Biochem Flashcards
1
Q
What are the main proteins involved in anchoring actin to the extracellular matrix?
A
Dystrophin (largest gene in the body) connects to a,b-Dystroglycan (part of the sarcoglycan complex in the sarcolemma) which links to laminin via O and N-linked sugars (Perlecan and agrin proteins also present)
2
Q
What causes scar tissue build up in DMD patients?
A
The lack of dystrophin causes the muscle cells to tear, and fibroblasts fill in the space with collagenous scar tissue
3
Q
Defects in a,b-dystroglycan or O and N-linked sugars leads to what?
A
Milder forms of MD
4
Q
What two molecules are important in regulating the polymerization of actin?
A
ATP and Mg2+
5
Q
Which proteins control the ordering of F actin into higher order structures? What causes the biconcave shape of red blood cells?
A
Actin nucleating and actin binding proteins
F actin and spectrin
6
Q
Describe actin treadmilling
A
ATP-Gactin binds to the + end of the F actin and polymerizes. The ATP becomes hydrolyzed leaving Pi and ADP. When the Pi leaves the actin molecule (leaving only ADP), the Gactin leaves the chain (depolymerizes)
7
Q
Thymosin B4, cofilin, profilin, tropomodulin, and CapZ
A
Cofilin binds to F actin whose subunits contain ADP. It breaks off a chunk of actin from the minus end which creates more minus ends making depolymerization easier. Profilin facilitates the exchange of ADP for ATP on G actin. Thymosin B4 sequesters ATP G actin if its concentration gets too high and releases it for polymerization when necessary. CapZ binds the plus end of actin to prevent polymerization. This action is modulated by signal transduction molecules. Tropomodulin binds to the minus end and stabilizes F actin
8
Q
What is formin?
A
Dimeric protein (2 horseshoes) which stimulates the assembly of long, unbranched F actin (good for stress fibers and contractile rings)
9
Q
Where are branched actin filaments found? What protein stimulates the branched formation? What activates it?
A
On the leading edge of migrating cells; WASp activates Arp2/3 by causing a conformation change. This induces the branched formation of actin (the whole process is controlled by signal transduction).
10
Q
Osteogenesis Imperfecta (OI)
A
Caused by genetic disease that causes malformation of collagen. Characterized by easily broken bones
11
Q
What is the amino acid sequence in collagen that causes the triple helix structure? What are the two other amino acids usually?
A
Gly-X-Y. The X and Y are usually proline and hydroxyproline. Proline prevents a-helixes and hydroxyproline increases the thermal stability of collagen
12
Q
Are there intrachain or interchain h bonds in collagen triple helixes?
A
Interchain, no intrachain (no a-helixes)
13
Q
What does scurvy do to your hair?
A
Perifollicular hemorrhage and corkscrew hairs (as well as anemia, bleeding gums, and general weakness)
14
Q
What are the fibril forming collagens? Where are they found?
A
Types I,II, and III. Type I is most abundant and found in connective tissue. Type II is in cartilage and vitreous humor. Type III found in extensible connective tissues (lungs, blood vessels, skins, etc.)
15
Q
Desmosine and Isodesmosine
A
Cross-link proteins that allow elastin to return to its original conformation
16
Q
What are the four steps of cell migration?
A
Extension (lamellipodium juts out), adhesion, translocation (cell mass), and deadhesion and endocytotic cycling
17
Q
Describe the mechanism by which a cell migrates. What are the roles of growth factors, Rad, Cdc42, Arp2/3, and Rho?
A
A growth factor acts as the chemotactic molecule. It binds to a receptor which causes a conformational change in Rad and Cdc42 receptors allowing GTP to bind. This causes a signal cascade which activates Arp2/3 causing branching and polymerization at the leading edge of the cell (lamellopedia). At the back of the cell, Rho binds GTP and activates foramin to polymerize stress fibers. It also activates Rho kinase which turns on myosin II and causes cell contraction.
18
Q
What do fimbrin, a-actinin, and spectrin all have in common? What are their individual responsibilities?
A
They are all actin linking proteins. Fimbrin organizes actin in microfilaments. a-actinin organizes actin in stress fibers and muscle cells. Spectrin helps form a cytoskeletal meshwork below the plasma membrane.
19
Q
What protein links cytoskeletal proteins to the extracellular matrix and helps form focal adhesions?
A
Integrins
20
Q
What happens in hereditary spherocytosis?
A
RBCs become fragile due to a defect in spectrin, a-actinin, ankyrin, protein 4.1, or Band 3 and glycophorin. Can lead to enlarged spleen and gallstones due to breakdown of RBCs
21
Q
Describe Osteogenesis Imperfecta. What type of inheritance is it?
A
Autosomal Dominant disorder characterized by bones that break easily due to the body’s malformation of collagen. Symptoms include multiple bone fractures, triangle shaped face, blue sclera of the eyes, hearing loss that begins in the 20s, smooth skin, low muscle tone, scoliosis, loose joints, brittle teeth.
22
Q
What is the role of Vitamin C in collagen production?
A
Vitamin C acts as a cofactor (helping stabilize ferrous iron) for the 4-prolyl-hydroxylase mediated conversion of proline to hydroxyproline
23
Q
What makes Glycosaminoglycans hydroactive?
A
The water molecules interact with negative charges on the uronic acid and sulfate groups
24
Q
What are the general functions of the extracellular matrix?
A
Provide a barrier against cell migration, bind and release growth factors, and provide signals to cells during morphogenesis, would healing, and maintenance of the differentiated state
25
What are the proteoglycan and multiadhesive proteins that make up the basement membrane?
Perlecan, and fibronectin/laminin
26
What are the repeating disaccharide units of GAGs?
Uronic acid and an amino sugar
27
What is aggrecan’s special function in cartilage? What are the GAGs that make it up? What does it like to complex with? What growth factor does it bind? What is osteoarthritis?
Aggrecan complexes with HYALURONIC ACID and secures high levels of GAGs that retain water in the ECM. Under high pressure, the water is released then returns when pressure is released. This lubricates the joint.
Complexes with hyaluronic acid.
Binds TGF-B
Osteoarthritis is when the GAGs are broken down and released from the cartilage
28
What must occur for a cancer cell to metastasize in the ECM?
It must express proteinases that can break down collagen IV to get into basal lamina
29
What binds growth factors in the ECM?
Proteoglycans
30
Where is it found and what does it do— aggrecan, decorin, perlecan?
Aggrecan found in cartilage. Binds type II collagen, lubricates joints, binds TGF-B to inhibit ECM synthesis.
Decorin found in all types of ECM. Binds collagen type I and TGF-B to limit the size of collagen fibers.
Perlecan found in basal lamina. The GAGs attached to perlecan prevent proteins from leaving the serum into the urine
31
What kinds of binding sites does fibronectin have? What is the RGD sequence?
Has binding site for collagen I, fibrin, heparin sulfate, hyanluronate, and gangliosides. RGD (arginine, glycine, arginine) sequence is recognized by cell receptors for fibronectin— such as integrins
32
How do enzymes increase the rate of reaction? Does it change the equilibrium of a reaction?
The substrate lowers the energy required to achieve the transition state (makes the hill smaller). They are catalysts that increase the rate but DO NOT CHANGE THE EQUILIBRIUM
33
What is Vmax? What is the Michaelis-menton equation? What are the assumptions?
Velocity at infinite (or very high) substrate concentration (when all enzymes are saturated with substrate)
Vo = Vmax*[S]/Km + [S]
Assumes: [S] is much higher than [E]; concentration of [ES] does not change; [P] is low under initial velocity conditions
34
The (-) end of a microtubule is entirely ringed with which tubulin monomer?
a-subunit (+ end is ringed by B-tubulin)
35
Which polar end of microtubules has a lower critical concentration (for polymerization)?
The (+) end has a lower critical concentration. If the concentration is between the high and low concentrations, treadmilling occurs
36
What are the three types of specificity that enzymes exhibit?
Substrate specificity, reaction specificity, and TISSUE specificity
37
What does bond formation have to do with how enzymes work?
Enzymes make weak bonds (anything besides covalent) with a substrate and use that energy to put the substrate in a more favorable conformation for rxn.
38
Enzymes only change which energy state?
The transition energy state. the substrate and product energy states remain the same
39
The free energy of favorable rxns are positive or negative?
Negative (- change in G)
40
How do TAMIFLU and HIV protease inhibitors work? (Transition state analogues)
They mimic the perfect ‘transition state’ molecule and thus bind enzymes very tightly. TAMIFLU binds neuraminidase as a competitive inhibitor and stops the flu from separating from its host and spreading.
41
What is a transition state?
It is the highest free energy point in a rxn. At this point, the formation of substrate or product is equally as likely.
42
Oxidoreductases catalyze which type of rxn?
Transfer of electrons (from carbs or fats to NADs and FADs, for example)
43
What are the ‘mobile electron carries’ and what are the ‘protein bound electron carriers’?
M- NAD+, NADP+
PB- FAD+, FMN
44
Which atoms can be oxidized and reduced in our cells?
Carbon, nitrogen, and sulfur
45
How does Vo change with enzyme concentration? What about substrate concentration?
Vo increases linearly with changes in enzyme concentration. As substrate concentration changes, Vo increases but tapers off at a limiting value that is characteristic of each enzyme
46
What line is tangent to the reaction rate line?
The initial reaction rate
47
What is Km?
It is the [S] at which 1/2Vmax occurs. In a way, it demonstrates the affinity of the enzyme for the substrate. A lower Km means substrate more readily binds to the enzyme
48
Why does it make sense that Hexokinase I (brain) has a lower Km than Hexokinase IV (liver)
the brain can’t store glucose so it needs a high affinity in order to use glucose that is even in relatively low concentration. In the liver, glucose is only metabolized after a large meal (otherwise it gets stored)
49
What is kcat?
It is the rate constant that describes the limiting rate of any enzyme reaction. It equals Vmax/enzyme concentration (units are sec^-1). Basically, how many units of substrate are being reacted per enzyme per second
50
What is the lineweaver-Burke double reciprocal equation? What are the x and y intercepts? What is the slope?
Y=MX+B —> 1/Vo= (Km/Vmax)(1/[S]) + (1/Vmax). x intercept is -1/Km and y intercept is 1/Vmax. Slope is Km/Vmax
51
Competitive inhibition
Competes with the substrate in the active site itself. Increasing substrate concentration reverses inhibition
Km gets larger (1/Km comes closer to zero) with this inhibition, but Vmax DOES NOT CHANGE
52
Mixed inhibition
Inhibitor binds away from active site to either E or ES complex
Km increases (1/Km gets closer to zero) and Vmax decreases (1/Vmax gets farther from zero)
53
Uncompetitive inhibition
Inhibitor binds away from active site only to ES complex
Km gets smaller (1/Km gets farther from zero) and Vmax decreases (1/Vmax gets farther from zero)
54
Noncompetitive inhibition
Special kind of mixed inhibition in which Km does not change but Vmax decreases (1/Vmax gets farther from zero)
55
Irreversible Inhibitor. What is a mechanism-based/suicide inhibitor?
An inhibitor that COVALENTLY binds to an enzyme thus inhibiting its active site
A suicide inhibitor is inactive until it gets activated by an enzyme to which it then covalently binds
56
Commitment step
A step/reaction that commits a metabolite to a certain fate (ex, the first step of glycolysis. Prior to this, glucose could become many different things)
57
Allosteric inhibitors such as feedback inhibitors for glycolysis exhibit which type of inhibition? What type of inhibition do Organophosphate pesticides perform?
Mixed inhibition
Noncompetitive inhibition (special case; both E and ES have same affinity for binding inhibitor)
58
Serpins are which type of inhibitor? Give two examples
Suicide inhibitors
A1-antitrypsin inhibits trypsin and elastase to prevent too much break down of elastin in the lungs and liver. If someone is deficient in A1, they can develop emphysema at a young age.
Antithrombin III inhibits thrombin to prevent blood clot formation
59
How does allosteric regulation work?
Allosteric enzymes always have multiple subunits
Changes in one region of a protein affect the conformation (and ability to bind a substrate) in another region of the protein
Depending on the enzyme, this can be inhibitory or stimulatory
Often, the product of a metabolic pathway is the allosteric inhibitor for that pathway (mixed inhibition). An example is CTP as an inhibitor for the pathways that converts aspartate to CTP
60
Which side of the protofilament is negative in cilia/flagella? How do the microtubules generate movement? Where in the body is cilia found?
The minus end is anchored in the basal body while the plus end is projecting outward. Dynein proteins hold the A tubule as cargo and motor along the B tubule of the adjacent doublet.
Cilia is found in the respiratory tract, Fallopian tubes, spermatozoa, and middle ear
61
What is situs inversus totalis? Who has it?
When all the internal organs are mirror images of where they should be. 50% of people with primary ciliary dyskinesia have Kartegener syndrome
62
When is centrosome duplication complete? What is the spindle pole? What are Aster MTs?
Before mitosis begins
Spindle pole is the name of the MTOC during mitosis
Aster MTs are the first mitotic microtubules to appear after the interphase MTs have broken down. They anchor the spindle poles to the cell membrane
63
What happens during prometaphase?
The nuclear envelope breaks down and the microtubules capture the chromosomes by attaching the + end of the MTs to the kinetochore proteins of the chromosome and bring them to the cell equator
64
What happens during Anaphase? What are the two movements occurring?
Cohesins holding the chromosomes together at the centromere degrade. The kinetochore MTs start to disassemble which pulls the chromosomes towards the poles. Meanwhile, the poles are physically separating pulling the cell apart in two
65
What is the double movement occurring during prophase?
Dyneins anchored to the cell membrane walk towards the - end of the MTs attached to the spindle pole which pulls the pole towards the cell perimeter. At the same time, double headed kinesins in the center of the cell are walking towards the center which pushes the MTs apart
66
How do Kinesins and dyneins work to align chromosomes during metaphase?
If the chromosome needs to move to the right, the left MT will grow and kinesins holding the kinetochore as cargo will move along the growing + chain (pushing the chromosome to the right) while dyneins will move towards the - end on a shrinking right side microtubule (pulling the chromosome into position)
67
What happens during Anaphase A and Anaphase B?
Anaphase A, the kinetochore attached MTs shrink at both the + and - ends simulataneously thanks to kinesin 13s (which destabilize the MT). This pulls the chromosomes towards the spindle poles
In Anaphase B, the same process that occurred in prophase (the double movement pushing the spindles apart) continues to occur
68
What does the critical concentration of tubulin dimers mean?
When placed together, a and b tubulin monomers spontaneously form dimers. At a certain concentration, dimer formation plateaus because microtubule formation starts to occur spontaneously
69
Which is more stable GTP bound or GDP bound B tubulin? Which occurs at which end?
GTP is more stable and GTP bound B tubulin is found at the + end which is why disassembly usually occurs at the - end
70
What happens to the GTP cap of a microtubule if the concentration of GTP bound B tubulin falls below the critical concentration of the + end?
The GTP cap is not maintained, the GTP becomes hydrolyzed, and catastrophe occurs (splaying of the + end of the microtubule)
71
Is microtubule polymerization GTP powered?
No, but B tubulin monomers must be GTP bound to polymerize to the new microtubule
72
When does “treadmilling” occur in MTs?
When the concentration of dimers is > the Cc+ but < Cc-
73
How is Taxol used to treat cancer?
It binds to B-subunits of tubulin and stabilizes it (makes it so microtubules can’t destabilize).
74
What are MT associating proteins (MAPs)? What are the basic and acidic domains? What happens if you phosphorylate the basic domain?
MAPs are proteins that help stabilize MTs by neutralizing its negative charge. The basic domain is positively charged and attaches to the negative side chains of tubulin. The acidic domain is negatively charged and helps maintain the distance between MTs (the distance maintained is characteristic of each MAP). Phosphorylation would neutralize the basic domain making it less likely to bind MTs. MTs become destabilized.
Example: MAP2 and TAU
75
Kinesin 13, Stathmin, Katanin
MT destabilizing proteins. Kinesin 13 bends the protofilaments at the + end promoting catastrophe to occur
Stathmin also bends the protofilament but removes two dimers at once
Katanin breaks through the middle of a MT creating newly exposed GDP tubulin which promotes catastrophe
76
Colchicine, Vinblastine, Podophyllotoxin, Nocodazole
All drugs used to treat cancer by affecting MTs
Colchicine- sequesters tubulin dimer pool to prevent polymerization of MTs and promote catastrophe
Vinblastine/Podophyllotoxin- destabilize MTs
Nocodazole- prevents spindle assembly during G2 prior to mitosis
77
How do neurofibrillary tangles form in Alzheimer’s disease? What does Pin 1 have to do with it?
In neurons, the TAU protein is a MT stabilizing protein that can aggregate if its basic domain becomes hyperphosphorylated. The hyperphosphorylation causes TAU to change shape and form tangles. Pin 1 is a molecule that changes cis proline residues on the phosphothreonine-proline motif back to trans (trans is much less likely to be phosphorylated). If Pin 1 is defective or not present, cis TAU proteins are more likely to occur and create tangles.
78
MTOC, spindle pole, centrosome, basal body
All different names for the point of origination of microtubules in a cell
79
How do kinesins accomplish anterograde movement?
The motor heads bind to the B tubulin subunits and swivel from subunit to subunit with each step requiring hydrolysis of an ATP molecule
80
How do dyneins accomplish retrograde movement?
Dyneins have a stem, head, and stalk (stalk attaches to the MT). The head has ATPase function and when it hydrolyzes an ATP molecule it causes a conformational change that moves the stalk and allows it to rebind farther down the MT
81
What is the handoff that occurs during exocytosis?
Kinesins move vesicles to the cell periphery and handoff the cargo to myosin proteins on microfilaments that take it the rest of the way
82
What are the first five reactions of glycolysis called? What do they require? What do they yield?
Preparatory phase. Require 2 ATP. Yields two Glyceraldehyde-3-phosphate molecules
83
What is the enzyme of the first reaction in glycolysis? What is it called in the liver? How do their Km values compare?
Hexokinase. This enzyme converts glucose to Glucose-6-phosphate. In the liver it is called glucokinase and it has a higher Km (lower affinity) than elsewhere
84
What is the commitment step of glycolysis? What enzyme facilitates the reaction? What is required for the reaction to occur?
Step 3 which is the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate. ATP is required to phosphorylate and push the rxn uphill. Up until this point, glucose-6-phosphate could feed into the Pentose phosphate pathway or glycogen synthesis, but after this step it is bound for glycolysis. The enzyme is phosphofructokinase-1 (PFK-1)
85
What is substrate flux? Why is it relevant in regards to glycolysis?
When a reaction is delta G positive (or close to zero), the product or reactant could form just as readily, but if the product is used up quickly it shifts the equilibrium to favor forming the product. In glycolysis, the second step (formation of fructose-6-phosphate from glucose-6-phosphate) and the fourth step (DHAP isomerization to G3P) are both substrate flux reactions
86
Muscle fat liver pancreas brain
This is where we talk most about metabolism
87
What it is enzyme responsible for the conversion of fructose 1,6-bisphosphate to G3P and DHAP?
Aldolase
88
What is step 6 of glycolysis? What is the enzyme? Byproduct? How many times does the rxn happen?
Oxidation of G3P to 1,3-bisphosphoglycerate (requires inorganic phosphate).
G3P dehydrogenase
Requires NAD+ and makes an NADH
1 glucose makes 2 G3P so it happens twice
89
What is the high priority rxn occurring in step 7 of glycolysis? What is the enzyme involved?
Substrate level phosphorylation (ADP converted to ATP by breaking a high energy phosphate bond from another substrate) as 1,3-bisphosphoglycerate is converted to 3-phosphoglycerate.
Enzyme is phosphoglycerate kinase
(Remember this rxn happens twice!)
90
What is step 9 of glycolysis?
Dehydration of 2-phosphoglycerate by enolase to form the very high energy phosphoenolpyruvate
91
What is the last step of glycolysis? What enzyme is used? What can be said about the relative energy states of the substrate and product?
Phosphoenolpyruvate is hydrolyzed to pyruvate with the help of pyruvate kinase. ATP is made from ADP and is considered a lower energy molecule than phosphoenolpyruvate.
Remember that this rxn happens twice per 1 glucose molecule!
92
Where does ALL red blood cell energy production come from?
Anaerobic respiration/glycolysis (because they don’t have mitochondria)
93
Why might blood hemorrhaging lead to someone becoming light headed?
Loss of blood (or decreased function of RBCs) would cause hypoxia (low oxygen conditions) which means that cells would halt oxidative phosphorylation and instead convert pyruvate to lactate via anaerobic respiration. In brain cells that require constant supply of glucose, this pathway is highly inefficient. Brain cells don’t get enough ATP and light headedness ensues.
94
Where are GLUT 1, 2, 4, and 5 found? What are their Km’s?
GLUT 1- every tissue. 1-2mM (means it is always very active)
GLUT 2- found only in liver and pancreatic tissue. 15-20mM (needs high blood glucose)
GLUT 4- muscle and fat cells. 5mM (insulin sensitive though)
GLUT 5- small intestine mucosa and spermatozoa. 10mM (fructose transport)
95
How does GLUT 2 influence insulin secretion from pancreatic cells? Are GLUT 2 receptors influenced by the insulin secretion?
GLUT 2 receptors take up glucose when blood glucose is high (Km=15-20mM). That glucose becomes converted to ATP. The ATP binds to K+ channels causing them to shut. Because K+ can’t leave, the membrane becomes depolarized. This depolarization causes voltage gated Ca++ channels to open. The influx of Ca++ triggers the release of insulin vesicles.
GLUT 2 is not influenced by insulin secretion
96
How do anti-hyperglycemia drugs (sulfonylureas, meglinitides) and anti-hypoglycemia drugs (Diazoxide) function?
Anti-hyperglycemia drugs bind the K+ channel from the outside closing it. This results in insulin release.
Anti-hypoglycemia drugs keep the K+ channel open inhibiting insulin release
97
What is type I diabetes? What happens when a type I diabetic doesn’t take their insulin?
Type I diabetes is complete absence of insulin production
Ketoacidosis occurs. If insulin isn’t released into the blood, muscle and fat cells don’t activate GLUT 4 receptors and instead of making pyruvate via glycolysis, triglycerides are broken down then sent to the liver where they are converted into ketone bodies (which are acidic)
98
How do Hexokinase I-III and Hexokinase IV differ?
Hexokinase I-III have a very small Km so they very readily convert glucose to G6P. They are regulated by G6P via allosteric regulation
Hexokinase IV has a higher Km consistent with the higher Km of GLUT 2 receptor. When gluconeogenesis or glycogenolysis need to occur, PFK-1 in the liver is switched off causing fructose-6-phosphate to build up which causes glucokinase to be sequestered in the nucleus
99
How is PFK-1 regulated?
ATP and citrate are allosteric inhibitors of PFK-1 while ADP and AMP are activators. Fructose 2,6-bisphosphate is the most potent activator of PFK-1
100
What is the bifunctional enzyme that ultimately regulates the actions of PFK-1 and thus the third (committing) step of glycolysis?
The PFK-2/FBPase-2 enzyme has two domains. Glucagon phosphorylates the enzyme causing activation of the FBPase-2 domain which converts Fructose 2,6-bisphosphate back to Fructose-6-phosphate which causes inhibition of PFK-1 and activation of FBPase-1 (converts F1,6-bisphosphate back to F6P).
Insulin causes dephosphorylation of the enzyme which activates PFK-2 thus causing fructose-6-phosphate to be converted to Fructose 2,6-bisphosphate which activates PFK-1 causing F6P to become F16BP and thus glycolysis goes forward
101
How is pyruvate kinase regulated?
Upregulated by the presence of substrates such as ADP and PEP.
Upregulated by Fructose 1,6-bisphosphate (the product of the third step of glycolysis)
Downregulated by molecules representing well-fed state (ATP, acetyl-coa, long chain fatty acids)
Downregulated by Alanine which can be converted from pyruvate
Inactivated in liver tissue by phosphorylation as a result of glucagon-PKA signaling
Activated in liver tissue by dephosphorylation as a result of insulin-phosphatase signaling
102
Which enzyme is most responsible for hemolytic anemia?
Pyruvate kinase
103
What is the role of 2,3-bisphosphoglycerate? Why is its formative path significant?
2,3BPG causes hemoglobin to have a lower affinity for oxygen (so it dumps more oxygen in the tissues). 2,3BPG increases when someone is in an area with lower pO2.
Formation of 2,3BPG skips the ATP production step that occurs when 1,3BPG is converted to 3-phosphoglycerate. If a RBC is too ATP deficient then it can cause malformation of the cell and hemolytic crisis
104
How do 2-deoxyglucose, iodoacetamide, arsenate, and fluoride function to inhibit glycolysis?
2-deoxyglucose— goes through first step of glycolysis but cannot be converted to fructose-6-phosphate
Iodoacetamide— reacts with free sulfylhydryls G3P dehydrogenase and inhibits its function
Arsenate— phosphate analogue that undergoes G3P dehydrogenase rxn then breaks down
Fluoride— inhibits enolase rxn
105
What do fimbrin, a-actinin, and spectrin do?
Fimbrin organizes actin into microvilli
A-actinin stabilizes actin in stress fibers
Spectrin creates a cross-linking network of actin filaments to support the plasma membrane
106
What do integrins do?
Cross-link cytoskeletal proteins to the extracellular matrix. Anchor cells to the ECM via focal adhesions. Generate intercellular signals
107
Where is desmin, GFAP, vimentin, and lamin found?
Intermediate filaments. Found in muscle, glial cells, mesenchymal cells, and nuclei.
108
What are the major players of calcium sensitization (physiology)
Normally, myosin light chain is activated by MLCK and inactivated by MLCP. Inhibiting MLCP can prolong the effects of an IP3/Ca2+ mediated muscle contraction by leaving myosin light chain activated. CPI-17 and ROK inhibit the MLCP. Extracellular signals like vasopressin, catelcholamine, angiotensin induce this pathway.
109
What are the 4 enzymes specific to gluconeogenesis that allow it to circumvent the irreversible steps of glycolysis? What are their reactions? Where do they occur?
Pyruvate kinase converts pyruvate to oxaloacetate. It requires biotin as a cofactor and input of ATP and CO2. This occurs in the mitochondria
PEP carboxykinase converts oxaloacetate to PEP and requires GTP. Occurs in cytosolic and mitochondria.
Fructose 1,6-bisphosphatase converts F1,6BP back to Fructose-6-phosphate. Occurs in cytosol.
Glucose 6 phosphatase converts G6P back to glucose. Occurs in the ER.
110
Where can gluconeogenesis occur?
Kidneys, liver, intestinal epithelium (adipose tissue has two of the four necessary enzymes)
111
During gluconeogenesis, what keeps the newly formed glucose from undergoing glycolysis?
Hexokinase IV has a Km that is too high to bind such small amounts of glucose substrate, therefore the glucose simply leaves the cell
112
Most of the glucose made during gluconeogenesis comes from which Amino acid?
Alanine (which can be converted pyruvate)
113
What is the total energy requirement of gluconeogenesis?
2 pyruvate + 4ATP + 2 GTP + 2 NADH + 2H+ + 2H2O
1 ATP and 1 GTP required for the first two steps (pyruvate to PEP). 1 ATP required to take 3 phosphoglycerate to 1,3BPG. 1 NADH to take 1,3BPG to G3P.
114
What effect does glucagon have on gluconeogenesis and glycolysis?
Glucagon triggers phosphorylation of stuff which lowers F2,6BP (reducing PFK-1 activity), and phosphorylation of pyruvate kinase leads to an increase in PEP (which is much more energetically preferred for gluconeogenesis).
115
What effect does Acetyl-CoA have on gluconeogenesis?
Acetyl CoA is an allosteric activator of pyruvate carboxylase (first step of gluconeogenesis)
116
What effects do insulin/glucagon have on gene transcription?
High insulin to glucagon ratio upregulates transcription of glycolysis enzymes like Hexokinase, PFK-1, and pyruvate kinase
High glucagon to insulin ratio stimulation transcription of gluconeogenesis enzymes like PEP carboxykinase, Fructose 1,6 bisphosphatase, and Glucose 6 phosphatase.
117
What is glyceroneogenesis? Where does it occur?
Adipose (and liver) contains the first two enzymes of gluconeogenesis and uses them to make glycerol 3 phosphate. This glycerol can esterify fatty acids to make triglycerides.
This is important in regulating the amount of free fatty acid in the blood because it can reduce the body’s sensitivity to insulin.
118
What does muscle not do in regards to glycogen storage that liver cells do?
Muscle cells do not have Glc 6 phosphatase so they cannot elevate blood glucose. Instead the glycogen is broken in G6P which goes straight into glycolysis
119
What is the first step of glycogen synthesis? What is the enzyme involved? Is it reversible?
Conversion of G6P to G1P via phosphoglucomutase. Yes it is easily reversible for making glucose
120
When is glycogen synthesis most likely to occur?
During the well fed state when glucose levels are high
121
What are the linkages in glycogen? Where is new glucose added?
a-1,4 linkages make long chains and a-1,6 linkages create branches. New glucose gets added to the non-reducing end in an a-1,4 linkage
122
How is glucose “activated” in the second step of glycogen synthesis? What is the enzyme involved? Where does the energy for this rxn come from?
Glucose 1 phosphate is activated by UDP pyrophosphorylase. UTP comes along, gets attacked by G1P creating UDP glucose (activated) and pyrophosphate (PPi). The pyrophosphate is hydrolyzed which drives the rxn forward.
Net rxn: G1P + UTP —> UDP Glc + 2 Pi
123
What is the third step of glycogen synthesis? What enzyme catalyzes this step? What happens if there is a problem with this enzyme?
Glucose is added to a glycogen chain at the 4 carbon end of the chain (non-reducing side). Catalyzed by glycogen synthase.
If problem with glycogen synthase, you have Type 0 glycogen storage disorder. Liver glycogen goes down causing blood glucose to go down and ketone bodies to increase (leads to death).
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Fourth step of glycogen synthesis, enzyme, and disease if deficient? Why is this step important?
Glycogen chains are branched. Glycosyl 4,6 transferase moves a few glucose molecules down and attaches them via an a-1,6 linkage.
Increases the number of non-reducing ends for rapid addition or cleavage when needed
Increases number of glucose molecules stored for potential energy use
Makes the molecule more soluble
Deficiency in Glycosyl 4,6 Transferase is called Andersen’s storage disease which causes hepatomegaly, splenomegaly, and break down of muscle cells which results in myoglobin in the urine.
125
De novo glycogen synthesis starts with which protein?
Glycogenin. Has intrinsic enzymatic activity
126
What is the first step of glycogenolysis? Enzyme? Cofactors? Deficiency?
Breaking G1P molecules off of glycogen via Glycogen phosphorylase (pyridoxal phosphate, PLP, is a cofactor). Deficiency in muscles causes McArdle (Type V) syndrome and in the liver causes Hers (Type VI) syndrome. Leads to muscle cramps and muscle pain and hepatomegaly
127
What is the second step of glycogenolysis? Enzyme? Deficiency?
Debranching enzyme debranches the a-1,6 linkages.
2 functions: 1) when the residue has 4 glucose molecules left, Glucosyl 4,4 transglycosidase takes the first three and puts them on another long chain. 2) a-1,6 glucosidase hydrolyzes the one branched residue into GLUCOSE (not G1P).
Deficiency in debranching enzyme causes Cori or Forbes (Type IIIa) disease. Results in hepatomegaly and myopathy. A liver specific deficiency in this enzyme causes Type IIIb and results in hepatomegaly in infants
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What is the last step of glycogenolysis?
G1P gets converted back into G6P by phosphoglucomutase
129
What is Pompe’s disease?
Lysosomal glucosidase is defective. Affects skeletal and cardiac muscle and causes death within two years in infants (in adults, it looks like muscular dystrophy)
130
What is Von Gierke’s?
Glycogen storage disease in which Glucose 6 Phosphatase is defective in the LIVER. Causes an enlarged liver and kidney failure (this affects the last step of glycogenolysis in which G6P gets converted to Glucose)
131
What is the mechanism by which glucagon (epinephrine) and insulin regulate glycogen synthesis and glycogenolysis?
Glucagon triggers PKA to phosphorylate Phosphorylase Kinase which then phosphorylates Glycogen Synthase (inactivating it) and Glycogen Phosphorylase (activating it).
Insulin triggers dephosphorylation and thus reverses the roles, activating G synthase and inactivating G phosphorylase in the well-fed state
132
When glucagon and epinephrine trigger phosphorylation of Glycogen synthase and Glycogen phosphorylase, what happens to the resultant product? What allosteric activators can upregulate this process?
Glucose 6 Phosphate immediately undergoes glycolysis in muscle cells or gets converted to Glucose by hepatic cells and sent into the blood.
Ca2+ and AMP are both allosteric activators along the signal transduction path leading to the phosphorylation of both enzymes
133
How do Insulin and Glucose act to dephosphorylate glycogen phosphorylase and glycogen synthase?
Insulin signaling stimulates Phosphorylase phosphatase 1 (PP1) whose job it is to dephosphorylate Glycogen Phosphorylase (inactivating it).
In the LIVER, the serine residues on Glycogen Phosphorylase are well protected, but Glucose can bind Glc Phosphorylase and cause a conformational change that spreads out the serines making them more susceptible to PP1
134
What activates/upregulates Glycogen Synthase? What inactivates it?
Insulin dephosphorylates glycogen synthase, activating it.
Glucose and G6P are allosteric activators of Glycogen Synthase
Glucagon/epinephrine cause Glycogen Synthase Kinase 3 to phosphorylate and inactivate it.
135
What is the role of the core protein in a proteoglycan?
The core protein attaches certain GAGs in the ECM that bind ligands. The core protein is embedded in the plasma membrane and transmits the signal to the interior of the cell
136
What is the most abundant multiadhesive matrix protein? What are some others? What are their main features?
Fibronectin. It binds type I collagen, fibrin, heparan sulfate, and integrins. It plays a large role in wound healing.
Laminin. Important in basal lamina. Made by epi- and endothelial cells. First multiadhesive protein to show up during development.
VonWillebrand factor is another one
137
How many genes code for laminin? What can it bind?
8 genes code for laminin like proteins.
It is shaped like a cross and has domains that can bind collagen, sulfated lipids, neurite outgrowths (dendrites, axons), and LG domains that bind carbs and integrins (like when it binds a,b-dystroglycan
138
What proteins make up the basal lamina?
Fibronectin, laminin, perlecan, type IV collagen, entactin
139
What is a CAM? How many different types are there? Which are homophilic reactors and which are heterophilic?
Cell adhesion molecule (these interact with multiadhesive matrix proteins).
140
What role do selectins and integrins play in WBC extravasation (leaving the capillary to get into the tissues)?
Platelet activating factor (PAF) is released by platelets and causes Selectins to be exocytosed from capillary endothelial cells. WBCs recognize the PAF and bind to the cells via Selectin—>Glycoprotein interaction. The WBC is secured by binding to Integrins as well. The WBC can then extravasate between two endothelial cells.
141
What’s up with a and b subunits on integrins?
Integrins are heterodimers with a and b subunits. 19 different a’s and 8 different b’s. The binding specificity of an integrin depends upon its a and b subunits (which are both embedded in the plasma membrane via a SINGLE TRANSMEMBRANE HELIX). Many integrins require a specific amino acid motif be present in their ligand (like RGD in fibronectin)
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What are the possible integrin binding domains?
Collagen, laminin, fibronectin, leukocytes specific
143
Are integrins always active? What is their role in communication and cell motility?
No. In the example of WBC extravasation, they had to be activated by a selectin binding a glycoprotein.
After exposing a binding site and binding a ligand, the Integrin communicates in a bidirectional manner.
144
What affect does insulin have on muscle tissue metabolism? Which enzymes are involved?
Increased glucose uptake— GLUT 4 transporter
Increased glycogen synthesis— glycogen synthase
Decreased glycogen breakdown- inhibition of glycogen phosphorylase
Increased glycolysis/acetyl-CoA production— dephosphorylation of PFK-2 which makes F-2,6-BPG which stimulates PFK-1 (glycolysis moves forward); stimulates PDH complex which makes Acetyl CoA for the citric acid cycle
