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

Core Biochemistry

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.

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 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 experimental glycoside PRZ-3256 has been shown to be effective as a reversible male contraceptive treatment. The primary antifertility effect appears to be directly on sperm cells (spermatozoa) within the cauda epididymis.

Highly toxic adverse effects have precluded human studies of this drug but the mechanism of action of PRZ-3256 was studied in male horses in order to develop less toxic analogues.

Radiolabelled substrates were used to elucidate where the drug was having an effect on metabolism. The substrates are labelled U-14C which indicates that all the carbon atoms in the molecule are labelled uniformly rather than one specifically. The following experiments were carried out.

Experiment 1

Spermatozoa were washed in buffer (horse spermatozoa washed, HSW) and incubated for 4 hours with U-14C glucose (15mM) or U-14C fructose (12mM) in the presence of PRZ-3256 (0.5mM). Aerobic respiration was determined by the measurement of oxygen uptake. Radioactivity recovered from released CO2 resulting from the of oxidation of the substrate was also measured. Both oxygen uptake and released CO2 were decreased to 50% in samples treated with PRZ-3256 compared to untreated control samples. There was no accumulation of lactate.

Experiment 2

Incubation of HSW with U-14C pyruvate (3mM) or U-14C lactate (3mM) needed PRZ-3256 at a concentration of 10-50mM to cause a similar inhibition in oxygen uptake or in CO2 released as observed in experiment 1.

Experiment 3

Further incubations of HSW with PRZ-3256 only, where no additional substrates were added, such that endogenous substrates (likely lipid) are required for oxidation, resulted in negligible inhibition. Even when higher concentrations of up to 100mM PRZ-3256 was added the oxygen consumption was decreased by only 15%.

Experiment 4

HSW were incubated with fructose (10mM) in the presence and absence of PRZ-3256 (5mM) and concentrations of glycolytic intermediates were measured. The results for samples treated with PRZ-3256, expressed as a percentage of control samples measured in the absence of PRZ-3256, are shown in Table 1.

Table 1: Concentrations of Intermediates of Glycolysis following incubation of HSW with PRZ-3256 .

Concentration	% of control value
glucose-6-phosphate	48 +/-3
fructose-6-phosphate	51 +/-2
fructose-1,6 bisphosphate	2830 +/-100
dihydroxyacetone phosphate	240 +/- 20
glyceraldehyde-3-phosphate	>280
3-phosphoglycerate	Not detected*
2-phosphoglycerate	Not detected*
phosphoenolpyruvate	Not detected*
pyruvate	3 +/-0.4
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

Extracts of purified glycolytic enzymes were incubated with HSW and their activity was determined. Addition of PRZ-3256 at final concentrations as high as 200mM to the experiment 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.) (15 marks)

2. What is the significance of the lack of accumulation of lactate in Experiment 1?
   (5 marks)
3. What two explanations can you offer for the effect of PRZ-3256 on the decrease in

O2 uptake and CO2 output? (10 marks)

4. Explain why the results of Experiment 2 point to some inhibition of the glycolytic pathway by PRZ-3256 . (10 marks)

5. Explain why the results of Experiment 3 confirm the findings of Experiment 2.
   (10 marks)
6. Suggest the probable site of PRZ-3256 inhibition from the data in Table 1, explaining your reasoning. (15 marks)

7. Give reasons why there is such a high amount of fructose-1,6 bisphosphate.
   (5 marks)
8. Suggest why PRZ-3256 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 PRZ-3256 acts in vivo? (10 marks)
10. What is the physiological explanation for the anti-fertility effect of PRZ-3256 ?
    (10 marks)

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.