biochemistry Flashcards
list stages of erythropoiesis starting with hematopoetic stem cell
hematopoetic stem cell megakaryocyte erythroid progenitor (MEP) proerythroblast (pronormoblast) early erythroblast intermediate erythroblast late erythroblast reticulocyte RBC
define Howell-Jolly Bodies and what do they indicate
nucleus/DNA fragments
indicative of spleen dysfunction
diameter of RBC vs diameter of capillary
RBC: 8 micrometers
capillary: 5-10 micrometers
how many days are RBC in circulation
90-120 days
list the function of the following in RBC: Band 3 Glut1 aquaporin 1 actin and tropomyosin spectrin/ankyrin/actin complexes
band 3: anion channel that mediates Cl-/HCO3 exchange, also tethers the membrane and spectrin protein substructure to provide elasticity
Glut1: glucose transporter
aquaporin 1: transports water
actin/tropomyosin: allow the membrane to be actively distorted in an ATP-dependent manner
s/a/a/ complexes: create a network for stability, deformability, and flexibility to the membrane (allows biconcave shape)
2,3-bisphosphoglycerate (BPG) shunt
synthesis of 2,3-BPG from 1,3-bisphosphoglycerate (intermediate of glycolysis) via bisphosphoglycerate mutase (BPGM)
function of 2,3-BPG in RBC
allows hemoglobin to hand-off oxygen to myoglobin by competitively binding hemoglobin and stabilizing the T sate
pentose phosphate shunt in RBC
glucose –> ribulose-5-phoshpate –> G3P –> F6P
makes NADPH to maintain/reduce glutathione to reduce H202 and oxygen free radicals
methemoglobinemia
Fe3+ (oxidized) is stabilized and bound to hemoglobin bc either hemoglobin or oxidizing agents are mutated
can be controlled by methemoglobin reductase and NADH, patients are treated with methylene blue which acts as a reducing agent
result of PK deficiency in RBC
insufficient ATP synthesis
result of G6P deficiency in RBC
insufficient NADPH production
How is hemoglobin able to pass O2 to myoglobin if they both have same affinity for O2
BPG
how does pH and carbon monoxide affect hemoglobin binding curve
acidic conditions (<7.4) right (lower affinity) basic conditions (>7.4) left (higher affinity)
carbon monoxide shifts to left, carbon monoxide binds (to the Nterminus of hemes) with higher afftinity and increases O2 affinity of hemoglobin because puts it in the R state, but will not reach as high of a max because will never be completely saturated with O2
sickle cell: mutation and result of mutation
glutamate (charged, -) mutated to valine (nonpolar, noncharged) results in HbS which creates hydrophobic patch and complementary binding to normal beta globin subunit on other hemoglobin causing polymerization
differentiate: homozygotic HbSS, HbS beta-0 thalassemia, HbSC disease, HbS/hereditary persistance of fetal hemoglobin (S/HP-HP)
homozygotic HbSS: sickle cell anemia, 100% HbS
HbS beta-0 thalassemia: severe double heterozygote for HbS and beta-0 thalassemia, almost indistinguishable from sickle cell anemia phennotypically (MCV low) (thalassemia is no synthesis or partial synthesis)
HbSC disease: double heterozygote for HbS and HbC, with intermediate clinical severity (HbC causes defects in beta, as well, also resulting in lower soulbility of hemoglobin)
HbS/hereditary persistence of fetal hemoglobin (S/HP-HP): mild form or symptom free because fetal hemoglobin reduces effects of the mutant beta subunits of hemoglobin in the HbS
describe the goal of the different approaches for treating RBC diseases: hydroxyurea, endari, voxoletor, bone marrow transplantation, gene therapy and gene editing
hydroxyurea: increases HbF and hemoglobin production
Endari: L-glutamine (precursor for glutatione), boosts the production of NADH
voxelotor: increases hemoglobin’s affinity for oxygen, blocks polymerization of HbS
Bone marrow transplant: can cure the disease
gene therapy and editing: use CRISPR to cut specific sequences in DNA to potentially cure the disease
RQ. what proteins compose the membrane and substructure of erythrocytes, and what are key functions of the RBC membrane?
proteins: spectrin, ankryin, actin, tropomyosin, tropomodulin, band 3, Glut1, aquaporin, Na+/K+ ATPase, CA2+ ATPase, GPA, GPB, GPC/D, Duffy, Kell, etc.
gas exchange and flexibility to fit through capillaries
RQ. what metabolic pathways are used in erythrocytes and what are the key modifications with respect to normal pathways?
glycolysis and pentose phosphate pathway
BPGM converts 1,3 BP glycerate –> 2,3 BPG (this induces release of O2)
RQ. what is the structure, function, and regulation of hemoglobin?
tetramer of heme, carries oxygen, regulated by BPG, pH, and carbon monoxide
how do diseases like SCD impact hemoglobin and RBC function?
SCD creates hydrophobic pocket in hemoglobin which binds to normal beta subunit of other hemoglobin resulting in polymerization, this polymerization of hemoglobins caused a sickle shape in the red blood cell –> decreased flexibilityto fit through capillaries
based on biochemistry, what clinical observations would you make concerning patients with SCD?
high counts of reticulocytes in circulating blood, swollen spleen, ischemias due to blocked capillaries, higher risk of infection, autosplenectomy
what are current and future therapuetics for SCD and RBC diseases?
hydroxyurea, endari, voxelotor, bone marrow transplant, gene therapy and gene editing, avoid low oxygen situations
glycolysis in RBC: steps and fates of products
glucose –> G-6-P –> F-6-P–> F-1,6-DP –>GA3P –>1,3-DPG –>3-PG –>2-PG–> PEpyruvic acid –> pyruvic acid –> lactic acid (dumped in liver) (step pyruvic acid –> lactic acid oxidizes NADH back to NAD+)
OR use 2,3-bisphophoglycerate shunt to convert 1,3 DP GLY to 2,3-BPG which is used for unloading oxygen to muscle cells
steps of pentose phosphate pathway in RBC
glucose –> 6-p-gluconolactone –> 6-p-gluconate –> ribulose 5-P –> ribose -5-P –> GA3P –> F6P
*NADP is reduced to NADPH by G6P –> Ribulose 5P step
structure of flutathione
Glu, Cys, Gly
structure of embryonic Hb, where is it made
2zeta/2epsilon; Hbepsilon
yolk sac
fetal Hb: structure and production site
HbF, 2alpha/2gamma
liver and spleen
adult Hb: structure and production site
HbA1: 2alpha/2beta (most common)
HbA2: 2 alpha/2delta
bone marrow
histidines and coordinating O2, Fe binding
there are 2 histidine molecules per heme
proximal histidine binds Fe which forces Fe out of its plane which allows O2 to bind then distal His stabilizes O2 through H-bond
BPG (DPG) affect on hemoglobin
decreases O2 affinity, increases offloading, and promotes T-state
binds the beta-beta interface (its negative charges bind positive charges of heme which stabilize T state)
what is the significance of fetal Hb having higher O2 affinity than adult Hb
fetus is able to strip O2 from maternal RBC due to its higher affitnity to O2
competitive antagonism
molecules competing for same binding site
CO2 –> bicarb equation
CO2 + H20 –> carbonic acid (via carbonic anhydrase- zinc dependent)
carbonic acid –> bicarbonate ion + H+ (no enzyme needed, pH naterually will remove this ion)
CO2 and heme’s affinity to O2
CO2 decreases O2 affinity but binds to different site on the heme
Bohr effect of O2 affinity
1.) lower pH –> less O2 affinity –> release of O2
higher pH –> higher O2 affinity
2.) CO2 reversibly covalent modifies terminal amino groups of alpha and beta chains which forms carbamino hemoglobin and reduces the hemes affinity for O2
both of these mechanisms make up the Bohr Effect
nitric oxide
acts as a potent vasodilator
produced by nitric oxide synthase from arginine and released to smooth muscles
hemoglobin facilitates transport of NO, NO binds to thiol of Hb when in the R state (oxygenated), NO is transported by glutathione otherwise (X-S-NO transporter)
when bound to Hb, its actions are inhibitted, thus NO is inactive in oxygenated states
source of heme precursors
succinyl CoA is precursor of heme which is product of TCA
rate limiting step of heme synthesis
d-aminolevulinic acid (ALA) synthase
converts succinyl CoA to delta-aminolevulninic acid (ALA)
only in mitochondria and has a short half life
steps of heme synthesis
mitochondira:
- succinyl CoA –> delta-ALA
- delta-ALA synthase
cytosol:
- delta-ALA –> porphobilinogen (BPG)
- ALA dehydrogenase - BPG –> hydroxymethyl bilane
- BPG deaminase - hydroxymethyl bilane –> uroporphyrinogen III and uroporphyrinogen I
- uroporphyrinogen III synthase - uroporphyrinogen III –> coprorphyrinogen III
- uroporphyrinogen decarboxylase
mitochondria:
- coproporphyrinogen III –> protoporphyrinogen IX
- coproporphyrinogen III oxide - protoporphyrinogen IX –> heme
- ferrochelatase
how are GCPR able to trigger different effects in different cell types
differences in intracellular proteins
describe a fast vs a slow GPCR mediated reaction
slow: altered protein synthesis (effects gene expression in the nucleus)
fast: altered protein function (proteins already exist)
GPCR activation of G protein (conformation changes)
antagonist binding to GPCR causes conformational changes in H3, H5, and H7 which causes a rotation in H6 which opens the G-protein interacting cleft in its C terminus
the open cleft binds and activates the G protein
cAMP activation of PKA
cyclic AMP (quickly syntehsized by adenyl cyclase) binds the 2 regulatory subunits sequestering 2 pKA subunits causing them to release and thereofre activate PKA
action of PKA in GPCR signaling
PKA enters nuclear pore and activates CREB to which promotes gene expression
list mechanism of cardiac beta 1 adrenergic GPCR signaling
adrenaline/noradrenaline/pharmacological agonist activates cardiac B1-adrenergic GPCR
GPCR activates Gs protein
Gs activates AC to produce cAMP
cAMP activates PKA
PKA phosphorylates SR and cell membrane Ca2+ chanels
phosphorylated channels open and rapidly increase CA2+ concentration in cytosol and increase in heart rate and contraction force
phsophorylated SERCA pumps Ca2+ into SR to make relaxation rapid
airway smooth muscle B2 adregernic receptor mechanism
agonist binds B2 adrenergic GPCR activating exchange of GDP –> GTP on Gs
Gs activates AC –> cAMP –> PKA
PKA –> decreased Ca2+ concentration through increased uptake by SERCA
myosin light chains are dephosphrylated
smooth muscles relax
dilation of airway
mechanism of cardiac muscarinic acetylcholine GPCR
vagus nerve release acetylecholine
acetylcholine binds muscarinic receptor
Gi protein activated and inhibits AC and activates K+ channel
rate and force of heart contraction decreased
mechanism of bacterial toxins (ex. cholera) on GPCR
cholera toxin ADP-ribosylates alpha subunit of Gs
alpha subunit cannot hydrolyze GTP and permanently active
AC –> cAMP–> PKA
phosphorylates chloride channels in intestinal epithelial cells causing efflux of Cl- and water into gut
severe diarrhea
Gq protein in GPCR signaling
active Gq activates phospholipase C-B which splits diacylglycerol and IP3 from eachother
IP3 initiates Ca2+releasae from ER
Diacylglycerol remains on membrane and binds protein kinase C which is activated by the released Ca2+
carbon donor for cholesterol synthesis
acetate
rate limiting step of cholesterol synthesis
HMG-COA reductase converts HMG-CoA –> mevalonate
describe steps of mevalonate –> activated isoprene in cholesterol synthesis
3 steps of adding phosphates from ATP creating a good leaving group to remove a CO2 and phosphate leaving behind a double bond in the activates osoprenes
describe steps of activated isoprene –> squalene in cholesterol synthesis
6 activated isoprenes are sequentially added together, kicking off 2 phophates each time. final is reduced and 2 phosphates are removed resulting in 30 carbon long squalene
describe steps from squalene to cholesterol
cyclase closes rings in a multistep process results in 27 carbon cholesterol
ATP and acetyl CoA requirements of cholesterol synthesis
18 acetyl CoA, 18 ATP
where is cholesterol primarily made
liver
how does statin affect cholesterol synthesis
statin blocks HMGCoA therefore blocks choelsterol synthesis
how does SREBP effect chollesterol synthesis (also how is SREBP regulated)
high levels of cholesterol block SREBP from entering the nucleus but during low levels of cholesterol it enters the nucleus and binds to enhancer region which is an activator to the promotor region of genes encoding HMG-CoA reductase
aka: SREBP promotes cholesterol synthesis when cholesterol is low
regulation: rapidly degregated by ubiquitylation and proteasome; SCAP cleaves SREBP allowing its release from the ER (this is blocked by sterol)
SREBP also upregulates LDL receptor expression so promotes cholesterol uptake
regulation of cholesterol synthesis by AMPK
AMPK is activated via phosphorylation by LKB1 +AMP (low ATP), and CAMKK + Ca++ (which are high during muscle contraction)
AMPK is dephosphorylated (and deactivated) by PP2C which is inhibited by PP1
AMPK phosphorylates HMG-CoA reductase which makes it inactive
AMPK inhibits cholesterol synthesis
regulation of cholesterol synthesis by cAMP
glucagon and epinephrine both increase production of cAMP
cAMP phosphorylates PKA, PKA phosphorylates PP1, PP1 inhibits HMG-cOA reductase phosphatase from removing phosphate from HMG-CoA reductase, HMG-CoA reductase remains inactive
cAMP inhibits cholesterol synthesis
cholesterol regulation by ACAT
ACAT is activated in high cholesterol levels
ACAT enhances esterfication of cholesterol for storage
define amine
compounds and functional groups that contain basic nitrogen and a lone pair of valence electrons
alpha amino acid group
NH2 functional group of amino acids
ammonia
NH3
in the human body, ammonium (NH4+) is formed from ammonia (mainly in the ammonium form in the body which cannot cross membranes)
what enzyme transfers NH3+ from amino acid alanine to alpha keto gluterate
ALT (alanine amino transferase)
results in glutamate and pyruvate
what coenzyme is needed for all aminotransferases
pyridoxal phosphate (PLP)
what enzyme transfers NH3+ from aspartate to alpha keto gluterate
AST (apartate amino transferase)
makes glutamate and oxaloacetate
net result and mechanismof malate-aspartate shuttle
moving NADH into the matrix for oxidative phosphorylation
malate-alpha KG transporter: moves malate in and alpha KG out
glutamate-aspartate transporter: moves aspartate out and glutamate into the matrix
in cytosol: aspartate is converted to oxaloacetate and then malate; glutamate converted back to alpha ketoglutarate by AST
in matrix: opposite of cytosol (when malate –> oxaloacetate NADH is released into matrix)
how is NH3+ from muscle transported in the blood to the liver
alanine (done by adding NH3 from glutamate to pyruvate via ALT)
what removes NH4+ from glutamate and where in liver
GDH (glutamate dehydrogenase) removes ammonium from glutamate via oxidative deamination (uses NAD –> NADH) in the liver yeilding alpha KG
how is NH4+ from extrahepatic organs (not muscle) transported through the blood to the liver
GS (glutamine synetase) uses ATP to add a NH4+ to glutamate making glutamanine which travels to the liver
how is NH4+ removed from glutamine in the liver
in the mitochondria, GLS (glutaminase) removes NH4+ from glutamine, yeilds glutamate
formation of carbamoyl phosphate
NH4+ added to HCO3- via carbamoyl phosphate synthetase I (CPSI)
how does NH4+ leave the mitochondria to enter urea cycle in cytosol
formed into carbaboyl phosphate which is added to ornithine via OTC (ornithine transcarbamoylase) in mitochondria to make citrulline and citrulline can exit via ORC1 (orthanine enters mito through ORC1)
what activates CPSI
N-acetylglutamate which is activated by arginine (an intermediate of the urea cycle)
list steps of urea cycle that occur in cytosol
citrulline –> arginosuccinate via arginosuccinate synthetase and aspartate and ATP
arginosuccinate –> arginine and fumarate via arginosuccinase
arginine –> ornithine and urea via arginase
what is the only reversible step of urea cycle
arginosuccinase converting arginosuccinate to arginine and fumarate
what links the CAC to urea cycle
apartate-arginosuccinate shunt of citric acid cycle
citrulline from urea cycle + aspartate from citric acid cycle = argininosuccinate via argininosuccinate synthetase
arginosccinate is broken into fumarate (which is converted into malate via cfumarase and reenters CAC) and arginine which stays in urea cycle
what mechanism catches NH4+ that passes the periportal hepatocytes
perivenous hepatocytes are scavenger cells that convert NH4+ into glutamine via GS which can be secreted
what happens to glutamine during metabolic acidosis
glutamine is used for glucose synthesis in metabolic acidosis, broekn into bicarb and NH4+ in kidney which nutralizes acid
how do BUN levels change in both liver and kidney disease
BUN is low in liver disease
BUN is high in kidney disease
define essential amino acids
essential amino acids must be obtained as dietary protein
non essential amino acids
S-serine A- alanine N-asparganine D- aspartate E- glutamate
*pneumonic: SAND-E
essential amino acids
T- threonine V- valine W- tryptophan M- methionine I- isoleucine L- leucine K- lysine F- phenylalanine H- histidine
*pneumonic: TV W/ MILK F(or) H(im)
define and list only glucogenic amino acids
can be converted to glucose arginine glutamine alanine cysteine glycine serine glutamine glutamate histidine proline methionine valine aspartate asparginine
define and list the only ketogenic amino acids
can be converted into ketone bodies
leucine
lysine
which amino acids are both ketogenic and glucogenic
isoleucine threonine phenylalanine tyrosine tryptophan
4 types of passive transport
diffusion
facilitated diffusion
filtration
osmosis
NA/K- ATPase
binds 3 Na+ inside the cell ATP binds and changes conformation releases 3 Na+ to outside binds 2 K+ on outside of cell dephosphorylation chanfes conformation release 2 K+ inside the cell
proximal tubule: transporters on apical surface
Na/H antiporter
Na/x symporter (glucose, aa, lactate, phosphate, chloride)
CL/base antiporter (HCO3, oxalate, formate, and OH)
aquaporins
SNAT (glutamine/Na symporter)
Ca channel
proximal tubule: transports on basal side
Na/K ATPase Na/HCO3- symporter aquaporin chanels for glucose, aa, lactate, phosphate, chloride K/Cl symporter Na/Ca symporter Na/glutamine symporter
how is glutamine/NH3 handled in early proximal cells
glutamine is absorbed from lumen and interstium via SNAT (Na/glutamine symporter) glutamine enters mitochondria where NH4 is removed by glutaminase (GS) and NH4 is removed frome glutamate by glutamate dehydrogenase (GDH)
NH4 –> NH3 + H and both enter the lumen
glutamate –> alpha KG –> succinyl CoA–> oxaloacetate –> 2PEP –> glucose: enters interstitium via GLUT
descending loop of Henle
water reabsoprtion aquaporins
UT-A2 transporter urea excretion
NH3 excretion
ascending loop of Henle
Na/K ATPase
CLC-K1: chloride channel on both membranes (extremely high Cl- permeability and absorbtion in this area)
impermeable to H20
tons of ion reabsorbtion: Na, Cl, HCO3, K, Ca, Mg
Na/K/2Cl cotransporter (apical)
ROMK (apical, returns K back in lumen)
Na/H antiporter
Bartter syndrome
- NA/K/2Cl mutation
defective sodium, chloride and potassium reabsorption
hypokalemia, hypochloremia, metabolic alkalosis, and hyperreninemia with normal blood pressure
excessive urinary losses of Na, Cl, K - ROMK mutation
inability to recycle potassium from the cell back into lumen so inhibits Na/K/2Cl with same effects as above - CLC-kb mutation
inability of Cl to leave the cell into the blood, same effects as above
early distal tubule
Na+/K+ atpase basal Na+/Cl- sympoter apical impermeable to water TRPV5: Ca channel apical Na/Ca antiporter basal (Na in, Ca out) Ca ATPase: basal (Ca out) CLC-kb channels (basal)
principal cells in late distal tubule and collecting duct
K+/Na ATPase ENAC : Na channels apical ROMK: apical (K out) AQP2 aquaporins controlled by ADH K+ channels basal (K out) passive transcellular Cl
alpha intercalated cells distal tubule and collecting duct
H+ ATPase apical (H out) H/K ATPase apical (H out) H/Na antiporter apical (H out) Cl- channel apical (Cl- out) HCO3/Cl antiporter basal (HCO3 out) Na/K ATPase K and Cl channels basal, out NH4/Na ATPase (NH4 in)
Hensin/DMBT1
deletion of Hensin/DMBT1 prevents conversion of beta to alpha intercalated cells and induces distal renal tubular acidsis
beta intercalated cells distal tubule and collecting duct
pendrin: apical Cl/HCO3 exchanger, apical RhCG: secrete NH3 apical Na and K channels apical Cl- channel basal (Cl out) H+ ATPase basal (H out)
aldosterone regulation of principal cells
enhances Na/K ATPase and ENAC
aldosterone regulation in intercalated cells
activates H+ ATPase (increases H secretion)
Aldosterone H+ ATPase stimulation pathway
aldosterone –> G alphaq + PLC + Ca2 –> PKC +DOG –> ERK + Ca –> H+ATPase
or
aldosterone + cAMP–> PKA –> PKC +DOG –> ERK + Ca –> H+ATPase
which aquaporins are apical and which aquaporins are basal
apical: AQP2
basal: AQP3/4
osmotic diuretics
inhibit reabsorption of H2O in proximal tubule
ed. mannitol
carbonic anhydrase inhibitors
inhibit Na+ aborption by blocking carbonic anhydrase (bc Na/H antiporter)
ex. acetzolamide
loop diuretics
inhibit Na,Cl, K symporter to reduce Na absorption in loop of Henle
ex. furosemide, bumetanide and ethacrynic acid
thiazide diuretics
inhibit Na aborption by blocking Na and Cl- symporter in early distal tubule
ex. chorothiazide and metolazone
potassium sparing diuretics
inhibit K secretion by antagonizing Na channel or aldosterone action (ENAC in late distal tubule and collecting ducts)
ex. sprionolactone, amiloride, triamterene
