Problems Added to the 10th edition, 2015. (Answers are at the bottom, after the last question.)
Questions:
6-24. Some G proteins are not involved in signaling.
These G proteins are activated by GPAFs (G protein activating factors). You have
an in vitro system with a G protein and a GPAF plus ATP and GTP. (No other
proteins present.)
A. To activate the G protein, you need the GPAF and (ATP) (GTP)
(both) (neither) (either one).
B. Suppose you use labeled nucleotides (either ATP and/or GTP)
and the G protein becomes radioactive when it is activated. (The labeled
nucleotides have radioactivity in all 3 phosphates.) You wait until the G
protein is inactivated. Then you isolate the inactivated G protein, and put it
into fresh solution with unlabeled GTP and ATP. Then you add GPAF and (re)activate
the G protein. When the G protein is (re)activated, what radioactive material(s)
will be released into the solution?) (GTP) (GDP) (GMP) (phosphate) (PPi) (ATP)
(AMP) (cAMP) (ADP) (none -- nothing radioactive will be released). Circle all
correct answers, and explain.
Note: Nothing was removed from the 9th edition, but some of the problems were renumbered. The problem numbered '6-24' in the 9th edition is numbered '6-26' in the 10th edition.
6-25. In normal fat cells, protein Q binds to lipid droplets and prevents the fats in the droplet from being hydrolyzed. Epinephrine, acting through a GPCR, activates PKA and triggers phosphorylation of protein Q. This allows HSL (hormone sensitive lipase) to bind to protein Q and start hydrolyzing the lipid. You have an in vitro system with HSL, protein Q and PKA. No other proteins are present. You measure the binding of HSL to protein Q. You get binding if you add epinephrine, ATP, GTP, and cAMP. You should get just as much binding if you omit (epi) (GTP) (ATP) (cAMP) (none of these – you need them all). Circle all correct answers and explain your choices.
12-23. BND (bad news
disease) is an inborn error of metabolism. It is recessive; people with the BND
genotype (homozygotes) have inherited two defective copies of the BND4 gene.
Many people with the BND genotype have a normal phenotype (no symptoms), but
some develop disease symptoms, including problems with the brain such as
psychiatric problems and ataxia (lack of balance).
The BND4 gene is not expressed in the brain, but it is highly
expressed in the kidney and in the intestine. It codes for a Na+/amino
acid co-transporter for neutral amino acids. It does not transport charged amino
acids.
Normal people, and people with BND, also have a H+/dipeptide
co-transporter (HDCT) in the intestine and they have intracellular proteases
that can hydrolyze intracellular dipeptides. Because of the HDCTs, most people
with the disease genotype have normal levels of amino acids in their plasma,
whether they have symptoms or not.
A. For each part of A,
fill in the blank with all correct choices from the following list. Choices are:
(the afferent arteriole) (the peritubular capillaries) (the glomerular
capillaries) (the proximal tubule) (the distal tubule) (the ascending loop of
Henle) (the descending loop of Henle) (the collecting ducts) (none of these)
(all of these).
A-1. In the kidney, the BND4 protein should be made in the
cells of _________________________.
A-2 An enhancer of the BND4 gene should be located in the
cells of _________________________.
B. Consider a person with
the BND genotype.
B-1. In the USA, people with a BND genotype usually do not
develop symptoms. Why not ? Probably because they eat a diet that is (low in
protein) (high in protein) (high in carbohydrates)
(low in carbohydrates) (high in salt) (low in salt) (any of these – diet should
have no effect).
B-2. The level of neutral AAs in their urine should be
(higher than normal) (lower than normal) (normal, which is zero) (normal, which
is nonzero) (can't predict – depends on phenotype).
B-3. The level of the AA aspartic acid in their urine should
be (higher than normal) (lower than normal) (normal, which is zero) (normal,
which is nonzero) (can't predict).
C. In a person with the BND genotype, the concentration of neutral AAs in the filtrate entering the collecting ducts of the kidney should be (about the same as in their plasma) (lower than in their plasma) (higher than in their plasma) (can't predict – depends on the person's phenotype).
D. If a person with the
BND genotype stops secreting ADH, then what will happen?
(i) In the urine, the concentration of neutral AAs
will (increase) (decrease) (stay the same) (can't predict).
(ii) In the urine, the amount of neutral AAs will
(increase) (decrease) (stay the same) (can't predict).
*E-1. Suppose you could
take neurons from the brain of a person with BND symptoms, and measure neutral
AA uptake into the cells. The maximal rate of uptake, measured in vitro,
should be
(higher than normal) (lower than normal) (normal, which is zero) (normal, which
is nonzero) (can't predict).
*E-2. When people develop brain symptoms in BND, the most likely explanation is (inability to transport AAs properly in brain) (low levels of neutral AA in the plasma) (tubular secretion of toxic byproducts of AAs in the kidney) (production of toxic byproducts from neutral AAs remaining in the intestine) (toxic compounds from either tubular secretion or intestinal metabolism) (any one of these – all are equally likely) (something else – none of these could explain the symptoms).
Answers:
6-24. A. To activate the G
protein, you need the GPAF and GTP.
B. When the G protein is (re)activated,
you will release radioactive GDP .
Activation of a G protein requires GTP for replacement of a GDP with a GTP.
Deactivation occurs by hydrolysis of the GTP to GDP. In part B, the phosphate
has already been hydrolyzed off, and washed away, but the inactive enzyme still
has a bound GDP. To reactivate, you have to exchange the GDP for a GTP, not
phosphorylate the GDP to GTP.
6-25. You should get just as much binding if
you omit GTP & epinephrine (epi).
(1) You need cAMP to activate PKA, so you can't omit it. (There
is no adenyl cyclase to generate it.)
(2) You need ATP to phosphorylate protein Q -- PKA is a kinase
that requires ATP as a substrate.
(3) You do not need GTP or epi, because (a) you already have
cAMP, and (b) they are useless without a signal transduction system (GPCR, G
protein & adenyl cyclase). In other words, you have the end product, cAMP, so
you don't need to carry out the steps to produce cAMP in response to epi, and
you don't have the proteins to do it anyway.
12-23. Note: BND (bad news disease) is based on Hartnup disease.
A-1. In the kidney, the BND4 protein should be made in the
cells of the proximal tubule. BND protein is an amino acid transporter.
All transport of AA takes place in the proximal tubule.
A-2 An enhancer of the BND4 gene should be located in the
cells of all of these. An enhancer is a cis-acting regulatory DNA
sequence. The enhancers in every cell are the same.
B-1. In the USA, people with a BND genotype usually eat a
diet that is high in protein. A person with the BND genotype must eat a
high protein diet to maintain a normal level of neutral AAs in the plasma –
there must be a high rate of neutral AA uptake from the intestine (using HDCTs)
to balance the high rate of neutral AA loss in the kidney (due to the absence of
BND protein).
B-2. The level of neutral AAs in their urine should be
higher than normal. If BND protein is missing, any neutral AAs that are
filtered into the kidney tubule cannot be recovered – they will be lost in the
urine. The kidney does not have any other proteins that can substitute for the
transport function of the missing BND protein. However, the situation is
different in the intestine, which has multiple proteins (hydrolases,
transporters, etc.) for absorbing nutrients from food. So even if BND protein is
missing, neutral AA can enter the plasma, because dipeptides containing neutral
AAs can be absorbed from the intestine using HDCTs.
B-3. The level of the AA aspartic acid in their urine should
be normal, which is zero. Charged AAs are transported by a different
protein, in both intestine and kidney, not by the BND protein. So the charged AA
will be transported normally, even if BND protein is missing. Why is the normal
AA level zero? If normal carriers are present, all the AAs will be removed from
the urine and reabsorbed by the body. AA will only appear in the urine if the
level of AAs in the plasma is very high, or there is something wrong with the
kidney, such as the absence of BND protein.
C. Higher than in their plasma. When the filtrate enters the kidney tubule, the [neutral AA] is the same as in the plasma. If there is no BND protein, all the neutral AAs will remain in the filtrate. In the descending loop of Henle, water will be removed, increasing the [neutral AA]. There are changes in Na+, and changes in osmolarity, but the question only asks about the concentration of neutral AA.
D. (i) In the urine, the concentration of neutral AAs will decrease & (ii) the amount of neutral AAs will stay the same. Secretion of ADH increases water reabsorption and decreases the volume of urine, but it does not affect the transport of neutral AA. (Therefore, if ADH secretion stops, the reabsorption of water will decrease, and the volume of urine will increase, diluting the concentration of neutral AAs.)
E-1. Uptake of neutral AA in
vitro should be normal, which is nonzero. Uptake in the brain is
necessary, as amino acids from the plasma are needed by all cells for growth and
maintenance. (Neutral amino acids are especially important since trp is needed
to make NAD, and cells cannot make trp themselves.) The blood-brain barrier does
not prevent transport of essential molecules into the brain, but it does affect
how transport occurs. For small molecules to reach brain cells, the molecules
must be transported by carrier proteins out of the capillaries; they can’t just
diffuse out through the pores between the endothelial cells. Since the BND gene
is not expressed in brain, meaning BND protein is not made in the brain, there
must be a different carrier for transport of neutral AAs in the brain. The
actual carrier for neutral amino acids should be normal in brain cells from a
person with BND.
E-2. The most likely explanation is production of toxic
byproducts from neutral AAs remaining in the intestine.
There are only two possibilities here – (a) that a lack of BND protein
causes a buildup of toxic byproducts that reach the brain, or (b) that a lack
BND protein results in a shortage of neutral AA in the brain. Possibility (a)
is thought to be correct, since BND protein is not made in the brain, and the
plasma levels of neutral AAs are usually normal* in Hartnup disease. (However,
the urine levels are elevated, as explained above.) Since toxic
byproducts probably cause the problem, as explained above, and since any toxic
byproducts in the kidney would be excreted in the urine, the source of the toxic
byproducts is probably the intestine.
More details: In the absence of BND protein in the GI tract, neutral amino
acids will build up in the stomach or intestine. These can be converted to
toxic byproducts. Some will be lost in the feces, but some could enter the
plasma, and be carried to the brain. Toxic products might also accumulate in the
kidney filtrate, but if they are secreted into the tubule, they would end up
leaving the body in the urine; they wouldn’t get into the plasma, so they
couldn’t reach the brain.
*Given the normal Western high protein diet. On a low protein diet, the levels of neutral AA in the plasma would be below normal, and then a lack of neutral AA in the brain could be the problem, and ‘inability to transport AAs properly’ would be a reasonable answer.