolism can be used to identify biochemical lesions.

· To develop your understanding of the glycolytic pathway.

Learning Outcomes

At the end of this exercise you should be able to:

· Understand what is meant by a U-14C label and how it can be used.

· Be very familiar with the glycolytic pathway.

· Understand some of the principles of the activation and action of toxicants.

· Have developed your ability to analyse data and think about metabolism logically.

Introduction

The glycoside beta drummin has been shown to be an effective reversible male anti-fertility agent in ram and boar. The primary contraceptive effect appears to be directly on spermatozoa within the cauda epididymis.

Adverse side effects have precluded human trials and it was decided to investigate the mechanism of action of beta drummin in order to develop less toxic anti-fertility analogues.

Radiolabelled substrates were used to elucidate where the compound was having an effect on metabolism. The substrates are designated U-14C which indicates that a proportion of the C atoms are the 14C isotope, U indicating that all the carbon atoms in the molecule are labelled rather than one specifically. The following experiments were carried out.

Experiment 1

Ram spermatozoa were washed in buffer (ram spermatozoa washed, RSW) and incubated for 3 hours with U-14C glucose (15mM) or U-14C fructose (10mM) in the presence of beta drummin (0.1mM). Oxygen uptake, as an indicator of aerobic respiration, was measured. Radioactivity recovered in the CO2 released as a result of oxidation of the substrate was also measured. Both were decreased to 50% in treated samples compared with control values. There was no accumulation of lactate.

Experiment 2

Incubation of RSW with U-14C pyruvate (5mM) or U-14C lactate (5mM) needed beta drummin at a concentration of 10-100mM to cause a 50% inhibition in oxygen uptake and 50% inhibition in CO2 released.

Experiment 3

Further incubations of RSW with inhibitor only, such that endogenous substrates (presumably lipid) are required for oxidation, resulted in negligible inhibition. Even when 100mM beta drummin was added the oxygen consumption was decreased by only 20%.

Experiment 4

RSW were incubated with fructose (15mM) in the presence and absence of beta drummin (3mM) and concentrations of glycolytic intermediates were measured. The results for samples treated with beta drummin, expressed as a percentage of control, are shown in Table 1.

Table 1: Concentrations of Intermediates of Glycolysis following incubation of RSW with beta drummin.

Concentration % of control value
glucose-6-phosphate 50 +/-2
fructose-6-phosphate 52 +/-3
fructose-1,6 bisphosphate 3,280 +/-100
dihydroxyacetone phosphate 280 +/- 20
glyceraldehyde-3-phosphate >300
3-phosphoglycerate Not detected*
2-phosphoglycerate Not detected*
phosphoenolpyruvate Not detected*
pyruvate 2 +/-0.1
lactate 0

* The assay method used could not detect these compounds in control or test incubates. In a separate experiment it was found that if two control incubates were pooled and the pooled volumes reduced by 50%, the assay method could just measure these compounds.

Experiment 5

Purified glycolytic enzymes were prepared from RSW and assayed for activity. Addition of beta drummin at final concentrations as high as 300mM to the reaction cuvettes had no effect on the activity of any of the glycolytic enzymes assayed including hexokinase, phosphofructokinase (PFK), aldolase, triose phosphate isomerise, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase and lactate dehydrogenase (LDH).

Questions

1. In just a few sentences explain how the 14C in the labelled glucose and fructose is incorporated into the CO2 released. (Only an overview, precise details of the pathways are not required.)

(10 marks)

2. What is the significance of the lack of accumulation of lactate in Experiment 1?

(10 marks)

3. What two explanations can you offer for the decrease in O2 uptake and CO2 output in the presence of beta drummin?

(10 marks)

4. Explain why the results of Experiment 2 point to some blockage of the glycolytic pathway by beta drummin.

(10 marks)

5. Explain why the results of Experiment 3 confirm the findings of Experiment 2.

(10 marks)

6. Suggest the probable site of beta drummin inhibition from the data in Table 1, explaining your reasoning.

(10 marks)

7. Give reasons why fructose-1,6 bisphosphate should be relatively so high.

(10 marks)

8. Postulate why beta drummin is effective as an inhibitor only when added to crude extracts but not when added to purified enzymes.

(10 marks)

9. What is the probable mechanism of inhibition shown when beta drummin acts in vivo?

(10 marks)

10. What is the physiological explanation for the anti-fertility effect of beta drummin?

(10 marks)

Guidelines

1) The total word count for the document is 1000 words.

2) The assignment should be typed, single-sided, double spaced in size 12 font. Please leave a standard margin on all edges (approximately 2.5 cm) and number the pages.

3) To facilitate anonymous marking please ensure your student number and word count are included in the header/footer of the assignment.

4) You should submit a document that has only the answers to each question (do not include the questions themselves).

5) You should submit a file that has only your student number as the filename.

6) Your name should not appear anywhere in the document.

7) Read through the information given and the questions at least twice before attempting your answers.

Failure to follow these guidelines will result in 5% being deducted from your overall mark.

The information is presented in a logical step-by-step way. Only consider the data for the specific experiment in each question, building on what you have already discovered, without including assumptions from later experiments.

4

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