-
metabolism
chemical changes (conversions) required to sustain life
-
anabolism
- biosynthesis
- gluconeogenesis: generation of glucose in response to decreased blood glucose levels metabolic opposite of glycolysis
-
catabolism
- biodegredation
- glycolysis: breakdown of glucose into pyruvate for energy (RBCs have no mitochondria)
- urea cycle: breakdown of protein into nitrogenous wastes for excretion
-
enzymes
- protein catalysts
- increase the rate at which a reaction reaches equilibrium but do NOT alter the ratios of a chemical reaction's products and substrates
- not altered themselves by the reaction in which they participate
-
chemical alterations involved in metabolism
- substrate availability: high concentration necessary
- allosteric modulation of enzyme activities: alters the dynamics at an enzyme's active site by binding with a secondary site on the molecule
- covalent modification: usually phosphorylation; alters an enzyme's activity
- changes in enzyme protein levels
-
well fed state
- glucose & amino acid delivery: absorbed by the intestine, carried by portal vein to liver
- fat packed by enterocytes in the gut as chylomicrons, carried by lymphatics into general circulation
-
primary constituents of metabolism
carbohydrates, lipids, and proteins
-
"energy charge"
energy availability as represented by levels of ATP, NADH, & NADPH
-
glucose metabolism during well fed state
- liver: glycogenesis will store glucose as the polymer glycogen for easy access later (through glycolysis)
- in other tissues: adipose tissue (fat); muscle (storage & energy); brain (ATP production); RBCs (glycolysis)
- lactate & pyruvate from glucose metabolism by other tissues incorporated into fatty acids by the liver
-
protein metabolism during well fed state
- hydrolyzed in the intestine so they can be absorbed as amino acid monomers or short polypeptides (asp, asn, glu, gln)
- the rest pass through the liver & will be used throughout the body for protein synthesis
- excess amino acids will be processed into fatty acids in lipogenesis or processed into urea & excreted
-
endogenous fat
predominantly manufactured in the liver & exported as very low density lipoprotein (VLDL)
-
exogenous fats
absorbed by the intestines and packaged into chylomicrons
-
lipoprotein lipase
breaks down VLDL and chylomicrons into fatty acids (triacylglycerols) for storgage in adipocytes
-
fasting state
- liver responsible for maintaining blood glucose levels
- glycogenolysis will release glucose from glycogen
- lipogenesis is curtailed
- gluconeogenesis will produce glucose in preparation for the depletion of liver glycogen stores (~12 hours of fasting)
-
sources for glucose synthesis
- glycerol from lipolysis in adipose tissue (minor source for gluconeogenesis)
- amino acids from muscle protein proteolysis (major contributor)
-
fat metabolism during fasting state
- large increase in lipolysis resulting in increased levels of fatty acids in the blood
- fatty acids are used by many tissues in preference to glucose (brain needs glucose)
- liver will increase fatty acid oxidation
-
overall metabolic changes that occur during starvation
- blood glucose decreases to a constant lower level (minimum requirement)
- free fatty acid levels increase somewhat
- ketone bodies increase dramatically in the blood
- total urinary nitrogen decreases
- urinary ammonia increases as a way to pull protons from the blood & counter the acidity of ketone bodies
-
common genetic disorders
- hereditary hemochromatosis (1 in 300)
- adult polycystic kidney disease (1 in 900)
- a1-antitrypsin deficiency (1 in 1700)
- cystic fibrosis (1 in 3000)
- neurofibromatosis 1 (1 in 3000)
-
direct to consumer marketing of DNA testing
- an increasing number of companies can provide DNA testing direct to consumers
- testing can be for paternity, disease gene identification or even for the appropriate diet
- it is not clear how that information will be used or even whether it will be understood
-
phenotype
the observed expression of the genes of an individual (expression can be biochemical, physiological or morphological)
-
genotype
- the genetic constitution of an individual
- the alleles present at a particular locus
-
alleles
alternative forms of a gene, or of a DNA sequence, at a specific position on a chromosome
-
gene locus
a specific location on a chromosome; often a specific gene
-
homozygous
both alleles at a locus are identical
-
heterozygous
if the alleles at a locus are different
-
human genetic variability
- 99.6% of DNA sequence is identical between two people
- the difference between any two people is about 24 million bp
- sequence variations are polymorphisms
- changes in the DNA that are pathologic are mutations
-
polymorphisms
sequence variations
-
mutations
changes in DNA that are pathologic
-
SNP
- single nucleotide polymorphism (>1% abundance)
- human genome has ~3 million SNPs distributed randomly
-
genetic heterogeneity
- different mutations can cause different or similar phenotypes
- allelic heterogeneity: different mutations at the same gene cause the same phenotype (cystic fibrosis)
- locus heterogeneity: different mutations at different loci cause the same phenotype (breast cancer BRCA 1 & 2)
-
penetrance
- the frequency with which the observable expression of a mutant gene manifests itself
- affected by: modifier genes, carcinogens, response to DNA damage & hormonal/reproductive factors
-
variable expressivity
- caused by mutations in genes that affect several organ systems & functions
- may involve modifier genes that alter the affect/expression of the mutation
- ex - Neurofibromatosis 1 (severity of affected parent does not indicate extent to which child will be affected)
-
examples of variable expressivity
- neurofibromatosis 1
- myotonic dystrophy
-
examples of genetic disorders/diseases
- single gene: CF, achondroplasia, hemophilia
- chromosomal: trisomy 21
- complex: diabetes, heart disease
-
monogenic (single gene) disorders
- mendelian diseases are the result of a mutation in a single gene
- the mutation alters the protein product in such a way that in can no longer function normally or may even be toxic
- the mutation can arise spontaneously (de novo) or it can be inherited
-
genetic traits
eye color, hair color, weight
-
symbols used in pedigrees
- open square: unaffected male
- open circle: unaffected female
- open square with diagonal line: deceased male
- open circle with diagonal line: deceased female
- any symbol with an arrow pointing to it: propositus
- filled-in square: affected male
- filled-in circle: affected female
- circle with a dot in the middle: carrier for X linked condition
- diamond: sex unspecified
- single line: mating
- double line: consanguineous mating
-
modes of inheritance
- autosomal: inheritance of a gene located on any of the 22 chromosomes other than X or Y
- X linked: inheritance of a gene located on the X chromosome
- Y linked: inheritance of a gene located on the Y chromosome
-
human genome project
- identifying all the genes in humans
- sequencing the DNA in the human genome (6 billion bases)
- studying the genomes of model organisms
- identifying the ethical and social issues caused by genomics
-
P4 medicine
- personalized, predictive, preventive, and participatory
- better diagnoses and earlier interventions
- more efficient drug development
- more effective therapies
-
nutrigenomics
genetic influence on response to certain foods, vitamins, etc.
-
microbiomics
- microbial flora can affect our body and health
- normal microbial flora in the human body outnumber human cells by 10:1
-
proteomics
large scale study of proteins
-
metabalomics
study of chemical processes involving metabolites
-
pharmacogenomics
- drug dosing/use based on genetics
- genetic variations in enzymes that metabolize the pro-drug into the active drug
- enzymes that metabolize drugs into a form that is inactivated/elimination
- variation in target receptors
-
laws of heredity
- heredity is determined by units or "factors"
- the units come in pairs
- the units come in different variant forms
- a form can be either dominant or recessive
- different factors are inherited independently of one another
-
autosomal dominant inheritance
- conditions that are expressed when just one allele is present (Aa)
- therefore, these conditions are expressed in heterozygotes
- direct transmission from an affected parent to an affected child
- transmission can occur from affected father to affected son
- approximately a 1:1 ratio of affected verse unaffected progeny with one affected parent
- equally transmitted by men & women
- no skipped generation
- each child has a 50% chance of inheriting the mutation
-
examples of autosomal dominant disorders
- achondroplastic dwarfism
- adult polycystic kidney disease
- Marfan's syndrome
- polycystic kidney disease
- neurofibromatosis 1 & 2
- polydactly
- Huntington's disease
-
achondroplasia
- affected individuals often choose similar mates (selective mating)
- when two affected individuals mate, there is a 50% chance of affected child, 25% chance of homozygous lethal, 25% chance of child with normal stature
- most common cause of dwarfism
- mutation in the fibroblast growth factor receptor 3 gene (FGFR3)
- normal intelligence
- recurrence risk is low if parent is not affected
-
adult dominant polycystic kidney disease
- autosomal dominant
- adult onset
- one of the most common inherited disorders in humans (1:900)
- accounts for 10% of all dialysis patients
- cysts form throughout the kidney
- causes progressive renal dysfunction and ultimately renal failure
-
clinical symptoms of adult dominant polycystic kidney disease
- symptoms begin in mid adulthood
- pain in the abdomen
- blood in the urine
- hypertension
- intense pain from kidney stones
- ~50% of patients with ADPKD have hypertension at the time of diagnosis
-
adult dominant polycystic kidney disease pathophysiology
- hepatic cysts (focal expression)
- cerebral aneurysms
- cardiac abnormalities
-
adult dominant polycystic kidney disease mortality/morbidity
- subarachnoidal hemorrage
- hypertension & stroke
- mortality due to intracranial aneurysms
- stroke
-
symptoms of neurofibromatosis 1
- neurofibromas: two or more, or one plexiform neurofibroma
- cafe au lait macules: six or more measuring 1/5 cm in their greatest dimension
- freckling: in axillary or inguinal areas
- sphenoid dysplasia: thinning of the cortex of the long bones
-
Marfan syndrome
- connective tissue disorder - molecular defect in Fibrillin 1 gene
- tall with long extremities
- archnodactyly
- hyperextensible joints
- dissecting aortic aneurysms
- subluxation of lens
-
autosomal recessive inheritance
- phenotype only seen in individuals homozygous for the mutant allele
- males & females equally affected
- most often seen - two individuals who are clinically normal produce an affected child
- heterozygous individuals are called "carriers"
- 1 in 4 chance of producing an affected child
-
examples of autosomal recessive disorders
- cystic fibrosis
- phenylketonuria
- hemochromatosis
- sickle cell anemia
- Tay-Sachs disease
- thalassemia
-
hereditary hemachromatosis
- most common genetic disease (13% of all Americans are carriers)
- about 1 in 200 people are affected
- iron overload syndrome: excess iron deposition in liver, pancreas, heart & other tissues
- no physiologic process to reduce excess iron
- symptoms begin at 30-50 years
- men more likely to be diagnosed and are affected at a younger age
- develop type 1 diabetes, liver sclerosis and cardiovascular problems
-
sickle cell anemia
- autosomal recessive inheritance
- most common genetic disease of African Americans (1/700)
- caused by mutation in hemoglobin B subunit
- skeletal problems
- infarctions
- anemia & repeated infections
-
symptoms of sickle cell anemia
- abdominal pain
- bone pain
- breathlessness
- fatigue
- fever
- delayed growth
-
sickle cell mutation
glutamate --> valine substitution (point mutation)
-
X inactivation
- in order to achieve balanced dosage for X linked genes, one of the two X chromosomes is inactivated
- begins as early as the 8 cell stage
- is always random, unless there is a structural defect in one of the Xs
- once the pattern of inactivation is set, it is preserved in subsequent cell divisions
- can be visualized as a Barr body
- female mammals are mosaics
-
X linked recessive disorders
- sons of carrier females have a 50% chance of inheriting the mutant allele
- examples include hemophilia, G6PD deficiency, Duchenne Muscular Dystrophy, and red/green color blindness
-
characteristics of X linked recessive disorders
- more affected males than affected females
- affected grandfather to affected grandson transmission through a carrier female intermediate
- no male to male transmission
-
hemophilia
- X linked recessive inheritance
- primarily males affected
- caused by mutations in either factor VIII or factor IX
- clotting disorder that causes bleeding into soft tissues
-
Duchenne muscular dystrophy
- most common form of MD
- mutation in the dystrophin gene
- largest gene
- males have 50% chance of being affected
-
Duchenne Muscular Dystrophy
- fatigue
- muscle weakness (begins in legs & pelvis, but occurs less severely in arms, neck & other areas)
- progressive difficulty walking (lose ability by age 12)
-
X linked dominant inheritance
- 50% of the children (both sexes) of an affected mother will be affected
- NONE of the sons of an affected man will be affected (they get the Y from him)
- males are usually more affected than females
-
hypophosphatemic rickets
- X linked dominant disorder
- most frequently encountered form of rickets
- consists of a genetic defect in the handling of phosphate in the proximal tubules of the kidney
- short stature, leg bowing, tooth anomalies
-
pathophysiology of hypophosphatemic rickets
- decreased reabsorption of phosphatate by the renal tubule (otherwise renal function is normal - BUN and Cr)
- decreased absorption of calcium & phosphorous from the GI tract
-
Rett Syndrome
- X linked dominant neurodevelopmental disorder
- male fetuses rarely survive
- slowed growth (microcephaly)
- loss of movement & coordination
- abnormal hand movements
- unusal eye movement
- irritability, seizures & scoliosis
-
mutations with sex-modified expression
genes involved have distinct function in males and females (ex. prostate cancer & milk production)
-
Y linked disorders
- passed from father to sons
- genes on the Y chromosome
-
mitochondrial segregation
- mitochondrial homoplasmy: all mutant or all normal
- mitochondrial heteroplasmy: mixture of mutant & normal
-
mitochondrial DNA diseases
- mitochondrial genome codes for only 37 genes and containing only about 16,600 base pairs
- mutation rate is about 20X higher than for nuclear genome (limited DNA repair capacity)
- damages: brain, heart, liver, skeletal muscles, kidney (tissue that have high energy capacity)
-
symptoms of mitochondrial DNA diseases
- nervous system: seizures, tremors, developmental delays, deafness, dementia, stroke before age 40, poor balance, problems with peripheral nerves
- heart: cardiomyopathy (heart failure, conduction block)
- liver: liver failure uncommon except in babies with mitochondrial DNA depletion
- kidneys: fanconi syndrome (loss of essential metabolites in urine)
- eyes: drooping eyelids (ptosis), inability to move eyes from side to side (external ophthalmoplegia), blindness (retinitis pigmentosa)
- skeletal muscle: muscle weakness, exercise intolerance, cramps
- digestive tract: acid reflux, vomiting, chronic diarrhea, intestinal obstruction
- pancreas: diabetes
-
dynamic mutations
- trinucleotide expansion disorders
- tandem repeats of three base pairs (trinucleotides) that often change size upon transmissioin to children
- Fragile X syndrome: outside the gene
- Huntington's disease: middle of gene
- Machado-Joseph Disease: intron
- myotonic dystrophy: end of gene
-
anticipation
- trinucleotide repeats expand with each successive generation
- age of onset becomes younger with each successive generation
- severity of disease increases with each generation
-
Huntington's Disease
- autosomal dominant inheritance (1/20,000)
- late age of onset (usually in 40s)
- symptoms: dementia, memory loss, choreic movements
- death of cells in the caudate nucleus
- mutation is a triplet repeat expansion of CAG
- diagnosis with PCR
- new mutation rate is 3%
-
Fragile X syndrome
- the most common heritable form of moderate mental retardation
- affected individuals have "fragile site" at Xq27.3 that can be observed with cytogenetic analysis
- moderately retarded males & mildly retarded females
- patients have behavior problems, hyperactivity and autistic features
- triplet repeat is CGG and is in the 5' untranslated region of the gene
- most physical features develop after adolescence; long face with prominent jaw, large ears & macroorchidism
- normal lifespan
-
Friedreich's ataxia
- inherited as autosomal recessive
- trinucleotide repeat of GAA
- symptoms begin in adolescence
- dysarthria: slowness and slurring of speech
- ataxia: impaired muscle coordination starts in legs & spreads to arms and trunk
- heart problems, muscle weakness & fatigue
- degenerative of peripheral nerves and spinal cord
-
fundamental structure of amino acids
- 20 common amino acids in proteins
- amino group (NH3+): protonated at physiological pH
- carboxyl group (COOH): unprotonated at physiological pH (forms the negatively charged carboxylate ion COO-)
- hydrogen ion
- R groups: side chains; distinctive for each amino acid; most important for charge characteristics of proteins
-
classification of amino acids
- nonpolar or hydrophobic
- polar or hydrophilic
- acidic (proton donors - negatively charged)
- basic (proton acceptors - positively charged)
-
nonpolar (hydrophobic) amino acids
- GAVLIMP-FW
- glycine: gly, G (simplest & smallest, only non-chiral)
- alanine: ala, A (methyl group side chain)
- valine: val, V
- leucine: leu, L
- isoleucine: ile, I
- methionine: met, M (sulfur side chain)
- proline: pro, P (secondary amine group; changes ability to fold)
- phenylalanine: phe, F
- tryptophan: trp, W (biggest side chain)
-
amino acids with uncharged polar side chains
- serine: ser, S
- threonine: thr, T
- tyrosine: tyr, Y
- asparagine: asn, N ("carboxamide" amino carbonyl)
- glutamine: gln, Q ("carboxamide" amino carbonyl)
- cysteine: cys, C (forms disulfide bond, interacts with water)
-
acidic amino acids
- aspartic acid: asp, D
- glutamic acid: glu, E
- side chains act as proton donors
- fully ionized (negatively charged) at physiological pH 7
-
basic amino acids
- histidine: his, H (largely uncharged)
- lysine: lys, K
- arginine: arg, R
- side chains act as proton acceptors
- at physiological pH, lysine & arginine are positively charged
-
sickle cell anemia
- hemoglobin is a tetramer of two alpha & two beta subunits
- point mutation (glutamate to valine) at position 6 in the beta-globin chain causes sickle cell anemia
- RBCs become misshapen & too rigid to move in the microvasculature
-
optical properties of amino acids
- chiral carbon (optically active): carbon that has four different moieties attached to it (all amino acids but glycine)
- D & L forms (mirror images): optically active pair called stereoisomers, optical isomers & enantiomers
- L form: eukaryotes
- D form: some bacterial proteins
-
essential amino acids
those amino acids that cannot be synthesized by humans & must be obtained from diet
-
which amino acids are hydroxylated?
serine, threonine & tyrosine
-
which amino acids are acidic?
glutamate & aspartate
-
which amino acids are basic?
arginine, lysine & histidine
-
which amino acids contain sulfur?
methionine & cysteine
-
which amino acid does NOT have a free amino group?
proline
-
which amino acids are nonpolar?
- GAVLIMP-FW
- glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan
-
which amino acids can form a disulfide bond?
ONLY cysteine
-
which amino acid has the smallest R group?
glycine (just a hydrogen)
-
which amino acids are polar?
serine, threonine, tyrosine, asparagine, glutamine, cysteine
-
acid/base properties of amino acids
- amino acids have a weakly acidic alpha carboxyl and weakly basic alpha amino group
- acidic & basic amino acids have ionizable side chains
- free amino acids & those in proteins can act as buffers
-
buffer
- a solution that resists changes in pH after addition of a strong acid or strong base
- created by mixing a weak base (A-, proton acceptor) & its conjugate acid (HA, proton donor)
- system capable of resisting pH changes
-
maximum buffering capacity
- when pH = pKa
- effective when pH of solution is +/- 1 of the pKa
-
dissociation constant
- Ka = [H+][A-] / [HA]
- a weak acid is mostly intact (weakly ionized)
- a strong acid is mostly dissociated
- bigger Ka: stronger acid
- smaller Ka: weaker acid
-
Henderson-Hasselbach Equation
- pH = pKa + log [A-]/[HA]
- pH: -log of [H+]
- pKa: -log of Ka
-
application of Henderson-Hasselbach equation
- useful in diagnosing metabolic or respiratory acidosis and alkalosis
- determining when a drug is charged will help determine best route of administration/dosing
-
primary protein structure
- the sequence/chain of amino acids linked by peptide bonds
- crucial in determining a protein's structure
- abnormal sequence can lead to misfolding/lack of function/disease
-
secondary protein structure
- primary structures linked by hydrogen bonds
- ex: alpha helices, B strands & B pleated sheets, B turns, non repetitive structures, supersecondary structures
-
peptide bond formation
condensation reaction: condensation of amino group (NH3+) of one amino acid with the carboxyl group (COO-) of another amino acid; removal of H2O molecule
-
peptide bond characteristics
- "partial double bond"
- no free rotation around peptide bond
- rigid & planar
- almost always trans
- uncharged but polar (hydrogen bonds possible)
- R group rotation around alpha-carbon is possible (exception - proline)
-
determining amino acid composition of proteins
- HCl hydrolysis: at high temp, breaks all peptide bonds (alters/destroys some amino acids)
- cation exchange chromatography: separates amino acids based on charge; eluted with solution of increasing ionic strength and pH
- heat with ninhydrin: measure purple color with spectrophotometer
-
Edman degradation
- beginning with N-terminal of protein, react with phenylisothiocyanate (Edman's reagent)
- forms unstable phenylthiohydantoin (PTH) amino acid derivative that can be released from protein without interupting other peptide bonds
- different PTH-aa derivatives can be identified
- reaction is sequentially repeated to ID first 100 aa in protein (NOT COMPLETE SEQUENCE)
-
preparation of overlapping peptide fragments
- enzymatic cleavage or chemical cleavage - specificity of peptide bonds broken
- trypsin: carbonyl (carboxyl) side of lysine & arginine (basic residues)
- chymotrypsin: carbonyl side of tryptophan, tyrosine, phenylalanine, methionine, leucine (hydrophobics)
- elastase: carbonyl side of alanine, glycine, serine (small side chains)
- cyanogen bromide (CNBr): breaks bond on carbonyl side of methionine
-
carbonyl
- carbon double bonded to an oxygen
- ex: aldehyde, keton, carboxylic acid
-
native gel electrophoresis
- used to determine whether there are differences in charges of proteins/peptides
- molecules placed in an electric field - separated by charge at pH used
- no denaturation
-
isoelectric focusing
- set up a pH gradient in a gel (or chromatography column)
- different proteins will stop migrating in an electric field when they reach their isoelectric point
- denaturation not necessary
-
Western Blotting
- separates proteins by their size using gel electrophoresis
- denaturation
- uses antibodies to ID proteins involved in HIV, Lyme disease & BSE, etc.
-
alpha helix
- most common secondary structure
- spiral structure, tightly packed, coiled
- held together by H bonds that are parallel to helical axis
-
alpha-helix breakers
- proline: imino structure; introduces "kink"
- highly charged amino acids: aspartate, glutamate, histidine, lysine, arginine introduce ionic bonds that disrupt the helix
- amino acids with bulky side chains: tryptophan physically impair formation of helix
- amino acids with branched side chains: valine, isoleucine physically impair formation of helix
-
beta-sheet
- composed of beta-strands
- stabilized by H bonds perpendicular to polypeptide backbone
- antiparallel: N terminal next to C terminal of other strand
- parallel: N terminals next to one another, C terminals too
-
interchain H bonds
hydrogen bond between two different polypeptide chains, each containing their own N & C termini
-
intrachain H bonds
hydrogen bond between amino acids in the same polypeptide chain
-
beta bend
- B-turn
- reverses the direction of a polypeptide chain
- helps form compact globular shape
- almost exclusively found on surface of protein
- composed mainly of charged amino acids - glycine & proline
-
nonrepetitive secondary structures
less regular coils or loops
-
motifs
- regular packing of other higher order structures
- "supersecondary structures"
- ex: beta-alpha-beta unit, Greek key, beta-meander, beta-barrel
-
allosteric inhibitors
preferentially bind to the T state
-
allosteric activators
preferentially bind to the R state
-
2,3 BPG
- 2,3 bisphosphoglycerate
- most common organic phosphate in RBCs (alternative intermediate in glycolysis)
- decreases oxygen affinity of hemoglobin
- binds to deoxyhemoglobin NOT to oxyhemoglobin
- stabilizes T form of hemoglobin
- released when oxygen binds
-
Bohr effect
- oxygen released from hemoglobin increased by lower pH or higher pCO2
- causes decreased O2 affinity & right shift of dissociation curve
- higher pH & lower pCO2 increases O2 affinity of hemoglobin
-
-
-
heme-heme interactions
- last oxygen is bound with 300X higher affinity than first oxygen
- lets more oxygen get to tissues with smaller change in pO2
-
oxygen binding by globular proteins
- myoglobin: binds one O2; hyperbolic curve
- hemoglobin: binds 1-4 O2; sigmoidal curve
- amount of bound oxygen depends on partial pressure of O2 (pO2)
-
forms of hemoglobin
- T: taut, deoxyhemoglobin; dimers bound tightly together (ionic, H bonds); low affinity for O2
- R: relaxed, oxyhemoglobin; less tightly bound, more movement; high affinity for O2
-
structure & function of hemoglobin
- RBCs: transport oxygen from lungs to capillaries; brings CO2 from tissues to lungs
- HbA: major adult form; tetramer of 4 polypeptide chains (alpha-beta)2 in a quaternary structure
- heme binding pocket & alpha helical structure of each subunit similar to myoglobin
- binds allosteric effectors & exhibits cooperative binding
-
structure & function of myoglobin
- heart & skeletal muscle: reservoir & transporter of oxygen in tissue
- one polypetide chain - 80% alpha helic (8 stretches)
- interior: all nonpolar amino acids; heme sits in crevice
- surface: charged amino acids
- proximal histidine: binds to iron in heme
- distal histidine: stabilizes binding of oxygen to heme
-
heme
- tightly bound prosthetic group
- a porphyrin with bound ferrous iron (Fe2+)
- six bonds of iron: four bind to nitrogens in porphyrin ring; 2 available
-
prion diseases
- creutzfeld jakob (humans)
- scrapie (sheep)
- bovine spongiform encephalopathy (cows)
- infectious agent - misfolded PrP protein
-
amyloidoses
- abnormal proteolytic cleavage can generate protein fragments that form beta pleated sheets & then associate to form long fibrous aggregates called amyloids
- ex. Alzheimer's & neurofibrillary tangles
-
protein denaturation
chemical reactions, heat, changes in solvent, heavy metals - disrupt the interactions of the secondary & tertiary structures
-
quaternary structure
- relationship of subunits to each other
- MUST HAVE MORE THAN ONE POLYPEPTIDE
-
protein folding
- all proteins are synthesized as linear sequence of amino acid residues
- N terminal to C terminal
- some polypeptide chains fold during synthesis to take up the native conformation
- protein folding can be complicated - may require chaperone proteins
-
interactions that stabilize tertiary structure
- disulfide bonds
- hydrophobic interactions
- hydrogen bonds
- ionic interactions (salt bridges)
-
tertiary proteins
- final 3D shape of protein (single polypeptide chain) built from secondary structure elements
- hydrophobic side chains interiorly, hydrophilic side chains on exterior (soluble proteins)
-
domains
combination of motifs that form globular units of the protein
-
carbaminohemoglobin
- form of hemoglobin that carries CO2 from tissue to the lungs
- when CO2 is expelled in the lungs, Hb reverts to R form
-
carbon monoxyhemoglobin
- CO binds tightly to iron in hemoglobin (affinity is 220X greater than O2)
- stabilizes R form (high O2 affinity) of hemoglobin - but does NOT release oxygen as it should
- O2 binding curve is shifted to the left
-
types of hemoglobins
- HbA: major adult form
- HbA2: minor adult form; (alpha-delta)2
- HbA1c: minor adult form to monitor glucose; (alpha-beta - GLUCOSE)
- HbF: fetal (alpha-gamma)2
- HbS: sickle cell
-
HbA1c
- nonenzymatic glycosylation occurs by Schiff's base formation between the alpha amino group at the N terminus of Hb beta chains & C1 carboxyl group of glucose
- C2 hydroxyl of glucose allows a subsequent Amadori rearrangement to irreversibly link glucose to the protein
-
globin gene organization
- alpha gene family: cluster; a1, a2
- beta gene family: cluster; embryonic & fetal
-
synthesis of globin mRNAs
- three exons (protein coding regions) are spliced together
- removal of two introns (noncoding sequences)
- spliced mRNA codes for globin proteins
-
hemoglobin C disease
- glu 6 -> lys 6
- makes HbC migrate slower than HbA and HbS
- mild, chronic hemolytic anemia
-
hemoglobin SC disease
- double mutant: one HbS beta chain gene & one HbC beta chain gene
- usually undiagnosed until infarctive crisis occurs (childbirth or surgery)
- can be fatal
-
methemoglobin
- oxidation of ferrous (Fe2+) iron to ferric (3+)
- cannot bind O2, binds H2O instead
- caused by environment (drug) or inherited mutation
- leads to formation of HbM - methemoglobinopathy (BLUE PEOPLE)
- can be reduced by NADH cytochrome b5 reductase (can now bind O2)
- BINDS CYANIDE
-
chocolate cyanosis
- methemoglobin is dark in color leading to brownish blue color in skin and membranes
- blood is chocolate colored
- symptoms: anxiety, headache, dyspnea
-
thalassemias
- hereditary hemolytic diseases (common in humans)
- synthesis of defective alpha or beta chains (unbalanced hemoglobin chain synthesis)
- gene deletions, substitutions or deletion in nucleotides, defect in splicing
-
beta thalassemias
- decreased of absent beta chains - only two copies of beta chains (only seen after birth; shift from fetal to adult hemoglobin)
- beta thalassemia minor: one normal, one defective beta chain gene (not life threatening)
- beta thalassemia major: both genes defective; develop severe anemia by age 2; iron overload (hemosiderosis), death b/w 15-25 years
-
alpha thalassemias
- decreased or absent alpha chain synthesis
- four copies of alpha gene --> severity of disease depends upon the number of defective genes
- 0 defective: normal
- 1 defective: silent carrier of alpha thalassemia; no symptoms
- 2 defective: alpha thalassemia trait; mild symptoms
- 3 defective: moderately severe hemolytic anemia
- 4 defective: hydrops fetalis (fetal death)
-
population genetics
- looks at the statistical distribution of genes in a particular population
- looks at how the frequency of genes is maintained or changes
- is the basis for understanding how different disease genes are more common in different populations
-
CCR5 gene
- encodes a cell surface cytokine receptor which is the entry point for HIV
- a 32 bp deletion results in nonfunction protein
- homozygotes for the mutation do not have the receptor and are resistant to HIV infection - autosomal recessive
- the normal allele can be distinguishes from the 32 bp deletion using PCR
-
Hardy Weinberg Equation
- p+q=1
- p2 + 2pq + q2 = 1
- p = frequency of dominant allele
- q = frequency of recessive allele
- 2pq = frequency of heterozygote
-
Hardy Weinberg in autosomal recessive disease
- can be used to help estimate the frequency of carriers in a population
- usually put all disease-causing alleles together (whether compound heterozygotes or homozygotes)
-
rare genetic disorders
- for rare recessive traits, p = 1 (approximately)
- 2pq is approximately 2q
- the number of carriers in the population is much larger than the number of homozygous affected individuals
-
Hardy Weinberg theorem
- 1 - random mating
- 2 - a large population size
- 3 - no mutations
- 4 - no new alleles are introduced/lost
- 5 - no natural selection
- if all these assumptions are correct, then the population is in genetic balance (Hardy Weinberg equilibrium)
-
circumstances in which Hardy Weinberg law may fail to apply
- mutation
- gene flow
- genetic drift
- nonrandom mating
- natural selection
-
human population exceptions to Hardy Weinberg
- human populations do NOT randomly mate
- populations are large, but often distributed in small groups
- frequently have new mutations
- human populations are highly mobile and introduce new alleles
- populations are under a variety of natural selection stressors (malaria, HIV, etc.)
-
non-random matings
- stratification: when a population has subgroups that are relatively genetically separate (Cajuns, Amish)
- assortive matings: choice of mate is made based on possessing a particular trait (deafness & achondroplasia)
- consanguinity & inbreeding: higher risk of producing homozygous offspring
-
genetic drift
- affects the genetic makeup of the population but, unlike natural selection, through an entirely random process
- mechanism of evolution that does not work to produce adaptations
-
population bottlenecks
- occur when a population's size is reduced for at least one generation
- genetic drift acts more quickly to reduce genetic variation in small populations
-
founder effect
- occurs when a new colony is started by a few members of the original population
- the small population size means that the colony may have reduced genetic variation from the original population & a nonrandom sample of the genes in the original population
-
gene flow
- any movement of genes from one population to another
- gene migration includes people moving to new cities or countries
- important source of genetic variation
-
QTL Analysis
- quantitative trait loci: traits whose phenotypic characteristics vary and are caused by two or more genes
- quantifiable traits (bp, height, etc) that may not be genes, but are tightly linked by SNPs
- statistical methods are used to determine the degree of association
- can be used to identify candidate genes in that region
-
GWAS
genome wide association studies: look for variation across the entire human genome in order to identify genetic associations with observable traits
-
copy number variants
large scale variations in gene copy number are common & suspected to underlie genetic diversity & susceptibility to certain diseases
-
fibrous proteins
- very regular, organized linear secondary structures that is crucial to its function
- ex. collagen, elastin, keratin
- unique mechanical properties
-
collagen
- three alpha chains wound in a triple helix (NOT an alpha helix)
- most abundant protein in body; rigid, insoluble
- gel: extracellular matrix (ECM), vitreous humor (eye)
- parallel fibers: tendons, great strength
- cornea: stacked to allow light transmittal
- bone: angled arrangement to prevent shearin any direction
-
types of collagen
- twenty types, differ in structure & function
- three alpha chains for a triple helix held together by H bonds
- fibril forming collagen
- network forming collagen
- fibril associated collagen
-
fibril forming collagen
- type I: high tensile strength
- type II: cartilage
- type III: less rigid
- forms regular overlapping fibril that contribute to tensile strength
-
network forming collagen
- type IV: major component of basement membranes that support adjacent cells
- type VII
- form meshlike supports
-
fibril associated collagen
- type IX & XII: located on the surface
- link collagen fibrils together
-
structure of collagen
- amino acid sequence (gly-X-Y)333
- X: usually proline
- Y: usually hydroxyproline or hydroxylysine
- triple helical - R groups on outside
- posttranslations modifications: hydroxylation of proline (prolyl hydroxylase - requires ascorbate and oxygen) & glycosylation
-
biosynthesis of collagen
- made in fibroblasts, osteoblasts & chondrocytes
- enzymatically modified
- aggregate & cross link
- secreted into extracellular matrix
-
degradation of collagen
- numerous collagenases degrade collagen to allow remodeling of the ECM
- high collagenase expression is seen in metastatic cancer cells
-
collagen diseases
- lack necessary tensile strength
- defects in synthesis, mutations in collagen genes, etc.
- Ehlers-Danlos syndrome
- Osteogenesis imperfecta
-
Ehlers-Danlos syndrome
- heterogenous group of disorders arising from defects in the synthesis or mutations in collagen types I, II & V (fibrillar collagens)
- characterized by stretchy skin, loose joints & thin transparent skin
-
osteogenesis imperfecta
- brittle bone syndrome
- various enzyme deficiences and mutations in type I collagen
- mostly autosomal dominant; some spontaneous cases
- blue sclera, slow wound healing, bones bend easily & fracture
-
osteogenesis imperfecta tarda
- type I: decreased production of alpha1 and alpha2 chains
- fractures early in infancy, sometimes predicted in utero (long bone bowing seen on ultrasound)
-
osteogenesis imperfecta congenita
- type II: severe mutations involving the substitution of bulky amino acids for glycine in the proalpha1 or proalpha2 chains
- death in utero or as neonate from pulmonary hypoplasia
-
elastin
- precursor: tropoelastin
- lots of proline, lysine, but no hydroxylysine
- found in lungs, walls of large blood vessels, ligaments
- stretch 2-4 times normal length; recoil to original length
- interacts with fibrillin
-
Marfan's Syndrome
- mutations in fibrillin
- irregular interchain crosslinks (lysine involved)
- symptoms: tall, loose jointed, heart murmur, etc.
-
desmosine
- a ringed structure made of lysine/allysines
- joins separate elastin polypeptides
- allows stretching and bending in all directions (elasticity)
-
alpha1 antitrypsin
- alpha1 antiproteinase: inhibits family of proteases that degrade protein (includes trypsin)
- inhibits neutrophil elastase: degrades elastin
- protects lung alveolar elastine & prevents loss of lung elasticity
- mutation in alpha1 antitrypsin may cause inherited emphysema
-
overview of blood clotting
- proper balance between clot formation and dissolution is required for hemostasis (maintenance of correct blood flow)
- platelets form a "plug" and send out pro-clotting signals (begins when tissue is damaged)
- a cascade of proteolytic reactions occurs that results in thrombin production and a "hard" blood clot (begins when tissue is damage)
- clotting cascade is inactivated, clot is dissolved (begins when tissue is damaged!)
-
zymogen
- proenzyme: an inactive enzyme precursor
- enzyme is in a self-inhibitory conformation
- may be partially activated by contact (with another molecule/surface) or fully activated by proteolysis
-
pathways of clotting cascade
- extrinsic: blood contacts tissue in injury; requires extravascular component
- intrinsic: initiated by damaged blood vessels endothelium - all necessary components are present in blood
- common: common to both intrinsic & extrinsic pathways
- pathways activate proteolytic zymogens in a cascade that greatly amplifies procoagulation signals upon blood vessel damage
- result in production of thrombin (serine protease) that converts fibrinogen to fibrin & "feeds back" into the coagulation cascade pathways
-
clotting factors
- I: fibrinogen
- II: prothrombin (SP)
- III: tissue factor/thrombokinase
- IV: Ca 2+
- V: proaccelerin
- VII: proconvertin (SP)
- VIII: antihemophilic factor
- IX: christmas factor (SP)
- X: stuart factor (SP)
- XI: plasma thromboplastin antecedent (SP)
- XII: hageman factor (SP)
- XIII: fibrin stabilizing factor
- prekallikrein/kallikrein (Fletcher factor - SP)
- HMWK
- vWF
-
factor I (fibrinogen)
- soluble plasma glycoprotein synthesized by the liver
- 340kDa hexamer consisting of 2 sets of alpha, beta & gamma chains linked to each other by disulfide bonds
- the fibrinogen alpha & beta chains have small peptides that prevent fibrinogen from forming polymers (FpA + FpB)
- thrombin cleaves the alpha & beta chains releasing FpA, FpB creating fibrin
- fibrin associates end-to-end to create fibrils & then makes lateral associations to create fibers and then a fibrin "gel"
- the transglutaminase factor XIII then creates covalent crosslinks between fibrin monomers using lysine & glutamine residues
-
factor II (prothrombin)
- serine protease zymogen precursor of thrombin synthesized in liver
- glycoprotein - contains gamma carboxyglutamate residues (become vitamin K later)
- gla residues bind to Ca2+ and allow membrane association
- two cleavage events are required to generate thrombin
- thrombin's degradation of fragment 1.2 is a means of self regulation
- inhibited by antithrombin, a serpin, that plugs active site (competitive inhibitor) - strongly potentiated by binding to heparin
-
antithrombin
- serpin inhibitor of thrombin (factor IIa)
- glycoprotein produced in the liver
- plugs active site (competitive inhibitor)
- inhibits IIa, IXa, Xa, VIIa, XIa, XIIa, kallikrein
- strongly potentiated by binding to heparin
-
factor III (tissue factor, thrombokinase)
- membrane bound glycoprotein found in subendothelial tissue, activated platelets & WBCs
- bind to and partially activates factor VII
-
thromboplastin
factor III (tissue factor, thrombokinase) + surrounding membrane
-
factor V (proaccelerin)
- plasma protein cofactor
- activated by thrombin
- degraded by active protein C: protein S
- Factor V Leiden: mutation resistance to protein C:S cleavage; activated Protein C resistance
-
factor VII (proconvertin)
- serine protease zymogen produced in the liver
- contains gla residues (gamma carboxyglutamate) necessary for binding calcium & membranes
- partially activated by binding to factor III (tissue factor) in context of negatively charged membrane
- fully activated by thrombin
- can auto-activate to VIIa
- inactivated by tissue pathway factor inhibitor ONLY when Xa is present
- also inactivated by antithrombin
-
factor VIII (anti-hemophilic factor)
- binds to von Willebrand factor which stabilizes VIII
- upon cleavage by thrombin, VIIIa dissociated from vWF and forms a complex with IXa
- inactivated proteolytically by protein C:S complex & IXa
- deficiency results in hemophilia A (X linked recessive)
- Rx: recombinant VIII
-
factor IX (christmas factor)
- serine protease zymogen synthesized in liver
- contains gla residues (gamma carboxyglutamate) for calcium & membrane binding
- converted to IXa by factor XIa and VIIa/TF
- inhibited by antithrombin
- deficiency: hemophilia B (Christmas disease - X linked recessive)
-
factor X (stuart-prower factor, prothrombinase)
- serine protease zymogen synthesized in the liver
- contains gla residues (gamma carboxyglutamate)
- converted into active Xa by IXa/VIII (intrinsic pathway) and VIIa/TF (extrinsic pathway)
- inhibited by tissue factor pathway inhibitor (TFPI), antithrombin & protein Z dependent protease inhibitor
- deficiency is rare autosomal recessive
-
common pathway of procoagulation
Xa, Va, IIa, and I
-
Factor XI (plasma thromboplastin antecedent)
- serine protease zymogen produced in the liver
- coverted to XIa by XIIa, thrombin & autocatalysis
- binks to high molecular weight kininogen which brings XI to negative surfaces
- inhibited by protein Z dependent inhibitor but does not require protein Z (like Xa inhibition)
- deficiency: hemophilia C (autosomal recessive; esp. Ashkenazi Jews)
-
Factor XII (Hageman factor)
- serine protease zymogen synthesized in the liver
- partially activated by negatively charged surfaces
- fully activated by kallikrein & autocatalysis
- XIIa & kallikrein are important for fibrinolysis (convert plasminogen to plasmin which cleaves fibrin & vWF)
- deficiency is autosomal recessive; essentially asymptomatic
-
Factor XIII (protransglutamidase)
- heterotetramer composed of 2 B chains and 2 A chains
- activation by thrombin - results in dissociation of B chains (leaving catalytic A chain homodimer)
- platelets only have XIIIa (2 A chain form)
-
gla residues
- gamma carboxyglutamate
- "two headed" glutamate
- perfect for grabbing Ca2+ ions which bind to negatively charged membrane surfaces
- necessary for proper membrane association of clotting factors
- Factors II, VII, IX, X & protein C:S complex & Protein Z
-
warfarin
- coumadin
- inhibits the necessary reduction of vitamin K
- competitive inhibitor of VKOR (Vitamin K epoxide reductase)
-
other coagulation inhibitors
- heparin: highly sulfated glycosaminoglycan; binds to & activates antithrombin; complex inhibits Xa, IIa, VIIa, XIa, XIIa, kallikrein, plasmin
- dabigatran atexilate: direct thrombin inhibitor
- edoxaban: Factor Xa inhibitor (Phase III trial in Japan)
-
disseminated intravascular coagulation
- widespread clotting in the bloodstream
- symptoms: impaired coagulation (thrombosis then ischemia), organ failure and bleeding
- causes: bacterial infections (sepsis), major trauma, malignancies, others
- tissue factor (III) exposed to the bloodstream activates extrinsic pathway (then intrinsic)
- uses up clotting factors & platelets (thrombocytopenia) resulting in predisposition for hemorrhage -- hard to treat hemorrhages & thromboses simultaneously
-
review of procoagulation
- blood vessel damage exposes circulating plasma factors and platelets to collagen, tissue factor & some negative charges (damaged membranes)
- factor VII binds to tissue factor (III) and is partially activated making some Xa
- Xa finds factor V and is able to make some thrombin
- thrombin then converts VII --> VIIa, V --> Va, XI --> XIa, VIII --> VIIIa, XIII --> XIIIa
- VIIa/TF converts IX --> IXa and X -->Xa
- platelets are activated & provide negative membrane for collecting Gla-containing coagulation factors
- IXa/VIIIa and Xa/Va on membranes make LOTS of thrombin
- thrombin cleaves fibrinogen to fibrin
- XIIIa (activated by thrombin) crosslinks Lys & Gln residues in fibrin
-
normal platelet aggregation inhibitors
nitric oxide (NO) & prostacyclin (PGI2) produced by the endothelium
-
platelet aggregation
- upon endothelial cell injury, collagen & vWF are exposed
- platelets adhere to vWF via GP Ib/X/V and collagen via GP VI
- adhesion activates platelets resulting in shape change, release of ADP, TxA2, thrombin, vWF and activation of integrins GPIIb/IIIa (bind to fibrinogen)
- PF3 moves to outer leaflet of platelet membrane (negative surface)
- when blood vessels are injured, platelets adhere to molecules in the subendothelial tissues
- aggregate to form the primary "plug"
- platelets stimulate the local activation of other platelets and plasma coagulation factors
-
von Willebrand Factor
- glycogprotien present in plasma, endothelium, subendothelial connective tissue
- processed by ER and Golgi (furin) into very large multimers
- domains that bind to platelets (GP Ib/X/V and GP IIb/IIIa), heparin, collagen, Factor VIII
- deficiency: von Willebrand Disease; most common hereditary coagulopathy
-
types of antiplatelet drugs
- cyclooxygenase inhibitors: aspirin
- ADP receptor inhibitors: clopidogrel (plavix), prasugrel (effient), ticlopidine (ticlid)
- phosphodiesterase inhibitors: cilostazol (pletal)
- glycoprotein IIb/IIIa inhibitors (IV use only): abciximab (ReoPro), eptifibatide (integrillin), tirofiban (aggrastat)
- adenosine reuptake inhibitors: dipyridamole (persantine)
-
specific anticoagulant drugs
- heparin: IV, short acting coagulant; binds to & activates antithrombin III which inactivates IIa, IXa, Xa, XIa, XIIa and plasmin
- coumarins: vitamin K analogue; blocks the action of vitamin K (causes gamma glutamyl carboxylation of clotting factors)
- plavix: ADP receptor agonist; blocks ADP mediated platelet activation
- ReoPro: blocks GPIIb-GPIIIa which bind fibrinogen
- aspirin: cyclooxygenase inhibitor; COX required for TxA2 production (increases platelet aggregation & vasoconstriction)
-
clot dissolution
- clot ultimately dissolved by plasmin - activated by a cascade
- plasminogen activator inhibitor I inhibits clot dissolution
- with time, tissue Plasminogen Activator (tPA) becomes active
- tPA and urokinase convert plasminogen --> plasmin
- plasmin converts fibrin --> fibrin degradation products
- clot dissolves; replaced by connective tissue (scar)
-
therapeutic applications for the treatment of thrombosis
- recombinant tPA used to treat patients with thrombosis
- streptokinase (bacterial protein) inactive by itself; complexes with plasminogen & converts it to plasmin (then dissolves the clot)
- urokinase (uPA) can also be used
-
Hemophilia A
- X linked recessive: only males typically affected
- defective fibrin formation
- symptoms: joint & muscle hemorrhage, easy bruising, prolonged bleeding from wounds
- high in European royalty - inbreeding causes recessive genes to be manifested more often in homozygous state
- absence of deficiency in factor VIII (antihemophilic factor)
-
Hemophilia B (Christmas disease)
- X linked
- defective fibrin formation
- same symptoms as Hemophillia A
- mutations in Factor IX gene
-
von Willebrand Disease
- most common coagulopathy; autosomal chromosome 12 mutation
- mild form: deficiency in factor
- severe form: abnormal factor produced
-
types of von Willebrand Disease
- type I: quantitative deficiency in vWF (mild, heterozygous)
- type II: qualitative deficiency (normal levels, but poor/unusual function); may bind GP too tightly and not stabilize VIII
- type III: qualitative or quantitative homozygous - no function or no detectable vWF (severe)
- autoimmune form: body develops antibodies against vWF
-
coagulopathy treatments
- replace missing factor by injecting with factor isolated from donor blood
- problem: possibility of transmitting viruses through contaminated blood
- recombinant protein: produced in bacteria from human gene sequence; good alternative but very expensive
-
coagulation factor measurements
- bleeding time (BT)
- prothrombin time (PT)
- thrombin time (TT)
- activated partial thromboplastin time (aPTT)
- Russell's viper venom test (RVVT)
- clot stabililty
- ELISA assay
-
bleeding time
- the "instantaneous" plugging of a hold in a blood vessel wall (primary hemostasis)
- vasoconstriction plus platelet adhesion & aggregation (does not require fibrin clot formation)
- requires independent platelet count for interpretation of defects
- abnormally long in thrombocytopenia (low platelet count) & von Willebrand disease (platelet function disorders)
- normal in hemophilia
-
prothrombin time
- measure the functional state of both the extrinsic and common pathway
- plasma - add thromboplastin plus Ca2+ - measure time to clot formation
- this reaction requires vitamin K, often used to monitor coumadin treatment
- also used in pre-op screening
-
thrombin time
- functional evaluation of fibrinogen concentration and polymerization
- add purified thrombin to plasma and measure time to clot formation
- heparin in the plasma of the patient prolongs TT
-
activated partial thromboplastin time
- measure intrinsic & common pathway components
- stage 1: citrate anticoagulated blood is preincubated with a surface activator to produce activated factor XII
- stage 2: Ca2+ is added to initiate activation of Factor XI and the rest of the intrinsic pathway
- aPTT is prolonged in hemophilia A, B (Christmas disease), and C, von Willebrand Disease & other deficiencies in the intrinsic pathway
-
Russell's viper venom test
- dilute RVVT
- venom directly activates Factor X
- test detects deficiencies in X, V, I (fibrinogen) and II - the common pathway
-
clot stability test
- put patients clotted plasma into an acidic solution
- if clot rapidly dissolves, there is a XIII deficiency
-
Ristocetin cofactor assay
- ristocetin mimics endothelial substratum
- causeds GP Ib to bind to large multimers of vWF in plasma
- assesses vWF function by measuring binding of vWF to GP Ib/IX/V on platelets
- mix ristocetin, patient plasma & formalin fixed platelets
- measure amount of platelet aggregation (will require vWF)
- detects: Bernard-Soulier syndrome, Glanzmann's thrombasthenia
-
Bernard Soulier syndrome
- deficiency in GP Ib/IX/V
- autosomal recessive
-
Glanzmann's thrombasthenia
- autosomal recessive disease
- patient lacks GP IIb/IIIa
- platelets cannot bind to fibrinogen
-
classes of enzymes
- oxidoreductase: catalyzes oxidation-reduction reaction
- transferase: catalyzes transfer of C-, N-, or P-containing groups
- hydrolase: catalyzes cleavage of bonds by the addition of water
- lyase: catalyzes cleavage of C-C, C-S, and certain C-N bonds
- isomerase: catalyzes racemization of optical or geometric isomers
- ligase: catalyzes formation of bonds between carbon and O,S,N coupled to hydrolysis of high energy phosphate
-
enzyme
- protein catalyst that increases the velocity of a reaction without being consumed in the reaction
- rarely for RNA = ribozymes
-
properties of enzymes
- active site: a cleft or pocket lined with amino acids that form a 3D surface that is complementary to the substrate
- catalytic efficiency: faster reactions than without enzymes
- turnover number: Kcat, the number of substrate molecules converted to product per enzyme molecule per second **
- specificity: a single molecule or a group of structurally (closely) related substrate molecules
- cofactors: non protein component needed for enzyme activity
- regulation: positively or negatively
- subcellular localization: separates reaction pathways, allows macromolecular enzyme complexes to form that favor overall pathways
-
holoenzyme
the enzyme protein plus its non-protein component
-
apoenzyme
enzyme protein without its non-protein component
-
cofactors
- metal ions (Ni++)
- coenzymes - organic molecules (TPP)
- cosubstrate - organic, changed
- prosthetic groups - very tightly (usually covalently) attached to the protein
-
free energy of activation
- energy barrier separating the reactants and the products
- the energy difference between that of the reactants and a high energy intermediate that occurs during the formation of product
- represents the transition state
-
transition state
- T*: structure that reactant must be converted into to allow product formation
- formation of the transition state requires input of free energy of activation (slow reaction if uncatalyzed)
-
rate of reaction
- proportional to number of molecules in T*
- the lower the energy of activation, the faster the reaction proceeds
- enzyme catalyzed rate of reaction is increased, but the equilibrium of the reaction is not changed
-
active site chemistry (factors responsible for catalytic efficiency)
- stabilization of the transition state: enzyme binds substrate in a structure that resembles T*; lowers free energy of activation & puts more molecules into T* form --> accelerates reaction rate
- catalytic groups: help T* formation; acid/base catalysis (donation & acceptance of protons) or form covalent enzyme-substrate complex
-
factors affecting reaction velocity
- substrate concentration: more substrate present, more product formed; can be saturated (maximal velocity)
- temperature: increased velocity with increased temp (provides energy); denatured at too high temp
- pH: each enzyme has characteristic pH optimum suited for its environment; extreme pH can denature enzyme
-
enzyme velocity
number of substrate molecules converted to product per unit time (umol P formed/min)
-
Michaelis-Menten equation
- describes how intial velocity varies with substrate concentration
- can ONLY be used with simple (hyperbolic) enzyme curves, NOT with allosteric sigmoidal curves
-
shape of enzyme velocity curve
- hyperbolic: simple binding kinetics (Michaelis-Menten kinetics)
- sigmoidal: allosteric binding (like hemoglobin)
-
assumptions of the Michaelis-Menten equation
- the concentration of substrate is MUCH greater than the concentration of enzyme
- the reaction is at a STEADY STATE
- the initial velocity is used in the analysis of enzyme reaction (k2 is VERY SMALL and ignored)
-
conclusions about the Michaelis-Menten kinetics
- Km: the substrate concentration at which the initial rate is half the maximal velocity
- small Km: reflect high affinity of the enzyme for substrate (low concentration of substrate needed to half saturate enzyme)
- large Km: reflects low affinity of enzyme for substrate (high concentration of substrate needed to half-saturate the enzyme)
- reaction rate is directly proportional to the enzyme concentration at all substrate concentrations
- order of reaction can be determined
-
first order reaction
when concentration of substrate is much less than Km, the velocity of the reaction is roughly proportional to the concentration of substrate
-
zero order reaction
when the concentration of substrate is much greater than Km, the velocity (reaction rate) is constant and equal to Vmax
-
Lineweaver-Burke plot
- linear transformation of Michaelis-Menten equation
- double reciprocal plot
- x axis: plot 1/[S]
- y axis: plot 1/Vo
- x intercept: -1/Km
- y intercept: 1/Vmax
-
inhibition of enzyme activity
- slows down the velocity of the enzymatic reaction
- may be reversible or irreversible
- competitive and noncompetitive most common
-
competitive inhibition
- inhibitor binds reversibly to the same site on the enzyme that binds the substrate (active site)
- competes with substrate binding to the enzyme
-
effects of competitive inhibition
- Vmax: unchanged in competitive inhibition; effect of inhibitor reversed by adding more substrate
- Km: increased; [S] must be higher to achieve same 1/2Vmax as uninhibited reaction
- Lineweaver-Burke plot: y axis intercepts are the same for the inhibited & uninhibited reaction (Vmax is unchanged)
-
example of competitive inhibition
- statin drugs (antihyperlipidemic drugs)
- inhibitors of the binding HMG CoA substrate to the rate-limiting enzyme of cholesterol biosynthesis (HMG CoA reductase)
-
noncompetitive inhibition
- inhibitor & substrate bind at different sites
- the noncompetitive inhibitor can bind either to free E or to the ES complex
- ex: ferrochelatase
-
effect of noncompetitive inhibition
- Vmax: lowered (enzyme cannot reach Vmax; noncompetitive inhibition cannot be reversed by adding more substrate)
- Km: no effect
- Lineweaver-Burke plot: y intercept changes; x intercept stays the same (Km is unchanged)
-
enzyme inhibitors as drugs
- five of the 10 most commonly prescribed drugs in the US are enzyme inhibitors
- aspirin: irreversible inhibitor of cyclooxygenase used to synthesize prostaglandins
- NSAIDS: other non-steroidal anti-inflammatory drugs; reversible inhibitors of cyclooxygenase
-
regulation of enzyme activity
- concentration of substrate for many enzymes is near Km
- changes in reaction rates affected by alterations in substrate levels
- allosteric binding sites: homotropic effectors & heterotropic effectors
- covalent modification: phosphorylation, dephosphorylation
- induction & repression of enzyme protein synthesis
-
allosteric binding sites
- bind effectors, modifiers, or modulators
- binding of effectors can change binding affinity (Km), maximum enzyme activity (Vmax) or both
- negative effectors reduce enyme activity
- positive effectors increase enzyme activity
-
homotropic effectors
- substrate itself (binding at a different site than the active site) affects enzyme activity on other substrate molecules
- most often this is positive effector
- positive cooperativity is shown - sigmoidal curve seen for enzyme
- at high [S], allosteric effector stimulates its own metabolism to a faster rate
-
heterotropic effectors
- the effector is different from the substrate
- VERY OFTEN the product of a reaction pathway causes feedback inhibition
- most often occurs at an early step in the reaction path involving the rate-limiting, commited step (enzyme) in the biochemical pathway
-
covalent modification
- phosphorylation: protein kinases (usually highly regulated themselves), use ATP as phosphoryl group donor
- dephosphorylation: phosphoprotein phosphatases (usually constituitive); hydrolases that remove phosphate group
-
enzymes in clinical diagnosis
- cells are damaged & lyse due to disease
- serum levels of intracellular enzymes increase above the normal range
- plasma enzyme levels in serum correlates to severity of the disease
- many enzymes are specific for a certain tissue - helps pinpoint site of disease
-
isoenzymes
- enzymes that catalyze the same reaction but have different physical properties
- different organs have different isoenzymes
- can be distinguished physically by electrophoresis
- CK: creatine kinase
- LDH: lactate dehydrogenase
- (S)GOT: serum glutamate oxaloacetate transaminase
- commonly used to diagnose myocardial infarction
-
quaternary structure forms of CK
- different subunits (B & M chains) are mixed in different combinations; have different electrophoretic mobilities
- CK1: BB
- CK2: MB
- CK3: MM
-
diagnosis of myocardial infarction
- heart tissue - only tissue with significant amount of CK2 (MB form of CK)
- CK2 increases at 4-8 hr after chest pains; peaks at 24 hrs
- LDH peaks at 2-3 days after a myocardial infarction (better indicator after 48 hrs)
- NEVER DIAGNOSE BASED SOLELY ON SERUM ISOZYME LEVELS
-
newer MI markers
- troponin T & troponin I: cardiac specific contractile proteins
- high levels of these proteins indicate more severe disease and poorer prognosis than CK2
-
classification of mutations
- loss of function mutations: mutated product has a reduced or no function
- gain for function mutations: mutated product has a new and abnormal function
-
missense mutations
- replace one amino acid with another in the same gene product
- alters the protein product & its function
-
nonsense mutation
- replace an amino acid with a stop codon
- result in production of a truncated protein
-
frameshift mutation
produced by deletions, insertions or splicing errors
-
splice site mutation
create or destroy splice donor or acceptor, altering RNA splicing
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silent mutation
changes the codon but does not alter the amino acid encoded
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dynamic mutations
tandem repeats that often expand in size
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classes of proteins associated with mendelian diseases
- enzymes involved in metabolism
- transport proteins
- cell structure
- extracellular homeostasis
- development
- growth & differentiation
- metabolism & communication
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enzyme defects
- amino acid metabolism: phenylalanine hydrogenase (PKU)
- carbohydrates: G-1-uridyl transferase (galactosemia)
- complex lipids: hexomonidase A (Tay Sachs)
- purines: HPRT (Lesch-Nyan syndrome)
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general concepts of enzyme deficiencies & disease
- enzyme defects nearly always inherited in an autosomal recessive fashion
- the defect causes either an accumulation of the substrate or a deficiency of the product
- there can be loss of multiple enzyme activities: enzymes may share the same cofactors, a common subunit or activating protein, or may be processed by a common modifying enzymes
- phenotypic homology: diseases due to enzymes in the same area or metabolic pathway can cause very similar phenotypes
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phenylketonuria (PKU)
- autosomal recessive mutation in phenylalanine hydrogenase (PAH - chromosome 12)
- significant allelic heterogeneity & ethnic/geographic differences in incidence & mutation
- PAH converts phenylalanine into tyrosine
- PKU patients are unable to degrade phenylalanine --> accumulate in tissues
- hyperphenylalanemia: toxic to developing neurons; can cause mental retardation
- Rx: dietary restriction of phenylalanine intake
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maternal PKU: risk to fetus
- congenital heart diseasae
- microcephaly (small head size)
- low birthweight
- mental retardation
- slow development
- language deficit
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Tay Sachs
- autosomal recessive mutation in the gene for the alpha subunit of hexosaminidase A gene
- fatal disease: affected infants appear normal until about 6 mos; progressive neurological deterioration & eventual death around 2-4 years
- carrier testing: HexA levels in WBCs
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Tay Sachs symptoms
- seizures
- noticeable behavior changes
- feeding difficulties
- cherry red spot on retina
- abnormal body tone
- loss of motor skills
- blindness
- deafness
- loss of intellectual skills
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albinism
- autosomal recessive
- enzyme deficiency caused by mutation in tyrosinase
- visual acuity is not normal & cannot be corrected completely (lack of development of the fovea)
- more prone to skin cancer
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G6PD deficiency
- glucose 6 phosphate dehydrogenase: enzyme normally involved in producing NADPH
- X linked recessive: more common in AA males
- most common human enzyme deficiency
- results in hemolytic anemia
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G6PD symptoms
- hemolytic anemia in states of oxidative stress
- paleness especially in mouth (lips, tongue)
- rapid breathing or shortness of breath
- extreme tiredness
- rapid heartbeat
- jaundice
- enlarged spleen
- dark, tea-colored urine
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familial hypercholesterolemia
- defect in LDL receptor protein
- autosomal dominant inheritance of mutation in gene that codes for LDL receptor
- Aa & AA develop premature heart disease
- patients have xanthomas (cholesterol deposits in skin & tendons)
- gene dosage effect: homozygotes have symptoms earlier
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transport protein defects
- cystic fibrosis: chloride transport
- Menke's/Wilson's syndrome: copper transport
- hemoglobin thalasemmia: iron transport?
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cystic fibrosis
- cystic fibrosis transmembrane regulator (CFTR): chloride ion channel
- mutation (deletion) in codon 508 is present on 70% of CF chromosomes
- more than 900 mutations IDed
- carrier incidence in caucasians is about 1 in 25
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organs affected by cystic fibrosis
sinuses, lungs, skin, liver, pancreas, intestines, reproductive organs
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Duchenne Muscular Dystrophy
- X linked recessive inheritance
- elevated creatine kinase levels (100X elevated)
- modest decreaase in IQ
- develop muscle weakness age 3-5; progressive muscle degeneration that begins with hip girdle muscle
- patients usually die of respiratory or cardiac failure
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dystrophin
- the Duchenne Muscular Dystrophy gene
- largest gene known in any species
- high mutation rate
- structural protein
- roles: maintaining muscular cell membrane integrity & required for assembly of synaptic junction
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osteogenesis imperfecta
- AKA "brittle bone" disease
- autosomal dominant inheritance of defect in type I collagen gene
- eight types
- severity depends on which procollagen chain is affected & the location of mutation
- mildest: type I due to decreased production
- most severe: type II due to structure defect in procollagen
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bioenergetics
the study of energy transformations that occur in biological systems
-
laws of thermodynamics
- 1: the total energy of a system & its surroundings is constant
- 2: the total entropy of a system & its surrounding always increases for a spontaneous process
- 3: as temperature of a system is decreased, entropy of the system is decreased
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change in free energy
- the sign of deltaG predicts the direction of a reaction
- less than zero: there is a net loss of energy by the system to the surroundings; the reaction occurs spontaneously (exergonic)
- greater than zero: there is a net gain of energy by the system from its surroundings; the reaction does NOT proceed spontaneously (endergonic)
- equals zero: the reactants are in equilibrium
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standard free energy change
- is predictive ONLY under standard conditions (concentration of products and reactants at 1mol/L, pH = 7 & P = 1 atm)
- Keq = 1: standard free energy equal zero (reaction is in equilibrium)
- Keq greater than 1: standard free energy is negative (reaction will proceed A --> B; spontaneous forward)
- Keq less than 1: standard free energy is positive (reaction will proceed A <-- B; spontaneous reverse)
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