A rat liver (2 gms) was dissected, homogenized under optimal conditions in appropriate buffer (50 ml), pH, temperature & supplemented with protease inhibitors. The protein level in a 100 μl sample of the homogenate (H) was determined using standard procedures, & was estimated to be 2 mg/ml. Peepo is a medical student interested in purifying a protein that transports copper (Cu) at the expense of ATP hydrolysis into ADP and inorganic phosphate (Pi). Thus, prior to purification Peepo must develop an assay/test that confirms the presence of this protein. Kindly select one answer for Questions 1-10 Question -1 Compared to a control which of the following may serve as a possible assay verifying the presence of a Cu sensitive protein that hydrolyzes ATP:
A rat liver (2 gms) was dissected, homogenized under optimal conditions in appropriate buffer (50 ml), pH, temperature & supplemented with protease inhibitors. The protein level in a 100 μl sample of the homogenate (H) was determined using standard procedures, & was estimated to be 2 mg/ml.
Peepo is a medical student interested in purifying a protein that transports copper (Cu) at the expense of ATP hydrolysis into ADP and inorganic phosphate (Pi). Thus, prior to purification Peepo must develop an assay/test that confirms the presence of this protein.
Kindly select one answer for Questions 1-10
Question -1
Compared to a control which of the following may serve as a possible assay verifying the presence of a Cu sensitive protein that hydrolyzes ATP:
- Add Cu to the H and measure the level of Pi
- Add ATP to H and measure the level of copper
- Vary copper level, add inorganic phosphate to H & measure residual Pi
- Vary Cu, add ATP to H and measure level of inorganic Pi
Question -2
A good control for this assay would be adding all assay components
- to boiled H followed by estimation of Pi
- to H excluding ATP, followed by estimation of ATP
- to H excluding Cu , followed by estimation of ATP level
Peepo then determined the unit activity of the ATPase in the H as 10 units.
Question -3
The specific activity (SA) units/mg of the copper sensitive ATPase in H is
- a. 1000
- 100
- 50
- Not possible to determine from the given data
Peepo proceeded by centrifuging the homogenate at 5000 rpm for 15 min. which yielded a supernatant (S) (45ml) and a pellet (P) that was re-suspended in 15 ml of buffer. He also determined the
protein levels in S & P, which were estimated at 1.4 mg/ml and 2.0 mg/ml respectively.
ATPase activity was measured in S & P and estimated as 3 units and 21 units respectively.
H
50 ml, total protein 100mg
Total activity =5000 units
S P
45 ml, total protein = 63 mg 15 ml, total protein = 30 mg
Total activity = 1350 units Total activity = 3150 units
Question - 4
Which fraction should Peepo proceed with in order to purify the ATPase? Should he choose the fraction with
- Higher protein yield
- Greater total unit ATPase activity
- Higher specific activity though lower protein yield
- Lower specific activity though higher protein yield
Question -5
The % yield of the ATPase in S and P is approximately
- 63 & 30 % respectively
- 27 & 63 % respectively
- 90 & 10 % respectively
Question -6
How many fold has centrifugation purified the ATPase in S and P respectively?
- 20 & 84 fold
- 0.4 & 2 fold
- 0.27 & 0.63 fold
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