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2013-01-31 00:28:42
Biochemistry Block

Biochemistry Block A
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  1. metabolism
    chemical changes (conversions) required to sustain life
  2. anabolism
    • biosynthesis
    • gluconeogenesis: generation of glucose in response to decreased blood glucose levels metabolic opposite of glycolysis
  3. catabolism
    • biodegredation
    • glycolysis: breakdown of glucose into pyruvate for energy (RBCs have no mitochondria)
    • urea cycle: breakdown of protein into nitrogenous wastes for excretion
  4. 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
  5. 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
  6. 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
  7. primary constituents of metabolism
    carbohydrates, lipids, and proteins
  8. "energy charge"
    energy availability as represented by levels of ATP, NADH, &  NADPH
  9. 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
  10. 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
  11. endogenous fat
    predominantly manufactured in the liver & exported as very low density lipoprotein (VLDL)
  12. exogenous fats
    absorbed by the intestines and packaged into chylomicrons
  13. lipoprotein lipase
    breaks down VLDL and chylomicrons into fatty acids (triacylglycerols) for storgage in adipocytes
  14. 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)
  15. sources for glucose synthesis
    • glycerol from lipolysis in adipose tissue (minor source for gluconeogenesis)
    • amino acids from muscle protein proteolysis (major contributor)
  16. 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
  17. 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
  18. 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)
  19. 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
  20. phenotype
    the observed expression of the genes of an individual (expression can be biochemical, physiological or morphological)
  21. genotype
    • the genetic constitution of an individual
    • the alleles present at a particular locus
  22. alleles
    alternative forms of a gene, or of a DNA sequence, at a specific position on a chromosome
  23. gene locus
    a specific location on a chromosome; often a specific gene
  24. homozygous
    both alleles at a locus are identical
  25. heterozygous
    if the alleles at a locus are different
  26. 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
  27. polymorphisms
    sequence variations
  28. mutations
    changes in DNA that are pathologic
  29. SNP
    • single nucleotide polymorphism (>1% abundance)
    • human genome has ~3 million SNPs distributed randomly
  30. 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)
  31. 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
  32. 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)
  33. examples of variable expressivity
    • neurofibromatosis 1
    • myotonic dystrophy
  34. examples of genetic disorders/diseases
    • single gene: CF, achondroplasia, hemophilia
    • chromosomal: trisomy 21
    • complex: diabetes, heart disease
  35. 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
  36. genetic traits
    eye color, hair color, weight
  37. 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
  38. 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
  39. 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
  40. P4 medicine
    • personalized, predictive, preventive, and participatory
    • better diagnoses and earlier interventions
    • more efficient drug development
    • more effective therapies
  41. nutrigenomics
    genetic influence on response to certain foods, vitamins, etc.
  42. microbiomics
    • microbial flora can affect our body and health
    • normal microbial flora in the human body outnumber human cells by 10:1
  43. proteomics
    large scale study of proteins
  44. metabalomics
    study of chemical processes involving metabolites
  45. 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
  46. 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
  47. 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
  48. examples of autosomal dominant disorders
    • achondroplastic dwarfism
    • adult polycystic kidney disease
    • Marfan's syndrome
    • polycystic kidney disease
    • neurofibromatosis 1 & 2
    • polydactly
    • Huntington's disease
  49. 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
  50. 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
  51. 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
  52. adult dominant polycystic kidney disease pathophysiology
    • hepatic cysts (focal expression)
    • cerebral aneurysms
    • cardiac abnormalities
  53. adult dominant polycystic kidney disease mortality/morbidity
    • subarachnoidal hemorrage
    • hypertension & stroke
    • mortality due to intracranial aneurysms
    • stroke
  54. 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
  55. Marfan syndrome
    • connective tissue disorder - molecular defect in  Fibrillin 1 gene
    • tall with long extremities
    • archnodactyly
    • hyperextensible joints
    • dissecting aortic aneurysms
    • subluxation of lens
  56. 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
  57. examples of autosomal recessive disorders
    • cystic fibrosis
    • phenylketonuria
    • hemochromatosis
    • sickle cell anemia
    • Tay-Sachs disease
    • thalassemia
  58. 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
  59. 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
  60. symptoms of sickle cell anemia
    • abdominal pain
    • bone pain
    • breathlessness
    • fatigue
    • fever
    • delayed growth
  61. sickle cell mutation
    glutamate --> valine substitution (point mutation)
  62. 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
  63. 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
  64. 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
  65. 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
  66. Duchenne muscular dystrophy
    • most common form of MD
    • mutation in the dystrophin gene
    • largest gene
    • males have 50% chance of being affected
  67. 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)
  68. 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
  69. 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
  70. 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
  71. 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
  72. mutations with sex-modified expression
    genes involved have distinct function in males and females (ex. prostate cancer & milk production)
  73. Y linked disorders
    • passed from father to sons
    • genes on the Y chromosome
  74. mitochondrial segregation
    • mitochondrial homoplasmy: all mutant or all normal
    • mitochondrial heteroplasmy: mixture of mutant & normal
  75. 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)
  76. 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
  77. 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
  78. anticipation
    • trinucleotide repeats expand with each successive generation
    • age of onset becomes younger with each successive generation
    • severity of disease increases with each generation
  79. 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%
  80. 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
  81. 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
  82. 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
  83. classification of amino acids
    • nonpolar or hydrophobic
    • polar or hydrophilic
    • acidic (proton donors - negatively charged)
    • basic (proton acceptors - positively charged)
  84. nonpolar (hydrophobic) amino acids
    • 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)
  85. 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)
  86. 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
  87. 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
  88. 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
  89. 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
  90. essential amino acids
    those amino acids that cannot be synthesized by humans & must be obtained from diet
  91. which amino acids are hydroxylated?
    serine, threonine & tyrosine
  92. which amino acids are acidic?
    glutamate & aspartate
  93. which amino acids are basic?
    arginine, lysine & histidine
  94. which amino acids contain sulfur?
    methionine & cysteine
  95. which amino acid does NOT have a free amino group?
  96. which amino acids are nonpolar?
    • glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan
  97. which amino acids can form a disulfide bond?
    ONLY cysteine
  98. which amino acid has the smallest R group?
    glycine (just a hydrogen)
  99. which amino acids are polar?
    serine, threonine, tyrosine, asparagine, glutamine, cysteine
  100. 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
  101. 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
  102. maximum buffering capacity
    • when pH = pKa
    • effective when pH of solution is +/- 1 of the pKa
  103. 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
  104. Henderson-Hasselbach Equation
    • pH = pKa + log [A-]/[HA]
    • pH: -log of [H+]
    • pKa: -log of Ka
  105. 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
  106. 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
  107. secondary protein structure
    • primary structures linked by hydrogen bonds
    • ex: alpha helices, B strands & B pleated sheets, B turns, non repetitive structures, supersecondary structures
  108. 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
  109. 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)
  110. 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
  111. 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)
  112. 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
  113. carbonyl
    • carbon double bonded to an oxygen
    • ex: aldehyde, keton, carboxylic acid
  114. 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
  115. 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
  116. Western Blotting
    • separates proteins by their size using gel electrophoresis
    • denaturation
    • uses antibodies to ID proteins involved in HIV, Lyme disease & BSE, etc.
  117. alpha helix
    • most common secondary structure
    • spiral structure, tightly packed, coiled
    • held together by H bonds that are parallel to helical axis
  118. 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 
  119. 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
  120. interchain H bonds
    hydrogen bond between two different polypeptide chains, each containing their own N & C termini
  121. intrachain H bonds
    hydrogen bond between amino acids in the same polypeptide chain
  122. 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
  123. nonrepetitive secondary structures
    less regular coils or loops
  124. motifs
    • regular packing of other higher order structures
    • "supersecondary structures"
    • ex: beta-alpha-beta unit, Greek key, beta-meander, beta-barrel
  125. allosteric inhibitors
    preferentially bind to the T state
  126. allosteric activators
    preferentially bind to the R state
  127. 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
  128. 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
  129. acidosis
    pH < 7.4
  130. alkylosis
    pH > 7.4
  131. 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
  132. 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)
  133. 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
  134. 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
  135. 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
  136. 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
  137. prion diseases
    • creutzfeld jakob (humans)
    • scrapie (sheep)
    • bovine spongiform encephalopathy (cows)
    • infectious agent - misfolded PrP protein
  138. 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
  139. protein denaturation
    chemical reactions, heat, changes in solvent, heavy metals - disrupt the interactions of the secondary & tertiary structures
  140. quaternary structure
    • relationship of subunits to each other
  141. 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
  142. interactions that stabilize tertiary structure
    • disulfide bonds
    • hydrophobic interactions
    • hydrogen bonds
    • ionic interactions (salt bridges)
  143. 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)
  144. domains
    combination of motifs that form globular units of the protein
  145. carbaminohemoglobin
    • form of hemoglobin that carries CO2 from tissue to the lungs
    • when CO2 is expelled in the lungs, Hb reverts to R form
  146. 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
  147. 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
  148. 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
  149. globin gene organization
    • alpha gene family: cluster; a1, a2
    • beta gene family: cluster; embryonic & fetal
  150. synthesis of globin mRNAs
    • three exons (protein coding regions) are spliced together
    • removal of two introns (noncoding sequences)
    • spliced mRNA codes for globin proteins
  151. hemoglobin C disease
    • glu 6 -> lys 6
    • makes HbC migrate slower than HbA and HbS
    • mild, chronic hemolytic anemia
  152. 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
  153. 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)
  154. chocolate cyanosis
    • methemoglobin is dark in color leading to brownish blue color in skin and membranes
    • blood is chocolate colored
    • symptoms: anxiety, headache, dyspnea
  155. 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
  156. 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
  157. 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)
  158. 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
  159. 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
  160. Hardy Weinberg Equation
    • p+q=1
    • p2 + 2pq + q2 = 1
    • p = frequency of dominant allele
    • q = frequency of recessive allele
    • 2pq = frequency of heterozygote
  161. 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)
  162. 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
  163. 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)
  164. circumstances in which Hardy Weinberg law may fail to apply
    • mutation
    • gene flow
    • genetic drift
    • nonrandom mating
    • natural selection
  165. 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.)
  166. 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
  167. 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
  168. 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
  169. 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
  170. 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
  171. 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
  172. GWAS
    genome wide association studies: look for variation across the entire human genome in order to identify genetic associations with observable traits
  173. copy number variants
    large scale variations in gene copy number are common & suspected to underlie genetic diversity & susceptibility to certain diseases
  174. fibrous proteins
    • very regular, organized linear secondary structures that is crucial to its function
    • ex. collagen, elastin, keratin
    • unique mechanical properties
  175. 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
  176. 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
  177. fibril forming collagen
    • type I: high tensile strength
    • type II: cartilage
    • type III: less rigid
    • forms regular overlapping fibril that contribute to tensile strength 
  178. network forming collagen
    • type IV: major component of basement membranes that support adjacent cells
    • type VII
    • form meshlike supports
  179. fibril associated collagen
    • type IX & XII: located on the surface
    • link collagen fibrils together
  180. 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
  181. biosynthesis of collagen
    • made in fibroblasts, osteoblasts & chondrocytes
    • enzymatically modified
    • aggregate & cross link
    • secreted into extracellular matrix
  182. degradation of collagen
    • numerous collagenases degrade collagen to allow remodeling of the ECM
    • high collagenase expression is seen in metastatic cancer cells
  183. collagen diseases
    • lack necessary tensile strength
    • defects in synthesis, mutations in collagen genes, etc.
    • Ehlers-Danlos syndrome
    • Osteogenesis imperfecta
  184. 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
  185. 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
  186. 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)
  187. 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
  188. 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
  189. Marfan's Syndrome
    • mutations in fibrillin
    • irregular interchain crosslinks (lysine involved)
    • symptoms: tall, loose jointed, heart murmur, etc.
  190. desmosine
    • a ringed structure made of lysine/allysines
    • joins separate elastin polypeptides
    • allows stretching and bending in all directions (elasticity)
  191. 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
  192. 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!)
  193. 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
  194. 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
  195. 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
  196. 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
  197. 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
  198. 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
  199. factor III (tissue factor, thrombokinase)
    • membrane bound glycoprotein found in subendothelial tissue, activated platelets & WBCs
    • bind to and partially activates factor VII
  200. thromboplastin
    factor III (tissue factor, thrombokinase) + surrounding membrane
  201. 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
  202. 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
  203. 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
  204. 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)
  205. 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
  206. common pathway of procoagulation
    Xa, Va, IIa, and I
  207. 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)
  208. 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
  209. 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)
  210. 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
  211. warfarin
    • coumadin
    • inhibits the necessary reduction of vitamin K
    • competitive inhibitor of VKOR (Vitamin K epoxide reductase)
  212. 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)
  213. 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
  214. 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
  215. normal platelet aggregation inhibitors
    nitric oxide (NO) & prostacyclin (PGI2) produced by the endothelium
  216. 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
  217. 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
  218. 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)
  219. 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)
  220. 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)
  221. 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
  222. 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)
  223. Hemophilia B (Christmas disease)
    • X linked
    • defective fibrin formation
    • same symptoms as Hemophillia A
    • mutations in Factor IX gene
  224. von Willebrand Disease
    • most common coagulopathy; autosomal chromosome 12 mutation
    • mild form: deficiency in factor
    • severe form: abnormal factor produced
  225. 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
  226. 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
  227. 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
  228. 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
  229. 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
  230. 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
  231. 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
  232. 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
  233. clot stability test
    • put patients clotted plasma into an acidic solution
    • if clot rapidly dissolves, there is a XIII deficiency
  234. 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
  235. Bernard Soulier syndrome
    • deficiency in GP Ib/IX/V
    • autosomal recessive
  236. Glanzmann's thrombasthenia
    • autosomal recessive disease
    • patient lacks GP IIb/IIIa
    • platelets cannot bind to fibrinogen
  237. 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
  238. enzyme
    • protein catalyst that increases the velocity of a reaction without being consumed in the reaction
    • rarely for RNA = ribozymes
  239. 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
  240. holoenzyme
    the enzyme protein plus its non-protein component
  241. apoenzyme
    enzyme protein without its non-protein component
  242. cofactors
    • metal ions (Ni++)
    • coenzymes - organic molecules (TPP)
    • cosubstrate - organic, changed
    • prosthetic groups - very tightly (usually covalently) attached to the protein
  243. 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
  244. 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)
  245. 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
  246. 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
  247. 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
  248. enzyme velocity
    number of substrate molecules converted to product per unit time (umol P formed/min)
  249. 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
  250. shape of enzyme velocity curve
    • hyperbolic: simple binding kinetics (Michaelis-Menten kinetics)
    • sigmoidal: allosteric binding (like hemoglobin)
  251. 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)
  252. 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
  253. 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
  254. zero order reaction
    when the concentration of substrate is much greater than Km, the velocity (reaction rate) is constant and equal to Vmax
  255. 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
  256. inhibition of enzyme activity
    • slows down the velocity of the enzymatic reaction
    • may be reversible or irreversible
    • competitive and noncompetitive most common
  257. 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
  258. 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)
  259. 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)
  260. noncompetitive inhibition
    • inhibitor & substrate bind at different sites
    • the noncompetitive inhibitor can bind either to free E or to the ES complex
    • ex: ferrochelatase
  261. 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)
  262. 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
  263. 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
  264. 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
  265. 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
  266. 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
  267. 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
  268. 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
  269. 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
  270. 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
  271. 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)
  272. 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
  273. 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
  274. missense mutations
    • replace one amino acid with another in the same gene product
    • alters the protein product & its function
  275. nonsense mutation
    • replace an amino acid with a stop codon
    • result in production of a truncated protein
  276. frameshift mutation
    produced by deletions, insertions or splicing errors
  277. splice site mutation
    create or destroy splice donor or acceptor, altering RNA splicing
  278. silent mutation
    changes the codon but does not alter the amino acid encoded
  279. dynamic mutations
    tandem repeats that often expand in size
  280. classes of proteins associated with mendelian diseases
    • enzymes involved in metabolism
    • transport proteins
    • cell structure
    • extracellular homeostasis
    • development
    • growth & differentiation
    • metabolism & communication
  281. 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)
  282. 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
  283. 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
  284. maternal PKU: risk to fetus
    • congenital heart diseasae
    • microcephaly (small head size)
    • low birthweight
    • mental retardation
    • slow development
    • language deficit
  285. 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
  286. 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
  287. 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
  288. 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
  289. 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
  290. 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
  291. transport protein defects
    • cystic fibrosis: chloride transport
    • Menke's/Wilson's syndrome: copper transport
    • hemoglobin thalasemmia: iron transport?
  292. 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
  293. organs affected by cystic fibrosis
    sinuses, lungs, skin, liver, pancreas, intestines, reproductive organs
  294. 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
  295. 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
  296. 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
  297. bioenergetics
    the study of energy transformations that occur in biological systems
  298. 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
  299. 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
  300. 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)