Titheradge 10

Question 1

Action of Glycogen synthase

Answer 1

Adds UDP glucose residues to existing 4+ length glycogen chain. By a1,4 linkage. Phosphorylation DECREASES synthase activity

Question 2

Glycogenin protein and a tyrosine residue in it are:

Answer 2

the base on which glycogen is constructed

Question 3

key enzyme in glycogenolysis, and its function

Answer 3

(Glycogen) Phosphorylase. breaks a-1,4 glycosidic bonds (by phosphorolysis) up to 4 residues from a branch point. produces glucose-1-phosphate. (does not use ATP)

Question 4

(Glucosyl)Transferase activity of debranching enzyme, function in glycogenolysis

Answer 4

transfers group of 3 glucose residues from a 4 long branch of glycogen to a longer branch.

Question 5

a-1,6 glucosidase activity of glycogen debranching enzyme

Answer 5

removes final glucose residue from a glycogen side chain, breaking a-1,6 glycosidic bond (by hydrolysis)(forming normal glucose)

Question 6

Phosphorylase b conformations (in dynamic equilibrium)

Answer 6

Tense (inactive), stabilised by signs of high energy such as ATP, G-6-P (and glucose in hepatocytes). Relaxed (active) stabilised by low energy indicators AMP and Pi, as well as phosphorylation (which converts it to phosphorylase A.)

Question 7

Structure of Phosphorylase

Answer 7

Homodimer, allosteric enzyme. So each subunit has N-terminal domain with 2 subdomains: glycogen binding subdomain (with a glycogen storage site) and interface subdomain (that binds the other subunit and also is location of allosteric site)

Question 8

Why is the buried active site of Phosphorylase important?

Answer 8

It forms a hydrophobic pocket that excludes water and so stops hydrolysis of glycosidic bonds occurring, so that phosphorolysis can produce high energy glucose-1-phosphate instead.

Question 9

How are the glycogen storage site (of the glycogen binding subdomain of the n-terminal domain) and the active site linked?

Answer 9

by a groove, which uses glycogen like a track, so phosphorylase runs along like a train. it accommodates about 4/5 glucose residues, hence why it can’t remove the last four residues.

Question 10

Phosphorylase B kinase function

Answer 10

Calcium dependent. Phosphorylates serine 14 residue at n-terminus of glycogen phosphorylase B. converts to phosphorylase A which spend most of its time in the relaxed (active) conformation. This makes it also independent of the energy status of the cell (AMP presence etc)

Question 11

How does AMP inhibit the action of protein phosphatase 1 (PP-1)?

Answer 11

Its binding to the allosteric site on a phosphorylated n-terminus domain of phosphorylase A causes the phosphate group to be hidden internally, sheltering it from the action of PP-1.

Question 12

Stimulation and inhibition of adenylate cyclase by adrenaline

Answer 12

B-adrenergic receptor coupled to Gs protein (whose alpha s subunit activates adenylate cyclase). alpha 2 adrenergic receptor coupled to Gi protein (whose alpha i subunit inhibits adenylate cyclase).

Question 13

Alpha 1 adrenergic receptor function

Answer 13

Gq protein coupled receptor. binding of adrenaline to receptor causes assocation to Gq which causes Gqa subunit to swap GDP for cytoplasmic GTP. Gqa subunit dissociates and then activates Phospholipase Cbeta in membrane.

Question 14

Role of active phospholipase Cbeta?

Answer 14

hydrolyses PIP2 into IP3 and diacylglycerol.

Question 15

Role of IP3?

Answer 15

releases Calcium from endoplasmic reticulum stores by binding to IP3 dependent calcium channels

Question 16

Role of Diacylglycerol?

Answer 16

Membrane bound. Activates PKC

Question 17

Location of alpha1 adrenoceptor?

Answer 17

Smooth muscle in blood vessels. Plentiful. Vasocontriction

Question 18

PKC structure and function?

Answer 18

Hydrophobic regulatory domain, has a pseudosubstrate region at N-terminus. C2 region binds Ca2+ and C1 binds DAG and Phosphatidylserine (by C2) in the membrane. Hydrophilic catalytic domain (C3 binds ATP, C4 catalytic domain binds pseudosubstrate.)

Question 19

Response of PKC to calcium binding

Answer 19

Calcium binds to C2 domain, moves to membrane where it meets PS, positive charge of Ca2+ allows binding of C2 to anionic PS. Also meets DAG, which binds C1. Overall this causes release of pseudosubstrate from catalytic site.

Question 20

Phorbol ester (tomour promotor) effect

Answer 20

Mimics DAG (diacyl glycerol)–> activates PKC constantly, which causes cleavage at calpain cleavage site (hinge site) of PKC, leaving constituently active catalytic region of PKC free. This stays active for 12 hours before degrading.

Question 21

Calmodulin structure-function

Answer 21

4 Ca2+ binding sites, calcium binding reveals hydrophobic region which allows it to bind to its target protein

Question 22

How does increased intracellular calcium (for example from muscle contraction or a1 adrenoceptor activation) cause glycogenolysis?

Answer 22

Through activating phosphorylase kinase, via its 4 calmodulin delta subunits. This phosphorylates phosphorylase b using ATP, activating it (–>R form)

Question 23

Draw phosphorylase kinase:

Answer 23

Question 24

How does phosphorylation of phosphorylase kinase change its response to calcium?

Answer 24

Can be activated by lower calcium concentrations (e.g. normal physiological levels) when phosphorylated (on its alpha and beta subunits) because it needs to only bind calcium ions to its 2 high affinity calmodulin (delta subunits), but not its low affinity d subunits.

4Ca2+ to each calmodulin

Question 25

General structure of PKA (inactive to activated)

Answer 25

(cAMP dependent protein kinase.) 2 regulatory subunits, bound to 2 catalytic subunits.

2 cAMPs (when present) bind to each regulatory subunit

This causes conformational shift in regulatory subunits, causing them to dissociate from catalytic subunits.

Question 26

How does PKA effect PP1?

Answer 26

Active PKA (cAMP dependent protein kinase) phosphorylates Inhibitor-1 (I-1), activating it.

Active I-1 then inhibits PP-1 by binding to it.

Question 27

Effect of theophylline

Answer 27

Inhibits phosphodiesterase conversion of cAMP to AMP.

Thereby increasing (preventing decrease) of cAMP levels.

Thereby potentiating cAMP mediated adrenaline effects.

Question 28

2 Glycogenesis enzymes

Answer 28

Glycogen synthase, adds UDP glucose by a-1,4 glycosidic bonds to existing 4 glycosyl residue long chains. Branching enzyme transfers 4 units from long chain to form new a-1,6 linkage branch.

Question 29

Regulation of glycogen synthase

Answer 29

Phosphorylation of glycogen synthase I to glycogen synthase D (by multiple kinases cf.phosphorylase) reduces its activation. D is for “dependent” on G-6-P for activation as an allosteric regulator. (I for independent of G-6-P)

G-6-P increases Vmax. Dephosphorylation mediated by PP-1

Question 30

PKA phosphorylates which residues?

Answer 30

Serine/Threonine, because they have hydroxyl groups.

Question 31

What is CREB? What is its function?

Answer 31

cAMP Response Element Binding Protein. A transcription factor.

When phosphorylated by PKA (catalytic subunit) in the nucleus it binds to CRE DNA sequences in several genes. (normally in pairs)

Recruits CBP/P300 coactivators that acetylate histones, causing DNA to uncoil and transcription to increase.

PP-1 also dephosphorylates CREB.

Question 32

How can cAMP have different effectors and effects in different tissues?

Answer 32

Specific receptors for specific hormones on specific tissues that all work through cAMP.

Presence of different enzymes that can be phosphorylated in different tissues.

Question 33

Binding of hormone to G protein coupled receptor causes:

Answer 33

Conformational change in receptor that increases its affinity for G-protein, they associate and GDP released. Replaced with GTP (as there’s more of it).

This causes a conformational change in the alpha subunit, decreasing its affinity for the beta gamma subunits and increasing its affinity for the adenylate cyclase, which it binds to then activates.

Question 34

What happens to G-protein receptor complex after alpha subunit dissociates?

Answer 34

Conformational change in receptor causing it to release hormone!

Question 35

How does the dissociated alpha subunit of a g-protein return to the beta gamma subunits?

Answer 35

Because of its GTPase activity. It only has high affinity for adenylate cyclase whilst GTP is bound, and after a finite time it hydrolyses GTP to GDP + Pi, causing it to dissociate from adenylate cyclase and reassociate with the Beta gamma subunits, reforming the original structures. It is a cycle.

Question 36

Effect of cholera toxin on GTPase cycle (of G-protein alpha subunit):

Answer 36

Cholera toxin causes inhibition of the GTPase activity of Gstimulatory alpha subunit by ADP-ribosylation. (using NAD, [leaving nicotinamide]). This causes any GTP bound Gs alpha subunits to not breakdown, causing permanent activation of AC (adenylate cyclase).

This activates ion and water channels in gut, causing profuse diarrhea.

Question 37

What are dimerisation loops?

Answer 37

Loops on receptor tyrosine kinase monomers that interact to form dimers upon binding of ligand (growth factors)

ligand binding causes conformational change

Question 38

What binds to phosphorylated tyrosine residues on c-terminal end of active receptor tyrosine kinases?

Answer 38

SH2 domains of proteins including: GEF’s adaptor protein GRB2 (grab2), Phospholipase C gamma, GAP, p85 regulatory subunit of PI-3 kinase

Question 39

Why is GRB2 like a plug adaptor?

Answer 39

Adaptor protein.

it’s SH2 domains bind phosphorylated tyrosine residues, and then its 2 SH3 domains bind proline rich sequences of GEF (guanine nucleotide exchange factor, aka SOS, GNRP)

Question 40

RAS is analogous to?

Answer 40

alpha subunit of G protein.

Farnesylated (fatty acid chain) attaches it to membrane. Exists inactive attached to inside of plasma membrane, bound to GDP, GEP(akaSOS) exchanges GDP for GTP, activating it

Question 41

Function of GAP?

Answer 41

GTPase activating protein

Activates GTPase activity of RAS, speeding the breakdown of bound GTP to GDP and Pi. (to about a minute)

(Unlike alpha subunits of Gs, which are much faster without assistance)

Question 42

Molecular switch function/structure of RAS?

Answer 42

GTP bound, 3rd phosphate of GTP interacts with an amino acid in each of the switch I and switch II regions, keeping it in closed conformation, and activated.

(Thr-35 Switch I.

Gly-60 in switch II)

Question 43

How does SOS (aka GEF) activate RAS?

Answer 43

Sos inserts an alpha helix into the structure of RAS, opening up its GDP binding site, releasing bound GDP.

GTP (in higher conc.) can then bind to replace the GDP and displace the alpha helix of Sos.

Question 44

What are 14-3-3 proteins?

Answer 44

Dimerised proteins that bind to phosphorylated Raf at either end (N-terminal regulatory and C terminal catalytic/kinase domains) and keep it inactive.

Question 45

How does Ras(-GTP) activate Raf?

Answer 45

Interacts with N-terminal regulatory domain and displaces 14-3-3 protein from N-terminus, by dephosphorylating it.

THis allows Raf to uncurl, exposing C-terminus to phosphorylation by as yet unidentified kinases.

Question 46

MEK, what does it do and what is unusual about it?

Answer 46

MAPKK(inase) It is a serine/threonine, and a tyrosine kinase!

Activated by S/T phosphorylation by Raf.

ACtivates MAPK by threonine and tyrosine phosphorylation

Question 47

How is MAPK activated?

Answer 47

Mitogen activated protein kinase is activated by dual phosphorylation by MEK (MAPKK)

On both threonine and tyrosine residues!

Question 48

Functions of activated MAPK?

Answer 48

Turn on glycogenesis, and off glycogenolysis through phosphorylation of PP-1G through activating p90rsk protein.

Goes to the nucleus where it switches on gene expression for cell growth and division related genes

Question 49

2 types of phospholipase C (PLC) and what are they involved in?

Answer 49

PLCgamma is activated by binding to phosphorylated receptor tyrosine kinases.

PLCbeta is activated by the Gq-protein alpha subunit. alpha-1 adrenoceptor is its g-protein coupled receptor.

Both go on to hydrolyse membrane-bound PIP2 into IP3 [releases Ca2+, to activate calcium dependent kinases etc] and DAG [activates PKC]

Question 50

How can receptor tyrosine kinase activation increase cellular Na+ and alkalinity?

Answer 50

PLCgamma activated by binding to phosphorylated RTK, PLCgamma hydrolyses PIP2 into IP3 and DAG. DAG activates PKC when it reaches the membrane.

PKC activates Na+/H+ exchange pumps.

Question 51

Why do activated receptor tyrosine kinases activate multiple pathways? give example proteins.

Answer 51

Each phospho-tyrosine residue on the C-terminal end recruits a different protein with a slightly different SH2 domain.

E.g. PLCgamma, Ras (via GRB2 to GEF), GAP, PI3K p85 domain.

Question 52

What is PI3K?

Answer 52

A kinase whose catalytic p110 subunit phosphorylates PIP2 to PIP3. (using ATP)

[PIP3 recruits PDK1 and other proteins to the membrane through their PH domain.]

PI3K also has a p85 regulatory domain with an SH2 region that binds to phosphorylated receptor tyrosine kinase.

Activation of PI3K also enhanced by Ras-GTP.

Question 53

PH domain?

Answer 53

Pleckstrin homology domain.

Present in proteins such as Phospholipid Dependent protein Kinase 1. PDK1.

Associates with inositol phospholipids phosphorylated at the 3 position of the inositol ring. PIP3

This recruits these proteins to the membrane, which PIP3 is attached to, it does not play a role in their activation but simply moves them to where their substrates are found.

Question 54

PKA, PKB, PKC

Answer 54

PKA = cAMP dependent protein kinase. 2 regulatory 2 catalytic subunits. Phosphorylates a + b subunits on Phosphorylase kinase. Phosphorylates many other things. And activates CREB –> transcription.

PKB = Akt, activated by PDK1 and 2 at the membrane. Has PH domain ==> associates with PIP3.

PKC = activated by DAG on membrane (and phosphatidyl serine). Ca2+ attracts to membrane. Phosphorylates many proteins, including glycogen synthase.

Question 55

How is PKB (Akt) activated?

Answer 55

Its PH domain associates with PIP3 (made by PI3K). THis partially activates it.

PDK1 at the membrane then phosphorylates its activation lip.

Phosphoinositide dependent protein kinase 2 then phosphorylates its C terminus, resulting in full activation.

Question 56

How does insulin increase Glut-4 translocation to the membrane?

Answer 56

Insulin receptor is a receptor tyrosine kinase.

Insulin binding on striated muscle, or adipose tissue cells causes activation of PI3K –> PIP3 –> PDK and PKB –> increased Glut-4 translocation. –> increases glucose uptake.

Question 57

General roles of PKB?

Answer 57

Generally: cell growth and division.

Increases Glut-4 translocation to the membrane.(therefore more glucose intake –>more energy)

Opposes apoptosis –>cell survival

Activates transcription factors

Protein synthesis.

Increases glycogen synthesis, (through inactivation of glycogen synthase kinase-3 by phosphorylation)

Question 58

How do MAPK and PI3K pathways work together to increase glycogen synthesis?

Answer 58

Following receptor tyrosine kinase activation (e.g. insulin receptor) both pathways are activated.

MAPK phosphorylates p90rsk which phosphorylates Protein phosphatase 1 (glycogen targeting) PP1G.

This phosphatase activates glycogen synthase and deactivates phosphorylase and Phosphorylase Kinase.

PI3K pathway activates PKB which increases Glut-4 translocation to the membrane. PKB also increases Glycogen synthesis by deactivating glycogen synthase kinase 3. –> activating glycogen synthase.

Question 59

Ectodomain shedding what and how?

Answer 59

Growth factors and cytokines, normally membrane bound.

THen activated by proteolysis they are released from the membrane to go and carry a signal.

therefore originally juxtacrine but now released they can be autocrine, paracrine, endocrine.

Question 60

What ‘triads’ are found around myofibrils?

Answer 60

T-tubules (invaginations of sarcolemma) bordered on either side by terminal cisternae of sarcoplasmic reticulum

Question 61

Structure of myosin thick filaments

Answer 61

Heavy chains: 2 alpha helices coiled around each other forming a coiled-coil structure. Tail region and hinge region.

Globular head regions containing ATPase, and bind actin. Attached to myosin light chains that control activity of myosin.

Question 62

Sequence of events at neuromuscular junction:

Answer 62

Depolarisation reaches synaptic knob of motor neurone. Opens voltage gated calcium channels.

Increase in intracellular calcium concentration is signal for ACh containing vesicles to move to and fuse with the cell membrane, releasing ACh into synaptic cleft.

ACh diffuses across and interacts with nicotinic ACh receptors which allow sodium into the cell. (also small efflux of potassium out)

This small depolarisation spreads down the t-tubules via voltage gated sodium channels.

Question 63

Structure of NAChR

Answer 63

pentameric pore forming transmembrane protein.

2 Alpha subunits have ACh binding sites.

All subunits (alpha, beta delta gamma) have roughly the same structure with 4 alpha helical transmembrane spanning domains (M1-M4)

M2 helices are amphipathic (combining both hydrophobic and hydrophilic amino acids). These form the walls of the pore. Other helical domains hydrophobic to sit within the membrane.

Channel wide at top, narrow at bottom as passes through membrane. Rings of negatively charged amino acids line tops and bottoms of channel, repelling anions.

Question 64

Response of NAChR to ACh binding

Answer 64

Induces rotation of the M2 subunits to twist bulky hydrophobic leucine residues out of the channel.

Replaced by smaller polar residues.

This increases

Question 65

Effect and mechanism of nitroglycerin (GTN)

Answer 65

Interacts with tissue thiols to form nitrothiols

Question 66

Structure of Dihydropyridine Receptor

Answer 66

Alpha1s, alpha2-delta, beta1 and gamma subunits.

alpha_1s subunit is key, composed of 4 homologous transmembrane domains. I-IV

Each has 6 transmembrane helices, S4 is voltage sensing

Ryanodine receptor binding site is a loop between II and III