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Enzymes of the Urea Cycle
Introduction
The urea cycle can be described as a metabolic pathway that uses carbon in CO and nitrogen present in glutamate and NH to synthesise urea; a mechanism for detoxifying NH. (Boyer, 00).
The transformation of NH4+ into urea requires a five-step pathway. This pathway utilises the enzymes Carbamyl Phosphate Synthetase, Ornithine Transcarbamylase, Arginine Synthetase and Arginase in varying amounts to catalyse reactions.
To assay Carbamyl Phosphate Synthetase, citrulline formation from ammonia, bicarbonate and Ornithine is measured, to assay Ornithine Transcarbamylase; citrulline formation from Ornithine and Carbamyl phosphate is measured.
Arginine Synthetase is assayed by measuring urea production from citrulline and Aspartate while Arginase activity can be estimated by measuring urea formation from Arginine. (Mosse, 00).
Aim
By assaying Carbamyl Phosphate Synthetase (CPS) and Ornithine Transcarbamylase (OTC), citrulline production may be measured, while assaying Arginine Synthetase (AS) and Arginase will enable urea formation to be estimated.
By assaying these various enzymes, we hope to become more informed on the reaction sequences of the urea cycle, as well as the rate of reactions at which the assayed enzymes perform in conjunction with their various cofactors.
Materials and Methods
This experiment was carried out according to Mosse, 00, and was conducted in four parts using prepared liver extract. The liver from a freshly killed rat was removed and homogenised in ice-cold trimethyl ammonium bromide (CTAB), 0.1% (w/v) in a ration of 1.5mL CTAB/g liver. Rats were starved for several hours prior to commencement of liver processing to enhance enzymatic activities within the liver.
A dounce homogeniser was used to disrupt liver tissues. The homogenate was centrifuged and the supernatant was retained and diluted to suit the following specifications
1g liver/10mL for both CPS and AS assays,
1g liver/50mL for OTC assay and 1g liver/500mL for Arginase assay.
A small amount of the liver extract was removed and boiled for use if required in the assay.
CPS Assay
5 reaction mixtures were prepared in 15mL glass centrifuge tubes, each containing differing ingredients. For a full list of these constituents, consult the BTH757, Cellular Metabolism Laboratory Manual, by Mosse, 00.
mL 0.5M HclO4 was added to Tube A, after which all tubes were incubated in a 8oC water bath for a pre-incubation period of 5 minutes.
0.mL enzyme extract (1g liver/10mL) was added to tube A. Precisely 1 minute after, 0.mL extract was added to tube B and so on until all tubes in the set contained the liver extract.
After a further minute, 0.mL boiled liver extract was added to tube E. All tubes remained in the water bath.
Precisely fifteen (15) minutes after the enzyme was added, tube A was removed from the bath. One-minute post removal, mL HclO4 was added to tube B. This addition and removal was repeated for tubes C-E inclusive at one-minute intervals.
Each tube was centrifuged for 5 minutes, 80% of top speed.
mL of each supernatants fraction was removed for Citrulline estimation.
OTC Assay
5 reaction mixtures were prepared in 15mL glass centrifuge tubes, each containing different items. For a full list of these constituents, consult the BTH757, Cellular Metabolism Laboratory Manual, by Mosse, 00.
5mL 0.5M Hclo4 was added to tube A.
All tubes were placed in a 8oC water bath for a pre-incubation period of 5 minutes.
0.5 mL Carbamyl phosphate (40mM) was added to tubes A-D at five-minute intervals.
Each tube was incubated for 15 minutes at 8oC. 5mL 0.5M HclO4 was added to tubes B-E inclusive upon completion of the 15-minute incubation period.
Each tube was centrifuged on low speed for five minutes.
1mL of each supernatant fraction was removed from each tube and set aside for citrulline assay.
AS Assay
6 reaction mixtures were prepared in 15mL glass centrifuge tubes, each containing different items. For a full list of these constituents, consult the BTH757, Cellular Metabolism Laboratory Manual, by Mosse, 00.
.0 mL 0.5M HclO4 was added to tube A. All tubes were placed in a 8oC water bath and pre-incubated for 5 minutes.
0.5mL extract (1g liver/10mL) was added to tubes A-E inclusive at one-minute intervals. 0.5mL boiled extract was also added to tube F.
Each tube was incubated for 5 minutes, after which time mL 0.5M HclO4 was added.
Each tube was centrifuged for 5 minutes, after which time mL of supernatant was removed for assay using the Archibald method. A more detailed explanation of this method may be found in Mosse, 00.
Arginase Essay
4 reaction mixtures were prepared in 15mL glass centrifuge tubes, each containing different items. For a full list of these constituents, consult the BTH757, Cellular Metabolism Laboratory Manual, by Mosse, 00
.0mL 0.5M HclO4 was added to tube A after which all tubes were placed in a 8oC water bath for a pre-incubation period of 5 minutes.
0.5mL liver extract (1g liver/500mL) was added to tubes A-D inclusive at one-minute intervals.
Each tube was incubated for a further 10 minutes at 8oC. Following this, mL 0.5M HClO4 was added to tubes B-D inclusive.
mL of each solution was then reserved for urea estimation by Timmerman’s Method.
A more detailed explanation of this method may be found in Mosse, 00.
Colorimetric Assay of Citrulline � To be used for solutions reserved from CPS and OTC Assays.
10 boiling tubes were numbered 1-10 inclusive. 0, 0.5, 1.0, 1.5 and .0mL of standard citrulline were placed into tubes 1-5 respectively.
Depending on the assay being performed, mL of the various supernatants from the CPS assay were placed into tubes 6-10, or 1mL supernatant from the OTC assay.
Water was added to each tube, making the final volume equal to 8.5mL.
mL HSO4/HPO4 acid mixture was added to each tube, and mixed adequately.
0.5mL diacetyl monoxime was then added to each tube.
Each tube was mixed with a long stirring rod, and each tube was numbered using masking tape and lead pencil. A marble was placed atop each tube, and the entire tube, plus marble, was wrapped in aluminium foil.
All tubes were heated for 15 minutes in a covered, boiling water bath.
After this time, all tubes were removed and placed in a half-filled beaker of boiling water. Care was taken to ensure tubes were not exposed to light during this time. For this reason, the beaker and tubes were placed within a dark cupboard and left undisturbed for five minutes.
Absorbances of each solution were measured at 450nm using a bench top spectrophotometer. Again, care was taken to ensure as little light exposure as possible.
Calculations were performed on the absorbance figure for each solution to determine the amount of citrulline or urea in each.
A sample calculation for enzyme activity mmoles product formed/minute/g liver protein may be found in the appendix.
Results
Table 1 Carbamyl Phosphate Synthetase
Conditions Enzyme Activity
A) Zero Time 0.14
B) Full Test 0.50
C) Minus ATP 0.01
D) Minus Acetyl Glutamate 0.14
E) Boiled Enzyme 0.07
Table Arginase Essay
Conditions Enzyme Activity
A) Zero Time 10
B) Full Test 106
C) Minus Arginine 10
D) Minus Mn+ 76
Table Ornithine Transcarbamylase
Conditions Enzyme Activity
A) Zero Time .0
B) Full Test 1.0
C) Minus Ornithine 0
D) Minus Extract .65
E) Minus Carbamyl Phosphate 6.0
Table 4 Arginine Synthetase
Conditions Enzyme Activity
A) Zero Time 0.1
B) Full Test 0.4
C) Minus ATP 0.14
D) Minus Citrulline 0.0
E) Minus Aspartate 0.17
F) Boiled Enzyme 0.14
Discussion
Table 1 displays the results for the Carbamyl Phosphate Synthetase assay. As was predicted, the full test � tube B � demonstrated the highest amount of enzyme activity (mmoles formed/min/g liver protein).
This is because all constituents required for the proceeding reaction were present within the reaction mixture.
It can be seen that for Carbamyl phosphate Synthetase to function properly, ATP is required to power the reaction forward. In the absence of ATP, only 0.01 mmoles of product were formed per minute for each gram of liver protein � thus, the enzyme activity was so low it was almost non-existent.
This reaction can be allosterically controlled by acetyl glutamate as acetyl glutamate enhances the enzyme activity when present.
The actual role of carbamyl phosphate synthetase within the first reaction of the urea cycle is to catalyse the reaction between ammonia and bicarbonate ions. When boiled liver extract was used (as for tube E), there was very little enzymatic activity detected. This is due to the fact that heating of the extract caused the proteins to become denatured, and therefore useless in relation to this reaction.
As for reaction number 1 of the urea cycle, reaction , involving ornithine transcarbamylase, also occurs within the mitochondrial matrix.
Table , tube E shows that the amount of carbamyl phosphate directly affects the rate of transformation of ornithine into citrulline by ornithine transcarbamylase.
In the results, it may be seen that the reaction can proceed without carbamyl phosphate present, but in the full test the enzymatic activity is more than twice as high in the conversion of ornithine into citrulline. This is because, although there is no carbamyl phosphate present, there is CPS present � the enzyme used in the first reaction. This allows some carbamyl phosphate to be made from CPS, although there is not enough present to convert the ornithine.
In the absence of ornithine there was no enzyme activity noted. This is die to the fact that there was no substrate for the enzyme to act on, as well as no constituents present to make citrulline � the product of the reaction.
After the above reactions have occurred within the confines of the mitochondrial matrix, the citrulline is shuttled across into the cytoplasm where it is converted into argininosuccinate followed by arginine and fumarate by the enzymes argininosuccinate synthetase and argininosuccinate lyase. This was investigated in the AS assay. This reaction was found to be freely reversible, demonstrated by the fact that when there was no citrulline present (tube D), it was none the less created via the reverse reaction of the enzyme.
The reaction was also able to proceed (tube F) after the liver extract had been boiled and enzymes were rendered ineffective. Without the presence of aspartate (tube E), the reaction was still able to proceed, and the formation of urea was noted.
Although there was no aspartate present, there was, however, citrulline present within the liver extract. The citrulline, by providing all constituents missing from aspartate, allowed the reaction to proceed, albeit at a lower rate, with less mmoles of product formed than there were in the full test.
As predicted, the exclusion of ATP within the reaction mixture ensured that the reaction proceeded quite slowly. As for the CPS assay, ATP is required to ‘push’ the reaction and without it, the reaction occurs more slowly with less product being formed.
The Arginase assay is probably the most important part of the urea cycle, as it is the creator of the urea from the excess ammonia groups produced by the oxidation of the amino acids in the mitochondria. Arginine is important in this reaction, as the enzyme is more active when there is arginine present in the solution, than it is without. This can also be a control mechanism in that by quickly converting arginine into ornithine excess ammonia can be disregarded into urea.
Manganese chloride was added to each reaction mixture except for D. This is due to the fact that Mn+ is considered to be a cofactor in relation to the enzyme arginase. By excluding Mn+ from reaction mixture D, it was hoped that a reduction in enzyme activity might have been noted. This was not found to be the case. Throughout each of the experiments, whenever water was required in one of the reaction mixtures, distilled water was employed. It is now believed that Mn+ may have been present in trace amounts in the distilled water due to the fact that although Mn+ was purposefully excluded from the reaction mixture, obvious enzyme activity was still noted.
As with the OTC assay, it was found that when the substrate was expelled from the reaction mixture (in this case it is arginine) the reaction is almost at a standstill � having the same amount of enzyme activity as the zero time reading in the full test.
Since the urea cycle uses bicarbonate ions combined with ammonia ions, excess ammonia is disregarded from the body with ease. By breathing deeply and quickly we can cause an increase in the amounts of formed bicarbonate ions. In the onset of alkalosis, this is a very important mechanism employed by the body as it enables blood pH to be balanced.
The same mechanism that helps us to correct acidosis, therefore assists in the correction of alkalosis. Through employment of the urea cycle alkalosis may be corrected by reassigning the additional ammonia groups into urea, which may then be excreted.
Conclusion
By assaying the enzymes present in the urea cycle, it can be seen that acetyl glutamate levels predominantly control the cycle, which in turn controls carbamyl phosphate production.
This enables nitrogen levels within the body to be maintained. Excess nitrogen can be excreted through the urea cycle by inserting nitrogen into the cycle in the form of ammonia which can then be converted into carbamyl phosphate that will then go into the cycle until it is removed as urea.
Appendix
Sample calculation for enzyme activity in mmoles product formed/minute/g liver protein.
mmole/min/0.0g liver
Citrulline 0.4mM = 0.4mm/mL
0.4mM = 0.4mmole
Raw data from CPS assay
1 (Standard 0.0mL) 0.000
(Standard 0.5mL) 0.01
(Standard 1.0mL) 0.05
4 (Standard 1.5mL) 0.105
5 (Standard .0mL) 0.50
Tube A 0.010
Tube B 0.01
Tube C 0.01
Tube D 0.01
Tube E 0.010
Boyer, 00. Concepts in Biochemistry.
John Wiley and Sons, New York, United States of America.
Mosse, 00. BTH757, Cellular Metabolism, Laboratory Manual.
Centre for Learning and Teaching Support, Monash University, Australia.
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