Biochem
CENTRAL DOGMA - Prelearning
Nucleic Acid – composed of DNA & RNA
DNA
Forms inherited genetic material
Genes code for protein, determine physical traits
Doesn’t direct protein synthesis itself, uses RNA as intermediate
DNA transcribed into RNA to serve as template for protein translation
DNA transcripted into RNA → RNA translated to protein
RNA (detailed later)
DNA Structure – Nucleotides, Double Helix, Base Pair Bonding, Phosphodiester Bonds
Nucleotides
3 components: nitrogenous bases, 5-C sugar, phosphate group
2 types of nitrogenous bases: purines, pyrimidines
Purines = 2 ring base = guanine, adenine
Pyrimidines = 1 ring base = thymine, cytosine
Double Helix
Sugar phosphate backbone maximizes efficiency of base-pairing
3 forces needed to stabilize structure
H-bonds b/w base pairs
Base stacking
Parallel to each other
Expels water from b/w them (hydrophobic effects)
Base Pair Bonding
Held together by H-bonds (A-T, G-C)
A always pairs with T and G always pairs with C because these are the only combinations that allow for hydrogen bonding to occur
A-C cannot form H-bonds, neither can G-T
Chargaff’s Rule - # of purines = # of pyrimidines
Necessary to maintain shape of double helix, which requires there to be one purine and one pyrimidine in each base pair
Shape of DNA helix would change if we had purine-purine, pyrimidine-pyrimidine interaction
If two purines paired there would be four 'rings' and they would distort the structure of the DNA molecule. Two pyrimidines would not easily bond as their single ring structures would not normally span the distance between the two strands
Phosphodiester Bonds
Sugar-phosphate backbone
Nucleotides join together
Covalent phosphodiester bond
Neg charge b/w P groups w/in sugar-phosphate backbone
Adjacent neg charges repel each other, destabilize helix
Positively charged Mg2+ ions help stabilize neg charges (ionic interactions)
Use 5’ and 3’ to denote direction of strand
Summary:
The following bonds and forces contribute to the stability of the DNA helix:
Phosphodiester bond (sugar-phosphate backbone)
Ionic interactions
H-bonds b/w complementary base pairs
Hydrophobic interactions (base stacking)
DNA Condensation – Nucleosomes, Chromatin, Chromosomes
Nucleosomes
Structural unit for packaging DNA
Composed of 147 base pairs wrapped around histone core
Octamer of H2A, H2B, H3, H4
H1 Linker protein
Chromatin
Complex of DNA + tightly bound protein
Heterochromatin – densely packed
Euchromatin – dispersed
Found when cell is transcriptionally active
DNA must be exposed for genes to be transcripted
Chromosomes
Most condensed form of DNA
23 pairs (46 total)
1 copy of each from each parent (2n; diploid)
Maternal/paternal pair = homologous chromosomes (homologs)
Haploid cells
1 copy of each chromosome
Sperm, oocytes (mature egg)
Autosomal
1-22
Form homologous pairs (1 maternal, 1 paternal copy)
Sex Chromosomes
Determine biological sex
Non-homologous
Female = XX, Male = XY
Genes
Functional units of heredity
Segment of DNA w/ instructions to make particular protein
Exon – coding sequence; EXpressed; included in RNA transcript, translated into protein
Intron – non-coding sequence; INTerfering; not included in RNA
Removed via splicing after transcription
Non-Coding DNA
98.5% of genome doesn’t encode protein, instead regulates gene expression
Promoter & enhancer regions – bind transcription factors
Binding sites for factors that organize chromatin structures
Non-coding regulator RNA – eg. MiRNA
Mobile genetic elements (“transposons”) not well understood
Gene regulation and chromatin organization
RNA
RNA Structure
Polymer made of nucleotides link by phosphodiester bond (like DNA)
Chemically different from DNA
Ribose sugar vs. Deoxyribose
Uracil (U) base instead of thymine (T)
Structurally different
Single stranded (can fold into various shape) vs. double-stranded helix of DNA
Types of RNA – Coding
mRNA
Messenger RNA = transcript of DNA transcribed into RNA as template for protein translation
Directly codes for proteins
Initially pre-mRNA then undergoes processing into mature mRNA
Types of RNA – Non-Coding
Sequences of DNA transcribed into RNA, not translated to proteins
Serve as enzymatic, structural, or regulatory components of cell processes
snRNA
Small nuclear RNA; Functions in spliceosome (needed to removing introns, splicing, from pre-mRNA)
Associated w/ protein subunits to form small nuclear ribonucleoproteins (snRNPs) which form core of spliceosome
rRNA
Ribosomal RNA; Needed for basic structure of ribosome complex
Catalyzes peptide bond b/w AAs
tRNA
Transfer RNA; carry correct AA to polypeptide chain
Unique cloverleaf shape
Anticodon:
3 consecutive nucleotides that pair with complementary codon in mRNA
Amino acid binding site:
Short, single-stranded region at 3’ end of tRNA
Binds AA that corresponds to anticodon on tRNA
Wobble Hypothesis
64 possible combos of nucleotides into 3 nucleotide codon, but only 20 AAs
Demonstrates redundancy of genetic code
More than 1 possible tRNA for many AAs
some tRNA molecules can base-pair w/ more than one codon
Some tRNA only require accurate base pairing of first 2 positions of codon and can tolerate mismatch (aka wobble) in 3rd position
Explains why so many alternative codons for an AA differ only in 3rd nucleotide
Micro RNA (miRNA)
Regulates gene expression via post-transcriptional silencing
Blocks/prevents translation of specific mRNAs; promote degradation
Small interfering RNA (siRNA)
Reduce gene expression
Direct degradation of specific mRNA
Long non-coding RNA (lncRNA)
Regulate gene expression
Increase or decrease transcription
Involved in X-chromosome activation
Types of RNA
Name
Function
mRNA
Messenger RNA
Codes for proteins
rRNA
Ribosomal RNA
Important constituents of ribosomes. Catalyzes protein synthesis
tRNA
Transfer RNA
Adaptor between mRNA and amino acids
snRNA
Small nuclear RNA
Splicing of pre-mRNA
miRNA
Micro RNA
Regulate gene expression: block translation of specific mRNA & promote its degradation
siRNA
Small interfering RNA
Regulate gene expression: direct specific mRNA degradation
lncRNA
Long non-coding RNA
Regulate gene expression: can increase or decrease transcription
CENTRAL DOGMA – In Class
Transcription
Transcription: process of synthesizing RNA molecule from DNA template to dictate synthesis of a protein
Occurs in cell nucleus
Transcriptional Unit
Promotor region – contains consensus sequence
Coding region – DNA transcribed into mRNA
Terminator region – specifies end of transcription; tells us when to stop
Anything before TSS is upstream, anything after is downstream
Template Strand
Strand of DNA that is transcribed into RNA; used to code
Anti-sense strand
Non-template strand – complimentary partner to template strand; not used in coding
Sense-strand
RNA Polymerase
Main enzyme for transcription; moves along DNA
Unwinds DNA helix before active site
Catalyzes new phosphodiester bond on newly-forming strand of RNA*
Works in 5’ --> 3’ direction
Makes more mistakes than DNA polymerase
OK since only making proteins; AA sequence can make up for it
Mistake is in protein, not genome like DNA’s would be
Steps of Transcription
Initiation
Initiation factors help RNA polymerase recognize where to start
Prokaryotes: only one, Sigma Factor
Eukaryotes: many! TFII (transcription factor 2) is a general transcription factor
TFII binds consensus sequence in promotor region
TATA box (consensus sequence); located upstream from TSS
TFIID is the specific TFII that binds the consensus sequence (TATA)
Within it is the TATA binding protein (TBP)
Other transcription factors join
RNA Polymerase II joins
Transcription initiation complex completes; transcription begins
Summary: First part of transcription is to form the transcription initiation complex (which is composed of TFIID, other TFIIs, and RNA polymerase)*
Regulation
Repressor proteins bind upstream silencers (aka negative regulatory elements)
Inhibit gene transcription
Transcriptional activator proteins bind upstream enhancers (aka positive regulatory elements)
Increase transcription rate by attracting RNA Polym II
If there was a mutation in either of these, transcription would speed up or slow down, giving us more or less protein in the end, influencing cell function
Ex. of how mutations don’t need to be in genes to influence amount of protein made
Elongation
TFII released once RNA Polym begins transcribing DNA
TFs then available to initiate another round of transcription
RNA moves downstream, transcribes codon region
Elongation factors needed; reduce likelihood of RNA Polym dissociating from DNA*
Eukaryotes require:
Chromatin remodeling complexes to help RNA navigate chromatin structure
Histone chaperones disassemble and reassemble nucleosomes as RNA Polym passes through
Supercoils generated as RNA Polym moves along
DNA topoisomerase removes supercoiling by breaking phosphodiester bond
Allows two sections of helix to rate freely, relieves tension
Phospho bond reforms as DNA topo leaves
know that you need RNA Polymerase and DNA Topoisomerase for Elongation
Processing (specific to Eukaryotes) and Termination
preMRNA transcript processed in 3 ways:
- Capping 5’ end
Modified guanine nucleotide added to 5’ end of transcribed preMRNA*
Occurs early
Facilitates export of mRNA out of nucleus; involved in translation
- Splicing
Introns and exons transcribed into RNA
Introns removed by RNA splicing
Performed by spliceosomes
Require snRNA (small nuclear RNA) and proteins complexed into snRNPs (small nuclear ribonucleoprotein)
SnRNP = snRNA + proteins
Allows ONE gene to produce variety of diff. Proteins
Mutation can occur; too much or too little intron could be removed → folding impacted → structure impacted
- Polyadenylation of 3’ end
3’ end of mRNA is specified by signals encoded in DNA → transcribed into RNA → bind to proteins that facilitate cleavage of mRNA from RNA Polym
Once termination signals are transcribed, mRNA is cleaved from RNA Polym
Once mRNA is cleaved, poly A tail added (~200 nucleotides added)
Poly A tail protects mRNA from degradation; facilitates export from nucleus
First termination, then Poly A binding proteins then bind poly-A tail to 3’ end
Once complete, transcript is mRNA
Prokaryotes
Steps are same, except:
No processing required (no 5’ cap, splicing, or poly-A tail)
No export from nucleus (translation beings ASAP)
Transcript is polycistronic
Multiple genes w/in transcript; transcript codes for more than one protein
In Eukaryotes, one transcript for one gene
Translation
After processing & termination, mature mRNA exported from nucleus
Once in cytosol, translated into protein
MRNA decoded in sets of 3 nucleotides called codons (ex. AUG is a codon); correct sequence depends on correct reading frame
In Eukaryotes, AUG is first sequence
In Prokaryotes, it is Shine Dalgarno sequence (ribosome binding site)
Additional recognition sequence needed for ribosome binding
If a nucleotide was accidentally inserted into middle of a gene, it would mess up subsequent codon, end up w/ wrong AAs
Molecules needed for translation:
MRNA transcript
“charged” tRNA (bound to correct AAs)
Ribosomes (large and small subunit
Preparing tRNA
Cell makes a variety; each corresponds to one of 20 AAs
Aminoacyl-tRNA synthetase catalyzes attachment of correct AA to tRNA
Basic enzyme name – each AA has it’s own specific tRNA synthetase name (ex. Tryptophanyl tRNA synthetase)
Once tRNA is bound to codon in RNA, tRNA is charged (need aminoacyl tRNA synthetase)
Ribosomes
Where protein synthesis is performed
Maintains correct reading frame; ensures accuracy of codon-anticodon interaction
Composed of ribosomal proteins + ribosomal RNA (rRNA)
Large subunit – involved in catalyzing new peptide bond (has E, P, A binding site)
Small subunit – mRNA binding site w/in small unit
know we need: mRNA transcript, charged tRNA (w/ aminoacyl tRNA synthetase), and ribosomes (rRNA + protein)
Steps of Translation
Initiation
AUG, methionine, is first codon translated on mRNA; start codon
*all newly made proteins begin w/ methionine as their first AA @ N-terminus
Met-tRNAi - Forms initiator tRNA-methionine complex
Loaded into small ribosomal subunit P site w/ initiation factors (eIFs)
Small ribosomal subunit binds to 5’ end of mRNA
modified guanine cap recognized 5’ end
Small ribosome moves along mRNA from 5’ to 3’ scanning for AUG
Requires ATP hydrolysis; expensive energy-wise
Once AUG found, initiation factors dissociate
large ribosomal subunit assembles; complete ribosome complex
*Met-tRNAi binds P site of small subunit → small s.u. binds to 5’ cap → scans mRNA until AUG → large ribosome s.u. added → complex complete
Elongation
tRNA binding
Charged tRNA binds to A-site of ribosome complex
Peptide bond formation
Carboxyl end of chain released from tRNA at P site → joins AA linked to tRNA at A site
Catalyzed by peptidyl transferase in large ribosomal s.u.
Large subunit translocation
Large r.s.u. moves relative to mRNA held by small s.u.
Two tRNAs are shifted to E and P sites
Keeps shifting; what was the A site becomes the P site
Small subunit translocation
Small s.u. shifts by 3 nucleotides
TRNA in E site is ejected
Cycle repeated for new incoming aminoacyl-tRNA
Elongation factors help; coupled w/ GTP Hydrolysis
*charged tRNA binds w/ A site → peptide bond formed (peptidyl transferase) → large subunit translocation → small subunit translocation
Termination
End of translation marked by STOP codon
UAA, UAG, UGA
Not recognized by tRNA; do not specify an AA
Release factors bind to ribosomes w/ STOP codon at A site
Peptidyl transferase catalyzes addition of H20 molecule rather than AA
Frees carboxyl end, releases polypeptide
Ribosome releases mRNA, dissociates into large and small subunits
Subunits recycled for new round of protein synthesis
mRNA is now a polypeptide w/in cytosol
Polysomes
Synthesis of proteins occurs on polyribosomes (group of ribosomes)
Multiple initiations take place on each mRNA molecule being translated
As soon as preceding ribosome moves out of way, new ribosome complex is formed
Helps speed up rate of protein synthesis
Post Translational
Protein folded into specific 3D shape
Structure dictates function; proper folding important
May be modified in ER (proteins, sugars added)
Sent to proper cell location (cytosol, nucleus, etc.)
Regulation of Protein Synthesis
Cell can regulate amount of protein available to cell by regulating:
Amount of transcription that occurs
Stability of mRNA transcript
Location of protein
Destruction of protein (later)
Transcription Regulation
Histones
Highly dynamic; regulated by many nuclear proteins
Chromatin remodeling complexes – reposition nucleosomes on DNA to expose or obscure gene regulatory elements (ex. Promoters)
Chromatin writer complexes – carry out histone modifications (methylation, acetylation, phosphorylation)
Histone acetylation – opens chromatin & increases transcription
Performed by histone acetyltransferases (HAT)
Euchromatin (active)
Deacetylation reverses changes; promotes chromatin condensation
Heterochromatin (inactive)
Histone methylation – promotes transcriptional activation/repression (DNA and RNA)
DNA methylation results in transcriptional silencing
MRNA stability
Longer mRNA in cytosol, more protein made via translation
Non-coders (miRNA, siRNA) promote destruction of mRNA transcript, OR
Specific proteins bind to mRNA, prevent degradation, resulting in more protein synthesis
Ex. Transferrin – brings iron into cell
If low iron levels, mRNA is stable, transferrin receptor made
If high iron levels, mRNA degrades, no receptor made
We decrease amount of transferrin protein by increasing degradation
Targeting cell location
Cell can limit protein to particular location
If needed for intracellular use, translated on free ribosomes in cytosol
If needed for nucleus, mitochondria, or peroxisomes:
Specific AA signal targets protein for correct location
If needed for lysosomes, ER, cell membrane or secretion:
Directed to rough ER co-translational transfer
Translation begins on free ribosome in cytosol
Signal peptide sequence translated, binds signal recognition particle (SRP)
Binding SPR stops translation, directs ribosome to RER, binds to SRP receptor
Translation restarts, polypeptide moves into lumen of RER, signal peptide sequence removed
If protein needs to be in cell membrane, will contain stop transfer sequence
Translation is paused when it comes in contact with translocator
Translocator discharges polypeptide into phospholipid bilayer of ER
Translation resumes until polypeptide is complete
PROTEINS + ENZYMES 1 (Pre-Learning)
Protein Classification
Shape
Fibrous Protein
Globular Protein
Long and rod-shaped
Compact & spherical
Generally has structural function
Provides strength
Generally has dynamic function
Eg. Enzymes to catalyze reactions
Eg. Carrier proteins
Often insoluble in water
Often soluble in water
Eg. Keratin, Collagen
Eg. Enzymes, albumin, hemoglobin
Composition
Simple – composed only of AAs
Conjugated – composed of protein portion + non-protein portion
Protein portion – contains only AAs
Non-protein portion – prosthetic group
Conjugated protein w/o prosthetic group is called “apoprotein”
Protein Structure
Primary
Polypeptide chain
Linear sequence of AAs
AAs held together by peptide bond
Secondary
Repeating backbone formed by H-bonds b/w carboxyl and amino groups
Alpha helix
Each COOH group H-bonds w/ amino group 4 AAs away
Rigid, rod-like structures
Beta pleated sheet
2+ polypeptide lined up side-by-side
Held together by H-bonds between distant carboxyl and amino groups
Tip points in C terminal direction
Super-secondary structure are a combo of alpha helices and/or beta-pleated sheet
Tertiary
3D structure created by side chain interactions
H-bonds
Salt bridges
Disulfide bridges
important in extracellular proteins
Strong bonds: help protect protein from denaturation during changes in blood pH or salt concentrations
Insulin held together by disulfide bonds
Hydrophobic interactions
Quaternary
Association of all multiple polypeptide subunits
Functional protein
Ex. Hemoglobin
4 subunits: 2 alpha, 2 Beta
Final 3D shape of protein dictates its function
Dimer – protein composed of 2 subunits
Oligomer – protein composed of several subunits (ex. Hemoglobin)
Multimer –protein composed of many subunits
Protomer – repeating structural unit w/in multimeric protein
Hemoglobin has a pair of αβ protomers
Protein Folding
After translation, proteins fold into secondary, tertiary, and quaternary shapes
Chaperones – proteins that help others
Fold into correct shape
Get to correct cell locations
Heat Shock Protein (HSP) is a common one
Binds and stabilizes portions of protein not yet folded
Eventually released via ATP hydrolysis
Refold proteins partially unfolded due to stress
PROTEINS + ENZYMES 1 (in class)
Protein Denaturation
Protein Denaturation
Occurs when bonds holding protein together are disrupted
Loss of protein structure (disruption in folding/shape)
Disrupts secondary and tertiary structures
Bonds w/in proteins can be disrupted, and proteins denatured, by using:
Strong acids or bases, or reducing agents:
These add or remove hydrogens
Organic solvents, detergents:
Disrupt hydrophobic, polar and charged interactions
Salts:
Disrupt polar and charged interactions
Heavy metal ions:
Ex: Mercury (Hg+2) and Lead (Pb+2)
Based on their positive charge, they can bind to negative amino acids with acidic R groups
Denature proteins by disrupting salt bridges
Have high affinity for thiol/sulfhydryl (SH) groups
Alters shape of protein
Basis of “lead poisoning”
Enzymes
Protein catalysts that speed up rxns and remain unchanged* by the rxn
Speed up by lowering Activation Energy Ea of the rxn, without changing:
Standard free energy (ΔG)
Equilibrium
Activation Energy (Ea)
Minimal amount of energy to make/break bonds necessary for rxn to occur
Also defined as amount of energy needed to reach transition state (TS)
TS = highest energy config. Formed when changing from reactants → products
Transient and not isolated
If enzyme present, aka catalyzed rxn, Ea is lowered; if no enzyme, Ea is higher.
Specificity
Enzymes = highly specific
Only substrate of correct size and shape can enter into active site
Active site – special cleft in enzymes, forms by precise quaternary structure of protein
AAs in active site participate in substrate binding and catalysis
Once inside site, substrate binds to enzyme forming enzyme-substrate (ES) complex
Induced fit model – binding of substrate induces conformational change in shape of E
Mechanisms
Induced fit b/w substrate and enzyme allows for:
Electrostatic interactions to form b/w them
Correct positioning of catalytic groups
Catalytic groups speed up rxns in two main ways:*
Acid-base catalysis
Covalent catalysis
Acid-Base Effects
Side chains of certain AAs add/remove H+’s by acting as acids or bases, making substrate more reactive
Ex. Histidine
Functions as both an acid or base (pKa of 6)
Often a catalytic acid w/in an enzyme to assist w/ acid/base effect
Adding an acid catalyst speeds up rxn, adding base catalyst speeds up rxn, adding both an acid and a base speed it up even more
catalytic AAs can either add or remove H+’s to speed up rxns
Covalent Catalysis
Nucleophilic side group in enzyme active site forms temporary covalent bond w/ substrate
Ex. Asp and Glu (R-COO-)
Ser (R-OH) and Cys (R-SH)
Only weakly nucleophilic; enhanced by presence of other AAs that can remove H
Bond forms temporarily, then goes back to normal
Role of Cofactors and Coenzymes
Cofactors and coenzymes help enzymes speed up rxns in three main ways:
Help position substrate on active site of enzyme
Stabilize negative charges on substrate or TS to make easier for nucleophilic attack to occur
Can accept/donate e- in redox rxns
Cofactors – metal cations, Mg2+, Zn2+
Mg2+ neutralizes negative charges on ATP
Helps position ATP on enzyme active site
Coenzymes – derived from vitamins
B2 – FADH2 – redox rxns
B3 – NADH – redox rxns
Can accept or donate electrons in redox rxns
B6 – PLP – transamination, decarboxylation
B9 – 1C transfers (eg. Methylation)
Cofactors and coenzymes only assist the enzyme, they don’t carry out their functions
Effect of temperatures or pH
Temperature
Optimal temperature for enzyme = temperature of organism
pH
Changing pH changes protonation state of enzyme (E) and/or substrate (S)
Disrupts:
H bonds
If H removed, no bond can be formed
If H added, bond might form where it is not supposed to
Electrostatic interactions
Will disrupt salt bridges/ionic bonds
Adding H can turn COO- into COOH, Removing H can turn NH3+ into NH2
Lysosome – intracellular enzymatic degradation for many molecules; breaks down proteins
Active at pH 4.5
Regulation
Enzyme activity controlled in four main ways
Genetic
Enzyme transcription can be induced/repressed based on cell’s needs
Induced in times of need
Enhancer regions of DNA
Repressed when in excess
Ex. Silencer regions of DNA
Other examples: transcription regulation (enhancer/silencer regions), modifying histones (chromatin remodeling and rewriting complexes, histone acetylation, histone methylation), mRNA stability
Slow process; in times of excess we increase transcription. In times of abundance, we decrease
Ex. Regular consumption of high sugar food → high insulin → increased transcription of genes for glucokinase, PFK-1, and pyruvate kinase → increased translation → higher amount of glucokinase, PFK-1, and pyruvate kinase in cytosol → more efficient conversion of glucose to pyruvate
Covalent modification
Involves altering the structure of enzyme (“proenzyme”) by making/breaking covalent bonds
Two types:
Reversible
Addition/removal of group to enzyme that causes it to convert to active or inactive form
Phosphate group commonly does this
Can activate or inactivate the enzyme
Methyl and acetyl groups also common
Glycogen metabolism
Main enzyme in glycogenesis is de-activated by phosphorylation while main enzyme in glycogenolysis is activated by phosphorylation
Catalyzed by protein kinase A (PKA)
Prevents both pathways from running @ same time
By simply adding P group, we activate one and inhibit other
Know that adding/removing P group is an example of reversible covalent modification and can either activate or inhibit an enzyme*
Irreversible
Cleavage of peptide bonds in proenzymes or zymogens to form active enzyme
Ensures enzyme isn’t used until in correct location and needed
Can be turned into an active enzyme by being turned on and off
Allosteric Modification
Binding to allosteric site changes conformation and activity of enzyme
Changes binding affinity of substrate @ active site
Have more than one subunit
Allosteric site on one, active site on another
Binding of effector molecule to allosteric enzyme can either:
Increase binding of substrate to enzyme
Effector = activator
Decrease binding of substrate to enzyme
Effector = inhibitor
Consider glycolytic enzyme PFK-1
PFK1 is inhibited allosterically by high levels of ATP
When there is a lot of ATP in cell, PFK1 will decrease its affinity for F6-P
It means we have a lot of energy, so we are going to slow down how quickly we go through glycolysis b/c we don’t need this additional energy
PFK1 is activated allosterically by high levels of AMP
High AMP increases affinity because AMP is a sign the cell is low in energy. We want to go through glycolysis faster so we have more energy
Binding AMP to PFK-1 increases it’s activity
Compartmentalization via membrane-bound organelles
Allows for regulation by:
Separation of enzymes from opposing pathways into diff compartments, and selective transportation of substrates
Ex. Two opposing metabolic pathways compartmentalized in diff. Areas of the cell are fatty acid synthesis (cytosol) and beta oxidation (mitochondria)
Creation of unique microenvironments
Lysosomal enzymes function at pH around 4.5, while most others function at pH 7
*know the categories; be able to pick out an example. Ex. What ATP binds to this enzyme, it’s affinity for this substrate increases. (Answer: allosteric); OR. If we increased transcription of this gene, what type of regulation is that? Genetic
Which of the following is an example of enzyme regulation via reversible covalent modification?
a. Phosphorylation of glycogen synthase inhibits glycogenesis
b. A high carbohydrate diet increases the transcription of the gene for pyruvate kinase
c. Cleavage of a peptide bond in a proenzyme to form an active enzyme
d. Binding of AMP to phosphofructokinase-1 increases its activity
ENZYME KINETICS
Michelis-Menten Kinetics
Determine if an enzyme is physiologically useful based on its:
Maximum rate
Max speed that enzyme can convert a substrate into its product
Affinity for substrate (or inhibitor or coenzyme…)
Basically gives an idea of how likely the enzyme is to bind a substrate and will do so even if concentration is low
If affinity is low, you will need high concentration of your substrate before enzyme can bind
Can be applied to certain rxns:
First order reactions
Concentration of a single substrate is directly proportional to rate of reaction
Straight line: [s] is proportional to rate; substrate concentration is proportional to the rate of the rxn
Many physiological rxns involve more than a single substrate
A single substrate can control the rate even when there is more than one substrate involved
Ex. You have enzyme that has two substrates, A and B
One A and many Bs
Only one B enzyme would be able to bind to A substrate
If you added more As to the solution, you will increase rxn rate
If you added more Bs, nothing would happen because we already have enough
This is a case where if you have two substrates, only one of them will affect the rate of enzyme
Called a pseudo first order rxn
Only one substrate affected the rate, but really, there are two substrates
In a fumarate to malate reaction, it is a hydration reaction
The substrate ignored in M & M kinetics is water (because adding more water would do nothing to the rxn)
In a acetylcholine to acetate and choline rxn, it is a hydrolysis rxn (using water to break molecule)
The substrate ignored again is water; acetylcholine is the single substrate that will affect the rate
Zero order rxn
Rate stops increasing even though substrate concentration continues to increase
Enzyme is already saturated so adding more substrate will not increase rxn rate
Equation
Vo: initial rate of rxn
S is high, P is low, therefore no reverse rxn takes place
Km: indication of how well the enzymes bind given a molecule
Small Km = high affinity; large Km = low affinity
Graphing S against rxn rate determines:
Vmax (max. Rate)
Km (enzyme affinity)
Lineweaver-Burk found it was not so easy to get info out of graph with that equation
Created reciprocal of M & M equation that creates a straight-line graph, easier to extrapolate
Y intercept = 1/Vmax
Extrapolated x-intercept = -1/Km
On a graph, the LOWEST intercept line is going to be the highest Vmax (because the Y axis is 1/Vmax)
On a graph, the furthest from the XY intercept line is the lowest Km value, therefore greatest affinity
Enzymes Part 2 - Kinetics
Application of Km and Vmax: Glucokinase and Hexokinase
Hexokinase (most tissue) contrasts w/ glucokinase (liver) by the following:
Hex has lower Vmax, lower Km, greater affinity for glucose
Turned off by high concentrations of glucose 6-P
Both HexoK and GlucoK catalyze Glucose → Glucose 6-P
Glycolysis breaks down glucose
Glycogenesis stores glucose
Glucokinase higher Km tells us it has lower affinity for glucose, compared to hexokinase
Hexokinase is active during fasting
Greater affinity, can convert even small amounts of glucose into energy
Glucokinase only becomes active after a high-carb meal
Lower affinity, needs a lot of glucose to be activated
Main role in glycogenesis because you want to store this excess
Liver
Where GK is found
Nutrients absorbed from intestines go here first; GK converts excess glucose from a meal to glycogen
Other tissues cant convert excess glucose to glycogen because Glucose 6-P inhibits Hexokinase (product inhibition)
Hexokinase only works until you have made enough G6-P to serve body’s energy needs
HK feeds into glycolysis; explains low Km because even though you have little glucose, HK binds to it because it has a greater affinity
Makes sense that GK has a higher Km because we do not want it storing glucose unless we have already satisfied our energy needs
HK is inhibited by G6-P because it is mediating glycolysis; once you’ve made enough G6-P to fulfill you energy needs, HK turns off and GK turns on to store as glycogen for later use. Once glucose levels fall, GK turns off because it has a low affinity for glucose
Vmax
High Vmax = high capacity to turn substrate into product
Allows GK to phosphorylate glucose to glucose 6-P after a meal to store as glycogen
Adding P to glucose means it gets trapped in the cell
Km
Helps determine enzyme efficiency
How well it binds (affinity) and how quick it converts to product
Kcat/Km
Kcat measures speed of product formation once ES has been made
Km measures binding affinity of E and S to make ES
E + S
First you have E and S bind, then you spit out product
Larger value of Kcat and smaller value of Km, the greater efficiency
Greater speed of product formation and higher affinity
Enzyme efficiency
Larger value of Kcat/Km = more efficient
Enzymes in body are close to catalytic perfection; very efficient
Carbonic anhydrase – bicarbonate buffer
Triose phosphate isomerase – glycolysis
Fumarase – CAC
Acetylcholinesterase – muscle contraction; breaks acetylcholine
Enzyme inhibition
Physiological feedback mechanisms
Ex. Turning off HK when Glucose 6-P is too high
Clinical therapies
Use of pharmaceuticals to inhibit viral enzymes from replicating in HIV
Types:
Reversible
Competitive
Reversible binding of inhibitor to active site of enzyme
Ex. Methotrexate used for chemo
Inhibits enzyme that helps convert folate B9 into its coenzyme form*
Folate coenzymes normally feed into purine/pyrimidine production used to make DNA
Helps prevent spread of cancer cells by inhibiting DNA from replicating cell; puts break on ALL cell division
Vmax doesn’t change; affinity appears worse in presence of inhibitor (higher Km) Kmapp = Km apparent
If you go high enough in your enzyme concentration in the presence of a competitive inhibitor, you basically shove it out of the way
Uncompetitive
Reversible binding of inhibitor to ES; binds in one place
Rare - Inhibitor binds enzyme after substrate has bound
Vmax is lower with inhibitor; affinity of enzyme increases
You don’t get up to the same speed with an inhibitor no matter how much of the substrate you put in, because the inhibitor can always bind no matter how much substrate you have
Appears affinity increases because you have 2 chances for E & S to be bound
Noncompetitive
Reversible binding of inhibitor to E or ES
Ex. Product inhibition – G6-P inhibition of hexokinase
Can bind in 2 places: before the substrate does, or after
Always option for inhibitor to bind, no matter how much substrate
Lower Vmax, Km (Ki in this case) doesn’t change
In sum:*
Competitive: V max doesn’t change, affinity worse
Uncompetitive: Vmax is lower with inhibitor, affinity better
Noncompetitive: Vmax lower with inhibitor, affinity doesn’t change
Binding to E appears to lower affinity
Binding to ES appears to increase affinity
Be able to identify and justify why:
Competitive, Uncompetitive, Noncompetitive
Irreversible (no longer M & M Kinetics)
Occurs when inhibitor forms covalent bond with active site of enzyme
Compared to competitive inhibition, the difference is the type of bond; they are still binding in the same place
Ex. Penicillin – blocks enzyme required for synthesis of bacterial cell walls
Lead poisoning – Lead binds to SH groups, changes shape of hemoglobin so no longer functions. Structure dictates function
Inhibition of allosteric enzymes
Multi-subunit Allosteric Enzymes
Allosteric means you have a site other than the active site you are going to bind
Binding place that is NOT the active site
Initially it is hard for substrate to bind. Once it does, it will change the shape of the remaining allosteric subunits to make it a little easier, so the second substrate can bind. Then once the second one binds, it changes the shape again so that the 3rd one binds the fastest.
Graph would be an S shape graph (Sigmoidal graph)
Inhibitor produces a right shift
Ex. PFK-1
Catalyzes Fructose 6-P to Fructose 1,6 bisphosphate
Creates sigmoidal graph
Allosterically inhibited by ATP
Means it is binding somewhere other than the active site
Inhibited means ATP would turn this off
Why? Kinase uses ATP. ATP is also a substrate because for this enzyme to work, you have two substrates (F6P and ATP) both have to bind
This is saying, in addition to being a substrate, it is an inhibitor. So in addition to finding an active site there will be an allosteric site
Usually the point of making the F 1,6BP is for energy (glycolysis). It is product inhibition.
ATP binds active site and allosteric inhibitor site
When binding to active site it is a substrate, better affinity, lower Km
When it binds to allosteric site, it is an inhibitor, lower affinity, higher Km
CLINICAL PHYS III January 27, 2023
Cardiac Exam
Chambers
Left atrium
Right atrium
Left ventricle
Right ventricle
Interventricular septum
Thick, muscular wall that separates the left and right ventricle
Vessels
Pulmonary trunk, left & right pulmonary arteries
Aorta
Superior and inferior vena cavae
Pulmonary veins
Valves
Atrioventricular valves – prevent backflow
Larger, floppy
Anchored by chordinae tendinae
Keeps them from flopping back (prolapse) into atria during ventricular contraction
Tricuspid valve (R), Mitral valve (L)
When open, the phase that involves the ventricle receiving blood is ventricular diastole
Atrium contracting into ventricle
Corresponding semilunar valve should be closed
Semilunar valves
Smaller, tighter
Do not require chordae tendinae
Pulmonary SV (R), Aortic SV (L)
b/w R Ventricle & pulmonary arteries; b/w L ventricle & aorta
When open, phase is ventricular systole; ventricles contract, increase pressure
Tricuspid and Mitral should be closed
Anterior Surface vs. Posterior Surface
Anterior – Major structures
Part of right atrium (auricle)
Right ventricle
Tip of left ventricle
Easiest place to palpate heart
Point of Maximal Impulse PMI
Superior, lateral side of left ventricle
Left coronary artery
Good landmark
Underneath is interventricular septum
Both SV valves anterior (in front of) to AV valves
Posterior Surface (mainly left side of heart)
Surface Anatomy
Using palpable anatomical landmarks during physical exam
Correlates sound w/ deep anatomical structure
Correlates palpation finding w/ anatomical structure
Where is:
The right border of the heart and what cardiac structures form it? Right atrium
The inferior surface of the heart what structures form it? Left ventricle
The left border of the heart what structures form it? Left atrium
The base of the heart = where the great arteries emerge from the superior aspect
Base is top, apex is bottom
Key locations for auscultation and palpation:
2nd intercostal space, left sternal border
Corresponds to the pulmonic valve
2nd intercostal space, right sternal border
Corresponds to the aortic valve
4th/5th intercostal space = right sternal border
Best place to hear sounds from the right ventricle and right AV valve
5th intercostal space = mid-clavicular line
*Best place to: hear left AV valve and left ventricular sounds, & palpate the PMI
Apex also here
Pulmonary goes up to left, aorta goes diagonally up to right
Systemic Pressures and the Cardiac Cycle
Heart sounds are caused by the
Closing of the atrioventricular valves (mitral on left)
Closing of semilunar valves (aortic valve on left)
Hardest pressure is what happens in atria (only during ventricular systole the pressure is higher than the atrium)
Systemic Pressures – Left Atrium
Systemic Pressures – Aorta
Systemic Pressures – Left Ventricle
Av valve opens as soon as pressure in ventricle is lower than pressure in atria
Semilunar valve opens as soon as pressure of ventricle higher than atria
Blood goes through aorta during ventricular systole
Types of heart sounds = hear the valves closing
Lub = AV valve closing - low frequency (big floppy valve)
Dub= semilunar valve closing – higher frequency (smaller tighter valve)
Uncommon to hear valve opening
Laminar flow= smooth orderly blood flow
Turbulent flow= rapid disorderly flow à valve abnormalities = murmurs, extra heart sounds
Valvular abnormalities
Stenosis= valve doesn’t open wide enough
Higher pressure needed to push blood through narrow valve
Higher pressure= turbulent flow = murmur
Noise = blood flowing across valve when it should be open
Sometimes valve scars over time dur to physical stresses --> narrowing
Regurgitation= valve doesn’t close fully
Backflow in chamber before it relaxes = turbulent flow = murmur
Noise= blood flowing across valve when it should be closed
Damage to heart valves can make them unable to fully close
Week 4 -PHYSIOLOGY CONCEPTS 1 (in class Jan 30, 2023)
The Cell Membrane – General Structure
Each component has unique function
Lipid components:
Glycerophospholipids
Fatty acid tail, 16-18 carbons (usually even number of Cs) Hydrophobic
Unbranched, may or may not be saturated
Glycerol backbone (part of hydrophilic head)
Ester linkage to FA tails
Phosphate “head” (hydrophilic)
R usually linked (ester) to another molecule
Choline (most common)
Ethanolamine, glycerol, inositol, serine
Cholesterol
Type of steroid
Found between phospholipids (with –OH closest to aqueous interface)
Impacts membrane fluidity
Smaller amounts stiffens membrane, decreased fluidity
Larger amounts interfere with interactions b/w lipid tails; increases fluidity
Sphingolipids
Different structure; sphingosine backbone, NOT glycerol
Diff shape can decrease membrane fluidity
Have sugar residues that:
Cellular signaling – formation of lipid rafts and myelin
Contribution to glycocalyx
Proteins (glycoproteins) and lipids bound to carbs that vary in size
More notable on external surface of cell membrane
Protective structural and signaling functions
Glycosphingolipid structures
Sphingolipid has sphingosine or ceramide backbone, and often we see carbohydrates attach*
Glycerol backbone has 3 carbon glycerol, sphingolipid has sphingosine
Structure and Function – Membrane Lipids
Amphipathic nature of lipids can form micelles or bilayers
Both thermodynamically favorable
Hydrophobic tails interact w/ each other and hydrophilic heads interact w/ aqueous environment
As concentration of phospholipids increase, bilayer formation more favourable than micelle
Bilayer is effective barrier to:
Charged and polar molecules (can’t pass through hydrophobic tail)
Medium-sized and large non-polar molecules (can't get through hydrophilic top layer)
Ineffective barrier to small, non-polar molecules
Integrity of plasma membrane is key to normal function of cell
Ionic and fluid homeostasis
Keeps vital molecules needed for cellular metabolism within the cell (ex. G6-P trapped in cell in glycolysis)
Cell movement and shape accomplished w/ interactions b/w cell membrane and cytoskeleton
Membrane system – plasma membrane, endomembrane, membrane-bound organelles – unique functions
Membrane = most important way these functions remain sequestered with these organelles
Loss of membrane integrity --> threatened cell survival
Cell membrane can reseal with MINOR mechanical disruptions
CANNOT reseal with MAJOR disruptions or stresses (like loss of ATP); leads to degradation
All membranes made of phospholipids and a bit of cholesterol
Membranes INSIDE the cell don’t need to:
signal to other cells
Protect cells from harsh environments
Form a glycocalyx
Therefore, no need for sphingolipids
Much less cholesterol in membranes of organelles
With exception of the ER (because all synthesis happens there, makes sense to have more cholesterol and sphingolipids)
Membrane Proteins
Wide range of functions
Signaling
Transport & general homeostasis
Protection
Structure & movement
Cellular Homeostasis
Diffusion
Movement of molecules from region of high to low concentration (no energy needed; spontaneous)
Thermodynamics --> spreading molecules (energy) as they collide against each other
Aspect of Gibb’s free energy? Entropy (S)
To keep molecules close (keep difference in concentrations) we have to do work (energy)
Osmosis
Diffusion of water through semi-permeable membrane
Allows water to pass through, but impermeable to at least one solute
U-tube
filled only w/ water and membrane is permeable to water but nothing else
Water free to move across membrane, no difference b/w concentration across it
Levels are equal
If you add sugar, membrane is impermeable to sugar, water levels rise on side w/ sugar
Region of lower solute to region of higher solute, so water balances out concentration of solutes; drives up surface of the water; doesn’t overflow because water will flow over, either until we have balanced out concentration of solutes on both sides OR we have balanced out hydrostatic pressure (pressure of gravity balanced against osmotic pressure – which is the pressure of water flowing through to the other side with osmosis)
If molecule moves from high to low concentration, it does NOT require energy; it is spontaneous
Relevance – Diffusion and Osmosis
Cell membranes are semi-permeable – water and certain substances can traverse them
Most cells express many aquaporins in the plasma membrane --> high water conductance
Membranes are impermeable to many solutes, esp larger ones, charged ions, etc.
Interior of cell has higher concentration of large solutes than exterior, so plasma membrane must expend energy to regulate solute concentration and cell volume
If interior has higher concentration than exterior, by osmosis, water will flow inside the cell, causing it to expand. Could burst if too much, so cell must regulate it.
Plasma membrane and organelle membranes rely on concentration gradients for important signaling and metabolic functions
Importance of the Sodium Potassium pump **** Na+/K+ ATPase
KEY plasma membrane transporter
Each cycle of transport involves:
3 Na+ OUT of cytosol, INTO extracellular fluid
2 K+ INTO cytosol, OUT of extracellular fluid
Hydrolysis of one ATP --> ADP + Pi
Responsible for up to 30% of ATP use in cells (expensive energetically; demonstrates importance of having ATP in cell)
Establishes gradient of Na and K across the membrane
Prevents cell swelling due to osmosis (we are sending more solutes out than in)
As soon as ATP falls to 10% of normal levels, cells start to swell
Because if we don’t have ATP, the pump stops working
Establishes gradient of charge across membrane
Na gradient can be used to transport other substances across membrane
We end up w/ high Na gradient externally, and high K gradient internally
Important cell signaling events depend on movement of charged particles (Na, K, etc.) across membrane
Changes the charge across membrane; can change cellular activity
Often sodium moves w/ it’s gradient and glucose against, creating a charge. Externally will be more positive than internally
Intercellular and Extracellular Fluid (ICF, ECF)
Na+; low ICF, high ECF
K+: high ICF, low ECF
Cl-: low ICF, high ECF (always mimics Na+)
Ca+2: very low ICF, higher ECF
HCO3: slightly higher ECF
Protein: higher ICF than ECF
Na, Cl, Ca, HCO3 have LOWER ICF
Only Potassium (K) and Protein have HIGHER ICF
Since Na in ECF is high, there is a diffusional force that drives it INTO the cell
can be used to transport other substances into or out of cell (co- or counter transport)
Transport and the Cell Membrane
Type of transport
Process
Example
Active
transport
A protein moves a substance(s) across a membrane against a concentration gradient using ATP
Na+/K+ ATPase
Sod Potass Pump
Passive
transport
A protein forms a channel that allows a substance across the membrane, along its concentration gradient
Aquaporins
Facilitated
transport
A protein carrier binds to a substance and transports it across a membrane, allowing it to follow its concentration gradient
Glucose transporter
(GLUT)
Co-transport
The transport of two substances (X and Y) are coupled using the same protein. The concentration gradient of X favours movement into the cell – Y is “pulled” along, even if the gradient for Y does not favour cell entry
Sodium-glucose co-transporter
(SGLT-1 and -2)
Counter-transport
X and Y move in opposite directions across the cell membrane – the gradient of one of the molecules supplies the energy to drive the transport
Cl-/HCO3 counter-transporter
Active – requires ATP because we are moving something against the gradient
Facilitated transport - key is that it is moving with the gradient with the help of a protein carrier
Co transport – essentially moving X down gradient, and Y AGAINST gradient, but moving in same direction
Ex sodium moves down gradient (high to low), glucose moves down as well even going low to high
Uses concentration gradient of another molecule for energy
Counter transport – still coupled to same protein. We move bicarb out, chloride in. Chloride moves with its gradient, helping bicarb against the gradient.
Difference w/ Co and Counter is: whether X and Y are going in same direction (co) or opposite (counter)
In either case, X is always moving down it’s gradient, and Y against. Energy of X moving down its gradient that supplies Y moving against.
Transport can be passive
Passive = always down gradient
Molecules move across membrane from high to low, driven by diffusion*
Channels or transporters required unless molecule is small and hydrophobic (ex. CO2, O2)
If it is a protein carrier it is facilitated transport
Transport can be active – energy required
Energy from hydrolysis of ATP
Active transport if ATP
Energy from gradient of another molecule
Co or counter transport
Requires integral membrane protein
Plasma Membrane and Signaling
Extracellular signals
Growth factors and anti-growth signals
Signals that change cell activity (hormones, neurotransmitters)
Most involve binding of a chemical (the signal) to a high-affinity protein receptor on the cell membrane, activates signal cascade
Most receptors have:
Hydrophobic domains that extend through lipid bilayer (alpha-helix)
Extracellular domain that binds to message (I.e. hormone)
Intracellular domain that amplifies the signal
Typical Extracellular Signal
Whether the cell responds to the signal (hormone) depends on whether it expresses the receptor
know that our cells have receptors that combine to extracellular signals and PROMOTE an intracellular cascade
Membrane Proteins – Structure
Important to overall structure of cell
Can link cell membrane to important extracellular structures
Can link cell membrane to cytoskeleton
Membrane proteins that link cell to extracellular structures are known as junctions
Tight Junctions
Separate cells into apical and basal compartments
Can be selective (leak-proof) or less selective (leaky)
regulated by cell; ex in digestive system
Purpose – commonly regulates movement across membranes and other epithelial structures
Between two cells
Anchoring Junction
Desmosome
Intracellular component – plaque formed associated w/ cadherins
Intermediate filaments bind to plaques
Between two cells of a tissue* (vs. Hemi-desmos between a cell and a membrane)
Extracellular component – cadherins on one cell interact w/ cadherins on neighboring
Purpose – structural integrity
*know it has intermediate filaments and cytoskeleton, and between cells it interacts w/ cadherins
Difference b/w tight junctions and desmosomes is that TJs can be regulated. Desmosomes are for structural integrity*
Hemi-desmosome
Similar to desmosome, but extracellular component involves protein integrin (instead of cadherin)
Plaque still binds to intermediate filament
Integrin commonly binds to basement membrane
Usually how epithelial cells stay anchored to basement membrane and underlying connective tissue
Adherens Junction
Contain plaque, may connect to:
Another cell – cadherins
basement membrane – integrins
do NOT connect w/ intermediate filaments
Connect w/ microfilaments formed from actin
*Desmosomes and Hemidesmosomes both interact w/ intermediate filaments. If interacting with another cell, desmo uses cadherins, hemi interacts w/ integrins. Adherens don’t interact w/ intermediate filaments, but microfilaments
Integrin = basement membrane
Cadherin = connecting two cells
Cytoskeleton
Cellular movement
Important for cells that move through space (macrophage, fibroblast) or can shame shape, perform work (muscle cells)
Organization of cellular components/organelles
During mitosis, meiosis (interaction of DNA)
Shuttling of organelles and membrane-bound vesicles (ER-->golgi-->vesicles)
Cellular structure
Strength (skin, keratin), shape (absorptive surfaces – actin builds high surface area)
Communication
Intracellular signals that regulate growth, general function
Structures:
Microtubules
Trafficking of organelles and cell division
Organization of overall cellular structure
Cellular movement
Molecule = tubulin (tubulin forms tubules, tubular structure)
Green in photos
Microfilaments
Cellular movement
Structural organization of plasma membrane
Molecule = actin
Red in photos
Intermediate filaments
Overall structural integrity (almost exclusively)
Variety of molecules – keratins, desmin
Blue in photos
Features:
Dynamic
Filament subunits (monomers, heterodimers) are constantly building themselves into polymer strands
Once monomers reach critical concentration and interact w/ nucleating factors --> polymer formation
Filaments can change in length; important in their function of movement
Microtubules and microfilaments (not intermediate filaments; stable for structural integrity)
Tightly regulated
Filament assembly and disassembly regulated by huge array of intracellular signals
Govern structure and formation of polymers
Can generate force
Move cell or organelles along axis of strands
Generate power or motion in cells through contraction
Particularly actin
Microfilaments - Actin
Monomer – G-actin; polymer – F-actin
2 F-actins together are a microfilament
*know that the stability of actin is dependent on a lot of proteins. If G actin is ATP, more likely to stay on. If ADP, more likely to fall off. Factors that can promote that. Quite modifiable and regulated
Nucleating factors stimulate formation of F-actin
When F-actin formed, spontaneously degrades (dynamic feature)
G-actin has ATP bound
Overtime, G-actin hydrolyzes ATP to ADP, makes it more likely to fall off F-actin strand
When its added, it is always as G-actin bound to ATP. Once the ATP is hydrolyzed into ADP, then G-actin is more likely to fall off the polymer
Stability of F-actin depends on:
Concentration of G-actin
“caps” that can be applied to F-actin that prevent disassembly
Tropomodulin, capping proteins
Capping proteins help prevent G-actin from falling off
Proteins that speed up or slow down rate that G-actin hydrolyzes ATP
if we speed up G Actin ATP hydrolysis, we speed up the breaking up of our filament (ADP more likely to fall off). If we prevent hydrolysis, less likely to fall off.
if it hydrolyzes ATP to ADP, it will break down
Nucleating factors or inhibitory factors that modify the formation of F-actin (I.e. formin)
In cellular structures:
Muscle cell
Needs stable, organized system of parallel fibers
Generates force
Long-lasting, precisely organized, strongly anchored to cell membrane and other proteins
Fibroblast
Needs to ‘crawl’ to area to deposit collagen/ECM; explores
Organizes actin filaments into meshes, fibers, and filopodium (feelers) under cell membrane
less stable in shape
Microtubules
More complicated than actin; share similar features
Protein monomer = tubulin
Alpha and beta form dimers
Organize in helical tube
Beta monomers hydrolyze nucleotide triphosphate
Tubulin cleaves GTP to GDP + Pi
After GTP cleaved, dimer falls off microtubule, falling apart
Dynamic instability
When tubulin (beta) is bound to GDP, more likely to fall off; when bound to GTP, more likely to stay on
Actin associates with ATP, tubulin with GTP
Microtubules and microfilaments have a lot of same characteristics; both dynamic
Important for:
Cell organization – formed from microtubules organizing center (MTOC)
Composed of two centrioles that form centrosome (where all microtubules originate)
Helps determine polarity of cell (if cell has side that faces lumen or basement membrane)
Cellular movement
Form cilia and flagella
Cell division
MTOC splits/pulls chromosomes to new daughter cells
Signaling – primary cilium
Found on most cells
Important for sensing stimuli in extracellular environment (think of whisker)
Microfilaments, Microtubules, Cellular Motors
F-actin and microtubules form network of dynamic filaments that ‘molecular motors’ can move along
F-actin --> myosin is a protein that can walk and advance along microfilaments
Multiple diff types of myosin exist
In muscle, myosin movement along an actin scaffold causes entire cell to contract
Microtubules --> dyneins and kinesins are other proteins that can move along microtubules and cause whipping movements of cilia and flagella
Dyneins and kinesins move opposite directions along microtubules
Use ATP to move along cytoskeleton; usually drag a structure along with them
Microfilaments promote movement of entire cell; microtubules promote movement of organelles w/in a cell
Both allow for movement, but need motors
*Things to know: Know monomer and polymer structures of actin and microfilaments. Know general functions of the cell. Know the motors they associate with. *
Intermediate Filaments
Commonly confer stability to cells
More diverse than actin and tubulin
70 diff genes
Long proteins w/ alpha helix that coil around monomers to form dimers
Dimers coil around each other as well; coiled coil
More stable than actin or tubulin
Don’t hydrolyze GTP or ATP, so don’t dissociate as readily
Change their structure in response to cellular needs
Types:
Lamins
Network under nuclear membrane
Keratins
Hair, nails; strong; limit water permeability
Vimentin,* (know it is a type of intermediate filament)
Neurofilaments; found in neurons
Note:
actin filaments are responsible for shape of microvilli
Desmin (intermediate filament) organized to provide strength across the cell
Microtubules have + and – end. + closer to nucleus, - closer to ends. Helps dictate shape
Build a table for the three skeletal elements: talk about monomers, functions, dynamic, stability. Know some general common functions, types, and features
Phospholipids – Post learning
Lipid broken down into two main classes:
Fatty acid structure
Isoprenoid structure
Fatty Acids
phospholipids
Hydrocarbon chain
Backbone
Glycerol
Phosphoglycerides
Phospholipids w/ glycerol backbone
Phosphatidate - if both fatty acids are linked to glycerol w/ ester link
Basic structure = phosphatidic acid
Two fatty acid tails, glycerol backbone w/ ester linkages, phosphate alcohol head group (OH) (glycerophospholipid)
Plasmalogen – if FA is linked to glycerol w/ an ester link, while the other has an ether link
*phophatidate more common
Ceramide
Sphingomyelin
Phosphate-alcohol head group
*Most Common Phospholipids in Cell Membrane
Phosphatidylethanolamine
Basic phosphatidate structure, but the sepcific head group is ethanolamine
Phosphatidylserine
Serine headgroup
Phosphatidylcholine
Choline headgroup
Looks like ethanolamine w/ three additional CH3 groups
Sphingomyelin (classified as both phospholipid and sphingolipid)
Ceramide backbone (instead of glycerol); also classified as a sphingolipid
Phospholipid Synthesis
Occurs on luminal surface of smooth ER and inner mitochondrial membrane
Basic steps of synthesis: (1 & 2 shared with triacylglycerol synthesis)
Make glycerol backbone
Attach fatty acids to backbone via ester linkage
Addition of head group
Exchange/modification of headgroup
Steps 1 & 2
Glycerol-3-phosphate is starting molecule
Derived from addition of P group to glycerol; only occurs in liver
Conversion of Glycerol-3-P from dihydroxyacetone phosphate (DHAP)
DHAP is an intermediate from glycolysis (and gluconeogenesis)
Two fatty acyl CoAs added to Glyc-3-P forming phosphatidic acid
Often FA C1 is saturated, C2 unsaturated
Step 3 – addition of headgroup
Activation of hydroxyl group by attachment of nucleotide, cytosine diphosphate (CDP)
Starting molecule is phosphatidic acid, attached to hydroxyl group, forms CDP
CMP displaced via nucleophilic attack and replaced by headgroup
Start with CDP-diacylglycerol, end up with glycerophospholipid
Example: start with CDP-diacylglycerol; Serine comes in and displaces CMP (nucleophilic attack) to form phosphatidyl serine
Another possibility is addition of CDP-headgroup to diacylglycerol (no phosphate group)
Staring with diacyl glycerol, CDP head group comes in, CMP leftover, phosphatidyl choline made
Step 4 – Modification/Alteration of headgroup
Phosphatidylserine and phosphatidylethanolamine interconverted in reversible head-group exchange reaction
Phosphatidylethanolamine can be converted to phosphatidylcholine by addition of 3 methyl groups
Donated by SAM; denotated as adoMet; need vitamin B9 and B12
Need 3 SAM molecules
Phosphatidylethanolamine and phosphatidylcholine are similar, only difference is methyl groups
Question: What nucleotide is used to help w/ addition of head group to Phosphatidic acid? Cytosine.
Phosphatidylinositol
Less common phospholipid in cell membrane, but plays important role in signaling
Next Steps:
Phospholipids need to get to other side of ER and to the cell membrane
Require special enzymes, flippase, to translocate across ER membrane to the cytosol
Polar phosphate alcohol head group cant flip over through the non-polar hydrocarbon chain center of our cell membrane. We require flippase to translocate the phospholipd
Pieces of sER membrane contain new phospholipids will bud off and inserted into plasma membrane
Cholesterol - Post –Learning
Isoprenoid
contain repeating 5C structural units called isoprene units
make up cholesterol
Cholesterol
Steroid; made from 6 isoprene units
Four fused rings w/ various substituents
If hydroxy group at C3, referred to as sterols (ex. Cholesterol)
Cell membrane contains large amounts of it (in addition to phospholipids)
Synthesis
Acquired through diet and synthesis w/in body
Most cells can perform cholesterol synthesis, but occurs mainly in liver and kidney
Inside cell, occurs in ER
Four steps:
Condensation of 3 acetyl CoA into mevalonate
Rate limiting step catalyzed by HMG CoA reductase (main regulatory enzyme in cholesterol synthesis pathway)
Statin medications work to lower cholesterol by blocking HMG enzyme
Requires NADPH (B3) generated by PPS
Formation of isopentenyl pyrophosphate (conversion of mevalonate into isoprenes)
3 P groups added to mevalonate to form ATP to form isopentenyl pyrophosphate
1 is immediately removed
3 ATP required to convert meva to delta3-isoP PyroP
*just know that mevalonate is converted into activated isoprene (isopentenyl pyrophosphate) with the addition of ATP
Creation of squalene
Condensation of 6 activated isoprene units to form squalene
Needs 18 acetyl CoA (each isoprene needs 3, 3x6=18); requires another NADPH
Formed from isomerization of Isopentenyl pyrophosphate
Uses 6 isopentenyl pyrophosphates
Cyclization of squalene into cholesterol
Ring closure; linear squalene converted to cyclic cholesterol
Requires another NADPH
Don’t need to know every structure, pathway or enzyme. Just know* the ones bolded and outlined in the red
Regulation
HMG CoA reductase is main regulatory step of cholesterol synthesis
Integral membrane protein in smooth ER
Inhibited by high intracellular levels of cholesterol (if we already have a lot, we don't need more)
Block transcription of HMG reductase gene
Gene will only be transcribed and translated if intracellular levels of cholesterol are low
High intracellular cholesterol levels promote esterification of cholesterol for storage in cell
HMG can also be regulated by reversible covalent modification
Exists in its inactive phosphorylated state
Insulin promotes de-phosphorylation of HMG, activating it, promoting cholesterol synthesis
Glucagon promotes phosphorylation of HMG, inhibiting it, inhibiting cholesterol synthesis
Sum: highly regulating enzyme catalyzing the rate limiting step of cholesterol synthesis is HMG CoA Reductase. Insulin activates this enzyme, glucagon inhibits it. High intracellular cholesterol levels will decrease transcription of HMG
PHYSIOLOGY CONCEPTS II
Flow Down Gradients – pre learning
Flow Down Gradients
Flow = movement of substance from one point (A) to another (B)
Measured by amount of substance (volumes, moles, charge) that moves over time (seconds, minutes)
Fluid measured in volume for example
Electrical particles it is the charge difference
Driving force for flow is energy gradient b/w point A and B
Amount of flow directly related to size of energy gradient
Greater gradient = greater flow
Every system has factors that resist flow
Important b/c life depends on movement of substances from one point in body to another
Fluids and gases constantly moving from A to B
Ex – flow of gases and fluids through “large tubes” determined by certain variables
Poiseuille's Law
Ex – molecular flow of gases, water, and solutes can be drive by diffusion, electrostatic interactions, or pressure gradients
Fick’s law, Ohm’s law
Movement of Gases and Liquids Through a Vessel
Can be described w/ the following parameters:
Hydrostatic pressure causes gas or liquid to flow from A to B
Physical structures resist flow (resistance)
Dimensions of tube that substance flows through
Substance characteristics that impact flow
Viscosity of fluid
Sometimes nature of the gas or liquid can aid flow or resist it
Rate of flow determined by Poiseuille’s law
Poiseuille’s Law
F = flow
Volume of liquid that passes through tube per unit time (I.e. ml/min)
P = hydrostatic pressure
Force that substance exerts on walls of container
If pressure increases, flow increases
R = radius
radius of tube that fluid moves through
If radius increases, flow increases
Resistance is inversely related to the 4th power of the radius
Resistance to flow increases if radius of vessel decreases
L = length of tube
If length increases, flow decreases
Fluid has to travel further; leads to reduced flow from friction
M = viscosity of fluid
If viscosity increases, flow decreases
The PRESSURE GRADIENT is the force that drives fluid movement per Poiseuille’s Law, not flow
For equations, if there is F on one side, and multiplication/division on the other side of the equation, it is an inverse relationship. If something in the denominator increases, it will decrease flow. For the numerator, as it increases it will increase flow.
In Sum:
Flow (of blood or lymph through vessels, air through airway) can be affected by:
difference in hydrostatic pressure b/w two points in tube/vessel
Cross-sectional size of tube/vessel (radius)
Biggest impact*
if tube gets small, resistance goes up, flow goes down
Distance b/w 2 points in tube (l)
How viscous the flowing substance is
For respiratory tract and CV system, clinically relevant to think of flow of gas or blood to tissues (mL/min)
Body controls flow through vessels by:
Controlling pressure in large vessels
Controlling radius of small vessels
Caveats:
Only accurate for rigid, simply-shaped tubes with non-turbulent fluid flow
Branched or irregularly shaped tubes make it harder to quantify resistance
Turbulent flow changes resistance
If tube flexible, like artery, law is not exact
For all the above, radius of tube is still most important determinant of resistance
Simplified equation that includes a measured (not calculated) resistance can also describe flow:
Ohm’s law is more for cardiovascular physiology
Diffusion
Movement of solute or gas from area of high to low concentration
usually occurs across a barrier composed of membrane
Fick’s Law
Quantifies how rate of diffusion is affected by various parameters:
Flow = flux = amount of solute moving across barrier per unit time
almost always a cell membrane
Force driving flux --> concentration gradient (C2 – C1)
Difference in concentration on either side of membrane
Diffusion isn't the force, concentration is the force.
Resistances:
Membrane surface area, membrane thickness
Permeability of membrane to the substance
Remember, inverse relationship to what is the in the denominator (t); if ‘t' increases, flux decreases
Which of the following will decrease flux via diffusion if it increases?
Surface area
Size of molecule
Solubility of molecule
Permeability of molecule
Summary:
Flux of solutes through capillaries, substances through cell membranes, O2 and CO2 from alveolus to blood… can be affected by the:
Concentration difference
Surface area available for solute/gas to cross
Permeability of the membrane
Solubility and molecular size of substance
Distance between two compartments
Tissue/cellular structure has adapted to meet the constraints of Fick’s law
Thickness of membrane/barrier to diffusion needs to be very small (less than .1mm)
membranes have adapted by creating many channels or transporters that increase permeability and solubility of a membrane
the need for channels/transporters depends on solubility of substance in membrane
Cells that are specialized for transporting large amounts of solutes have:
more transporters
structural features that increase surface area : volume ratio
Villi in intestines increase surface area
Our bodies manipulate concentration gradients all the time
Metabolism
Transporters that Increase gradients
Ex. Sodium potassium pump increases gradients all the time
Caveats
Mathematically accurate for gases diffusing across fluid barriers and close enough for other situations
Saturation of protein transporters will reduce flux
In most physiological situations diffusion happens so quickly that we don’t worry too much about the rate of flux
diffusion failure is a common theme in disease
Ohm’s Law
Movement of a dissolved, charged particle (ex. An ion) across a barrier (membrane) depends on:
Charge of the particle
Difference in “concentration” of charges across membrane (gradient known as voltage)
Type of potential energy --> how much work it takes to move charged particle through an electrical field
Permeability of membrane to charged particle
Defined as: rate of flow charges across membrane known as current (I)
Voltage – increases when space between particles is small, and where there are a lot of charged particles in the same area. Works over tiny distances like a cell membrane.
Summary:
Opposites attract – like charges repel
Particles move down a gradient of voltage according to their charge
Electric field of charged particle is responsible for establishing voltage
Resistance is anything that impedes the movement of the particle
In bio, Ohm’s law is most useful when thinking about unequal distributions of charges very close on either side of a membrane
Overall positive and negative charges are balanced in all physiologic compartments
Eletric field declines very rapidly as charges are separated by distance
Flow Down Gradients – in class
Combining Forces
Many situations where more than one force acts on the same substance
Filtration through a capillary --> diffusion and hydrostatic pressure
Distribution of ions across a membrane --> diffusion and electrostatic forces
Often these forces “pull” or “push” the same substance in opposite directions
Starling Forces
A capillary transports substances to and from tissues
Water -->
Hydrostatic pressure
Diffusion
“Everything else”
Diffusion
Protein-mediated transport
Endocytosis
Describe the movement of water, NOT the movement of everything else
Look at slides
Forces are difficult to measure experimentally
Value of the variables in different situations and in different locations is the subject of much debate
Flux vs. Flow
Flux = flow along defined membrane surface area
Describes tissue swelling in a wide variety of situations
Inflammation/infection
Changes in pressure w/in circulation
Nernst Potential
Charged particles can move across membrane based on electrostatic forces
Energy “powering movement along the gradient? Energy is voltage
Dissolved particles can move across a membrane based on their concentration gradient
Energy “powering movement along the gradient? Energy is diffusion gradient
We are looking at Ohm’s and Fick’s Law; we are talking about very close to surface of membrane
Equation for Nernst potential accounts for the following:
Diffusional forces and electrical fields are very small at large distances
Distribution of ions very close to either side of the membrane
Charge of the particle
Ration of particles concentration intracellular : extracellular
It does not include flow of ions (current) or resistance of the membrane to flow
Gives the energy gradient
we don’t care about flow. Its going to happen. We care about balance