C2S

Question 1

What makes glucose a good fuel source?

Answer 1

Oxidative phosphorylation of glucose produces lots of energy.
Broken down to pyruvate by glycolysis.
Efficiently stored as starch, glycogen
Soluble

Question 2

Aerobic/anaerobic break down of pyruvate

Answer 2

Aerobic: converted to Acetyl-CoA which enters the krebs cycle, produces 36 ATP
Anaerobic: converted into lactate, 2 ATP

Question 3

Metabolism
Catabolism
Anabolism

Answer 3

M: all the chemical reactions necessary for life in an organism
C: breaks down molecules, releases energy
A: builds complex molecules, uses energy

Question 4

Homeostasis (in this context)
Hyperglycaemia
Hypoglycaemia

Answer 4

Homo: blood sugar levels must constantly be kept within a range
per: high blood glucose
po: low blood glucose

Question 5

Insulin

Answer 5

Released from pancreatic Beta cells when blood glucose increases
Signals for the removal of glucose from the blood via increased uptake of glucose into fat and muscle
Increases glycogen synthesis in the liver by glycogen synthase
Inhibits glyconeogenesis in the liver/increased glycolysis by expression of glycolytic enzyme gene
Gylcolysis is the process of glucose being converted into 2 pyruvates, produces ATP

Question 6

Glucagon

Answer 6

Released from pancreatic alpha cells when blood glucose levels fall
Signals release of glucose from liver into blood
Stimulates gluconeogenesis; gluconeogenic enzyme gene expressed
Inhibits glycogen synthase in the liver but increased activity of glycogen phosphorylase
Triggers lipid breakdown

Question 7

Gluconeogenesis

Answer 7

A metabolic pathway that results in the generation of glucose from non-carbohydrate substrates such as lactate or amino acids

Question 8

Favourable

Answer 8

Both directions of the glucose/glucose-1-phosphate pathway are favourable. They happen simultaneously. So reciprocal regulation of enzymes needed. Which allows the system to react quickly to changes in blood sugar levels

Question 9

How are enzymes regulated

Answer 9

Changing rate of biosynthesis/degradation levels
Changing activity
Changing location

Question 10

Kinase

Answer 10

Enzymes which enable phosphorylation by the covalent addition of phosphate, transferred from ATP

Question 11

Phosphatases

Answer 11

Enables dephosphorylation by catalysing the removal of phosphate from a protein

Question 12

Types of kinases

Answer 12

1. Phosphorylate tyrosine residues
2. Phosphorylate serine/threonine residues

Question 13

Which enzymes are switched on/off in response to insulin and glucagon?

Answer 13

Insulin: Glycogen synthase - on
Glycogen phosphorylase - off
Glucose -> Glycogen

Glucagon: Glycogen phosphorylase - on
Glycogen synthase - off
Glycogen -> Glucose

Question 14

What controls enzyme regulation?

Answer 14

Levels (rate of biosynthesis/degradation)
Activity
Location

Question 15

Reversible covalent modification

Answer 15

Quickly regulates enzyme activity in response o a signal (eg hormone)
Most common form = phosphorylation

Question 16

Phosphorylation

Answer 16

The covalent addition of a phosphate, transferred from ATP by the action of kinase(s)
Process is reversible; removal of phosphate carried out by phosphatases

Question 17

How does phosphorylation affect enzyme activity?

Answer 17

Alters the 3D conformation of the protein by changing the electrical charge, from -ve (phosphoryl group) to +ve (salt bridges with arginine or lysine residue)

Question 18

Rate determining step

Answer 18

The slowest step of a chemical reaction that determines the speed/rate at which the overall reaction proceeds

Question 19

Rate limiting steps of glycolytic pathway

Answer 19

1. phosphorylation of glucose by hexokinase or glucokinase
2. the phosphorylation of fructose-6-phosphate to form fructose-1, 6-bisphosphate by phosphofructokinase (PFK-1)

Question 20

PFK-1 reaction

Answer 20

The main regulatory point of glycolysis and represents the true rate-limiting step .
Coupled to ATP hydrolysis and is essentially irreversible
Different pathway necessary to do the reverse conversion during gluconeogenesis

Question 21

Allosteric modulation

Answer 21

Allows enzymes in a metabolic pathway to respond to signals from other pathways. 2ndary form of control.
/
Enzyme regulation where other molecules bind to a site other than the active site (allosteric site), alters enzyme’s activity.

Question 22

Molecules which potentiate…

Answer 22

one direction (glycolysis) are often negative regulators of the other direction (gluconeogenesis)

Question 23

Type 1 diabetes

Answer 23

10% of diabetes cases
Hereditary
Caused by destruction of pancreatic Beta-cells due to an autoimmune process or unknown aetiology. Insulin is not produced.

Question 24

Type 2 diabetes

Answer 24

90% of diabetes cases
Caused by a combination of lifestyle factors and genetics
Results from a defect in insulin action, with insulin resistance as a root cause.

Question 25

Hibernation

Answer 25

Prior to hibernation, period of high caloric intake and low exercise, would cause Type 2 diabetes in human. Hibernating animals switch on/off insulin resistance in response to changing day length to ensure blood sugar homeostasis all year. Mediated by gene expression of 8 genes (Akt)

Question 26

Migratory birds

Answer 26

Put on an incredible amount of fat before migration (0.15g of triglyceride/day/g o body weight = 10kg/day in humans). Allows for muscle growth also. Fuels long distance flight

Question 27

First

Answer 27

Insulin was the 1st protein sequenced Nobel Prize for Fred Sanger

Question 28

Preproinsluin formation

Answer 28

1. Ribosome sees mRNA and starts to translate the polypeptide 2. Signal sequence recognised by the Signal Recognition Particle (SRP). Translation paused 3. SRP guides the whole complex to the ER membrane where SRP receptors bind SRP 4. SRP receptor passes ribosome to complex of proteins called Sec61 translocon complex. Translation resumes 5. Preoproinsulin peptide is co-translationally passed through membrane and into lumen of ER. Signal sequence cut off by signal peptidase 6. Proinsulin formed

Question 29

Pre > Pro > I

Answer 29

Preproinsulin is an unshapen polypepetide chain containing a signal sequence, insulin region, connecting polypeptide, insulin region. Signal sequence cleaved off, disulphide bonds form between insulin regions. Carabiner shaped. Proinsulin Removal of connecting polypeptide -> insulin

Question 30

RSSR Oxidative Folding

Answer 30

Disulphide bonds are important in the folding and stability of some proteins secreted into the extracellular medium. Since most cellular compartments are reducing environments, disulphide bonds are unstable in the cytosol

Question 31

Why is RSSR Oxidative folding important?

Answer 31

Newly synthesised insulin will only fold and be oxidised in the lumen of the ER. Disulphide bonds fold the molecules into its biologically active form. So the cell needs to separate insulin from the cytosol so it can adopt structure

Question 32

Pulse-chase experiments in ER

Answer 32

Pulse: Cells exposed to radioactive amino acids so newly made proteins are labelled Chase: Radioactive amino acids removed which allows labelled proteins to be tracked

Question 33

Secretion

Answer 33

Constitutive secretion is constant Regulated secretion is modulated

Question 34

Process of gene expression to insulin secretion, pt1

Answer 34

1. Preproinsulin translated on ribosomes and is inserted across the ER membrane 2. Inside the ER processing begins, the pre-sequence is cleaved by signal peptidase. Disulphide bonds form between A and B chains which stabilises the 3D structure. Proinsulin 3. Proinsulin trafficked to Golgi via vesicles

Question 35

Process of gene expression to insulin secretion, pt2

Answer 35

4. Proinsulin traffics through the Golgi, packed into secretory granules together with an enzymes called pro hormone convertase PC1/3, PC2, and Carboxypeptidase E. 5. Proinsulin cleaved into insulin and C-peptide by pro hormone convertase PC 1/3 and PC2. Carboxypeptidase E removes basic residues at the C-terminus. These enzymes are activated in the acidic environment of immature secretory granules

Question 36

Process of gene expression to insulin secretion, pt3

Answer 36

6. High levels of insulin packed into secretory vesicles by making a crystaline complex with Zn2+ ions selectively pumped into the secretory vesicles. Net result: very high insulin concentrations in specialised packages 7. In response to a suitable signal, secretory granules fuse with the plasma membrane and release their content (insulin and C-peptide)

Question 37

Why is the insulin process so complex?

Answer 37

Different compartments mediate different functions to correctly process insulin. Can packages into specialised 'secretory vesicles' at very high concentrations and regulate them easily. Compartmentalisation leads to specification and hence greater efficiency.

Question 38

Lets talk about charge baybee

Answer 38

1. Glucose enters beta-cell via GLUT2 2. Glycolysis and respiration lead to increase in ATP 3. Increased ATP closes ATP-sensitive K+ channels. Membrane depolarisation 4. Depolarisation opens voltage-gated Ca2+ channels allows calcium to enter the cell 5. Calcium triggers the fusion of insulin granules with the plasma membrane. Insulin secreted

Question 39

Glucose transporters are...

Answer 39

facilitative diffusion transporters. No ATP required

Question 40

Alternating conformation model

Answer 40

Refers to the conformation change for glucose transporters. Alternate between being outward and inward facing shapes. Glucose binds, conformation change, glucose released inside cell

Question 41

GLUT types and rates

Answer 41

12 types in humans 1 solute molecule at a time. Gluts have a turnover rate of 17000-20000/min 10^6 ions per second

Question 42

GLUT2

Answer 42

Low affinity glucose transporter expressed in the Beta-cell plasma membrane

Question 43

Michaelis-Menten Curve

Answer 43

Represents both a catalysed enzyme reaction But also a velocity/substrate concentration curve for a transporter protein

Question 44

V0 =

Answer 44

Vmax [S] / Km + [S] max velocity x glucose conc. / conc of substrate/enzyme where at 1/2 Vmax + glucose conc.

Question 45

High/Low Km

Answer 45

Low = high affinity - transporter binds glucose easily - works nearly at full speed even when glucose is low - not ideal for sensing change High = low affinity - transporter binds glucose only when at high conc - needs more glucose to work efficiently - good for sensing change (rate changes proportionally with glucose conc)

Question 46

GLUT3

Answer 46

Expressed in tissues out with the pancreas eg in brain Has a high affinity for glucose (low Km), ensuring neurons receive a constant glucose supply, even in hypoglycaemic conditions

Question 47

Why does GLUT2 have a high Km?

Answer 47

Low affinity necessary to ensure Beta-cells can release insulin proportionally to the rising glucose levels

Question 48

ATP relationship with insulin production

Answer 48

Glucose phosphorylation in beta-cells uses the low affinity/high Km enzyme glucokinase. Which is not saturated as physiological glucose, so phosphorylation rate directly proportional to intracellular glucose

Question 49

Hexo/gluco - kinase

Answer 49

Hexokinase: high affinity for glucose, constant activity. (Primarily phosphorylates glucose into glucose-1,6- phosphate) Glucokinase: low affinity which allows glucose sensing in Beta cells

Question 50

ATP and intracellular conditions of beta cells

Answer 50

1. ATP levels are lower in Beta-cells so ATP-sensitive potassium channels are usually open. +ve intracellular environment 2. As ATP rises the channels close and the plasma membrane depolarises 3. Which opens voltage-gated calcium channels, allowing rapid influx of Ca2+ ions 4. Which triggers the fusion of insulin-containing secretory vesicles with the plasma membrane

Question 51

Compartmentalisation

Answer 51

- Insulin only folds in the ER - Insulin is only correctly processed in secretory vesicles/granules - High concentration stored by Zn-mediated 'crystalline' array - Secretory granules are 'lined up' ready to be released - Ca channels are nearby, generating a rapid influx of Ca at exactly the right place

Question 52

Insulin receptors

Answer 52

Are found in fatty tissues, the liver and muscles, they're specific to insulin, transmembrane and held together by disulphide bonds. Made up of 2 extracellular alpha-subunits. Binding of insulin to these causes a conformational change in alpha. Which transmits a signal to the Beta-subunits. Activates an intrinsic tyrosine kinase within the cytosolic domain.

Question 53

Amplification

Answer 53

A signal molecules of insulin activates a kinase that can phosphorylates many, many target molecules

Question 54

Autophosphorylation

Answer 54

The receptor phosphorylates itself. Tyrosine kinase receptors phosphorylates specific Tyr residues within the receptor These p-Tyr residues recruit signalling molecules to the receptors which are then themselves phosphorylated Which forms a signalling complex, which selectively recruits molecules (SH2)

Question 55

SH2

Answer 55

Src Homology 2 domains bind to P-Tyr residues via 'pocket' Bind phosphotyrosine via second binding site pocket that recognise amino acids near it Specificity dictated by the surrounding sequence in the target (receptor) polypeptide.

Question 56

How is specificity achieved?

Answer 56

Specific ligand binds to specific receptor. Autophosphorylation of specific tyrosine residues Recruits SH2 domain-containing proteins to the receptor Each SH2 domain has a 2nd binding pocket that recognises the amino acids surrounding the P-Tyr Different cells express different SH2 proteins

Question 57

Akt

Answer 57

Key downstream target of SH2 activation. Activates glycogen synthesis

Question 58

GLUT4

Answer 58

Glucose transporters specific to fat and muscle cells. Only expressed in the presence of insulin. Normally exists inside specialised vesicles - GSVs. Insulin binding -> Akt-ivation -> GSVs move to cell membrane

Question 59

How to identify GLUT4 expression

Answer 59

GLUT4 fused to GFP on one of its intracellular loops fused to an epitope tag tambien Epitope tag = blue fluorescence

Question 60

Akt effect on fat and liver

Answer 60

Fat: Activates GLUT 4 trafficking to the cell surface. Lipid droplet formation Liver: Phosphorylates and activates protein phosphatase 1, which promotes glycogen storage

Question 61

Insulin modulates gene expression

Answer 61

Insulin signals through 1RS1 to a variety of transcription factors that control gene expression Insulin controls the activity of FOXO1 transcription factor (1 of many) in liver cells FOXO1 is phosphorylated by Akt, exported from the nucleus and gluconeogenesis is suppressed. Other transcription factors are regulated indirectly through Akt-dependent pathways.