Escolar Documentos
Profissional Documentos
Cultura Documentos
Semester 2, 2005
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Faculty of SCIENCE
School of MOLECULAR AND MICROBIAL BIOSCIENCES
BCHM2072 - HUMAN BIOCHEMISTRY
Duration: 3 hours
Reading time: 10 min
SEAT NUMBER:...................................................................................................................
FULL NAME:........................................................................................................................
SID:........................................................................................................................................
INSTRUCTIONS TO CANDIDATES
Students are permitted to bring in the coloured sheets of stimulus material
provided by the School of MMB. These sheets may be annotated but MUST
BE HANDED IN WITH THIS PAPER.
This paper is CONFIDENTIAL. No part of this paper may be removed from
the examination room.
Only University supplied calculators may be used
Answer Short Answer Questions in the spaces provided in this booklet
Answer Multiple Choice Questions on the answer sheet provided.
All Multiple Choice Questions are graded using the Partial Marking System.
Although each question has been designed to have only ONE option which
carries full marks, each option of each question may carry a partial positive or
negative mark. No negative marks are incurred for questions that are left
blank.
Section A covers the THEORY (lecture and assignment) Recommended time: 2 hours
Part I - SIXTY Multiple Choice Questions at 1.5 marks each.
Part II - SIX short answer questions (at 5 marks each)..
The 120 marks available in Section A will contribute 80-100% of your THEORY
mark. The remaining 0-20% is contributed from the marks that you chose to accept
from the assignments during the semester.
Section B covers the PRACTICAL (lab work) Recommended time, 1 hour
Part I - THIRTY FIVE Multiple Choice Questions at one mark each.
Part II ONE short answer question (6 marks)
The 41 marks available for this Section will contribute 50% of your PRACTICAL
mark for BCHM2072. The other 50% is contributed from the marks that you
obtained for practical reports and laboratory tasks during the semester.
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All marks are considered raw, and may be subjected to scaling, until approved by the
Faculty of Science.
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SECTION A - THEORY
PART I Multiple Choice Questions worth 1.5 marks each
1.
2.
3.
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Carnitine is a protein embedded in the cell membrane that allows fatty acids to
enter from the bloodstream
Fatty acids are covalently attached to Coenzyme A during the FAD/NAD
catalysed oxidation reactions
The oxidation reactions involving FAD/NAD occur only in the cytoplasm
Fatty acids attached to Coenzyme A can move freely across the mitochondrial
membrane
Carnitine is consumed (two carbons at a time) during fatty acid oxidation
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4.
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5.
6.
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Electrons can move down the electron transport chain even if proton pumping
from the matrix can not occur.
Protons are only pumped from the matrix if electrons are passed down the
electron transport chain.
ATP synthesis at the F1ATPase requires both ADP and phosphate
Protons will only come in through the F0F1ATPase if ATP is simultaneously
being made from ADP
Protons can pass freely across the outer mitochondrial membrane
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7.
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8.
Proton release occurs when electron carriers receive electrons from hydrogen
carriers
Cytochrome c carries only electrons
Ubiquinone carries hydrogens from Complex I to Complex III
Oxygen is consumed on the matrix side of the inner mitochondrial membrane
The actual protons that move out of the matrix during electron transport come
exclusively from the hydrogens on NADH
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Rate of ATP
Synthesis
Rate of Proton
Pumping
Size of Proton
Gradient
Rate of Oxygen
Consumption
STOPS
INCREASES
DISSIPATES
INCREASES
STOPS
STOPS
DISSIPATES
STOPS
STOPS
STOPS
STAYS HIGH
STOPS
CONTINUES
STOPS
STAYS HIGH
INCREASES
CONTINUES
CONTINUES
FALLS
SLIGHTLY
STOPS
Which situation (from A-E above) would you expect to result from the following
interventions?
9.
An uncoupler
10.
A lack of oxygen
11.
12.
13.
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14.
A
B
C
D
E
15.
A
B
C
D
E
16.
A
B
C
D
E
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Both indirect calorimetry and the doubly-labeled water (D2O18) methods can
be used to assess whole body energy expenditure. Which statement is
CORRECT?
Only indirect calorimetry can give an indication of which fuels are being burnt
The doubly-labeled water method measures oxygen production
The doubly labeled water method can be done over a shorter time frame than
indirect calorimetry
Only the doubly labeled water method can be used to determine basal
metabolic rate
Indirect calorimetry only works if the subject has consumed carbohydrates,
not fat.
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17.
A
B
C
D
E
18.
A
B
C
D
E
19.
A
B
C
D
E
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Which of the following scenarios would MOST likely lead to flatulence (ie
production of volatile short chain fatty acids and gases in the lower bowel)?
Consumption of sucrose by someone with lactase deficiency
Consumption of amylopectin starch
Consumption of amylose starch
Consumption of dairy products pre-treated with lactase
Consumption of glucose in association with an amylase inhibitor
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20.
A
B
C
D
E
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22
23
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Number
Number
2
Time (h)
2
Time (h)
Number
Number
2
Time (h)
2
Time (h)
Number
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2
Time (h)
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24.
A
B
C
D
E
25.
A
B
C
D
E
26.
A
B
C
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Which of the following enzymes is most likely to catalyse a rate limiting step
in a pathway
High Vmax enzyme that catalyses irreversible conversion of SP with the
prevailing [S] being about the same as the Km of the enzyme.
High Vmax enzyme that catalyses reversible conversion of SP with the
prevailing [S] being about the same as the Km of the enzyme
High Vmax enzyme that catalyses reversible conversion of SP with the
prevailing [S] being about 2-fold the Km of the enzyme
Moderate Vmax enzyme that catalyses reversible conversion of SP with the
prevailing [S] being about 2-fold the Km of the enzyme
Low Vmax enzyme that catalyses irreversible conversion of SP with the
prevailing [S] being about 20-fold the Km of the enzyme
D
E
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27.
A
B
C
D
E
28.
A
B
C
D
E
29.
A
B
C
D
E
30.
A
B
C
D
E
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31.
A
B
C
D
E
32.
A
B
C
D
E
33.
A
B
C
D
E
34.
A
B
C
D
E
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35.
A
B
C
D
E
36.
A
B
C
D
E
37.
A
B
C
D
E
38.
A
B
C
D
E
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Which treatment would LEAST likely affect the uptake of fatty acids into the
epithelial cells of the small intestine after a SINGLE fat meal?
Co-consumption of a drug which prevents emptying of the gall bladder
Co-consumption of a drug to prevent the formation of bile salts in the liver
Substituting 50% of the fat in the meal with Olestra
Co-consumption of a pancreatic lipase inhibitor with the meal
Co-consumption of a compound that prevents the formation of micelles
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39.
A
B
C
D
E
40.
A
B
C
D
E
41.
A
B
C
D
E
42.
A
B
C
D
E
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Which statement BEST DESCRIBES the fate of amino groups derived from
the catabolism of amino acids in muscle?
The amino groups are mainly excreted from the muscle as ammonia
The amino groups are mainly excreted from the muscle as urea
The amino groups become linked to pyruvate for transport to the liver
The amino groups are stored on pre-existing proteins by converting glutamate
residues in proteins to glutamine
The amino groups are stored on pre-existing polynucleotides by converting
thymine bases to cytosine
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43.
A
B
C
D
E
44.
A
B
C
D
E
45.
A
B
C
D
E
46.
A
B
C
D
E
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What would be the consequences of inhibition of lipolysis during the first few
days of starvation?
Blood ketone body concentration would rise
Blood glucose concentration would rise
Blood fatty acid concentration would rise
There would be fewer substrates for gluconeogenesis in the liver
Fatty acid oxidation in the muscles would increase
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47.
A
B
C
D
E
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12
200
100
1
Time (h)
Insulin (ng/ml)
Glucose (mM)
The figure below shows the normal response of blood glucose (solid line) and
insulin (dashed line) concentrations to a 50 g oral glucose load.
49.
50.
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100
0
1
Time (h)
100
0
0
1
Time (h)
200
100
0
1
Time (h)
12
200
100
12
Glucose (mM)
0
0
1
Time (h)
200
100
Insulin (ng/ml)
Glucose (mM)
12
0
0
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Insulin (ng/ml)
Glucose (mM)
200
1
Time (h)
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Insulin (ng/ml)
12
Glucose (mM)
200
Insulin (ng/ml)
Glucose (mM)
12
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Insulin (ng/ml)
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51.
A
B
C
D
E
52.
A
B
C
D
E
53.
A
B
C
D
E
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Which of the following amino acid sequences would be most likely to form
part of a signal peptide?
arg-glu-asp-asp-lys.
arg-arg-lys-lys.
pro-gly-pro-gly-ser.
val-val-leu-trp-ile.
cys-arg-asp-lys-asp.
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54.
A
B
C
D
E
55.
A
B
C
D
E
56.
A
B
C
D
E
57.
A
B
C
D
E
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58.
A
B
C
D
E
59.
A
B
C
D
E
60.
A
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D
E
One in which the G proteins found in intestinal cells have a unusually high
ability to hydrolyse GTP.
One in which the levels of NAD+ in intestinal cells are unusually high.
One with mutant G proteins, in which their arginine residues replaced by
lysine residues, with little effect on the structure of the proteins.
One in which protein kinase C is unusually active.
One is which biosynthesis of GPI-anchored proteins is defective.
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B
C
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SECTION A - THEORY
PART II Short Answer Questions worth 5 marks each
Answer these questions in the spaces provided below.
SAQ1.
What sort of weight change would be observed in an average person if there was 1%
mismatch between energy intake and energy expenditure averaged over the course of
a whole year? Show your working/logic. (2 marks)
List THREE ways of burning fuels without doing work. For each, give a ONE
sentence explanation of how the mechanism works. (3 marks)
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SAQ 2.
List FIVE ways (eg, drug or dietary intervention) in which blood cholesterol can be
manipulated. In each case give a ONE sentence description of how the intervention
works.
SAQ 3.
List FIVE ways in which an inhibitor of lipolysis might relieve the symptoms and
metabolic problems associated with Type I diabetes.
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SAQ 4.
The table below shows the effect of five different types/periods of exercise on the
relative rate of blood glucose oxidation (glucose uptake through to carbon dioxide)
and energy usage in leg muscles.
Type of exercise
A
B
C
D
E
At rest
One minute into a walk
30 minutes into a walk
30 minutes into a light jog
30 minutes into a run
Five seconds into a sprint
Relative Rate of
Muscle Energy Use
1
3
3
5
10
15
Explain the following. [Hint: focus what is limiting the rate of glucose oxidation in
each circumstance]
The difference between B and A
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SAQ 5.
List FIVE treatment strategies for Type II diabetes. In each case, give a single
sentence to describe the basis for the intervention.
SAQ 6.
With the aid of a labelled diagram, explain the signal transduction events which you
would expect to follow binding of a cytokine to its receptor.
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SECTION B - PRACTICAL
PART I Multiple Choice Questions worth 1 mark each
Questions on the design of an ENZYME LINKED METABOLITE ASSAY
You wish to measure the concentration of acetyl-CoA in some heart muscle samples.
You find an assay for the enzyme citrate synthase that you think you can manipulate
to become an Enzyme Linked Metabolite Assay (ELMA) for acetyl-CoA. The assay
is described as follows:
Small frozen pieces of hearts (~10 mg) are homogenized on ice in 10% (final w/v) homogenization
buffer containing (in mM) 20 TRIS, 10 EDTA, pH 7.4. Triton X-100 is then added to 1% (v/v) and,
after mixing, homogenates are then frozen and thawed three times.
The reaction is performed in 1 ml reaction buffer containing (in mM) 20 TRIS, 1 EDTA, 0.1 DTNB,
and 0.1 acetyl-CoA, pH 7.4 at 30C. The reaction is started by adding 0.05 mM oxaloacetate and
monitored at 412 nm for 3 min.
You find out that the assay is based on the reaction of CoA with DTNB (5,5-dithiobis -2-nitrobenzoic acid). As citrate synthase converts acetyl-CoA and oxaloacetate
into citrate, CoA is released and this quickly reacts with DTNB to make the a yellow
product.
Acetyl-CoA + oxaloactate citrate + CoA (catalysed by citrate synthase)
Then, DTNB (colourless) + CoA NTB-CoA (yellow) (fast reaction)
The extinction coefficient of the yellow product is 15 mM-1cm-1 at 412 nm.
DTNB does not absorb at this wavelength.
Your assistant resources all the chemicals needed and prepares a table of information,
including their recommendations for stock solutions and other useful comments.
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Chemical
Tris [Tris- (hydroxymethyl)-
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MW
121.14
aminomethan]
Source
Merck
Stock Solution
1.0 M
8382
Page 27 of
Comments
Adjust to pH 8.1 with HCl,
to obtain Tris-HCl buffer.
Make up using
2.4228 g in 20 ml
EDTA
372.2
(ethylenediaminetetraacetic
Sigma
water
50 mM
E-1644
Make up using
dihydrate,
186.1 mg in10 ml of
Triton X-100
646.87
Serva
water
10% (v/v)
37238
groups.
Add 10 ml to 90 ml
Oxaloacetic acid
132.1
Sigma
water
10 mM
O-4126
Make up using 6.6
DTNB [5,5`-dithiobis(2-
396.3
nitrobenzoic acid); 3-
Sigma
mg in 5 ml of water
1.01 mM
D-8130
TNB-S-S-TNB
(dithionitrobenzoic acid);
carboxy-4- nitrophenyl
Make up using 2 mg
irritant.
disulfide],
in 5 ml of water
Ellmans reagent,
Acetyl CoA (acetyl coenzyme
A), lithium salt,
compound
816.5
Sigma
12.2 mM
A-2181
Make up using 25
mg in 2.5 ml water.
Citrate synthase, CS
Sigma
Specific activity
C-3260
Supplied as a crystalline
Number.
suspension of 10 mg protein
dissolved in 1 ml 2.2 M
(NH4)2SO4, pH 7.
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61.
A
B
C
D
E
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What would be the most practical way of making the Tris solution
Weigh out 2.4228 g of Tris, dissolve in 20 ml of water, then adjust the pH to
8.1 by adding concentrated HCl
Dissolve 2.4228 g of Tris in 20 ml water, then adjust the pH to 8.1 by adding
dilute HCl
Dissolve 2.4228 g of Tris in 10 ml water, then adjust the pH to 8.1 with HCl,
then add water to 20 ml
Dissolve about 2.5 g of Tris in about 10 ml water, adjust the pH to 8.1 with
HCl, then add water to give a volume equal to 20 x (weight taken/2.4228)
Accurately measure 20 ml water and adjust its pH to 8.1 with HCl, then add
2.4228 g of Tris
You begin by confirming that you can get the citrate synthase assay system to work.
You want to set up 1 ml cuvettes so that they contain (in order):
20 mM TRIS, 1 mM EDTA, 0.1 mM DTNB, 0.1 mM acetyl-CoA,
0.05 mM oxaloacetate, followed by some citrate synthase enzyme suspension
62.
A
B
C
D
E
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To achieve the above, how much of the stock acetyl-CoA solution (as
recommended by your assistant) should you add to the cuvette?
120 l
8.2 l
1.2 l
100 l
2.5 l
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To confirm that the assay is working you want add enough citrate synthase to get a
fast, but measurable rate of between 0.3 and 1 absorbance units per min.
63.
A
B
C
D
E
64.
A
B
C
D
E
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If the absorbance change was 0.75 per min, how fast is the AMOUNT of
yellow product increasing in the cuvette?
75 mol per min
50 nmol per min
5 nmol per min
15 nmol per min
15 mol per min
You decide that you need 50 mU of citrate synthase (CS) in the cuvette. What
is the MOST PRACTICAL way of doing this?
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On adding all the reagents together in the order, you notice that the solution in the
cuvette shows a tinge of yellow on adding the acetyl-CoA. But, after adding the CS,
it rapidly turns dark yellow, even before you have a chance to put in the oxaloacetate.
This is shown in the diagram below.
CS
Dark yellow
EDTA
Light yellow
Tris
DTNB
Acetyl-CoA
Colourless
10
15
Time (min)
65.
A
B
C
D
E
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After some trouble-shooting, you have convinced yourself that you can now make the
citrate synthase assay work.
The scheme that you have come up with is to set up a cuvette containing
20 mM TRIS, 1 mM EDTA, 0.1 mM DTNB, 0.1 mM acetyl-CoA
and 50 mU of citrate synthase (total volume of 1 ml). Then you mix the cuvette
contents, put it in the spectrophotometer and zero its absorbance. Then you add
0.05 mM oxaloaceate and record the absorbance change with time.
Over the first 30 seconds, you find that the absorbance rises by about 0.35.
66.
A
B
C
D
E
Which statement is the MOST LIKELY prediction of what will happen next?
The rate of increase in absorbance will remain constant for at least 10 minutes
The absorbance will plateau when the acetyl-CoA runs out
The rate will fall after about 5 minutes as the citrate synthase becomes
degraded
The absorbance will fall as the all the DTNB gets used up
In less than five minutes all the oxaloacetate will run out
To make this system measure acetyl-CoA, you replace the stock acetyl-CoA with a
volume of an unkonwn sample (eg, a tissue extract)
67.
A
B
C
What else do you have to do to make this system useful as an assay for
acetyl-CoA?
D
E
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Now you try your assay system on a sample of heart extract. This extract has been
prepared as described in the original paper ie,
Small frozen pieces of hearts (~10 mg) are homogenized on ice in 10% (final w/v) homogenization
buffer containing (in mM) 20 TRIS, 10 EDTA, pH 7.4. Triton X-100 is then added to 1% (v/v) and,
after mixing, homogenates are then frozen and thawed three times.
68.
A
B
C
D
E
69.
A
B
C
D
E
70.
A
B
C
D
E
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How does the Triton X-100 and freezing/thawing help you get the most
accurate results from the tissue samples?
It activates citrate synthase
It increases the release of citrate synthase from the mitochondrial matrix
It causes the hydrolysis of acetyl-CoA
It increases the release of metabolites from the cell
It increases the turbidity of the extract
When you use the real tissue extract, you anticipate problems will occur.
Which is NOT likely to be a cause of such problems?.
Tissue extracts can contain pigments that absorb in the visible range
Tissue extracts may contain CoA
Tissue extracts contain citrate synthase
The acetyl CoA concentration in the tissue extracts may be too low
Tissue extracts can be cloudy
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71.
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What would give you the most confidence that your acetyl-CoA assay was
working well?
A
B
C
D
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Solution B:
Solution C:
1 mM tyrosine
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1
0.5
0.5
0
1000
0
2
0.5
0.5
100
900
0.21
3
0.5
0.5
200
800
0.39
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4
0.5
0.5
300
700
0.60
5
0.5
0.5
400
600
0.81
6
0.5
0.5
500
500
0.99
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Using this data, your assistant plots the following standard curve
72.
A
B
C
D
E
73.
A
B
C
D
E
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What is the approximate E450nm (mM-1 cm-1) for the coloured compound formed
in the assay
0.199
250
4
2
0.196
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74.
A
B
C
D
E
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20 l drop
Stretch to 1 cm
75.
A
B
C
D
E
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76.
A
B
C
D
E
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Your assistant repeats your set up but, although their absorbance readings
increase linearly from Tube 1 to Tube 6, the slope of their standard curve is
80% of yours (ie, their highest absorbance is 0.8). In your diagnosis of their
mistake, what can you RULE OUT (ie, what would NOT have caused this)?
They measured the samples at the wrong wavelength
They used a pipette for the tyrosine solution that consistently delivered 20%
less than it was meant to
They used a pipette for the tyrosine solution that consistently delivered 100 l
too little
They incubated their tubes for too short a time
They incubated their tubes for too long
One of your colleagues has made up a stock tyrosine solution but cannot remember its
concentration. You set up a series of tubes as follows:
Tube #
Solution A (ml)
Solution B (ml)
Unknown tyrosine sol (l)
Water (l)
Absorbance 450 nm
77.
A
B
C
D
E
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1
0.5
0.5
0
1000
0
2
0.5
0.5
10
990
0.41
3
0.5
0.5
50
950
1.85
4
0.5
0.5
250
750
2.05
5
0.5
0.5
500
500
2.1
6
0.5
0.5
1000
0
2.09
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You now use your assay to measure the concentration of tyrosine in various plasma
samples. Plasma is blood that has been centrifuged to remove red blood cells.
Plasma should be clear and colourless, but burst red blood cells can make it pink, and
excessive fat/lipoproteins can make it milky.
78.
A
B
C
D
E
How could you control for the extra problems associated with using plasma?
The complete absorption spectrum of each plasma sample needs to be
determined before the assay can be done
There is no need to introduce any extra controls because the pink colour or
milkyness will not affect the absorbance at 450 nm
If samples look pink or milky we should just use smaller volumes of these
plasma samples in the assay
Blanks containing plasma but no Solution A or B need to be set up
All the standards need to be spiked with plasma samples before assay
A
B
C
D
E
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Their samples of plasma are precious and they do not want you to use any
more than you need. What do you tell them?
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80.
A
B
C
D
E
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For the 2DG method to work properly, which of the following must be TRUE?
It is important that hexokinase works faster on 2DG that it does on glucose
The presence of trapped 2DGP should not affect the normal metabolism of
glucose
Muscle glucose transporters should behave differently towards 2DG than they
do towards glucose
2DGP should be able to desphosphorylate back to 2DG
2DGP should be able to move freely in and out of the muscle cells
[U-14C] 2deoxyglucose
50 Ci
0.2 mCi/ml
200 Ci/mol
81.
A
B
C
D
E
1812 A
Semester 2, 2005
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1812 A
82.
A
B
C
D
E
83.
A
B
C
D
E
84.
A
B
C
D
E
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Tube
Cell suspension (ml)
Buffer X (ml)
200 ng/ml Insulin solution (ml)
2DG/glucose mixture (ml)
1
0.5
0.5
0
0
2
0.5
0.4
0
0.1
3
0.5
0.4
0
0.1
4
0.5
0
0.4
0.1
5
0.5
0
0.4
0.1
Firstly, the cells, buffer and/or insulin were added to the tubes..
At time zero, the reaction in Tube #2 was started by adding 0.1 ml of the
2DG/glucose mixture.
At 5 min on the clock, Tube #2 was centrifuged for 30 seconds to form a pellet
of the cells. The supernatant was immediately aspirated (removed) and
discarded. The remaining pellet was counted for 14C for 10 minutes.
Tubes 4, 3 and 5 were treated in the same way when the clock read 7, 11 and
13 minutes respectively.
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You decide that you will need 1 ml of the radioactive 2DG/glucose mixture.
You dissolve 9 mg of glucose in 1 ml water to make a 50 mM solution.
85.
A
B
C
D
E
86.
A
B
C
D
E
87.
A
B
C
D
E
1812 A
How many dpm SHOULD be taken from the 2DG vial to make this glucose
solution the desired specific activity?
500
500,000
25 million
50,000
1 million
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88.
A
B
C
D
E
89.
A
B
C
D
E
90.
A
B
C
D
E
1812 A
Incubation
dpm in cell
time (min)
0
5
10
5
10
pellet
30
4151
7330
22030
42732
In Tube #4, what percentage of the total dpm put into the tube has been taken
up by the cells?
<1%
5 - 15%
20 - 30 %
50 80 %
>90%
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A
INCREASE
INCREASE
B
INCREASE
INCREASE
C
INCREASE
INCREASE
D
INCREASE
SAME
E
SAME
SAME
cell pellet
rate of 2DG/glucose
INCREASE
INCREASE
SAME
SAME
SAME
INCREASE
SAME
SAME
SAME
SAME
91.
92.
93.
Counting the cell pellet in the scintillation counter for twice as long
94.
95.
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SECTION B - PRACTICAL
PART II Short answer question worth 6 marks
A kibitzing* colleague suggests that your results are not valid because a significant
amount of free 2DG/glucose gets transferred into the scintillation vial. In other
words, they think that 2DG/glucose associated with the outside of the cells or solution
simply left with the cell pellet is interfering with your results. They also point out that
2DG/glucose uptake can still occur during the centrifugation and transfer steps.
Kibitz; to look on and offer intrusive comments and unwanted, usually meddlesome, advice to others.
.
They suggest the following improvements. Comment on each.
i)
You should re-suspend the pellet of cells in 1 ml fresh buffer and centrifuge
them again before you count them.
ii)
You should formally stop the reactions by adding something that will break
open the cells on demand (eg, strong acid).
iii)
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