Result of assignment
Ronnie01
Student188.docx16083796
The presence of oxygen promotes metabolic process where glucose is converted to pyruvate. This happens in the presence of enzymes hexokinase and pyruvate kinase which have both regulatory and catalytic roles. 14C is incorporated into the CO2 released by oxidation process where the uptake of oxygen leads to aerobic respiration. The radiolabelled substrates are broken down in the presence of oxygen with the glycolytic beta dummin as the inhibiting agent leading to the production of CO2. In this case, the process stops when oxygen supply is reduced by the beta drummin to 50% of the control values. The desired amounts of fructose and glucose are broken down at 50% O2 supply and 50% CO2 release. 1/10 Comment by Pryank Patel: You need to be more specific about when CO2 is released - in which particular reaction in metabolism? Comment by Pryank Patel: You have not fully understood the question. CO2 is released in the link reaction, when pyruvate is decarboxylated to form acetyl CoA. Acetyl CoA feeds into Krebs', and undergoes oxidative decarboxylation which releases CO2 in two other steps of the Krebs' cycle.
The low levels of glycolytic beta drummin inhibits the activity of enzyme phosphofructokinase by decreasing its affinity for fructose 6-phospate an intermediate formed during the glycolysis process. At 0.2mM of beta drummin, the sperm were unable to convert U-14C glucose (15mM) or U-14C fructose (10mM) to lactic acid. In order to produce the lactate more of the inhibitor and respiration supported by endogenous substances (presumably lipids) were required. Lactate could only be formed with insufficient oxygen supply to allow anaerobic respiration to take place. However, even the highest levels of beta drummin as observed from the experiment could only inhibit up to 20% oxygen supply. Aerobic respiration took place and only the final products of the metabolism were produced with no intermediates. 6/10 Comment by Pryank Patel: This is not what is happening in experiment 1. Comment by Pryank Patel: Good. Comment by Pryank Patel: Right. Comment by Pryank Patel: You go off-topic in places and take into consideration the information from the wrong experiments.
Beta drummin served as an inhibitor that affected the supply of oxygen. In return, when less O2 was present, metabolisms of the radiolabelled substrates diminished thus leading to decreased generation of CO2. The decrease of O2 uptake would have diminished because the required amounts of the substrates had been broken down thus the process stopped to prevent further breakdown thus reducing the amount of CO2 generated as well. The conclusion is that the presence of beta drummin inhibits the respiration process by affecting the enzymes and also cutting down the O2 uptake. 0/10 Comment by Pryank Patel: A vague, and sometimes confusing, answer. Beta drummin is acting as an inhibitor during glycolysis or the TCA cycle. If it was affecting the electron transport chain there would be a build-up of NADH, leading to lactate formation.
When U-14C pyruvate (5mM) or U-14C lactate (5mM) were used as substrates, the affinity for oxygen increased because it was required in higher percentage to breakdown the substrates. As a result, high level of beta drummin (10-100mM) was required to inhibit oxygen supply to 50% of the control values. This would in return affect the RSW enzymatic activity and thus overall effect in the aerobic respiration which would lead to the formation of some intermediate products and less ATP when the substrates were broken down. More oxygen would be required by the RSW enzymes for the substrates to be fully broken down to provide energy for the cells. With less energy the sperm mobility is slightly decreased (Gomes,1970). 1/10 Comment by Pryank Patel: Another answer that suggests confusion and a lack of understanding. When glycolysis is bypassed by using labelled pyruvate or lactate the inhibitor has to be used at vastly increased concentrations to have the same effect. Therefore, beta drummin must be affecting metabolism at a point before the formation of pyruvate.
In the absence of U-14C pyruvate (5mM) or U-14C lactate (5mM) .The only substrate available was the endogenous substrate (presumably lipid) which is a complex compound that required more oxygen to be broken down to release energy for the cells. Even when 100mM beta drummin was added the oxygen consumption was decreased by only 20%. This is a clear confirmation that the drug effect was failing for experiment 2. The oxygen consumption by the RSW enzymes was slightly affected explaining that the mobility of the RSW was not affected. 0/10 Comment by Pryank Patel: No, it indicates that beta drummin is affecting glycolysis, since glycolysis is by-passed in this experiment.
The most probable site of the beta drummin inhibition was the phosphofructokinase that acts on fructose-1, 6 bisphosphate substrates. From the observed results, it is clear that the fructose-1, 6 bisphosphate registered the highest percentage of the control values. The administered beta drummin attached itself to the active site of the RSW phosphofructokinase to provide a competing medium for the fructose-1, 6 bisphosphate substrate leading to the low motility of the RSW. 0/10 Comment by Pryank Patel: Glyceraldehyde-3-phosphate dehydrogenase - the enzyme for which glyceraldehyde-3-phosphate is the substrate. There is a build up of intermediates before this point but very little of any metabolites afterwards
They are relatively in high amounts to help activate the RSW enzymes to enable them to keep high phase with the oncoming high flux of intermediates and as well overcome the inhibitory drug effect. The accumulation of high levels of fructose-1, 6 bisphosphate and glyceraldehyde-3-phosphate and lack of accumulation of 2-phosphoglycerate, 3-phosphoglycerate and phosphoenolpyruvate implicated glyceraldehyde-3-phosphate dehydrogenase and phosphofructokinase as the enzymes labile to inhibitor. 1/10 Comment by Pryank Patel: The step between fructose-1,6-bisphosphate and glyceraldehyde-3-phosphate is a reversible reaction. However the previous step in the pathway is irreversible. There is therefore a build-up of fructose-1,6-bisphosphate.
Purified enzymes activity cannot be stopped by the beta drummin because it simply forma suspension which cannot attach itself to the enzymes. The enzymatic activity with the substrates is as well not affected thus for the drug to work effectively, it requires the crude extracts which are impure. This explains why 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) (Luttge, 1975). 1/10 Comment by Pryank Patel: Beta drummin is a pro drug and is activated when added to the crude extract. The conditions required for activation are not present when the purified enzymes are used.
The assay method was used in the incubation of the RSW with the substrates.
In vivo, the rams are treated with beta drummin and the sample semen collected for 4 days. The levels of implicated glyceraldehyde-3-phosphate dehydrogenase and phosphofructokinase were found to have reduced to undetectable levels after first day of the treatment. Comment by Pryank Patel: You are simply repeating the results of the experiment, but not answering the question.
The presence of beta drummin hinders the activity of enzymes and as well cuts of oxygen supply. This causes the substrates not to be broken down to releases ATP. Levels of AMP signals for low energy state should be detected and thus serve as allosteric regulator. In return, the range of sensitivity of phosphofructokinase should increase so that the glycolysis process furnishes carbon skeletons for biosynthesis. This is not possible because of the drug 0/10 Comment by Pryank Patel: Does not answer the question. It is activated and then binds to the active site of glyceraldehyde-3-phosphate dehydrogenase, preventing the glyceraldehyde-3-phosphate binding to the active site.
There is reduced circulation in testosterone, FSH and LH. This leads to arrest of spermatogenesis at meiosis without affecting the accessory gland. Sperm count and sperm motility decreases leading to azoospermia. The reason is because TPI activity is greatly reduced to 4 % of the control values in the fourth day while Aldolase levels are also reduced to 1/3 of the control values. Theoretically, 26 days would be required to have sperm count and sperm motility return to normal when the drug was withdrawn in the vitro treatment system and it needs energy to travel. 0/10 Comment by Pryank Patel: Spermatozoa need energy in the form of ATP to swim towards the ovum and for fertilisation to take place. Inhibition of energy supplying pathways prevents this.
989
