introduction The glycoside beta drummin has been shown to be an effective revers
ID: 215552 • Letter: I
Question
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 CO2released.
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).
1. Postulate why beta drummin is effective as an inhibitor only when added to crude extracts but not when added to purified enzymes.
2. What is the physiological explanation for the anti-fertility effect of beta drummin?
biochemistry.
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
Explanation / Answer
1. This is probably because its a inhibitor of active metabolism i.e. while the enzymes are at work and are pocessing their substrate like glucose etc.
2. A reduced metabolism in the sperms would not be enough to be able to povide energy for sperm motility, causing infertility (anti-fertility)
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