1. Nucleotide Catabolism and Salvage; Membrane Signaling
Andy Howard Biochemistry Lectures, Spring 2019 Thursday 2 May 2019

2. Membranes and Metabolism
Nucleotides can be broken down, but they’re often recycled instead
Signal transduction is a critical part of what membranes do
05/02/2019
Nucleotide Catabolism; Signaling

3. What we’ll discuss Ribonucleotide reductase Membrane signaling
Thymidylate synthesis
Nucleotide catabolism and salvage
Reactions
Medical significance
Membrane signaling
G proteins
Adenylyl cyclase
PIP2 pathways
Sphingolipid- based signaling
Tyrosine kinases
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Nucleotide Catabolism; Signaling

4. iClicker question #1 (a) ATP (b) ITP (c) CTP (d) GTP
(e) none of the above.
1. Which of the following is an energy-providing cosubstrate in converting IMP to adenylosuccinate on the way to making AMP?
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Nucleotide Catabolism; Signaling

5. iClicker question #2 (a) CTP-binding proteins
(b) GTP-binding proteins
(c) ATP-binding proteins
(d) none of the above.
2. P-loop proteins are typically
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Nucleotide Catabolism; Signaling

6. Making deoxyribonucleotides
Conversions of nucleotides to deoxynucleotides occurs at the diphosphate level
Reichard showed that most organisms have a single ribonucleotide reductase that converts ADP, GDP, CDP, UDP to dADP, dGDP, dCDP, and dUDP
NADPH is the reducing agent
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Nucleotide Catabolism; Signaling

7. Ribonucleotide reductase heterotetramer
2 RNR1 subunits; each has
a helical 220-aa domain
10-strand 480-aa structure (thiols here)
5-strand 70-aa structure
E.coli RNR1 258 kDa dimer PDB 1R1R 2.9Å
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Nucleotide Catabolism; Signaling

8. RNR 2 2 RNR2 subunits; each has A diferric ion center
A stable tyrosyl free radical
E.coli RNR2 82 kDa dimer EC PDB 1PJ0, 1.9Å
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Nucleotide Catabolism; Signaling

9. Mechanism of RNR Y122 in RNR2 is converted to stable free radical
Radical transmitted to RNR1 cys439
Cys439 reacts with substrate 3’-OH to form free radical at C3’
Substrate dehydrates to carbonyl at C3’ and free radical at C2’; S- formed at Cys462
Disulfide formed between Cys462,Cys225; radical regenerated at Cys439
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Nucleotide Catabolism; Signaling

10. RNR : control I
ATP, dATP, dTTP, and dGTP act as allosteric modulators by binding to two regulatory sites on the enzyme
Activity site (A) regulates activity of catalytic site
When ATP binds at A, activity goes up
When dATP binds at A, activity inhibited overall
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Nucleotide Catabolism; Signaling

11. RNR Control II
Specificity site (S) controls which substrates can be turned over
ATP at A + ATP or dATP at S : pyrimidines only
dTTP at S : activates reduction of GDP
dGTP at S : activates reduction of ADP
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Nucleotide Catabolism; Signaling

12. dUDP to dUMP dTMP formed at monophosphate level (from dUMP)
dUMP derived three ways en route to dTMP:
dUDP + ADP  dUMP + ATP
dUDP + ATP  dUTP + ADP dUTP + H2O  dUMP + PPi
dCMP + H2O  dUMP + NH4+
dTMP formed at monophosphate level (from dUMP)
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Nucleotide Catabolism; Signaling

13. Thymidylate synthase
5,10- methylene THF
dUMP + 5,10-methyleneTHF  dTMP + 7,8-dihydrofolate
Unusual THF reaction in that cofactor gets oxidized as well as giving up a carbon
CH2 from 5,10-methylene group
extra H from C6
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Nucleotide Catabolism; Signaling

14. Thymidylate synthase, cont’d
So DHF must be reduced back to THF via DHFR and get its methylene back from SHMT
dihydrofolate
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Nucleotide Catabolism; Signaling

15. Thymidylate synthase
Generally the controlling step in DNA synthesis because [dTTP] < other [deoxynucleoside triphosphates]
Therefore a target for cancer chemotherapy and other therapies that target rapidly-dividing cells
Enzyme is a 2-layer sandwich
E.coli TS 64 kDa dimer with dUMP + GA9 EC PDB 2A9W, 1.7Å
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Nucleotide Catabolism; Signaling

16. Thymidylate synthase and drug design
Both folate analogs and dUMP analogs can interfere with (DHFR  SHMT  dTMP synthase  … ) cycle
5-fluorouracil is specific to thymidylate synthase
5-fluorouracil
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Nucleotide Catabolism; Signaling

17. DHFR as a drug target
Human DHFR inhibition: kill cancer cells faster than healthy cells
Eukaryotic DHFR also catalyzes folate  dihydrofolate
Prokaryotic DHFR doesn’t;
DHF derived by another mechanism in bacteria
So there are antibacterials based on the structural differences between prokaryotic and eukaryotic DHFR
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Nucleotide Catabolism; Signaling

18. Special case: protozoan enzyme
Bifunctional enzyme:
Thymidylate synthase
Dihydrofolate reductase
Presumably some entropic advantage
Maybe electrostatics too, allowing the negative charges on DHF to tunnel through; but cf. Atreya et al (2003) J.Biol.Chem. 278:28901.
Plasmodium falciparum DHFR-TS EC , kDa dimer PDB 1J3K, 2.1Å
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Nucleotide Catabolism; Signaling

19. Recovery pathway to dTMP
Deoxythymidine can be phosphorylated by thymidine kinase: deoxythymidine + ATP  dTMP + ADP
Labeled thymidine is convenient for monitoring intracellular synthesis of DNA because thymidine enters cells easily
Herpes simplex thymidine kinase 73 kDa monomer EC PDB 1E2K
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Nucleotide Catabolism; Signaling

20. iClicker question #3
3. Thymidylate synthase is particularly significant because
(a) Excess thymidine is toxic
(b) We cannot obtain thymidine from the diet
(c) [dTTP] < [other nucleoside triphosphates]
(d) none of the above.
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Nucleotide Catabolism; Signaling

21. Fates of polynucleotides
Poly- and oligonucleotides hydrolyzed to mononucleotides via nucleases
Mononucleotides are dephosphorylated via nucleotidases and phosphatases
Resulting nucleosides are deglycosylated
Resulting bases are sent either into salvage pathways or get degraded and excreted
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Nucleotide Catabolism; Signaling

22. Salvage pathways
Nucleotides can be fully broken down and excreted, but often they get recycled—especially purines
Typically the free base reacts with PRPP to form xMP and PPi, and the xMP is used; enzymes are phosphoribosyltransferases
Deficiencies in salvage pathways often result in severe problems
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Nucleotide Catabolism; Signaling

23. Salvage pathways: Why?
We can describe them, and we will: but why do they matter so much?
They provide energy savings relative to de novo synthesis (think of all the ATP we used in making IMP!)
Considerable medical significance to interference with these pathways
Intracellular nucleic acid bases are usually recycled; dietary bases are usually broken down and excess nitrogen excreted
05/02/2019
Nucleotide Catabolism; Signaling

24. Orotate phosphoribosyl transferase
Principal salvage enzyme for pyrimidines
Orotate + PRPP  OMP + PPi
OMP can then reenter UMP synthetic pathway (decarboxylation to UMP, then form UDP and CDP)
Yeast OPT 50 kDa dimer EC PDB 2PS1, 1.75Å
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Nucleotide Catabolism; Signaling

25. OPT, continued
Same enzyme can act on other pyrimidines to make nucleotides: Pyr + PRPP  PyrMP + PPi
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Nucleotide Catabolism; Signaling

26. Pyrimidine interconversions
All phosphorylations & dephosphorylations can and do happen
UTP can be aminated to CTP
CDP and UDP can be reduced to dCDP and dUDP
dCMP can deaminate to dUMP
Cytidine can be converted to uridine
dUMP can be methylated to dTMP
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Nucleotide Catabolism; Signaling

27. CMP and cytidine CMP’s phosphate can be hydrolyzed off
That’s followed by deamination of cytidine to make uridine
Catalyzed by cytidine deaminase
Another sandwich protein
Mouse cytidine deaminase 66 kDa tetramer EC PDB 2FR5, 1.48Å
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Nucleotide Catabolism; Signaling

28. Uracil to acetyl CoA; thymine to succinyl CoA
Reduced to dihydrouracil and dihydrothymine
Hydrated and ring-opened to ureidopropionate or ureidoisobutyrate
Eliminate bicarbonate and ammonium to yield -alanine or -aminoisobutyrate
Several reactions from there to acetyl CoA and succinyl CoA
Thermus Dihydro-pyrimidinase EC kDa hexamer PDB 1GKP, 1.29Å
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Nucleotide Catabolism; Signaling

29. Hydrolysis of U, dU & dT
Glycosidic bond in uridine or thymidine is hydrolyzed by phosphate:
Uridine + Pi  -D-ribose-1-P + uracil
Enyzme is uridine phosphorylase
Similar enzyme handles deoxyuridine
Similar reaction using thymidine phosphorylase yields thymine + -D-deoxyribose-1-P
E.coli Uridine phosphorylase EC kDa hexamer
Dimer shown
PDB 1RXY, 1.7Å
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Nucleotide Catabolism; Signaling

30. Purine salvage
Two phosphoribosyl transferases convert adenine, guanine, and hypoxanthine to AMP, GMP, and IMP
Adenine phosphoribosyl transferase is specific
HGPRT accepts both hypoxanthine and guanine
Toxoplasma gondii HGPRT EC kDa tetramer dimer shown PDB 1FSG, 1.05Å
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Nucleotide Catabolism; Signaling

31. Purine Interconnections
All phosphorylations and dephosphorylations can and do occur
ADP and GDP can be reduced to dADP and dGDP
AMP can deaminated to IMP (new)
IMP can be aminated to AMP
IMP can oxidized to XMP
XMP can be aminated to GMP
Guanine, adenine phosphoribosylated to GMP and AMP
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Nucleotide Catabolism; Signaling

32. Purine catabolism
Nucleoside or deoxynucleoside + phosphate  base + (D)-ribose 1-P
Hypoxanthine and guanine both lead to uric acid as a product
Uric acid is final excreted nitrogenous compound in primates, birds, some reptiles
Other organisms catabolize it further
Uric acid
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Nucleotide Catabolism; Signaling

33. Uric acid to allantoin
urate
Urate oxidase: urate + 2H2O + O2  allantoin + H2O2 + CO2
Allantoin is the excreted product in many mammals, turtles, some insects, gastropods
Teleost fish excrete allantoic acid (glyoxalate is recovered)
Cartilagenous fish & amphibia excrete urea
Allantoin
Allantoate
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Nucleotide Catabolism; Signaling

34. Urate oxidase
Catalyzes the decarboxylation of uric acid to allantoin via 5-hydroxyisourate intermediate
Aspergillus flavus urate oxidase 134 kDa tetramer monomer shown EC PDB 3P9O, 1.45Å
5-hydroxy-isourate
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Nucleotide Catabolism; Signaling

35. Lesch-Nyhan syndrome
Michael Lesch
William Nyhan
Complete lack of hypoxanthine-guanine phosphoribosyl transferase
So hypoxanthine and guanine are degraded to uric acid rather than being built back up into IMP & GMP
Leads to dangerous buildup of uric acid in nervous tissue
Neurological effects are severe and poorly understood
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Nucleotide Catabolism; Signaling

36. Gout
Accumulation of sodium urate and uric acid, both of which are only moderately soluble
Arises from inadequate (~10%) functionality of HGPRT, so that urate accumulates in peripheral tissues, particularly the feet
Benjamin Franklin (celebrated gout sufferer)
Sodium urate
Sodium urate crystals
accumulating
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Nucleotide Catabolism; Signaling

37. iClicker question #4 4. Gout could be regarded as
(a) A disease only suffered by the rich
(b) a mild version of Lesch-Nyhan syndrome
(c) a severe version of Lesch-Nyhan syndrome
(d) an entirely preventable disease
4. Gout could be regarded as
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Nucleotide Catabolism; Signaling

38. Transducing signals
Plasma membranes contain proteins called receptors that allow the cell to respond to chemical stimuli that can’t cross the membrane
Example: Bacteria can detect chemicals. If something useful comes along, a signal is passed from the receptor to the flagella, enabling the bacterium to swim toward the source
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Nucleotide Catabolism; Signaling

39. Multicellular signaling
Hormones, neurotransmitters, growth factors all can travel to target cells and produce receptor signals
Diagram courtesy Science Creative Quarterly, U. British Columbia
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Nucleotide Catabolism; Signaling

40. Extracellular signals
Internal behavior of cells modulated by external influences
Extracellular signals are first messengers
7-helical transmembrane proteins with characteristic receptor sites on extracellular side are common, but they’re not the only receptors
Image courtesy CSU Channel Islands
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Nucleotide Catabolism; Signaling

41. G-protein coupled receptors
Not all of these transmembrane receptors interact with heterotrimeric G-proteins, but a huge percentage do
These receptors are known as G-protein coupled receptors, or GPCRs
Studies of GPCRs earned Robert Lefkowitz and Brian Kobilka the 2012 chemistry Nobel Prize
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Nucleotide Catabolism; Signaling

42. Internal results of signals
Intracellular: heterotrimeric G-proteins are the transducers: they receive signal from receptor, hydrolyze GTP, and emit small molecules called second messengers
Second messengers diffuse to target organelle or portion of cytoplasm
Big picture: many signals, many receptors, relatively few second messengers
Often there is amplification involved
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Nucleotide Catabolism; Signaling

43. Roles of these systems Response to sensory stimuli
Response to hormones
Response to growth factors
Response to some neurotransmitters
Metabolite transport
Immune response
This stuff gets complicated, because the kinds of signals are so varied!
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Nucleotide Catabolism; Signaling

44. G proteins Transducers of external signals into the inside of the cell
These are GTPases (GTP  GDP + Pi)
GTP-bound protein transduces signals; GDP-bound protein doesn’t
Rat Gi, α subunit EC
41 kDa monomer PDB 1SVK, 2Å
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Nucleotide Catabolism; Signaling

45. Subunits of the G protein
Heterotrimeric proteins; association of b and g subunits with a subunit is disrupted by complexation with ligand-receptor complex, allowing departure of GDP & binding of GTP
Alfred Gilman
Martin Rodbell
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Nucleotide Catabolism; Signaling

46. GTP
GDP
G protein cycle
Inactive a
Active b a
Ternary complex disrupted by binding of receptor complex
Ga-GTP interacts with effector enzyme
GTP slowly hydrolyzed away
Then Ga-GDP reassociates with b,g g
GTP b g Pi a
GDP
Inactive
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Nucleotide Catabolism; Signaling

47. Cyclic nucleotides cAMP and cGMP: second messengers
Cyclic AMP
cAMP and cGMP: second messengers
Adenylyl cyclase converts ATP to cAMP + PPi
Trypanosoma adenylyl cylase 53 kDa dimer; monomer shown EC PDB 1FX2, 1.46Å
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Nucleotide Catabolism; Signaling

48. Adenylyl cyclase Integral membrane enzyme; active site faces cytosol
cAMP diffuses from membrane surface through cytosol, activates protein kinase A
PKA phosphorylates ser,thr in target enzymes; action reversed by specific phosphatases
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Nucleotide Catabolism; Signaling

49. Modulators of cAMP
Ordinarily cAMP is shortlived (15 min): it is hydrolyzed via cAMP phosphodiesterase, which catalyzes cAMP + H2O → AMP
Caffeine, theophylline inhibit cAMP phosphodiesterase, prolonging cAMP’s stimulatory effects on protein kinase A
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Nucleotide Catabolism; Signaling

50. Hormonal modulators of cAMP
Hormones that bind to stimulatory receptors activate adenylyl cyclase, raising cAMP levels
Hormones that bind to inhibitory receptors inhibit adenylyl cyclase activity via receptor interaction with the transducer Gi.
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Nucleotide Catabolism; Signaling

51. Inositol-phospholipid signaling pathway
PIP2
2 Second messengers derived from phosphatidylinositol 4,5-bisphosphate (PIP2)
Ligand binds to specific receptor; signal transduced through G protein called Gq
Active form activates phosphoinositide- specific phospholipase C bound to cytoplasmic face of plasma membrane
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Nucleotide Catabolism; Signaling

52. PIP2 chemistry
Phospholipase C hydrolyzes PIP2 to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol
Both of these products are second messengers that transmit the signal through the cell
Streptomyces PI-PLC EC kDa monomer PDB 3H4X, 1.23Å
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Nucleotide Catabolism; Signaling

53. IP3 and calcium
IP3 diffuses through cytosol and binds to a calcium channel in the membrane of the endoplasmic reticulum
The calcium channel opens, releasing Ca2+ from lumen of ER into cytosol
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Nucleotide Catabolism; Signaling

54. Calcium as a 2nd messenger
Ca2+ is a short-lived 2nd messenger too: it activates Ca2+-dependent protein kinases that catalyze phosphorylation of certain proteins
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Nucleotide Catabolism; Signaling

55. Diacylglycerol and protein kinase C
Diacylglycerol plasma membrane
Protein kinase C (in equilibrium between soluble & peripheral-membrane form) moves to inner face of membrane; it binds transiently and is activated by diacylglycerol and Ca2+
Protein kinase C catalyzes phosphorylation of several proteins
Mouse PkCδ, C1B domain EC kDa PDB 3UFF, 1.3Å
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Nucleotide Catabolism; Signaling

56. Control of inositol-phospholipid pathway
Figure courtesy Motifolio.com
After GTP hydrolysis, Gq is inactive so it no longer stimulates phospholipase C
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Nucleotide Catabolism; Signaling

57. Activities of 2nd messengers are transient
IP3 rapidly hydrolyzed to other things
Diacylglycerol is phosphorylated to form phosphatidate
Calcium gets used or sequestered
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Nucleotide Catabolism; Signaling

58. Sphingolipids give rise to 2nd messengers
Some signals activate hydrolases that convert sphingomyelin to sphingosine, sphingosine-1-P, and ceramides
Sphingosine inhibits Protein Kinase C
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Nucleotide Catabolism; Signaling

59. Other second messengers arising from sphingolipids
Ceramides activate a protein kinase and a protein phosphatase
Sphingosine-1-P can activate Phospholipase D, which catalyzes hydrolysis of phosphatidylcholine; products are 2nd messengers
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Nucleotide Catabolism; Signaling

60. Receptor tyrosine kinases
ligands
Receptor tyrosine kinases
exterior
Tyr kinase monomers
Most growth factors function via a pathway that involves these enzymes
In absence of ligand, 2 nearby tyr kinase molecules are separated
Upon substrate binding they come together, form a dimer
interior
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Nucleotide Catabolism; Signaling

61. Autophosphorylation of the dimer
Enzyme catalyzes phosphorylation of specific tyr residues in the kinase itself; so this is autophosphorylation
Once it’s phosphorylated, it’s active and can phosphorylate various cytosolic proteins, starting a cascade of events
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Nucleotide Catabolism; Signaling

62. Insulin receptor
Insulin binds to an a2b2 tetramer; binding brings b subunits together
Each tyr kinase (b) subunit phosphorylates the other one
The activated tetramer can phosphorylate cytosolic proteins involved in metabolite regulation
Sketch courtesy of Davidson College, NC
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Nucleotide Catabolism; Signaling

63. iClicker question #5
5. Which of the following have we not described as a second messenger?
(a) cyclic AMP
(b) Na+
(c) sphingosine-1-phosphate
(d) Ca2+
(e) all four of these are second messengers
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Nucleotide Catabolism; Signaling